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Chemical Biology

Edited by
Stuart L. Schreiber,
Tarun M. Kapoor, and Cunther Wess

Volume I
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Chemical Biology

From Small Molecules to Systems Biology

and Drug Design

Edited by
Stuart 1. Schreiber, Tarun M. Kupoor,
and Cunther Wess



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ISBN 978-3-527-31150-7

Preface XV

List of Contributors XVll

Volume 1

Part I chemistry and Biology - Historical and Philosophical Aspects

1 Chemistry and Biology - Historical and PhilosophicalAspects 3

Gerhard Quinkert, Holger Wallmeier,Norbert Windhab,and
Dietmar Reichert
1.1 Prologue 3
1.2 Semantics 4
1.2.1 Synthesis - Genesis - Preparation 4
1.2.2 Synthetic Design - Synthetic Execution 8
1.2.3 Preparative Chemistry - Synthetic Chemistry 9
1.3 Bringing Chemical Solutions to Chemical Problems 10
1.3.1 The Present Situation 10
1.3.2 Historical Periods of Chemical Synthesis 12
1.3.3 Diels-Alder Reaction - Prototype of a Synthetically Useful
Reaction IG
1.4 Bringing Chemical Solutions to Biological Problems 18
1.4.1 The Role of Evolutionary Thinking in Shaping Biology 18
1.4.2 On the Sequence of Chemical Synthesis (Preparation) and
Biological Analysis (Screening) 20
1.5 Bringing Biological Solutions to Chemical Problems 45
1.5.1 Proteins [99] 45
1.5.2 Antibodies 52
1.G Bringing Biological Solutions to Biological Problems 53
1.7.1 The Fossil Fuel Dilemma of Present Chemical Industry 54

Chemical Biology. From Small Molecules to System Biology and Drug Design
Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Cunther Wess
Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
vi 1 Contents

1.7.2 Two Lessons From the Wealth of Published Total Syntheses 55

Acknowledgments 58
References 59

Part II Using Natural Products to Unravel Biological Mechanisms

2 Using Natural Products to Unravel Biological Mechanisms 71

2.1 Using Small Molecules to Unravel Biological Mechanisms 71
Michael A. Lampson and Tarun M . Kapoor
Outlook 71
2.1.1 Introduction 71
2.1.2 Use of Small Molecules to Link a Protein Target to a Cellular
Phenotype 72
2.1.3 Small Molecules as Probes for Biological Processes 77
2.1.4 Conclusion 89
References 90

2.2 Using Natural Products to Unravel Cell Biology 95

Jonathan D. Gough and Craig M . Crews
Outlook 95
2.2.1 Introduction 95
2.2.2 Historical Development 95
2.2.3 General Considerations 96
2.2.4 Applications and Practical Examples 96
2.2.5 Future Development 109
2.2.6 Conclusions 109
Acknowledgments 110
References 110

3 Engineering Control Over Protein Function Using Chemistry

3.1 Revealing Biological Specificityby Engineering Protein- Ligand
Interactions 115
Matthew D. Simon and Kevan M. Shokat
Outlook 115
3.1.1 Introduction 115
3.1.2 The Selection of Resistance Mutations to Small-moleculeAgents
3.1.3 Exploiting Sensitizing Mutations to Engineer Nucleotide Binding
Pockets 126
3.1.4 Engineering the Ligand Selectivelyof Ion Channels 130
3.1.5 Conclusion 134
References 136
Contents 1 vii

3.2 Controlling Protein Function by Caged Compounds 140

Andrea Giordano, Sirus Zarbakhsh, and Carsten Schultz
3.2.1 Introduction 140
3.2.2 Photoactivatable Groups and Their Applications 140
3.2.3 Caged Peptides and Proteins I S 0
3.2.4 Caged Proteins by Introduction of Photoactive Residues via Site
Directed, Unnatural Amino Acid Mutagenesis 156
3.2.5 Small Caged Molecules Used to Control Protein Activity 159
3.2.6 Conclusions 168
References 168

3.3 Engineering Control Over Protein Function; Transcription

Control by Small Molecules 174
j o h n T. Koh
Outlook 174
3.3.1 Introduction 174
3.3.2 The Role of Ligand-dependent Transcriptional Regulators 175
3.3.3 Engineering New Ligand Specificities into NHRs 179
3.3.4 The Requirement of “Functional Orthogonality” 180
3.3.5 Overcoming Receptor Plasticity 180
3.3.6 Nuclear Receptor Engineering by Selection 183
3.3.7 Ligand-dependent Recombinases 184
3.3.8 Complementation/Rescue of Genetic Disease 186
3.3.9 De Novo Design of Ligand-binding Pockets 188
3.3.10 Light-activated Gene Expression from Small Molecules 189
References 191

4 Controlling Protein-Protein Interactions 199

4.1 Chemical Complementation: Bringing the Power of Genetics to
Chemistry 199
Pamela Peralta-Yahya and Virginia W. Cornish
Outlook 199
4.1.1 Introduction 199
4.1.2 History/Development 202
4.1.3 General Considerations 208
4.1.4 Applications 21 G
4.1.5 Future Development 222
References 223

4.2 Controlling Protein- Protein Interactions Using Chemical

Inducers and Disrupters of Dimerization 227
T i m Clackson
Outlook 227
viii 1 Contents

4.2.1 Introduction 227

4.2.2 Development of Chemical Dimerization Technology 228
4.2.3 Dimerization Systems 229
4.2.4 Applications 237
4.2.5 Future Development 245
4.2.6 Conclusion 245
Acknowledgments 246
References 246

4.3 Protein Secondary Structure Mimetics as Modulators of

Protein-Protein and Protein-Ligand Interactions 250
Hang Yinand Andrew D. Hamilton
Outlook 250
4.3.1 Introduction 250
4.3.2 History and Development 251
4.3.3 General Considerations 253
4.3.4 Applications and Practical Examples 255
4.3.5 Future Developments 264
4.3.6 Conclusion 265
Acknowledgments 2G5
References 265

5 Expanding the Genetic Code 271

5.1 Synthetic Expansion of the Central Dogma 271
Masahiko Sisido
Outlook 271
5.1.1 Introduction 272
5.1.2 Aminoacylation of tRNA with Nonnatural Amino Acids 274 Micelle-mediatedAminoacylation 275 Ribozyme-mediatedAminoacylation 276 PNA-assisted Aminoacylation 277 Directed Evolution of Existing aaRS/tRNA Pair to Accept Non-
natural Amino Acids 278
5.1.3 Other Biomolecules That Must Be Optimized for Nonnatural
Amino Acids 281 Adaptability of EF-Tu to Aminoacyl-tRNAsCarrying a Wide
Variety of Nonnatural Amino Acids 283 Adaptability of Ribosome to Wide Variety of Nonnatural Amino
Acids 283
5.1.4 Expansion of the Genetic Codes 284 Four-base Codons 285 “Synthetic Codons” That Contain Nonnatural
Nucleobases 286
5.1.5 In vivo Synthesis of Nonnatural Mutants 287
Contents I ix

5.1.6 Application of Nonnatural Mutagenesis - Fluorescence

Labeling 289
5.1.7 Future Development and Conclusion 291
Acknowledgments 291
References 291

Part Ill Engineering Control Over Protein Function Using Chemistry

6 Forward Chemical Genetics 299

StephenJ. Haggarty and Stuart L. Schreiber
Outlook 299
6.1 Introduction 299
6.2 History/ Development 302
6.3 General Considerations 307
6.3.1 Small Molecules as a Means to Perturb Biological Systems
Conditionally 307
6.3.2 Forward and Reverse Chemical Genetics 308
6.3.3 Phenotypic Assays for Forward Chemical-Genetic
Screening 3 12
6.3.4 Nonheritable and Combinations of Perturbations 316
6.3.5 Multiparametric Considerations: Dose and Time 318
6.3.6 Sources of Phenotypic Variation: Genetic versus Chemical
Diversity 318
6.3.7 The “Target Identification” Problem 329
6.3.8 Relationship between Network Connectivity and Discovery of
Small-molecule Probes 323
6.3.9 Computational Framework for Forward Chemical Genetics:
Legacy of Morgan and Sturtevant 325
6.3.10 Mapping of Chemical Space Using Forward Chemical
Genetics 326
6.3.11 Dimensionality Reduction and Visualization of Chemical
Space 330
6.3.12 Discrete Methods of Analysis of Forward Chemical-genetic
Data 334
6.4 Applications and Practical Examples 336
6.4.1 Example 1: Mitosis and Spindle Assembly 336
6.4.2 Example 2: Protein Acetylation 338
6.4.3 Example 3: Chemical-genomic Profiling 340
6.5 Future Development 344
6.6 Conclusion 347
Acknowledgments 348
References 349
X I Contents

7 Reverse Chemical Genetics Revisited 355

7.1 Reverse Chemical Genetics - An Important Strategy for the
Study of Protein Function in Chemical Biology and Drug
Discovery 355
Rolf Breinbauer, Alexander Hillisch, and Herbert Waldmann
7.1.1 Introduction 355
7.1.2 History/Development 356
7.1.3 General Considerations 361
7.1.4 Applications and Practical Examples 366
7.1.5 Future Developments 376
7.1.6 Conclusion 379
Acknowledgments 380
References 380

7.2 Chemical Biology and Enzymology: Protein Phosphorylation as a

Casestudy 385
Philip A. Cole
Outlook 385
7.2.1 Overview 385
7.2.2 The Enzymology of Posttranslational Modifications
of Proteins 387
References 401

7.3 Chemical Strategies for Activity-based Proteomics 403

NadimJessani and Benjamin F. Cravatt
Outlook 403
7.3.1 Introduction 403
7.3.2 History/Development 404
7.3.3 General Considerations 407
7.3.4 Applications and Practical Examples 415
7.3.5 Future Development 421
7.3.6 Conclusions 422
Acknowledgments 423
References 423

8 Tags and Probes for Chemical Biology 427

8.1 The Biarsenical-tetracysteine Protein Tag: Chemistry
and Biological Applications 427
Stephen R. Adams
Outlook 427
8.1.1 Introduction 427
8.1.2 History and Design Concepts of the Tetracysteine-biarsenical
System 429
Contents 1 xi

8.1.3 General Considerations 430

8.1.4 Practical Applications of the Biarsenical-tetracysteine System
8.1.5 Future Developments and Applications 453
8.1.6 Conclusions 454
Acknowledgments 454
References 454

8.2 Chemical Approaches to Exploit Fusion Proteins for Functional

Studies 458
Anke Arnold, India SielaJ NilsJohnsson, and Kailohnsson
Outlook 458
8.2.1 Introduction 458
8.2.2 General Considerations 459
8.2.3 Applications and Practical Examples 463
8.2.4 Conclusions and Future Developments 476
Acknowledgments 477
References 477

Volume 2

Part IV Controlling Protein- Protein Interactions

9 Diversity-orientedSynthesis 483
9.1 Diversity-oriented Synthesis 483
Derek S. Tan

9.2 Combinatorial Biosynthesis of Polyketides and Nonribosomal

Peptides 519
Nathan A. Schnarr and Chaitan Khosla

10 Synthesis of Large Biological Molecules 537

10.1 Expressed Protein Ligation 537
Matthew R. Pratt and Tom W. Muir

10.2 Chemical Synthesis of Proteins and Large Bioconjugates 567

Philip Dawson

10.3 New Methods for Protein Bioconjugation 593

Matthew B. Francis

11 Advances in Sugar Chemistry 635

11.1 The Search for Chemical Probes to Illuminate Carbohydrate
Function 635
Laura L. Kiessling and Erin E. Carlson
xii I Contents

11.2 Chemical Glycomics as Basis for Drug Discovery 668

Daniel B. Werz and Peter H. Seeberger

12 The Bicyclic Depsipeptide Family of Histone Deacetylase In-

hibitors 693
Paul A. Townsend, Simon]. Crabb, Sean M. Davidson, Peter W. M.
Johnson, Graham Packham, and Arasu Ganesan

Part V Expandingthe Genetic Code

13 Chemical Informatics 723

13.1 Chemical Informatics 723
Paul A. Clemons

13.2 WOMBAT and WOMBAT-PK Bioactivity Databases for Lead and

Drug Discovery 760
Marius Olah, Ramona Rad, Liliana Ostopovici, Alina Bora, Nicoleta
Hadaruga, Dan Hadaruga, Ramona Moldovan, Adriana Fulias,
Maria Mracec, and Tudor 1. Oprea

Volume 3

Part VI Forward Chemical Genetics

14 Chemical Biology and Drug Discovery 789

14.1 Managerial Challenges in Implementing Chemical Biology
Platforms 789
Frank L. Douglas

14.2 The Molecular Basis of Predicting Druggability 804

Bissan Al-Lazikani, Anna Gaulton, Gaia Paolini, Jerry Lanfar,
John Overington, and Andrew Hopkins

15 Target Families 825

15.1 The Target Family Approach 825
Hans Peter Nestler

15.2 Chemical Biology of Kinases Studied by NMR Spectroscopy

Marco Betz, Martin Vogtherr, Ulrich Schieborr, Bettina Elshorst, Su-
sanne Grimrne, Barbara Pescatore, Thomas Langer, Krishna Saxena,
and Harald Schwalbe
Contents I xiii

15.3 The Nuclear Receptor Superfamily and Drug Discovery 891

John T. Moore, Jon L. Collins, and Kenneth H . Pearce

15.4 The GPCR - 7TM Receptor Target Family 933

Edgar Jacoby, Rochdi Bouhelal, Marc Gerspacher, and Klaus Seuwen

15.5 Drugs Targeting Protein-Protein Interactions 979

Patrick Che'ne

16 Prediction of ADM ET Properties I003

UEfNorinder and Christel A. S. Bergstrom

Part VII Reverse Chemical Genetics Revisited

17 Computational Methods and Modeling 1045

17.1 Systems Biology of the JAK-STATSignaling Pathway 1045
lens Timmer, Markus Kollrnann, and Ursula Klingmiiller

17.2 Modeling Intracellular Signal Transduction Processes 1 061

Jason M. Haugh and Michael C. Weiger

18 Genome and Proteome Studies 1083

18.1 Genome-wide Gene Expression Analysis: Practical Considera-
tions and Application to the Analysis of T-cell Subsets in Inflam-
matory Diseases 1083
Lars Rogge and Elisabetta Bianchi

18.2 Scanning the Proteome for Targets of Organic Small Molecules

Using Bifunctional Receptor Ligands 1118
Nikolai Kley

Part Vlll Tags and Probes for Chemical Biology

19 Chemical Biology - An Outlook 1143

Giinther Wess

Index 1151
I xv


Small molecules are at the heart of chemical biology. The contributions in

this book reveal the many ways in which chemical biologists’ studies of small
molecules in the context of living systems are transforming science and society.
Macromolecules are the basis of heritable information flow in living systems.
This is evident in the Central Dogma of biology, where heritable information is
replicated via DNA and flows from DNA to RNA to proteins. Small molecules
are the basis for dynamic information flow in living systems. They constitute
the hormones and neurotransmitters, many intra- and intercellular signaling
molecules, the defensive and offensive ”natural products”used in information
flow between organisms, among many others. They are the basis for memory
and cognition, sensing and signaling, and, of course, for many of the most
effective therapeutic agents.
One dominant theme in many of the chapters concerns small molecules
and small-molecule screening. Together, these have dramatically affected life-
science research in recent years. Many of the contributors to Chemical Biology
themselves both provided new tools for understanding living systems and
affected smoother transitions from biology to medicine. The chapters they
have provided offer riveting examples of the field’s impact on life science.
The range of approaches and the creativity that fueled these projects are
truly inspiring. After a period of widely recognized advances by geneticists
and molecular and disease biologists, chemists and chemical biologists are
returning to a position of prominence in the consciousness of the larger
scientific community.
The trend towards small molecules and small-molecule screening has
resulted in an urgent need for advances in synthetic planning and methodology.
Synthesis routes are needed for candidate small molecules and for improved
versions of candidates identified in biological discovery efforts. Several
contributors give hints to the question: How do we synthesize candidate
structures most effectively poised for optimization? They note that planning
and performing multi-step syntheses of natural products in the past resulted
in the recognition and, often, resolution of gaps in synthetic methodology. The
synergistic relationship between organic synthesis planning and methodology

Chemical Biology. From Small Molecules to System Biology and Drug Design.
Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Giinther Wess
Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
xvi 1 Preface

is even more profound as synthetic organic chemists tackle the new challenges
noted above. The objects of synthesis planning, no longer limited by the
biochemical transformations used by cells in synthesizing naturally occurring
small molecules, require radically new strategies and methodologies.
Several contributors help us answer a related question that also influences
synthetic plannig: What are the structural features of small, organic molecules
most likely to yield specific modulation of disease-relevant functions? They
note that the ability to assess the performance of these compounds, and to
compare their performance to other small molecules such as commercially
available or naturally occurring ones, is possible through public small-molecule
screening efforts and public small-molecule databases (e.g., WOMBAT,
PubChem, ChemBank). These developments are reminiscent of the early
stage of genomics research, where visionary scientists recognized the need to
create a culture of open data sharing and to develop public data repositories
(e.g., GenBank) and analysis environments (e.g., Ensembl, UCSC Genome
Sometimes the line between small and macromolecules is blurred.
Oligosaccharides are often presented as a third class of macromolecules, yet
several contributions here reveal arguably greater similarities of carbohydrates
to small-molecule terpenes than to nucleic acids and proteins, both in terms
of their biosynthesis and cellular functions. Oligosaccharides are shown to be
synthesized by glycosyl transferases (analogous to isopentenyl pyrophosphate
transferases used in terpene biosynthesis) and, like the terpenes, are subject
to tailoring enzymes. Transferase enzymes are used to attach oligosaccharides
and terpenes to proteins, where they serve key functions (e.g., glycoproteins,
farnesylated Ras). Chemical biologists have illuminated and manipulated
oligosaccharides and the unquestionable member of the macromolecule
family, the proteins, with great aplomb. Several of our contributors are
pioneers in the revolution of protein chemistry and protein engineering, and
their chapters provide clear testimony to the consequences of these advances
to life science. Finally, in examing the similarities of and synergies between
chemical biology and systems biology, several of our contributors have perhaps
offered a glimpse into the future of these fields.

Stuart L. Schreiber, Cambridge January 2007

Tarun M. Kapoor, New York
Gunther Wess, Neuherberg
List of Contributors

Stephen R. Adarns Elisabetta Bianchi

Department o f Pharmacology lmmunoregulation Laboratory
University o f California, San Diego Department o f Immunology
310 George Palade Laboratories 0647 Institute Pasteur
La Jolla, CA 92093-0647 25, rue du Dr. Roux
USA 75724 Paris Cedex 15
Anke Arnold
Ecole Polytechnique Federale A h a Bora
de Lausanne (EPFL) Division o f Biocomputing
Institute o f Chemical Sciences University o f New Mexico
and Engineering School o f Med, MSC11 6445
1011 Lausanne Albuquerque, N M 87131
Switzerland USA

Rochdi Bouhelal
Christel A. S. Bergstrom
Novartis Institutes for
AstraZeneca R&D
BioMedical Research
Discovery Medicinal Chemistry
Lichtstrasse 35
15185 Sodertalje
4056 Basel
Marco Betz
Rolf Breinbauer
Center for Biomolecular
Institute o f Organic Chemistry
Magnetic Resonance
University o f Leipzig
Institute o f Organic Chemistry
Johannisallee 29
and Chemical Biology
041 03 Leipzig
Johann Wolfgang Goethe-
University Frankfurt
Max-von-Laue-Str. 7 Erin E. Carkon
60439 Frankfurt Department o f Chemistry
Germany University o f Wisconsin
1101 University Avenue
Madison, WI 53706

Chemical Biology. From Small Molecules to System Biology and Drug Design.
Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Gunther Wess
Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
xviii 1 List ofContributors

Patrick Chene Craig M. Crews

Oncology Research Yale University
Novartis Institutes for School o f Medicine
Biomedical Research 333 Cedar Street
4002 Basel New Haven, CT 06510
Switzerland USA

Benjamin F. Cravatt
Tim Clackson
Neuro-Psychiatric Disorder Institute
ARIAD Pharmaceuticals, Inc.
The Skaggs Institute for Chemical
26 Landsdowne Street
Cambridge, MA 021 39-4234
The Scripps Research Institute
BCC 159
10550 North Torrey Pines Rd.
Paul A. Clemons
La Jolla, CA 92037
Chemical Biology
Broad Institute o f Harvard & MIT
7 Cambridge Center Sean M. Davidson
Cambridge Center, MA 02142 The Hatter Cardiovascular Institute
USA 67 Chenies Mews
University College Hospital
Philip A. Cole London WC1 E 6DB
Department o f Pharmacology United Kingdom
Johns Hopkins School o f Medicine
725 N. Wolfe St. Philip Dawson
Baltimore, MD 21 205 Department o f Cell Biology
USA and Chemistry
The Scripps Research Institute
Jon L. Collins 10550 N. Torrey Pines Road
Discovery Research. La Jolla, CA 92037
GlaxoSmithKline Discovery Research USA
Research Triangle Park, NC 27709
Frank L. Douglas
Aventis Pharma
lndustriepark Hochst
Virginia W. Cornish 65926 Frankfurt
Department o f Chemistry
Columbia University
3000 Broadway, MC 31 67 Bettina Elshorst
New York, NY 10027-6948 Center for Biomolecular
USA Magnetic Resonance
Institute o f Organic Chemistry
Simon J. Crabb and Chemical Biology
School o f Chemistry Johann Wolfgang Goethe-
University o f Southampton University Frankfurt
Highfield Max-von-Laue-Str. 7
Southampton SO1 7 1 BJ 60439 Frankfurt
United Kingdom Germany
List ofcontributors I xix

Matthew B. Francis Jonathan D. Cough

Department o f Chemistry Yale University
University of California, Berkeley Department of Molecular, Cellular,
Berkeley, CA 94720-1460 and Developmental Biology
USA Kline Biology Tower 442
New Haven, CT 06520-8103
Adriana Fulias USA
Division of Biocomputing
University o f New Mexico Susanne Crimme
School of Med, MS C l l 6445 Center for Biomolecular
Albuquerque, N M 87131 Magnetic Resonance
USA Institute o f Organic Chemistry
and Chemical Biology
Arasu Canesan Johann Wolfgang Goethe-
School of Chemistry University Frankfurt
University o f Southampton Max-von-Laue-Str. 7
Highfield 60439 Frankfurt
Southampton SO1 7 1BJ Germany
United Kingdom
Dan Hadaruga
Anna Caulton Division of Biocomputing
Pfizer Global Research and University of New Mexico
Development School of Medicine, MS C l l 6445
Pfizer Ltd. Albuquerque, N M 87131
Sandwich, Kent, CT13 9NJ USA
United Kingdom
Nicoleta Hadaruga
Marc Cerspacher Division of Biocomputing
Novartis Institutes for University of New Mexico
BioMedical Research School o f Med, MS C l l 6445
Klybeckstrasse 141 Albuquerque, N M 87131
4057 Basel USA
Stephen J. Haggarty
Andrea Giordano Broad Institute of Harvard and MIT
European Molecular Biology 320 Bent Street
Laboratory Cambridge, MA 02141
Gene Expression Programme USA
Meyerhofstr. 1
691 17 Heidelberg Andrew D. Hamilton
Germany Department of Chemistry
Yale University
225 Prospect St.
New Haven, CT 06520-8107
xx I List ofcontributors

JasonM. Haugh Nils Johnsson

Department o f Chemical and Center for Molecular Biology
Biomolecular Engineering o f Inflam mat io n
North Carolina State University Institute o f Medical Biochemistry
Raleigh, NC 27695-7905 University o f Muenster
USA Von-Esmarch-Str. 56.
48149 Muenster
Alexander Hillisch Germany
Bayer Healthcare AG
PH-GDD-EURC-CR Peter W. M. Johnson
Aprather Weg 18a School o f Chemistry
42096 Wupperta! University of Southampton
Germany Highfield
Southampton SO17 1BJ
Andrew Hopkins United Kingdom
Pfizer Global Research and
Development Tarun M. Kapoor
Pfizer Ltd. Laboratory of Chemistry and
Sandwich, Kent, CT13 9NJ Cell Biology
United Kingdom Rockefeller University
Flexner Hall
Edgar Jacoby 1230 York Ave.
Novartis Institute for New York, NY 10021
Biomedical Research USA
Lichtstrasse 35
4056 Basel Laura L. Kiessling
Switzerland Department o f Chemistry
University o f Wisconsin
Nadim Jessani 1101 University Avenue
Department of Cell Biology Madison, WI 53706
Celera USA
180 Kimball Way
South San Francisco, CA 94080 Nikolai Kley
USA CPC Biotech, Inc.
610 Lincoln Street
Kai Johnsson Waltham, MA 02451
Ecole Polytechnique Federale USA
de Lausanne (EPFL)
Institute o f Chemical Sciences Chaitan Khosla
and Engineering Department o f Chemistry
1011 Lausanne Stanford U n iversi ty
Switzerland 381 North South Mall
Stanford, CA 94305
List ofcontrjbutors 1 xxi

Ursula Klingmiiller Bissan Al-Lazikani

German Cancer Research Center lnpharmatica Ltd.
(DKFZ) 60 Charlotte Street
Im Neuenheimer Feld 280 London, W1T 2NU
69120 Heidelberg United Kingdom
Ramona Moldovan
John T. Koh Division o f Biocomputing
Department o f Chemistry University o f New Mexico
and Biochemistry School o f Med, M S C l l 6445
University o f Delaware Albuquerque, N M 87131
Newark, DE 19716 USA
JohnT. Moore
Markus Kollmann Discovery Research
Physics Institute GlaxoSmithKline Discovery Research
Hermann-Herder-Str. 3 Research Triangle Park, NC 27709
79104 Freiburg USA
Maria Mracec
Michael A. Lampson Division o f Biocomputing
Laboratory o f Chemistry and Cell University o f New Mexico
Biology School o f Med, M S C l l 6445
Rockefeller University Albuquerque, N M 87131
Flexner Hall USA
1230 York Ave.
New York, NY 10021 Tom W. Muir
USA The Rockefeller University
1230 York Avenue
Jerry Lanfear New York, NY 10021
Pfizer Global Research and USA
Pfizer Ltd. Hans Peter Nestler
Sandwich, Kent, CT13 9NJ Sanofi aventis
United Kingdom Combinatorial Technologies Center
1580 East Hanley Blvd.
Thomas Langer Tucson, AZ 85737
Center for Biomolecular USA
Magnetic Resonance
Institute o f Organic Chemistry Ulf Norinder
and Chemical Biology AstraZeneca R&D
Johann Wolfgang Goethe- Discovery Medicinal Chemistry
University Frankfurt 15185 Sodertalje
Max-von-Laue-Str. 7 Sweden
60439 Frankfurt
xxii I ~ i s ofcontributon

Marius Olah Pamela Peralta-Yahya

Division o f Biocomputing Department o f Chemistry
University o f New Mexico Columbia University
School o f Med, M SC l l 6445 3000 Broadway, MC 3167
Albuquerque, N M 87131 New 'fork, NY10027-6948

Tudor 1. Oprea Barbara Pescatore

Division o f Biocomputing Center for Biomolecular
University o f New Mexico Magnetic Resonance
School o f Med, MS C l l 6445 Institute of Organic Chemistry
Albuquerque, N M 87131 and Chemical Biology
USA Johann Wolfgang Coethe-
University Frankfurt
Liliana Ostopovici Max-von-Laue-Str.7
Division o f Biocomputing 60439 Frankfurt
University o f New Mexico Germany
School o f Med, M SC l l 6445
Albuquerque, N M 87131 Matthew R. Pratt
USA Laboratory of Synthetic
Protein Chemistry
John Overington The Rockefeller University
lnpharmatica Ltd. New York, NY 10021
60 Charlotte Street USA
London, W1T 2NU
United Kingdom Ramona Rad
Division o f Biocomputing
Graham Packham University o f New Mexico
School o f Chemistry School of Med, MS C l l 6445
University o f Southampton Albuquerque, N M 87131
Highfield USA
Southampton SO1 7 1BJ
United Kingdom Dietmar Reichert
Degussa AG
Gaia Paolini Exclusive Synthesis & Catalysis
Pfizer Global Research and Rodenbacher Chausssee 4
Developme nt 63457 Hanau
Pfizer Ltd. Germany
Sandwich, Kent, CT13 9NJ
United Kingdom Lars Rogge
lmmunoregulation Laboratory
Kenneth H. Pearce Department of Immunology
Gene Exp. and Protein Chem. Institute Pasteur
GIaxoSmith Kline Discovery Research 25, rue du Dr. Roux
Research Triangle Park, NC 27709 75724 Paris Cedex 15
USA France
List ofcontributors I xxiii

Cerhard Quinkert Stuart L. Schreiber

lnstitut fur Organische Chemie Howard Hughes Medical Institute
und Chemische Biology Department o f Chemistry and
Johann Wolfgang Goethe Universitat Chemical Biology
Marie-Curie-Str. 11 Harvard University
60439 Frankfurt Broad Institute o f Harvard and M I T
Germany Cambridge, MA 02142
Krishna Saxena
Carsten Schultz
Center for Biomolecular
European Molecular Biology
Magnetic Resonance
Institute o f Organic Chemistry
Gene Expression Programme
and Chemical Biology
Meyerhofstr. 1
Johann Wolfgang Goethe-
691 17 Heidelberg
University Frankfurt
Max-von-Laue-Str. 7
60439 Frankfurt Peter H. Seeberger
Germany Laboratory for Organic Chemistry
Swiss Federal Institute o f Technology
Ulrich Schieborr Zurich
Center for Biomolecular ETH-Honggerberg
Magnetic Resonance HCI F315
Institute o f Organic Chemistry Wolfgang- Pa u Ii-Str. 10
and Chemical Biology 8093 Zurich
Johann Wolfgang Goethe- Switzerland
University Frankfurt
Max-von-Laue-Str. 7 Klaus Seuwen
60439 Frankfurt Novartis Institutes for
Germany BioMedical Research
Lichtstrasse 35
Nathan A. Schnarr 4056 Basel
Department o f Chemistry Switzerland
Stanford University Kevan M. Shokat
381 North South Mall Department o f Cellular and
Stanford, CA 94305 Molecular Pharmacology
USA UC San Francisco
600 16th Street, Box 2280
Harald Schwalbe San Francisco, CA 90143-2280
Center for Biomolecular USA
Magnetic Resonance
Institute o f Organic Chemistry hdia Sielaff
and Chemical Biology Ecole Polytechnique Federale
Johann Wolfgang Goethe- de Lausanne (EPFL)
University Frankfurt Institute o f Chemical Sciences
Max-von-Laue-Str. 7 and Engineering
60439 Frankfurt 1011 Lausanne
Germany Switzerland
xxiv I List ofcontributors

Matthew D. Simon Herbert Waldmann

Department o f Cellular and MPI of Molecular Physiology
Molecular Pharmacology University of Dortmund
UC San Francisco Otto-Hahn-Str. 11
600 16th Street, Box 2280 44227 Dortmund
San Francisco, CA 90143-2280 Germany
Holger Wallmeier
Masahiko Sisido Aventis Pharma Deutschland GmbH
Department o f Bioscience and Research &Technologies
Biotechnology lndustriepark Hochst, K801
Okayama University 65926 Frankfurt am Main
3-1-1 Tsushimanaka Germany
Okayama 700-8530
Japan Michael C. Weiger
Department o f Chemical and
Derek S. Tan Biomolecular Engineering
Laboratory of Chemistry and North Carolina State University
Chemical and Chemical Genetic Raleigh, NC 27695-7905
Sloan-Kettering Cancer Center USA
1275 York Ave. RRL 1317
New York, NY 10021 Daniel B. Werz
USA Laboratory for Organic Chemistry
Swiss Federal Institute o f Technology
lens Timmer
Physics Institute
Hermann-Herder-Str. 3
HCI F315, Wolfgang-Pauli-Str. 10
79104 Freiburg
8093 Zurich
Paul A. Townsend
School o f Chemistry Ciinther Wess
University o f Southampton GSF - Forschungszentrum fur
Highfield Umwelt und Gesundheit
Southampton SO1 7 1BJ Ingolstadter Landstr. 1
United Kingdom 85764 Neuherberg
Martin Vogtherr
Center for Biomolecular Norbert Windhab
Magnetic Resonance Degussa AG
Institute o f Organic Chemistry CREAVIS
and Chemical Biology Rodenbacher Chausssee 4
Johann Wolfgang Goethe- 63457 Hanau
University Frankfurt Germany
60439 Frankfurt
Sirus Zarbakhsh
List ofContributors

Hang Yin
Department o f Chemistry European Molecular Biology
Yale University Laboratory
225 Prospect St. Gene Expression Programme
New Haven, Meyerhofstr. 1
CT 06520-8107 691 17 Heidelberg
USA Germany

Chemical Biology. From Small Molecules to System Biology and Drug Design.
Edited bv Stuart L. Schreiber. Tamn M. Kauoor. and Gunther Wess
Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
Chemical Biology
Edited by Stuart L. Schreiber, Tarun M. Kupoor,and Gunther Wess
CoDvriaht 0 2007 WILEY-VCH Verlaa CmbH & Co KCaA. Weinheim


Chemistry and Biology - Historical and Philosophical
Gerhard Quinkert, Holger Wallmeier,Norbert Windhab,and Dietmar Reichert

Dedicated to Profs. Helmut Schwarz and Utz-Hellmuth Felcht on the occasion of their
respective GOth birthdays.


The reductionistic attitude of philosophers [ 11has given way to the emergence-

based thinking [2] of biologists. In place of the view that phenomena occurring
at a higher level in a complex system [3] with hierarchically structured levels of
organization can also be described by rules and in terms of concepts already
verified at a lower level, it has come to be accepted that some of these rules
or concepts may be altered or even gained in the transition from lower to
higher level. This applies even in the case of the structural and functional
basic unit of all biological systems: the living cell. The living cell is a protected
region in which diverse ensembles of molecules interact with one another
in a harmony achieved through self-assembly [4]. The reality of the cell, with
its overlapping functional networks [S] (for regulation of metabolism, signal
transduction, or gene expression, for example) can serve as a model. The
question of the hierarchical organization of such networks arises. Top-down
analysis proceeds in the direction of decreasing complexity of the biological
systems, a cell, a tissue, or even an organism, step by step all the way down to
the level of molecules underlying their intra- and intermolecular interactions.
From chemistry’s molecules and supermolecules bottom-up synthesis starts
in the direction of increasing complexity to reach the totality of the cell and
its higher organizations emerging through modular motifs and supramodular
functional units [6]. Bottom-upsynthesis and top-down analysis are signposts
for changes in complexity in emergent systems, lending themselves not only
to narrative representation of what is, but also to reflective conjecture on why
something is as it is.
The interdisciplinary union of the worlds of chemistry and of biology has
to begin with the different entry points to the two disciplines. In the world
of chemistry, for material atoms and its associated interactions within and

Chemical Biology. From Small Molecules to System Biology and Drug Design.
Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Gunther Wess
Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
I between moleculesthe crucial aid is the open sesame represented by the periodic
1 Chemistry and Biology - Historical and Philosophical Aspects

system of the chemical elements. In the world of biology, the fundamental

information flow and the associated ascent from the biochemical network
of metabolism to the biological network of genetic information transfer can
be deciphered by the Rosetta Stone that is the genetic code. Fundamental
to this is the understanding that in biology - as in cosmology'), but wholly
different in chemistry (and physics) - earlier historical events influence future
developments. It is a characteristic of historical events that they may have
been played out completely differently under other circumstances. In such
cases, it is reasonable to ask why questions. Why did Darwinian evolution
eventually come to entrust its further fate to the chemistries of two polymer
types, nucleic acids and proteins, and their later collaboration in a ribosome?
Why did the dice fall in favor of a genetic code with triplet character? Why
did protein genesis satisfy itself with the 20 canonical amino acids? For a
transdisciplinary perspective it is worth addressing such cases in which the
emergence of chemistry (or, more precisely, biochemistry) into biology (or,
more precisely, molecular biology) signifies a tipping point. This came about
with the appearance of macromolecules possessing the aptitude to store and
distribute information and to translate it into catalytic function [gal. It became
manifest as awareness grew of the double-faceted nature of protein synthesis:
as an enzymatic chain of chemical reaction steps in biochemical space and as
a genetic information transfer process in molecular biological space 191.
This essay deals with the structures and functions of material things
produced by chemical or biological means. While the products obtained
in both routes are comparable, if not identical, the production facilities
differ substantially.As facilities of human design, they happen to be formed by
machines in the laboratory or in the factory;as facilities of Darwinian evolution,
they start to exist in generative supermolecules of the living world. Having
distinguished the generation of natural products by supramolecular facilities
built up by self-assemblyof complementary molecules from the production of
materials in man-made facilities, it seems appropriate to add a brief excursion
into semantics.


Synthesis - Genesis - Preparation

By a chemical reaction, whether it takes place in a laboratory, in a factory, or in

a living cell, an educt is converted into a product. If the product is structurally

1) The developments of stars and galaxies offer

no analog to Darwinian evolution by natural
selection, of course [7].
1.2 Semantics
more complex than the related educt, the conversion is called a construction
(in biochemistry: an anabolic pathway). In contrast, the conversion is called
a degradation (in biochemistry: a catabolic pathway), if the product is less
complex than the related educt. According to another classification, one may
distinguish between synthesis, genesis, and preparation. While execution follows
a subtle plan in the first and instructions of a naturally selected program
in the second case, tinkering takes place in the last instance. That such a
differentiation may prove useful to the keen mind of a synthetic chemist is
demonstrated by the example of the natural dye, indigo.
While its first offspring is often popularly held to be urea, synthetic chemistry
actually began in the last quarter of the nineteenth century, with the production
of artificial indigo [lo]. This dissent can be resolved if consensus is reached
on what should be understood by the term synthesis in organic chemistry
[ll].If it is taken to mean an attempt to construct a previously decided upon
target molecule with a known structure from a suitable starting molecule (or
molecules) according to some plan [12],the choice has to be for indigo. Urea, in
contrast, was discovered by chance as an isomerization product of ammonium
cyanate by Wohler [13]in 1828, and was not in any way prepared intentionally
[14].This qualification, however, does not mean that the urea synthesis can be
discounted as inconsequential. On the contrary, Friedrich Wohler’s production
of artificial urea from hydrogen cyanate and ammonia in 1828 was a key
discovery for the dawning chemical sciences, and researchers at the ever-
advancing frontiers of the science have to this day venerated the narrative
connection between Wohler’s urea synthesis and their own new findings and
future perspectives. What historians like to unmask as a benign legend [14]
serves scientists as a rhetorical shorthand and metaphorical paraphrase.
In the industrially used Heurnann-Pfleger synthesis, N-phenylglycine 1,
readily accessible from aniline, is transformed through indoxyl2 into indigo 3
in a targeted fashion (Scheme 1-1).
This process represents the culmination of a development first set in motion
in the laboratories of the Munchen University under Adolf Baeyer. Baeyer
had begun his efforts to prepare indigo in the laboratory at a time (before
1883) when the constitution of indigo was not even known [lG],starting his

1 2 3

Scheme 1-1 Industrial production o f indigo 3 by the Heurnann-Pfleger synthesis [15]:

from 1 via 2 t o 3.
I endeavors with degradation products (aniline,anthranilic acid,isatin) obtained
7 Chemistry and Biology - Historical and Philosophical Aspects

by the application of one of the usual degradative methods (alkali melt, effect
of oxidizing agents) to the naturally occurring dyestuff. These degradation
products were treated with an extraordinarily broad range of chemicals in
a form of intuitive combinatorial process, to examine whether the resulting
products would contain 3. In this way, Baeyer and Emmerling succeeded in
transforming isatin 10 into 3 in 1870.The preparation of 10 (from phenylacetic
acid4: 1878)was however too elaborate to becomrnerciallyviable (Scheme 1-2).
As long as the constitution of a target molecule is unknown, the above
definition of a synthesis is inadmissible. The sequence of reactions depicted in
Scheme 1-2, however, characterizes a venture that serves for the preparation
of indigo. Two other pathways that afforded indigo in the laboratory were also
not industrially viable. A. von Baeyer encouraged BASF and Farbwerke Hoechst
to undertake a systematic search for an industrial synthesis of artijicial indigo
(the constitution of which had meanwhile been established) in competition
with one another. This was finally achieved in a strategicallyclear and tactically
flexible manner through the already mentioned Heumann-P’eger synthesis
(Scheme 1-1).It was envisaged that the artificial preparation of dyes from coal
tar should become a source of national wealth. Baeyer’s Miinchen University
laboratories and the two representatives of Germany’s flowering chemical

r 1

4 5
1 6

7 a 9

Scheme 1-2 Laboratory studies ofthe preparation of indigo 3 by A. (uon) Baeyer and his
1.2 Semantics
industry had exchanged ideas and experiences in a previously unknown scale
and had thus passed the test for a collaboration in partnership. In 1905, Adolf
von Baeyer was awarded the Nobel Prize for Chemistry for his contribution to
the development of organic chemistry and the chemical industry.
It has thus been demonstrated that the example of indigo is suitable for
conceptual differentiation between molecule construction according to a plan
(synthesis) and one without a plan (preparation). It can also provide an
illustration, based on the different character of the synthetic steps involved,
of differentiation between chemical and biological synthesis steps within the
overall indigo syntheses. Chemical synthesis steps [ 17a] can be understood
to include transformations achieved not only through the use of reagents or
catalysts prepared by chemists but also those in which enzymes, antibodies,
or even dead cells are used. Synthesis steps in which the synthetic capabilities
of living cells, either possessing their original genomes or new recornbinant
variants, are deployed in a targeted manner, are classified as a part of biological
synthesis [17a]. Indigo was synthesized biologically in 1983 (Scheme 1-3) [18].
Biological indigo synthesis made use of an Escherichia coli strain with a
recornbinant genome, being capable of converting aromatic hydrocarbons in
general into cis-l,2-dihydrodiols and, in particular, indole (obtained from
tryptophan 11 with the aid of tryptophanase) into cis-2,3-dihydroxy-2,3-
dihydroindol13. The recombinant E. coli strain was augmented with the genes
expressing naphthalene dioxygenase from Pseudomonas putida. The initially
produced oxidation product spontaneously loses water, and the resulting
indoxyl 2 is converted by aerial oxidation into 3, which can be taken up into
organic solvents.

&NH2 H cis-2,3-dihydroxy-

11 12 13

- 12
+ 13


- H2O

Air oxidation
3 - 2

Scheme 1-3 Formation of indigo 3 in a recombinant strain of E. coli.

1 Chemistry a n d Biology Historical and Philosophical Aspects
- --
8 -

glycerol- 12 2 3

Scheme 1-4 On the formation of indigo 3.

After the discussion on the biological synthesis of indigo with the aid
of a recombinant E. coli strain, one question still remaining relates to the
programmed genesis of indigo precursors in plants. Plants cultivated for indigo
production contain 2, stabilized by glycosylation (e.g., as indican = indoxyl
B-D-glucoside or as isatan B = indoxyl 5-ketogluconate) [19]. Indoxyl on its
part is produced from indole 3-glycerinephosphate [20] (Scheme 1-4) and that
in turn by the chorismate pathway.
This essay deals not only with preparation (intuitive) and synthesis (planned)
but also with genesis (programmed). Such (genetically and somatically
regulated) programs have arisen through Darwinian evolution. A plan for
a synthesis is devised by a synthetic chemist as designer and enacted by the
synthetic chemist as molecule maker. How is a synthesis planned?

Synthetic Design - Synthetic Execution

Unlike the bottom-up-oriented execution of a synthesis, involving real

molecules, the designing of a synthesis is a top-down event using virtual
structuresZ).Design begins with the target structure and moves through a
greater or lesser number of intermediate structures to the starting structure,
with the complexity generally decreasing. The starting structure is worthy of
that name, once it can reasonably be said to represent a comfortably accessible
starting molecule for the carrying out of the synthesis. E. J . Corey coined some
terms for top-down-oriented synthesis design which intended to highlight
the fact that retrosynthetic structure analysis and synthetic building up of the
molecule are concurrent processes. Whilst bottom-up synthesis takes place with
molecules and in synthetic steps through the deployment of suitable synthetic
building blocks, from the appropriate starting molecule to the resulting target
molecule, top-down retrosynthesis operates with structures and in transformation
steps through the identification of appropriate retron structure elements, from the
particular target structure to the resulting starting structure. Some of Corey’s
achievements through his endeavors in the logic ofsynthesis [21] include:
the fact that organic synthesis can be taught [22] even where it
is not actively practiced;

2) Differentiation between abstract structures

and concrete molecules will also pay for itself
in other circumstances.
1.2 Semantics
the availability of computer-aided synthesis planning [23]as a
procedure to generate a population of synthesis plans from
which the synthetic chemist can select the best one to
use; and
his being awarded the 1990 Nobel Prize for Chemistry for
development and methodology of organic synthesis.

Twenty-five years earlier, R. B. Woodward had been awarded the Chemistry

Nobel Prize for his outstanding achievements in the art of organic synthesis.
Woodward’scategorical imperative [12] - Synthesismust always be carried out by
plan - rapidly became the sign of the coming generation of natural products’
synthesis chemists. His qualifying statement in the following sentence can
easily go unremarked: “The synthetic frontier can be defined only in terms
of the degree to which realistic planning is possible”. This is probably the
reason for Woodward’scomment at the end ofhis essay on the total synthesis of
chlorophyll [24a].“At the beginning there was detailed synthetic planning. The
degree to which our plans proved realizable is very gratifying, but laboratory
discoveries and knowledge obtained from observation and experimentation
contributed at least as much to the advancement of our studies. We learned and
found out much that would previously not have been knowable or at best would
have been only approximately imaginable.” Elsewhere he sounds the Leitmotif
of natural products synthesis [24b]: “In our time many organic chemists
address themselves explicitly to mechanistic and theoretical problems - and
make outstanding contributions in so doing - it should not be forgotten that
questions too self-consciouslyasked of Nature may well receive subconsciously
determined answers - answers which only with difficulty contain more than
was presupposed in the questions. It is important to keep open the avenues
for innovation and surprise.”

Preparative Chemistry - Synthetic Chemistry

The terms preparative chemistry and synthetic chemistry are often used
synonymously. We wish to draw some distinction between them: in preparative
chemistry we see a rich fund of knowledge from which the synthetic chemist
can draw, gained from work on chemical reactions. The preparative chemist is
concerned with broadly aimed investigations geared toward the discovery of
chemical reactions and the development and improvement of already known
ones. A chemical reaction may qualify as “mature” [17a] if it is capable of
transforming a starting compound of not too restricted substrate specificity in
a predictable manner:
under easily maintainable reaction conditions;
as far as possible with the use of substoichiometric
proportions of effective catalysts;
I Chemistry and Biology - Historical and Philosophical Aspects
I without restriction to a particular scale;
with high chemical yield; and
with high regio- and stereospecificity

into an envisaged product. There is now such an extensive available reservoir

of preparatively useful reactions of this level of comprehensiveness that for the
construction of molecular skeletons it appears expedient to switch to a handful of
trusted reactions in the first instance [25]. In the introduction, modijication, and
elimination offinctional groups, the a priori restriction on only a few methods
is already becoming more difficult.
Organic synthesis presupposes a substantial body of knowledge, usually de-
veloped through bottom-up strategies ofthe structures and reactivities oforganic
molecules. In education, though, it is important to begin concurrently practic-
ing top-down approaches based on this knowledge and its extension and further
enrichment, as early as possible. As example speaks louder than a long discus-
sion of principles: to demonstrate the problem-solving potential of synthetic
chemistry, it would be useful to identify a molecule that has served for a long
time, commanding undiminished interest both in the past and in the present,
as a sought-after target molecule for a solid synthetic pathway. One such
molecule is estrone. If a particular target structure has been decided upon, it is
appropriate to select a particular synthetic pathway from the multitude ofvirtual
ones identifiable by combinatorial analysis (Scheme 1-5).In the process, it usu-
ally remains open whether the whole set of alternative synthetic pathways for
the particular decision is evaluated or intuitively only a part of it is considered.

Bringing Chemical Solutions to Chemical Problems

The Present Situation

At the beginning of the twenty-first century chemistry finds itself in the middle
of a phase of reorientation. In the chemical industry there is a clear trend
toward specialization and concentration. It cannot be ignored that traditional
organizational structures can be altered appreciably by investment and
disinvestment decisions, the maxim being away from the broadly diversified
chemical concern of yesterday toward the megacorporation of tomorrow,
with its focus on a few core competences. Measures adopted in established
organizations are disposition of particular branches, horizontal fusion of
adjoining core activities, and vertical integration of new high-tech ventures.
In the chemical sciences, progressive integration with chemical biology
and also with nanotechnology is underway. Self-organization of molecules
and modules into supramolecular and supramodular functional units plays a
prominent role in both fields of development, as is clear from research and
1.3 Bringing Chemical Solutions to Chemical Problems

N A Y D1
6 further planning variants


A B C D t C 4 further planning variants

Scheme 1-5 Virtual synthetic pathways single step of an AB (AC, AD, BC, BD, or
toward the steroid skeleton with rings A, 6, CD)-building block into the ABCD system;
C, and D. Top row: stepwise conversion of a bottom row: expansion in a single step of an
ring A (B,C, or D)-building block into the A (B,C, or D)-building block into the ABCD
ABCD system; middle row: expansion in a system.

teaching in the top academic institutions. That this has been possible is due to
the development of physical methods without the aid of which it would be im-
possible even to establish the existence or presence of systems with particular
properties. The core competence of chemistry, though, remains the provision
of new molecules through synthesis, a mission equally valid for synthetic
chemists in both industrial and academic environments. Both can point to
great successes in the past. Nonetheless, synthesis finds itself in a dilemma.
Academic synthetic chemists tended to give the highest priority to the
elegance of the design of a synthesis, and this veneration was passed on to
their students. For industry’s molecular engineers, the expediency with which
the synthesis could be carried out held center stage: a concept which new
graduates did not have to come to terms with until their entry into their
industrial careers. Meanwhile, the constructive tension between elegance and
efficiency was usurped by the dream of the perfect reaction and the ideal
synthesis. The perfect reaction can be summarized in Derek Burton’s utopian
view: 100%yield, 100%stereoselectivity [25a]. B. M. Trost [25b]seeks to advance
toward the ideal through observance of atom-economy, and M. Beller [25c]
I through transformation of multiple-component educts into single-component
7 Chemistry and Biology - Historical and Philosophical Aspects

products. The ideal synthesis conforms to the prescription of K. B. Sharpless

[26]: rather than being concerned with the innumerable synthetic methods in
the textbooks one should assemble a handful of “perfect” reactions that may
be used again and again by synthetic chemists in the many-step construction
of a molecular framework. A solution to this dilemma lies in a radical new
orientation, as the synthetic chemist begins to take on a role in chemistry
similar to those long played by the medical doctor in biology or the engineer
in physics [27]. In this way, the synthetic chemist provides assistance to the
fundamental scientist as a practicing technologist for mutual benefit and
being capable of demonstrating that, and in what way, fundamental chemical
knowledge may be applied in a targeted fashion to problem solving in synthesis.
There is still the matter of future target molecules for the synthetic chemist.
The times are gone when it was sufficient to synthesize a target molecule
just because it had not yet been synthesized in another laboratory. The
accent of interest in chemistry has shifted. There are two reasons for this:
one is that the structure space of supramolecular chemistry, unlike that
of molecular chemistry, is in many regions only thinly populated and awaits
selective filling. The attention of chemists has therefore moved from molecular
structure to molecular function [28]. Molecules that combine themselves into
supramolecular functional units attract particular attention from synthetic
chemists. A. Eschenrnoser’s vision [29] of creating synthetically accessible
supramolecular systems that will spontaneously assemble and may even be
capable of reproducing themselves, thus representing the first artificial models
of living systems, is heading in this direction, although far into the future.

Historical Periods of Chemical Synthesis

From a distance, scientific and technological advancements look like a

continuous stream, contributed to by many activists. On closer inspection,
though, discontinuities due to outstanding contributions by individuals are
unmistakable. If the development of chemical synthesis is reviewed, it is
possible informally to identify three phases, following on from one another in
the sense that a later phase is characterized by a greater degree of selectivity
than the earlier, with which it partially overlaps. It is easy to make out
prominent protagonists for each of the three phases. The example of the
female sex hormone estrone serves well to demonstrate how the synthetic
chemist has succeeded in meeting growing demands for selectivity. The pre-Woodwardian Era

The first phase of chemical synthesis, ending at about the beginning
of the Second World War, might be termed the pre-Woodwardian era.
1.3 Bringing Chemical Solutions t o Chemical Problems

The pre- Woodwardian era largely concerned itself with the collection and
classification of synthetic tools: chemical reactions suited to broad application
to the constitutional construction of molecular skeletons (including Kiliani’s
chain-extension of aldoses, reactions of the aldol type, and cycloadditions of
the Diels-Alder type). The pre- Woodwardian era is dominated by two synthetic
chemists: Emil Fischer and Robert Robinson. Emil Fischer was emphasizing the
importance of synthetic chemistry in biology as early as 1907 [30]. He was
probably the first to make productive use of the three-dimensional structures
of organic molecules, in the interpretation of isomerism phenomena in
carbohydrates with the aid of the Van’t Ho$ and Le Be1 tetrahedron model (cf.
family tree of aldoses in Scheme I-G),and in the explanation of the action of
an enzyme on a substrate, which assumes that the complementarily fitting
surfaces of the mutually dependent partners are noncovalently bound for a
little while to one another (shape complementarity) [31].
Robert Robinson looked for suitable reactions with the aid of which
constitutional modifications in a pathway to, for example, a steroid synthesis
might be achieved. He was probably the first to employ mechanistic

! c 7 cs



/ \ / Arabinose
\ / Xylose \ / \





Allose Altrose Glucose Mannose Gulose Idose Galactose Talose

Scheme 1-6 The family tree o f aldoses derived f r o m

(+)-glyceraldehyde. The Fischer projections of the corresponding
aldaric acids are, variously, chiral and asymmetrical (C,), chiral
and symmetrical (C?), o r achiral and symmetrical (G).
I considerations in the process. There is a tendency toward charge balancing
7 Chemistry and Biology - Historical and Philosophical Aspects

between anionoid and cationoid atom groups [32] through space and through
the bonds lying between them (charge complementarity). Robinson used a
transparent accounting system (curly arrows) to illustrate the direction of charge
displacement (Scheme 1-7).
Case Study Estrone: Elisabeth Dane’s attempts to produce estrone 24
(Scheme 1-8)synthetically [33], beginning with a Diels-Alder reaction that
might formally give rise to two regioisomeric adduct components, ended in
disappointment: whilst no adduct at all was obtained from an attempted
reaction between the Dane diene 1 4 and the monoketonic dienophile
15a, the reaction between 14 and the biketonic dienophile 19a resulted
in a mixture of rac-20a and rac-2la, in which rac-20a, with the steroidal
molecular skeleton, was present only as a minor component. It is thus no
surprise that the Dane strategy was consigned to the files, at the end of
the 1930s. The Woodwardian Era

In the second phase of organic synthesis, which could reasonably be termed
the Woodwardian era, beginning in 1937”, chemical reactions characterized
by diastereoselection in the construction of a molecular skeleton found favor.
Here as well, two synthetic chemists tower over all their contemporaries:
one, naturally, is R. €3. Woodward, who advanced the intellectualization of
organic synthesis like no one else. Woodward’s seminars set a new standard
for natural products chemistry4).The other is Albert Eschenrn~ser~), the sole


Me Me

Scheme 1-7 Analysis ofthe relative orientation o f Dane’s diene 14 and the
complementary dienophile following Robinson’s way.

3) Woodward graduated as a Doctor of Philosophy 4) I have no doubt that they ( Woodwards seminars
in 1937, after submission of his dissertation at at ETH Zurich)played a major role in stimulating
M I T (Cambridge, Mass.) (341. my ownpredilectioizforand enthrallment with the
synthesis of complex natural products; A. E.: in
5) See the concise Preface in [36a].
1.3 Bringing Chemical Solutions to Chemical Problems
I 15

14 15a: R =M e 16a: R =M e 17a: R = Me

15b: R = Et 16b: R = Et 17b: R = Et

18a: R = Me 19a: R = Me 20a: R = Me 21a: R =M e

18b: R = Et 19b: R = Et 20b: R = Et 21b:RZEt

22a: R = Me 23 24
22b: R = Et

Scheme 1-8 Collections o f formulae relevant to Dane’s concept o f a steroid synthesis

following the AB D + ABCD aufbau principle.

recipient of the privilege of a “collaborative competition” with Woodwurd

[35]. To master the demands of stereoselection it is necessary to know the
mechanism of the reaction used and its stereostructural consequences. In
particular, knowledge of a mechanism demands the capability to gauge the
diastereomorphic transition states of rival parallel reactions (see Scheme 36
in [37]).A necessary prerequisite for the acceptance of proposed ideas is that
they should be able to predict the sense of chirality of the main product
components, accurately.
Case Study (f)-Estrone (ruc-24): In 1991, [33c] the presumed dead Dane
strategy was resurrected by the use of Lewis acids as mediators. Compound 1 4
does in fact react with 15a between 0 “C and room temperature in CH2Cl2 - to
provide a mixture of (mainly) ruc-16a and (as a minor product) ruc-17a - as
soon as Et2AlCl is added [33d]. In the presence of TiC14 in CHzCl2 at -80 “C
an 89% yield of ruc-18a is obtained. The post-Woodwordian Era

Characteristic of the third phase of organic synthesis, which would logically
be termed the post- Woodwurdian era, is that the constitutional construction
of a molecular framework is now concerned not only with the problem
of diastereoselection but also with the more demanding problem of
I enantioselection [37]. Certain chemical reactions serving as key stages in
I Chemistry and Biology - Historical and Phi/osophical Aspects

multistep syntheses have been developed to perfection through the preparation

of tailor-made catalysts by Barry Sharpless6) (38a],R. NoyoVi [39]and E. J. Corey
[40],setting the standard for the further development of organic synthesis.
Case Study: (+)-Estrone 24. The “Dane-style estrone synthesis” provides a
classic example of stereoselective access to an envisaged target molecule. The
Diels-Alder reactions between 14 and 15a or 19a are chirogenic’’ reaction steps
or, put another way, the enantioselective access to the Diels-Alder adducts can
already be set at this stage. This requires, for example, the participation of a
nonracemic Lewis acid with the “right” sense of chirality. In the presence of a
Ti-TADDOLate [42], cycloadduct 20a was thus obtained from the Dane diene
14 and the bidentate dienophile 19a and was further transformed via 23 into
(+)-estrone 24*1 [33d].
Before leaving estrone, a synthetic model for oral contraceptives, as synthetic
biologicals (vide infia), it should be pointed out that each historical period of
chemical synthesis can be correlated with a characteristic synthetic level
amenable to conscious perception [37]. The resurrection [33c] of the Dane
strategy for estrone prompted synthetic chemists working on the design of
metal-free, chirality-transferring catalysts to use the chirogenic opening step
as a selection assay. In this context, acceleration of adduct formation and
changes in the ratios of the resulting regioisomers are encouraging signs
that enantioselection, which may be finished off here by recrystallization if
necessary, may be anticipated [33d]. M. W. Gobel and coworkers [43] and
E. J. Corey and coworkers [44]have reported on the application of amidinium
catalysts and oxazaborolidinium catalysts, respectively,for the enantioselective
treatment of the Dane diene 14 with 19a or with acyclic dienophile~~).

Diels-Alder Reaction - Prototype of a Synthetically Useful Reaction

The Diels-Alder reaction occupies a cherished place in the hearts of organic

synthetic chemists, not only in the synthesis of steroids [45]but far and wide
in the synthesis of structurally complex natural products [46].The Diels-Alder

6 ) Thebottomline in Scheme 1-6shows the eight 8) The (S,S)-configurated Ti-TADDOLate [42]

aldohexoses ofnatural origin; they all belong to complex with four phenanthren-9-yl residues
the D-series. Their L-configured enantiomers is used at -80°C in CH2C12: 65% chemical
have been synthesized by use of the abiotic yield, 93% ee or 78% chemical yield, and 85%
Sharpless catalyst (38bj. ee (2 or 0.2 equiv, respectively).
7) See [41] for the meaning of the term “chi- 9) With cyclic dienophiles, rings C and D in the
rogenic reaction step” and the usefulness of cycloadduct are joined in cis fashion. With
its application. acyclic dienophiles containing E-configured
C=C bonds, an adduct in which the atom
groups necessary for construction ofthe D ring
are oriented, trans is produced; see Chapter 3
in [33d].
1.3 Bringing Chemical Solutions to Chemical Problems

reaction comes closest to meeting the stipulations of K. B. Sharpless [26]

and B. M. Trost [25b] set out in Section 1.3.1. It only remains to comment
that, besides diverse instances of intermolecular examples, the intramolecular
version1o'of a Diels-Alder reaction was not left neglected in the synthesis of
estrone and its derivatives.
Scheme 1-9 summarizes the construction of a steroid framework by the
A D + AD + [AD]* -+ ABCD aufiau principle"'.
[AD]* 25a is a photoenol generated i n situ, and reacts under meticulously
determined conditions [48] by cycloaddition and subsequent dehydration to
provide the estrone derivatives 2Ga and 27a. The mixture of regioisomeric styryl
derivatives can be reduced to give 24 after temporary protection of the 17-keto
group. The photoenol 25a is produced by regioselective electronic excitation
of the Michael adduct 28a with light having wavelengths of >340nm. The
Michael adduct is accessible by treatment of the chiral enolate anion 30a with
the achiral acceptor 29 [49]. The strength (the trans fusion of rings C and D
is directly accessible) and weakness (there is still no solution to the problem
of substitution of the multistep procedure that delivers diastereoselection
for a shorter route proceeding in tandem with enantioselection) of the
photochemical synthesis of 24 have already been commented upon [36b].

Me0 \

Me0 &&

Me0 \



29 30
b: R = Et

Scheme 1-9 Collection offormulae relevant to a steroid synthesis following an

A D + AD + [AD]* + ABCD aufbau principle.

10) For further examples see the section "In- 11) Optimization of the reaction conditions was
tramolecular DielT-Alder Reactions" in carried out in the racemic series 1481. See 1491
Ref. [47]. for the synthesis ofthe enantiomerically pure
target compounds.
I I Chemistry and Biology - Historical and Philosophical Aspects

Bringing Chemical Solutions to Biological Problems

The Role o f Evolutionary Thinking in Shaping Biology

Biology is such a hugely diversified field that a historical guide hardly helps as
an aid to orientation. Given this, it might then be reasonable to consciously
pick out some particular partial aspect, as Theodosius Dobzhansky did in his
famous statement “Nothing in Biology makes Sense except in the Light of
Evolution”. With evolutionary biology as a compass, it is not hard to discern
three historical periods. The pre-Darwinian Era

One prominent event in the pre-Darwinian era is the Cuvier-Geofioy debate
(concerning the primacy of anatomical structure over anatomical function or
vice versa) before the Acade‘mie des Sceances in Paris in the spring of 18301*).Its
immediate focus involved opposed viewpoints in comparative anatomy, while
indirectly it represented endeavors to turn “the static Chain of Being into an
ever-moving escalator” [511. Cuvier represented the functionalist approach of
the designer: Formfollows Function. Geofioy Saint-Hilaire expanded the theme
and took the structuralist standpoint of the evolutionist: Functionfollows Form.
The public argument was unable to settle the difference between the two
adversaries, though it became clear that fundamental scientific discussions
would in future no longer take place in a neutral en~ironment’~). It was
also evident that evolutionary thinking in biology could no longer be kept in
its cage. The Darwinian Era

In the narrow sense, the Darwinian era began with the publication of The
Origin of Species in 1859 and ended at the beginning of the twentieth century
with the rediscovery of Gregor Mendel’s 1866 Versuche iiber Pflanzen-Hybriden
(Experiments in Plant Hybridization). Charles Darwin’s book “The Origin of
Species by Means of Natural Selection could be read as one long argument.
It supported the claims of science to understand the world in its own terms.
Animals and plants are not the product of special design or special creation.
Natural selection was not self-evident in nature, nor was it the kind of theory in
which one could say, “Look here and see”. Darwin had no crucial experiment
that conclusively demonstrated evolution in action. His whole concept of
natural selection rested on analogy”, an analogy between selective processes
taking place under either artijcial or natural conditions [53]. A series of

12) See [SO] for the Cuuier-Geofioydebate before 13) See [52]: Discussions between Goethe and
and beyond the Academie. Eckerrnann of the 2nd August 1830.
1.4 Bringing Chemical Solutions to Biological Problems

questions was left open; that of whether in the union of two gametes into
a zygote a mixture of the genes involved took place (blending inheritance),
occupied a key position. It could only be answered after:
Gregor Mendel [54]had set out statistical rules for the passing
on of particular hereditary characteristics from generation to
generation, which are useful for discussion on the complex
relationships in questions of heredity, and
Wilhelm]ohannsen [55] had coined the terms phenotype and
genotype, which made it possible to distinguish between a
statistically apparent type (the phenotype) of observable
properties and the corresponding genetic make-up (the
genotype) of an organism.
The distinction between genotype and phenotype facilitated the separation
between genetics and embryology. It is clear from this separation that the
differentiation between genetic and environmental causes in embryology and
the wider discipline of developmental biology is something to talk about. The post-Darwinian Era

The post-Darwinianera saw the vision of Darwinian evolution through natural
selection being accepted as a reality. Since then, evolution has been observed
in action in many living organisms and also in innumerable viruses [56, 571.
Through Manfied Eigen’s paper on the role of “Self-organization of Matter
and the Evolution of Biological Macromolecules” [58] Darwin’s ideas have
been placed on firm physical foundations and have been tested by in vitro
evolution experiments [59]. The Darwinian view of evolution has prompted
biologists to think in terms of dynamic populations while considering a species
[60].To avoid misunderstandings among nonbiologists, Eigen introduced the
term quasispecies. Because of mutability, self-replicating systems are always
ensembles of mutants and are not, in any circumstances, single species made
up of uniform individuals. To indicate quantitative proportional relationships
between quasispecies and their mutants, Eigen’s evolutionary model uses a
multidimensional representation (sequence space). In a nucleic acid space [61]
(protein space [62]14)),each nucleic acid (protein) sequence is represented
in the sequence space by a point and each change in the sequence by a
vector. If the points in a sequence space are assigned specific scalar fitness
values, a fitness landscape is obtained. The metaphor of a fitness landscape
(adaptive landscape) was introduced into evolutionary biology in 1932 by
Sewall Wright [64] and was afterwards used abundantly, if with a certain
breadth of interpretation, by theoretical biologist^^^). The picture conveyed

14) See [63]:Footnote 10. their professional colleagues. T. Dobzhan-

15) R. A. Fisher, /. B. S. Haldane, and S. Wright sky, G . G . Simpson, and E. Mayr successfully
count as mathematical biologists; their pub- interpreted the mathematically formulated
lications were understood only by some of theorems [65].
I by the metaphor is that of an evolving population subject to exclusion of
7 Chemistry and Biology - Historical and Philosophical Aspects

unfit mutants making uphill progress until a local peak is reached. For the
evolutionary process in the high-dimensional sequence space, local peaks in
the vicinity may readily be reached by small jumps, without the need to traverse
the valleys between them, and a continuous sequence of small jumps to reach
a global summit is a realistic prospect. To use Eigen’s own words: “Because
of frequent criss-crossing of paths in multidimensional sequence space, by
virtue of its inherent non-linear mechanism which gives the appearance of
goal-directednessthe process of evolution is steered in the direction of optimal
value peak” [8b]. In brief, biological evolution uses two processes: genetic
mutation (as a means of generating random diversity) and natural selection
(as a means to optimize the peak-jumping technique) in the environmentally
shaped fitness landscape.
Through the removal of subdisciplinary barriers, biology’s evolutionary
thinking has contributed on two occasions to enhance that science’s voice in
the choir of the natural sciences. In the 1940s and 1950s, a union of Darwinian
and Mendelian perspectives took place in Modern Synthesis [65], whilst at the
turn of the twentieth to the twenty-first century a union of developmental
and evolutionary biology into evolutionary developmental biology (Evo-Devo)
is taking place before our eyes in the New Synthesis [66].

O n the Sequence of Chemical Synthesis (Preparation) and Biological Analysis

In an ideal starting situation for the synthetic chemist the structure of the
target molecule is already given. In the real world of the search for active
substances, the matter of whether a target molecule is to be synthesized is
determined by its presumed profile of properties. If a management decision
is made in favor of a target molecule to be synthesized, the synthetic chemist
then looks for a way to relate molecular function back to molecular structure.
This is based on the supposition that a functional unit should contain at
least two structurally complementary molecules non-covalently bound to one
another in a supermolecule. The idea of supermolecules as supramolecular
functional units, nowadays preached and systematically further developed
most conspicuously by Jean-Marie Lehn [67], goes back directly to Emil Fischer
[31], who introduced the instructive lock-and-key metaphor as early as 1894.
Fischer’s metaphor, as the tip of the submerged model of molecular recognition,
traces the function of a supermolecule back to structural interactions between
its complementary constituents. Through this, the complementarity between
substrate and enzyme was to become the basis of enzymology. Paul Ehrlich
seized on the lock-and-key metaphor in his 1908 Nobel lecture [68], and the
goal of chemotherapeutic endeavor thereafter came to be regarded as the
activation or deactivation of a receptor through noncovalent binding of a
7.4 Bringing Chemical Solutions to Biological Problems

complementary effective substance. Structural complementarity of effector and

receptor accordingly represents the fundamentals of chemotherapy, similar to
the way in which complementarity of antigen and antibody is regarded as
central to immunology.
The goal of synthesizing a target molecule with particular properties can
be achieved with the aid of two problem-solving processes based on different
In one problem-solving process, illustrated by the image of the key and
its lock, the maxim is to m o d i h a designed target structure little by little
until the corresponding target molecule has the very properties of interest. It
involves an iterative procedure, usually of several rounds, based on trial and
error. It is trivial to note that the screening can take place only after the
In the other problem-solvingprocess, which can be illustrated by the image ofan
assortment of keys, hopefully containing the key that will be complementary
to a given lock, the maxim is to develop a parallel structured search method,
with the aid of which the matching key will befound, without it being necessary to
subject the whole ensemble of candidates to the totality of&nctional tests. This is
a procedure based on the principle of trial and selection. Since a distinction
has been drawn between synthesis and preparation (Section 1.2.1),some spin
doctoring should come as no surprise. After preparation is performed on a
microscale, screening will follow before the synthesis on a macroscale. For the
time being, we should come back to the traditional search for a biological, with
a very particular function. Single-componentConsecutive Procedure

In traditional single-component consecutive procedures, the synthetic chemist
each time focuses on a structure (a molecule) from a series of successive
candidates. The example of the total synthesis of estrone in Sections 1.3.2 and
1.3.3 demonstrates the adaptation of synthetic goals to the state of the art
in organic synthetics. The case studies described there have academic value
that should not be underestimated, though for industrial synthetic practices
they are not directly relevant because estrone will in general be commercially
more advantageously accessible through partial synthesis than through total
synthesis. In the search for an ovulation inhibitor outlined below, however,
total synthesis plays a commercially acceptable role, since partial synthesis
drops out as a serious contender from the second generation of inhibitors to
be discovered in future. Oral Contraceptives

Thanks to initiatives instigated by Margaret Sanger, probably the highest-
profile campaigner worldwide for family planning, a project geared toward
the development of an orally administrable contraceptive was initiated in the
I early 1950s under the reproductive biologist Gregory G. Pincus at the Worcester
I Chemistry and Biology - Historical and Philosophical Aspects

Foundation for Experimental Biological Research [69a].

It was known that progesterone established and maintained pregnancy as an
endogenous gestagen and so was able to act as a contraceptive. As progesterone
was not suited for oral application, a systematic search for the steroidal
structure space was carried out for an exogenous gestagen [69b] that - orally
administered - would bind to the progesterone receptor, hereby initiating
a series of molecular events culminating in the induction or repression of a
certain set of target genes. Binding of a gestagen to the progesterone receptor is
necessary but not sufficient for the former’s playing an active role as an agonist
in reproductive biology. This became clear as soon as an antigestagen like R LJ
486 [70] was found, which bound to the progesterone receptor, but - unlike
an agonist - was unable to trigger the gestagenic response. As it turned out,
there is no known parameter of effector binding that can predict differential
agonistic or antagonistic activity of a steroid.
If a metaphorical statement can ever reveal “how things are”, Emil Fischer’s
static lock-and-keymetaphor [31a]ought to be replaced with a dynamic one. This
was done by D. E. Koshland’s induced-jit concept [31b],which readily produced
the self-explanatory hand-and-glove metaphor. Binding of a given effector will
bring about a conformational change of the receptor that is favorable for
catalytic activity of the formed supermolecule.
G. G . Pincus and M . C. Chang investigated a diverse range of variants of
about 200 steroids [69b], which were in most cases not naturally occurring
compounds but products that had accrued in countless laboratories as a
result of arduous individual studies on their biological functions. They found
that combinations of a gestagenic and an estrogenic 19-nor-steroid exhibited
the desired effects. These findings from animal experiments (rabbit and
rat) were also confirmed in humans, in almost militarily planned (Pincus)
clinical studies (by the gynaecologists I. Rock and C. R. Garcia). In the
early 19GOs, a combination pill made up of norethindrone (prepared by
C. Djerassi at Syntex in 1951 [71]) and 17w-ethynylestradiol (prepared by
H . H . Inhofen at Schering AG in 1938 [72]) reached the market as the first-
generation pill.

Members of the First Generation

Norethindrone 31a, the gestagenic component in the combination pill, is
smoothly accessible from estrone-methylether by partial synthesis [71]. The
reaction sequence begins with a dearomatization (Birch reduction) and ends
with an ethynylation (Scheme 1-10), necessary for the oral applicability.
Technical production of estrone 24 (or estradiol) from inexpensive steroids
such as diosgenin or cholesterol by partial synthesis is also feasible. Pyrolytic
aromatization (Inhofen at Schering A G ) assists the transition from the steroid
to the 19-nor-steroid class (such as from androsta-1,4-dien-17~-01-3-one 32 to
estradiol33 [72]).
1.4 Bringing Chemical Solutions to Biological Problems



3, a: R = M e 33
b: R = Et

Me0 a: R = Me
35 b: R = Et


37 38

Scheme 1-10 Collection o f formulae relevant to Trogov's concept o f a steroid synthesis

following the AB +
D + ABD + ABCD aufbau principle.

Members of the Second Generation

Here the gestagen (-)-norethindrone 31a has been supplanted by (-)-
norgestrel 31b. The difference between the two molecular structures, minor
in itself, still has far-reaching consequences for biological action and synthetic
accessibility. The presence of the ethyl group in place of the methyl
group at C( 13) slows down the compound's metabolism, thereby increasing
bioavailability and also ordaining that total synthesis now has to take the
place of partial synthesis. This begins (Scheme 1-10)with the condensation of
(~)-l-vinyl-l-hydroxy-G-methoxy-l,2,3,4-tetrahydronaphthalene (rac-34)with 2-
ethylcyclopentane-l,3-dione(35b) [73]. The resulting seco-dione 3Gb, with
a meso configuration, can be reduced microbiologically to one of four
stereoisomers: the microorganism used (Saccharornycesuvarurn) approaches
the surface of the five-membered ring differentially from one of the
two diastereotopic half-spaces and selectively attacks only one of the two
enantiotopic carbonyl groups [74b]. The reduction product 37b can be
stereoselectively converted into (-)-38b (as reported by V. Torgov [74a]) and
finally ( H . Smith [75])into (-)-norgestrel 31b.
I I Chemistry and Biology - Historical and Philosophical Aspects

Members of Later Generations

The search for unnatural gestagens with improved properties by the trial and
error approach continues. Oral applicability (through ethynylation at C(17))
and at low dosages (thanks to slow metabolism because of the ethyl group
at C(13)) have already been achieved. A new, exogenous gestagen therefore
has prospects of being favored over already known preparations only if it
distinguishes itself in at least one of the three following aspects:
through a higher binding specificity to the complementary
receptor (i.e., biological);
through more economically advantageous accessibility (i.e.,
through some advantage arising from patent law
(i.e., commercial).

What this means in detail should become clear through illustration with
later-generation gestagens.
Gestoden 39 (Scheme 1-11) has the lowest ovulation inhibitory dose
of all gestagens known to date. It displays both antiestrogenic and
antimineralcorticoidal activity. A lower affinity to the androgen re-
ceptor is not sufficient to produce measurable anabolic androgenic
The pathway to 39 passes through compound 47 (Scheme 1-12) [7G] and
after microbiological introduction of an 0 function at C(15) (with the aid
of Penicilliurn ruistuickii), on through the stations 48 (R = H or Ac) and
49 [77]. Compound 31b, incidentally, can be easily obtained starting from
47 [78].
Desogestrel 40 (Scheme 1-11) is a progestagen that is transformed in
the intestinal mucosa and in the liver into the actual effective metabo-
lite 3-ketogestrel. The bioavailability is around 75%. Desogestrel, obtained
partially synthetically by chemists at Orgunon [79], displays minimal an-
drogenic and estrogenic activity. The long pathway from the 19-nor-
steroid estr-4-ene-3,17-dione includes a microbiological hydroxylation of

39 40 41

Scheme 1-11 Cestagens of the Pill of later generations: (-)-gestodene 39,

(-)-desogestrel40, and (-)-drospirenone 41.
1.4 Bringing Chemical Solutions to Biological Problems

42 43 44

<! 0 0 /

O A O E t
45 46 47

48 49

Scheme 1-12 Collection offormul ae relevant to syntheses of (-)-norgestrel 31b a nd

(-)-gestodene 39 in both cases via 47.

the steroid skeleton at C(11) and an intramolecular functionalization of

E. J . Corey et al. [80]reported a total synthesis (Scheme 1-13) beginning with
the reduction product 50, easily accessible from 42'"'.
Alkylation of the metallated enol derived from 52 with m-methoxy-
phenylethyl-iodide to afford the tricyclic P-keto ester 53, followed by
cationic cyclization of this to furnish the steroid derivative 54, warrants
particular attention. Corey and colleagues have recently published another
total synthesis of 40 [82], beginning with an enantioselective Diels-Alder
reaction between Dane's diene 14 and dienophile 61. An oxazaborolidinium
salt (see Section used as an efficient catalyst (Scheme 1-14).
Drospirenone 41 (Scheme 1-11),the latest of the exogenous gestagens,
differs from its antecedents in some characteristic ways:
16) The bicyclic, chiral, non-racemic building excess from the achiral triketone precur-
block 42 represents a milestone in the his- sor through a proline-catalyzed, intramolecu-
tory of organic chemistry. It is accessible lar aldol condensation (Hajos-Parrish-Eder-
in high chemical yield and enantiomeric Sauer- Wiechert reaction [76,81]).
I 7 Chemistry and Biology - Historical and Philosophical Aspects

54 55 56 57

58 59 60

Scheme 1-13 Collection offormulae relevant t o a synthesis of (-)-desogestrel40 opened

by the asymmetric Hajos-Parrish-Eder-Sauer-Wiechert reaction.

61 62 63 38

26 b 64 65

Scheme 1-14 Collection o f formulae relevant t o a synthesis of (-)-desogestrel 40 opened

by an asymmetric Diels-Alder reaction o f Dane’s diene 14 and dienophile 61.
I . 4 Bringing Chemical Solutions to Biological Problems
constitutionally, in that both angular positions are occupied
by methyl groups whilst the tetracyclic steroid skeleton is
endowed with three additional rings, and
biologically, in that 41 is an unnatural gestagen that both acts
as an aldosterone antagonist and at the same time displays
pronounced antiestrogenic and antiandrogenic
With this combination of activities in one and the same dosage, drospirenone
currently holds a leading position in hormonal contraception, although it
requires a higher dosage than gestagens with an ethyl group at C( 13).
The synthesis ofDrospirenone 41 (Scheme 1-15) [83]starts with the inexpensive
androstenolone 66, which can be converted microbiologically (Colletotrichum
h i ) into the 7a,lSa-dihydroxy derivative 67. A selective epimerization at C(7)
proceeds by way of the acetalG8. Methylenation of the intermediate (C=C) bond
appearing between C(15) and C(1G) is successfully accomplished with the aid
of dimethylsulfoxonium methylide to provide 71, and that of the (C=C) bond
between C(G) and C(7) through a Simmons-Smith reaction. The conversion
of 76 into 41 can be carried out in a one-pot procedure, with a Pd-catalyzed
hydrogenation being followed by a Ru-catalyzed oxidation and a hydrochloric
acid-induced dehydration.

66 67 68 69

70 71 72 73

74 75 76

Scheme 1-15 Collection o f formulae relevant t o a synthesis of (-)-drospirenone 41

starting from the easily accessible androstenolone 66.
I I Chemistry and Biology - Historical and Philosophical Aspects

Pinkus and Chang (Section,in their search for orally applicable
contraceptives, had decided upon norethindrone after some 200 steroidal
candidates had been examined one by one. Chemists at Schering AG had
stumbled upon drospirenone after some 600 newly prepared molecules
with antialdosterone activity had become available [84].It can be justifiably
stated that the hardly ineffectual pharmaceutical industry had finished up
in a Mind alley in its search for new active substances by using traditional
strategies [85].
The rapidly progressing expansion of the world market, where new suppliers
have arrived in great numbers (globalization), places serious decisions before
the management of every multinational company [86] (see Section 1.3.1).
These are not merely restricted to restructuring of portfolios of the products
manufactured; they also do not exclude the reorganization of the entire
company structure”). Under real pressure from financial analysts and
resumptive pressure from shareholders, questions have also been directed
toward the scientists involved: whether there might be new methods that
could afford more rapid access to new active substances. The answer was not
long in coming: with chirotechnologyI8)and the combinatorial acceleration of
the preparation and screening of whole populations of molecular candidates,
a new turn has been taken in the solution of biological problems through
chemical methods. Multicornponent Simultaneous Procedure

Darwinian evolution is kept in motion by a continual succession of newly
arising variation and its modification by natural selection. The search
for active substances proceeds through multiple-component simultaneous
procedures, in which a restricted variant population is prepared on a
microscale by a combinatorial strategy, to be subjected to the new form
of selection, that is, collective screening. After a successfully applied
unnatural selection of a particular variant with the desired properties,
synthesis on a macroscale can take place. In Section a static
variation is going to be prepared and screened for anti-inflammatory

17) The consequences arising from reorganiza- 18) One of the main challenges of synthetic
tion of the structure of a business may be chemistry in the post-Woodwardian era (see
guessed by careful market analysis. Most dif- Section is to find routes that sat-
ficult to predict is the reaction of employees. isfy the demands of industrial applicability
If the creative people among them are not to enantiomerically pure compounds [37].
convinced by the new orientation, or have In 1992, various international journals (Fi-
even been put off by the way in which it has nancial Times, Neue Ziircher Zeitung, Science,
been implemented, they may defect to the and Chemical & Engineering News), as if co-
competition, thus doubly weakening their ordinated by a global editor, touched on the
previous employer. phenomenon of chirality. C&EN even pre-
dicted that chirotechnology may progress in
the future as biotechnology had grown in the
1.4 Bringing Chemical Solutions to Biological Problems

activity of individual variants that might be useful in controlling asthmatic

The worldwide incidence, morbidity, and mortality of allergic asthma are
increasing. Asthma has become an epidemic, affecting 155 million individuals
throughout the world. It is a complex disorder characterized by local and
systemic allergic inflammation, mucus hypersecretion, and reversible airway
obstruction [88].The pathogenesis of asthma reflects the activity of cytokines
from T Hcells.
~ Without these cells there is no asthma. Animal models support
important roles for the cytokines IL-4, IL-5, and the recent IL-13 [89].The latter
is closely related to IL-4: they both bind to the same IL-4 receptor, to the
a-chain of that receptor, particularly.
The molecular biologist is interested in the molecular consequences
of allergen binding to the T-cell receptor. Experimental investigations
have revealed various signal-transduction pathways that link T-cell surface
molecules with nuclear transcription events. A [Ca2+]-dependentroute has
been discovered, emanating from the T-cell receptor, which can be blocked by
natural products of fungi: cyclosporine A (CsA) and FK 506 (Scheme 1-16).
Another signal-transducing pathway, independent of [Ca2+],emanates from
the IL-2 receptor and controls translational events on ribosomes. It can be
blocked by a third natural product, rapamycin, but not by CsA or FK 506.
Two signaling pathways have been targeted for pharmacological treatment
of unwanted immune responses. It is essential to realize that blocking
signal transduction leading to regulated transcription or regulated translation,
requires CsA or FK 506 on the one hand and rapamycin on the other to
be more than an inhibitor of a cognate target protein: calcineurin in the
former and fascilin related adhesive protein (FRAP) in the latter case. As a
matter of fact, the fungi-derived ligands in each case act as a “molecular glue”
that mediates the interactions of primary and secondary receptors, forming a
ternary receptor-ligand-receptor complex. Calcineurin is blocked by CsA and
by FK 506, but only, after the two ligands have been activated by each complex
primary receptor, cyclophilin A and FK-506 binding protein 12 (FKBP 12),
respectively. In a similar way, rapamycin, on forming a binary complex with
the primary receptor FKBP 12, is promoted to block the secondary receptor
called FRAP on ternary complex formation (Table 1-1).
An antigen bound by the receptor of a T cell sets in motion a long cascade of
signal carriers and subsequent proliferation of T cells. In allergic subjects, this
signal cascade can be initiated by allergens, which are by themselves actually
harmless, leading to undesired T-cell overproduction. For allergy sufferers,
therefore, it is desirable to specifically interrupt or slow down transcriptional
or translational signal cascades involved in T-cell production. Because FK
506, rapamycin, and CsA are effective immunosuppressants, they cannot be

19) Project of the G e m a n Federal Ministry of’

Education and Research (87a], initiated by
A. Kleemann, K. Brune. G . Quinkert; fordetails
see (631 and [87b]. Beginning: 1 July 1994.
1 Chemistry and Biology - Historical and Philosophical Aspects

FK 506 Rapamycin


Scheme 1-16 Natural immunosuppressants.

Table 1-1 Naturally occurring immunosuppressants (ligands)

and their receptor complexes

Ligand Primary receptor Secondary receptor

Cyclosporine Cyclophilin Calcineurin

FK 506 FKBP Calcineurin
Rapamycin FKBP FRAP
Binary complex
Ternary complex

considered suitable for long-term treatment of allergic patients. The search is

on for nonnatura120)ligands with a more specific action on the immune system.
A collection of non-natural ligands - synthesized independently in various
laboratories - has demonstrated an immense chemical production effort in
search of specific modulators of the immune system with significantly reduced
20) V. Prelog [90]has underlined the viewthat nat- argument that “natural products are biolog-
ural products hold a worthwhile message. H. ically validated starting points in structural
Waldrnann et al. [91] entertain the plausible space for compound library development”.
1.4 5r;nging Chemical Solutions to Biological Problems

molecular complexity. One can’t help wondering why the traditional method,
I 31

making one compound at a time, analyzing it, and evaluating it biologically

indubitably was applied by all synthetic groups involved. As the synthetic target
structures aimed at are represented by isolated points scattered irregularly
over a relatively small segment of structure space, a combinatorial approach
furnishing a focused variation, whose members ought to be represented by a
cluster of points in abstract structural space, would seem promising. Preparation and Screening o f a Static Variation

The combinatorial approach that was pursued in search of an antiasthma
drug based on a split-and-mix strategy [92] as a practical use of the operational
principle of parsimony was to get the most with the least; in this case, to get
343 different types of variants in only 21 reaction steps. Scheme 1-17 sketches

Scheme 1-17 Construction o f a encoding-decoding alternation (resulting in

binary-encoded [93]combinatorial variation a state with every bead carrying a single
using the split-and-mix protocol (resulting tripeptide sequence).
in an one-bead-one-variant state) and an
I how a biased variation of 343 members was obtained on resin-beads in three
7 Chemistry and Biology - Historical and Philosophical Aspects

preparative rounds, each round allowing for the parallel attachment of one out
of seven building blocks available.
The complete set of monomeric building blocks used in the construction
of the combinatorial variation of Scheme 1-17 is shown in Scheme 1-18.The
aesthetic elegance of the combinatorial strategy reveals itself when compared
with alternative strategies*’).
The bead-bound substrate variation was screened for binding to a biological
receptor (a fluorescence-conjugated immunophilin [87])by mixing a sample
of the charged beads with a buffer containing the complementary protein.
The beads that carry variants with affinity for the receptor are easily identified
by visual inspection under a microscope with a fluorescent illuminator and
removed with the aid of a (non-plastic) syringe. The sequence of each bead-
bound substrate variant has been determined indirectly but unambiguously
by Clark Still’s encoding-decoding alternation [93].
Molecular encoding: During each step of the construction of a focused variation
of tripeptides (see Scheme 1-17)tagging molecules are attached to the beads

Scheme 1-18 21 building blocks for the preparation o f t h e 343 tripeptides of

Scheme 1-17 (building blocks 6,10, and 11 were used as racemates).

21) A divergent approach would require 399

+ +
(7’ 7’ 7 3 ) reaction steps, a serial ap-
proach even 1029 (73+ 7’t 7’) reaction
steps to reach the same 343 variants [63, 871.
7.4 Bringing Chemical Solutions t o Biological Problems

that encode both the step number (one through 21) and the reagent (amino
I 33

acid or acid chloride, respectively) used in that step. A combinatorial encoding

of the 21 reaction steps requires altogether seven molecular tags (i.e., A, B, C;
AB, AC, BC; ABC in one round).
Molecular decoding: After screening the variation, the molecular tags22'can be
cleaved photochemically from each of the selected beads and analyzed by gas
chromatography [93].The specified on-bead selection test afforded a mixture
of ruc-77 and rac-78 (Scheme 1-19).
To explore its biological properties by various functional tests [94],
a substantial amount had to be synthesized. Instead of going for 79
(Scheme 1-19)the more distant compound 80 (Scheme 1-20)was aimed at, by
conventional synthesis technique.
The cause for replacement oftarget structure 79 with 80 was accidental. While
looking for linkers for solid-phase synthesis that can be cleaved enzymatically,
the substitution took place. Substitution of the B-methoxyethylamino residue
by the Z-protected lysine residue [87] led to higher biological activity in
various functional tests. Compound 80, recently, [94] has been considered
to be a promising candidate for the treatment of diseases accompanied by
immunological inflammation.
The combinatorial approach produces large variations of related molecules,
which can be exploited by appropriate screening techniques. As far as the
production ofthese variations and their screening are concerned, combinatorial
chemistry reminds one of the immune system. In the immune system,
antibodies recognize cognate antigens. Those antibody-producing cells that
are effective against a particular type of invader molecules preferentially evolve
from a huge population. If the invaders are pathogens or parasites, dynamic

6 OCH3

6 OCH3

78 79

Scheme 1-19 On-bead molecules (rac-77 and roc-78) selected from the variation of
Scheme 1-17. and the seeming target structure 79.

22) The molecular tags that were used are

composed of a series of electrophoric tags
(halophenol derivatives) plus a photolabile
linker [93].
I 1 Chemistry and Biology Historical and Philosophical Aspects

H 0

80 0 81

81 a)82
81 -bl

82+83 - - -

a) 6 0 ~ ~aq0 NaOH,
, dioxane, 90 %
b) MeOH. SOClp, 98 %
c ) 2-Chloro-1methylpyridiniumiodide, CH2Cl2.NEt3. 50 %
d) MeOH. 2.5 N NaOH, 74 %
e) 2-Chloro-1methylpyridiniumiodide, CH2Clp,NEt3. 86 %

Scheme 1-20 Collection of formulae relevant to a synthesis of the biologically active

candidate 80.

coevolution between them and the host may occur. There is, however, a
tremendous difference between a static variation and the immune system.
While the processes of preparation and screening of a static variation were
designed by chemists, what happens in immunology was not designed but
rather evolved.
The preparation of a dynamic variation (to be described in the following
section) is somewhat in between the two extremes, though very much closer
to the designer's end. Preparation and Screening of a Dynamic Variationz3)

In the previous section, a well-known method was applied to a long-standing
biological problem: the discovery of a new biologically active substance. With

23) For dynamic non-covalent chemistry see

1.4 Bringing Chemical Solutions to Biological Problems

the intention of finding such a substance displaying properties closest to a

I 35

setup profile, a static molecular variation was prepared (on microscale) and
screened (collectively) to afford a select variant qualifying as the candidate
for subsequent synthesis (on macroscale). In this section, we present the self-
assembly ofa variation ofthree sets ofconjugates from which an added receptor
selects a number of effectors by molecular recognition. This selection works
by way of the interactions of protein surfaces within the receptor-effector
supermolecule, the knowledge of which ought to be helpful in drug design.
The self-assembly to be introduced is based on three pyranosyl-RNA (p-RNA)
[96] single strands (a, b, and c, Scheme 1-21) associating in a Watson-Crick-like
manner, initially into binary and further on into ternary super molecule^^^). In

Scheme 1-21 Base-pairing dynamics of single strands a, b, and c.

24) Project of the G e m a n Federal Ministry of

Education and Research [97a];for details see
[87b][97b]. Initiated by A. Eschenmoser, U.-H.
Felcht, G. Quinkert [97c]. Beginning: 1 April
I addition to the H bridges, intercatenary n,n-stackingeffects make a substantial
I Chemistry and Biology - Historical and Philosophical Aspects

contribution to the stabilization of the resulting duplexes [9Ga, 9Gd].

In its current form, the self-assembly is based on three p-RNA single strands
with 7 (a and b) or 14 (in the case of c) nucleobases. The two short strands are
sequence complementary to the first seven or the last seven bases in the longer
strand. The pairing gives rise eventually to water-soluble ternary complexes
acb (Scheme 1-21). Strand c is involved in all the equilibria. Since strands a
and b are unable to pair with one another and as they bind to non-overlapping
regions of c, they do not compete with each other in binding to c. The unusual
designation acb is used to reflect the dominant role of the longer strand c in
complex formation.
The following equilibria, with five independent equilibrium constants25),
apply to the pairing of the complementary strands:

ci + aj *aj : ci,

Subscripts i,j , and k are used to distinguish various possible sequences

displaying the required complementarity.
Scheme 1-22 shows a network representation of the above set of equilibria.
The nodes in the network correspond to the individual strands involved in the
equilibria, while the lines represent their possible associations or dissociations.
Along a given line, the concentrations of a single strand or of several strands
vary between zero and the maximum disposable value. Each of the colored
lines corresponds to a single strand, whilst black lines relate to more than one
strand or to a binary complex. With the exceptions of a and b, which have only
two connections each, all other nodes have at least three available connections,
whilst the node for the ternary acb complex has as many as five. The network
here results from the superposition of the synchronous formation from a, b,
and c with the formation both from ac plus b and from cb plus a.

25) (1)and (2) form closed subsystems. As soon out of the three single conjugates. Since
as all three components are present, how- this corresponds to third-order kinetics, a
ever, the full system of equilibria (1-5) is process of this type is significantly less prob-
valid. Equilibrium (5) represents the syn- able than the purely bimolecular processes
chronous formation of the ternary complex (1-4).
1.4 Bringing Chemical Solutions to Biological Problems
I 37

I I ' I l \ rh
a acb b

Variation of [a]
Variation of [b]
~ Variation of [c]

Scheme 1-22 Network representation of equilibria (1)-(5)

In a three-dimensional representation, the strands and their complexes can

be arranged as the vertices of a trigonal bipyramid, its edges corresponding to
the equilibrium arrows from (l)-(S)26).Each state ofthe system is thus a point
within the trigonal bipyramid.
The stability of the complexes may be preserved when the pairing-capable
strands a, b, and c are extended into sets of conjugates2'' A, B, and C
(Scheme 1-23).
Coupling with a series of oligopeptides transforms the pairing system (self-
assembly system) with the three single strands a, b, and c into an exploring
system (molecular recognizing system) with the three sets of conjugates A,
B, and C. The equilibria (1)-(5) also apply to the conjugates, if the subscripts
i, j, and k are used to denote the oligopeptides employed. For the resulting
system there is a particular assignment of roles: the pairing system based
on the p-RNA strands a, b, and c serves to bring the peptide regions into
proximity with each other, thus supporting their joint function. The law of
mass action applies here not only to the self-assembly but also to molecular
recognition, ensuring that the full potential of the structural variation can be
As effectors, the triple peptide combinations are capable of entering into
specific interactions with a further component, a receptor R (Scheme 1-24).As
a selector of complementary oligopeptide combinations, the receptor enables
unnatural selection from the variation of conjugates.

26) I t should be pointed out that the transition 27) For the conjugates the following p-RNA se-
from ac to cb does not take place as a quences have been used: a = {CGGGGGNJ.
direct, single process, but should be regarded b = [NGAAGGG], and c = (CCCTCTNCC
only as a conflation of processes ac cf CCCG}. N is a tryptamine nucleoside [98],
a + c and cb c) c + b. The corresponding which serves to attach the oligopeptides
edge of the bipyramid thus - unlike the (discrete random variation of hexapeptides
other edges - does not symbolize a single composed of the amino acids C, E, F, H , K ,
equilibrium. L, N, R, S, T, W).
I 7 Chemistry and Biology - Historical and Philosophical Aspects

Scheme 1-23 Equilibria between members ofthe three sets o f

conjugates of types A, B, and C each with p-RNA moieties (gray)
t o make self-assembly possible and oligopeptide moieties (green)
t o allow molecular recognition.

The equilibria (1-5) described above now need to be supplemented, first

to take account of the receptor itself, and second to allow for the receptor
complexes with the various components of binary and ternary aggregates
shown in Scheme 1-23: altogether eight molecular species are now involved.
Scheme 1-25 shows the corresponding network of 8 nodes and 28 possible
equilibria, each of the nodes having 7 connections. As in Scheme 1-22, green,
red, and blue lines represent the possible binary equilibria, whilst black lines
denote potential ternary and quaternary equilibria.
In the interactions with a receptor, unlike in the case of the separate ternary
complex, there are several types of substitution equilibria in which conjugates
1.4 Bringing Chemical Solutions to Biological Problems
I 39

Scheme 1-24 Sketch o f molecular recognition of a receptor (R) by a complementary

effector (here by a discrete variant of type ACB).

are exchanged. There are three types of pure binary substitutions, and two
higher order substitutions where one conjugate is substituted for two others at
a time. Whether these simultaneous exchanges of several conjugates, as well as
the higher order associations and dissociations are relevant, though, remains
to be determined experimentally. The alternative of stepwise processes is
available in any case.
Topologically, the molecular species can be ordered into four levels of
complexity28’(Scheme 1-25). On the simplest level is the free receptor R. The
level above is represented by the binary complexes R:A, R B , and R C , the next
level by the ternary complexes RAB, RAC, and RBC, whilst lastly the level of
highest complexity is occupied by the quaternary complex R:ACB. Accordingly,
the participating species can be arranged as vertices of a cube. All possible
equilibria are now either edges, or face- or space-diagonals of the cube and the
system is, by definition, described by a point inside the cube at any time.
The cube-style representation shows, firstly, that pathways from one species
to another are possible either via both edges and diagonals, or exclusively via

28) The free ternary complex and its subsystems

are found on these levels likewise and are
continuously present over the full span of
equilibria. For the sake of clarity, however,
they are not explicitly taken into account here.
I 1 Chemistry and Biology - Historical and Philosophical Aspects

Scheme 1-25 Network representation of all artifacts of the two-dimensional

possible equilibria extending Scheme 1-24. representation. For the sake o f clarity, face-
The eight nodes are labeled by bold and space-diagonals ofthe cube are not
characters. All other intersections are shown.

edges or diagonals. Secondly, it also demonstrates the high syntactic symmetry

(equivalenceof the different types of interactions) of the system and underlines
the exchangeability of receptor and effectors.
To delineate pharmacological properties of members of the dynamic system
shown in Scheme 1-25, data of an enzyme-binding experiment from a real-
time biomolecular interaction analysis27)and data of an enzyme-inhibition
experiment from a photometric assay30)have been correlated (Scheme 1-26).
One can see that the strongest affinity (binding) does not give rise to the
greatest activity (inhibition). Affinity is not proportional to activity. Species
RAC shows the strongest affinity, whilst species RACB causes the greatest
activity. Since species RCB has the weakest affinity, it is clear that B makes
no cooperative contribution to affinity, but is important for effective activity.

29) The biotinylated conjugates (ACB, AC, BC, 30) The enzyme is mixed with its photolabeled
or C) are captured by a sensor chip, whose substrate S. Upon cleavage by the enzyme,
surface is coated with immobilized strept- the label is activated and fluorescence can be
avidin and which acts via surface plasmon detected. In case ofinhibition by the effector,
resonance as a tool for enzyme (R) binding cleavage does not occur and fluorescence is
experiments. not detected.
7.4 Bringing Chemical Solutions to Biological Problems

Obviously, there is no additivity of the individual conjugates’ contributions.

I 41

From the quantitative point of view this corresponds to non-linear behavior.

The influence on the enzymatic reaction has to be interpreted in terms of
either competitive inhibition (ACB:R)31), uncompetitive inhibition (ACB:RS),
mixed inhibition (ACB:R ACB:RS), or substrate capture by the conjugates.
It should be noted that interactions of A, B, and C with the receptor may
mutually influence one another in both cooperative or anticooperative fashion.
Furthermore, the coordinating role that conjugate C is playing in self-assembly
(Scheme 1-23) may be pushed into the background or may even be absent
entirely while interacting with the receptor.

Scheme 1-26 Correlation diagram of affinity (binding) and activity (inhibition) for some
nodes ofthe network of Scheme 1-25. Values for ACB are set to 100%.

31) Here, and in the other possibilities men-

tioned, ACB:R stands for any ofthe molecular
species from Scheme 1-25 containing the re-
I 7 Chemistry and Biology - Historical and Philosophical Aspects
1.4 Bringing Chemical Solutions to Biological Problems

For a screening experiment on enzyme inhibition (Scheme 1-27),a variation

I 43

of conjugates of types A, B, and C was formatted spatially addressable using

16 microtiter plates. One out of 1308 different C conjugates was given each in
a separate well, together with 1of 8 different A conjugates and 1 of 11 different
B conjugates, as indicated on the margins. In 99 of the remaining wells, the
single A or B conjugates were given as inactive blank controls. The last well
was filled with solvent and buffer, only. To each of the various mixtures the
enzyme used was added, together with its fluorescence-labeled substrate s.
In each well, the enzyme could either select the substrate or the conjugates
of Scheme 1-25. In the first case, the labeled substrate would be cleaved by
the enzyme and fluorescence observed. In the second case, inhibition of the
enzyme would occur and little or no fluorescence detected.
The color coding in Scheme 1-27 indicates the degree of inhibitory activity
found in each case. White and pale blue denote inactive substances, red and
violet denote strong inhibitory effects. In a separate measurement, an ICs0
value of 23 nM was found for the strongest inhibitor (position A 8 / B l l on the
plate in the fourth column, third row). Surprisingly, there are not only single
point hits but also whole clusters of hits in which the participating conjugates
display inhibitory activity. A closer inspection of, for example, all the wells in
which conjugate A4 is present, reveals that the majority indeed shows activity,
independently of the B and C conjugates added. This notwithstanding, not
all 16 plates show the same distribution of active and inactive triplets, even
though the A and B conjugates are the same in each plate. So, variation in
the C conjugate significantly influences the activity of the A and B conjugates.
This is especially apparent in the mixtures of A3 with B1 through B8 and of
A2 with B1, B3, and B5 through B7 in the plate of the second column, third
row. Only in the presence of a C conjugate do A and B conjugates contribute
to the observed activity in this case.
The law of mass action suggests to depart from the 1 : 1: 1 stoichiometry
in the search for maximum activity. On changing the concentrations of
individual conjugates, one shifts the molecular system parallel to edges or
planes of the cube (Scheme 1-25).The statistical weights of the contributions
of individual conjugates to the network of interactions are altered in the
process. Scheme 1-28 shows the results of a pilot experiment in which the
inhibitory activity was measured as a function of the concentrations of the A
and B c o n j ~ g a t e s ~The
~ ) .results are displayed as a hypersurface for a constant
concentration of conjugate C. The sigmoidal dose-activity relationship is clearly
evident with regard to both A and B. The stoichiometric composition with
[A] = [B] = [C] = 555 nM is represented by a point located on top of a ridge,
separating a flat region of the hypersurface from a descending slope. Starting
from the stoichiometric point, activity increases with the concentrations of A
and B. The strongest inhibition value was found at the bottom of the slope
32) Results relate to the second strongest in- the third row and the second column with
hibitor found in the screening. In Sche- the conjugates A3/B1. The results presented
me 1-27 it is to be found on the plate in in Scheme 1-26 refer to the same complex.
I 7 Chemistry and Biology - Historical and Philosophical Aspects

Scheme 1-28 Three-dimensional stoichiometric composition

(hypersurface) view ofenzyme-inhibition [A] = [B] = [C] = 555 nM is close t o a ridge.
activity o f a combination ofthree Increasing the concentrations o f A and B
conjugates, A, B, and C as a function of the enhances the activity.
concentrations o f conjugates A and B. The

with [A] = [B] = 5000 nM and [C] = 555 nM, where the properties of A and B
have a 10 times greater statisticalweight than those of C33).From the foregoing
discussion it can be directly inferred that the activity of a conjugate triplet is
not connected to a single molecular species from Scheme 1-25.
Given the dynamics of the supramolecular system described, one could
go a step further and transgress the confinements of molecular constitution.
It should be just as possible to use carbohydrates, steroids, terpenes or
even nonbiogenic substance classes - dendrimers, for example - in place
of the peptides. Through the addition of conjugates of different types of
constitution, the transition from one type to another could be studied in a
quasi-continuous way, opening up a further, new option for the determination
of structure-activity relationships.
The dynamics of the system allows it to adapt to changes in the environment.
Adaptation here means that the balance between the interactions inside the

33) Comparing Scheme 1-28 with Scheme 1-26,

one can see that the increase of activity on
going from C to ACB, from CB to ACB,
and from AC to ACB is consistent with the
topology ofthe hypersurface in Scheme 1-28.
1.5 Bringing Biological Solutions to Chemical Problems

effector (between the individual conjugates) on the one hand and those
I 45

between the effector and the receptor on the other hand, can change. Therefore,
depending on the prevailing conditions, different molecular species may be
responsible for the effects produced at the receptor. Particular combinations
of members of the three sets described may be used to map the affinity
profile of the receptor. In short: receptor profiling directly results from a
thorough investigation of the dynamic system under discussion. It reveals the
complementarity between the sites of the interacting surfaces of receptor and
effectors and suggests the design for a specific, biologically active substance
finally taking over from the analyzing effectors.
Ultimately, the potential ofbiologicallyactive substances can only be assessed
in actual biological systems by means of animal experiments (Scheme 1-29)
and confirmed by subsequent clinical studies. En route to this, however, the
dynamic system described here offers various options for the analysis and
optimization of pharmacological parameters like affinity and activity. It is the
heterobifunctional character of the dynamic system that allows the synthetic
chemist to influence both intrinsic self-assembly as well as extrinsic molecular
recognition in a controlled way.

Bringing Biological Solutions to Chemical Problems

Proteins 1991

Among the bio-macromolecules, proteins are distinguished all-round players.

As fibrous proteins they are used for structural purposes. As enzymes they
catalyze almost every chemical reaction in a cell with great power and high
specificity. As gene regulators they control gene expression in development
and evolution. As antibodies (immunoglobulins) they bind invading antigens.
As motor proteins they convert chemical energy into kinetic energy. As
transport proteins they mediate transmembrane movements of ions or
metabolites. A Look at Protein Structure and Generation from Different Angles

The chemist fills the void in structure space left by the physicist who dislikes
the integrated complexity of the molecular world. Even the chemist, for some
time, had been treating his structure space rather unevenly. According to the
Beilstein Doctrine341,macromolecules neglected by the organic chemist for a

34) Beilstein Handbook ofOrganic Chemistry,

an encyclopedia of known micromolecular
carbon compounds, does not concern itself
with macromolecular carbon compounds
I Chemistry and Biology - Historical and Philosophical Aspects

Scheme 1-29 Outlook: supramolecular network concept in pharmacology.

long time [17f],were finally taken up by the biochemist who could not afford
to ignore bio-macromolecules like nuclear acids and proteins any longer.
The bottom-up view of the biochemist eventually was complemented by the
top-down attitude of the (molecular) biologist. Quite a few of those scientists
who considered themselves molecular biologists entertained the idea [ 100aI
that “other laws of physics’ might be discovered by studying the gene”. This
search for the physical paradox [100b] remained an important element of the
psychological infrastructure of the creators of molecular biology. As a matter of
fact, the physicists among the new group were going to create a new approach
to biology [loll.
1.5 Bringing Biological Solutions to Chemical Problems

The Chemist’s Look (1021

I 47

The HofFneister-Fischer Theory of Protein Structure was made public in 1902

[103, 1041. Accordingly, proteins consist of polypeptide chains in which the
individual a-amino acids are linked to one another through amide (peptide)
bonds formed between the COOH group of one amino acid and the NH2
group of the next amino acid. The structure of proteins, Linus Pauling has
demonstrated, some time later, how deep knowledge of chemistry can lead
to general rules [105]. The nature of the strong peptide bond, the role of
weak hydrogen bonding, and the importance of complementarity [lo61 were
such rules used in model building: one of Pauling’s methods to work out the
structure of bio-macromolecules.

Stepwise protein synthesis normally requires [ 1071

protection of the amino group of the first amino acid and the
carboxy group of the next amino acid;
activation of the carboxy group of the amino acid carrying the
protected amino group to form a peptide bond; and finally,
removal of the protecting groups.

Polypeptide synthesis on insoluble polymer supports was pioneered by R. B.

Merriield [108].This method could be automated and has facilitated protein
synthesis enormously [ 1091. Chemical ligation of even unprotected peptide
segments has recently been reported [IlO].
To summarize: systematic variation of structure with the aim of developing
peptides for therapeutic use gives the synthetic chemist a good excuse for
chemical synthesis. a-Amino acids, obtained from natural sources or from
the synthetic chemist’s laboratory, play a trailblazing role in the gradual
growth of chemical biology. For the synthetic protein chemist they are
the obvious building blocks, for the teaching chemical generalist they are
ideal demonstration objects with an unmistakable structural profile: two
unlike functional groups and - with the exception of glycine - at least one
stereogenic center within the smallest possible space. Nearly 50 years were
to pass from Emil Fischer’s view that synthetic chemistry should contribute
to the solution of biological problems [30] to Du Vigneaud’s synthesis of the
neuropeptide oxytocin [ 1111. Preparative stumbling blocks in the selective
protection and/or activation of functional groups as well as in the effective
separation of complex reaction products, first had to be cleared from the path.
Methodological progress toward the achievement of automated solid-phase
synthesis, with or even without utilization of protecting group technology,
finally made peptide synthesis more or less a routine matter. Sophisticated
methods have been developed to ligate smaller peptide segments together to
make larger peptides. As far as larger proteins are concerned, the chemist’s
ability to control their structure (and functions) specifically is still in its
7 Chemistry and Biology - Historical and Philosophical Aspects

The Biochemist’s Look [112]

In his study of endergonic protein genesis,3s)the biochemist is driven by
the desire to understand how the energy barrier from the amino acids to
the peptide is overcome [113]. Paul C. Zamecnik, Mahlon Hoagland, and their
colleagues developed and used a cell-free system for the in uitro study of the
mechanistic details of protein genesis [114]. By the use of radioactive amino
acids, it could be shown that, in an initial step, enzymatic activation of the one
amino acid out of 20 induced by the hydrolysis of ATP took place following
the reaction:

Amino acid + ATP Enzyme, AMP-amino acid residue:enzyme


The resulting adenylated amino acid appears to be tightly bound to its specific
enzyme, the corresponding aminoacyl-tRNA synthetase. without leaving its
enzyme, the former, in a consecutive step, reacts with a low-molecular-weight
RNA (called soluble RNA = sRNA, later more logically known as transfer RNA
= tRNA) to afford an aminoacyl-tRNA [115,116].

AMP-amino acid residue:enzyme GTP Amino acid residue-tRNA

+tRNA + AMP + enzyme
This transacylation furnishes conjugates that structurally bridge the gap
between amino acids and their ordered arrangement in proteins. The Molecular Biologist’s Look [117]

Aminoacyl-tRNAs not only bridged the gap between activated amino acids
and their ordered arrangement in proteins but they also, rather dramatically,
brought together the experimental biochemist and the theoretical molecular
biologist [113, 1181. The biochemist, beyond biogenesis, takes a lively interest
in flow of matter and energy during metabolism. The molecular biologist takes
additional interest in the flow of genetic information during gene expression
on the one-way road: D N A + RNA + Protein. M. Hoagland [115] and
P. C. Zamecnik [116]with their sRNAs acted as the experimental biochemists
while Francis Crick, by offering his adaptor hypothesis [119], figured as the
theoretical biologist. Several years, before sRNAs were discovered, Crick had
already proposed 20 types of adaptor-RNAmolecules, which could line up along
an unspecified template-RNA, and each bind to a particular amino acid. In his
own words: “one would require twenty adaptors, one for each amino acid, and
separate enzymes would be needed to join each adaptor to its cognate amino

35) We distinguish in this essay products of

protein synthesis which were designed by man
from products of protein genesis which were
produced by evolution.
1.5 Bringing Biological Solutions to Chemical Problems

acid. Thus one is lead to suppose that after the activating step, discovered by
I 49

Hoagland and described earlier (vide supra), some other more specific step is
needed before the amino acid can reach the template”.
Which template? Several observations had excluded rRNAs from being
candidates for acting as templates. A cell, for example, could make a new type
of protein without making a new type of ribosome. The template-RNA was
finally disinterred as a class of unstable intermediates, self-explanatorilycalled
messenger-RNAs ( ~ R N A s ) ~When ~ ) . J . D. Watson informed the scientific
community “About the Involvement of RNA in the Synthesis of Protein”
[117a]he could begin with the sentence: “The ordered interaction of the three
classes of RNA controls the assembly of amino acids into protein”.
Now essential details in brief: protein genesis (translation) is the central event
in molecular biology. It takes place in the incredibly complex machinery3’)
of the ribosome [124], where the syntactic structure of ribonucleic acids is
translated into the syntactic structure of proteins. During the translation
process, the information contained in a triplet codon of mRNA is decrypted by
an anticodon of a tRNA molecule, according to the instructions of the genetic
code. The genetic code is an abstract scheme for the redundant correlation of 64
“words” (nucleoside triplets) in the language of nucleic acids with 20 “words”
(canonical amino acids) in the language of proteins. The synthetic chemist
accepts the limitation on the number of amino acid building blocks as the
price for his readymade use of the ribosomal protein generating system. The
undisputed leading actors in the translation process at the stage of information
transfer from ribonucleic acids to proteins are aminoacyl-tRNAs [ 1251. These
are conjugates made up of proportions of both biopolymer types (language
systems), produced through esterification of an amino acid with a tRNA. A
particular tRNA with its anticodon corresponding to a specific amino acid is
covalently coupled (esterified) with precisely this amino acid. The esterification
takes place through the help of an enzyme (an aminoacyl-tRNA synthetase)
capable of specifically recognizing and coupling that particular tRNA and its
cognate amino acid [126].Whilst the self-assembly of mRNA and tRNA during
translation is due to codon-anticodon interaction, based on Watson-Crick

36) Messenger-RNAs were the last of the RNA 37) In an urgent appeal, we are certainly going to
trio engaged in protein genesis, to be de- follow henceforth, Carl Woese [123] requests
tected [120]. A further type of RNA has been to stop looking at an organism as a molecular
discovered as a widespread, universal tool machine. The machine metaphor, according
in biology for gene regulation by means of to his view, overlooks much of what biology
antisense-like interactions [121]. It is called is. To understand living systems in any deep
inductive RNA (RNAi) and is produced from sense, “we must come to see them not
double stranded RNA in a cascade of enzy- materialistically, as machines, but as stable
matic processes by a set of specific RNAses. complex, dynamic organization”.
Several regulatory pathways involving RNAi
are known in many eukaryotes, including
plants and mammals. RNAi is used exten-
sively as a tool for research and its therapeutic
potential is getting more and more obvious
1 Chemistry and Biology - Historical and Philosophical Aspects
I pairing of complementary nucleobases, the mutual recognition of a tRNA and
its cognate synthetase during aminoacyl-tRNA formation is due to molecular
shape complementarity. The Genetic Code [127]

1.5.1 2.1 Cracking the Genetic Code

The genetic code was cracked in the early 19GOs, beginning with investigations
by Marshall Nirenberg and Heinrich Matthaei by using a cell-free E. coli
system. The N I H researchers, in an inaugural experiment demonstrated that
the homopolymer polyuridylic acid coded for the nonnatural protein poly-
phenylalanine [ 1281. Clearly, the natural system of protein genesis would
translate any appropriate message, natural or artificial, into a polypeptide
chain, natural or artificial [116]. Expanding the Genetic Code

By Natural Selection
The genetic code has the potential for 64 (=43) triplet codons, 61 of which
redundantly specify the 20 canonical amino acids. The methionine-specifying
triple code AUG may take on the role of a starting signal at the beginning
of protein synthesis: it thus has a double function. Three triplet codes in a
mRNA - UAA (ochre), UGA (opal), and UAG (amber) - known as nonsense
codons, specify no amino acids; that is, there are no tRNAs with complementary
anticodons for these codons. As a consequence, translation breaks off here.
The nonsense codons are also, therefore, termed stop signals (termination
codons). Broader roles in protein genesis, however, have also been established
for two of these three stop signals in recent years. In E. coli (and also in a
whole range of other organisms) the UGA codon may be redefined to perform
one of two different functions: either it may function as a stop codon and thus
end the elongation of the protein chain under construction, or further growth
of the polypeptide chain may carry on with incorporation of selenocysteine
[129],not a member of the standard set of canonical amino acids. Which of
the two instructions is followed by the translation system is dictated by the
secondary and tertiary structure of the mRNA to be decrypted (and possibly by
protein factors). Similarly, structural alterations in mRNA are able to modify
the programming of the UAG codon: once more, a codon that continues a
translation in progress, in this case through the incorporation of pyrrolysine
[130], is produced from a stop codon. The genetic code is thus naturally
expanded from the standard set. Instead of the original 20 amino acids, 22
amino acids specified by mRNA sequences are currently recognized. Further
as yet unrecognized extensions of the genetic code through natural selection
cannot be excluded. Why no sense codon has (yet) been found to be doubly
1.5 Bringing Biological Solutions to Chemical Problems

coded, is unclear. The discovery that the genetic code, as a result of natural
I 51

selection, already has more than 20 amino acid building blocks for protein
genesis in store, poses the question of whether the genetic code might also be
expandable by design; that is, whether amino acids not specified by the genetic
code in their original version might be introducible into a polypeptide chain
by translation.

By Design [131]
Peter G. Schultz, a leading protagonist of the movement to consider biology
an engineering discipline, is aiming at the construction of new proteins and,
eventually of new organisms with enhanced properties. Two alternatives for
site-specific in vivo incorporation into proteins, of amino acids not specified
by the genetic code in their original version, have been designed to achieve
that goal: systematic reassignment of three-base nonsense codons or use of
supersized codons.
The addition of a non-canonical amino acid to the genetic code requires - in
the first case - additional components of the protein producing system: a
noncanonical amino acid, an exogenous tRNA/aminoacyl-tRNA synthetase
pair, and an unique codon that specifies the amino acid of interest.
Orthogonality between the exogenous translational components (Scheme 1-30)
and their endogenous opposite numbers is the key feature of this approach.
With the effect
that the codon for the noncanonical amino acid should not
encode a canonical amino acid;
that the new tRNA or the cognate aminoacyl-tRNA synthetase
should not cross-react with any endogenous tRNA/synthetase
pair; and
that the new synthetase should recognize only the
noncanonical and not any of the canonical amino acids.
A completely autonomous bacterium with a 21 amino acid genetic code was
engineered. The bacterium can generate p-aminophenylalanine from basic
carbon sources and incorporate this amino acid into proteins in response to
the amber nonsense codon (1321.
As the restriction of non-coding triplet codons limits the number of non-
canonical amino acids, the question arises as to whether or not expansion of
the genetic code by use of a supersized codon and cognate tRNA with an ex-
panded anticodon loop might be possible. A study Exploring the Limits of Codon
and Anticodon Size [133] reveals that the E. coli ribosome is capable of using
codons of three to five nucleobases. The tRNAs that decode these codons are
most efficient with a Watson-Crick complementary anticodon containing two
additional nucleotides on either side of the normal-sized anticodon in the loop.
An orthogonal synthetase/tRNA pair was designed and constructed, which
site-specifically incorporates a noncanonical amino acid (L-homoglutamin)
into proteins of E. coli in response to the four-base codon AGGA [134].
J Chemistry and Biology - Historical and Philosophical Aspects

Scheme 1-30 Incorporation of (a) canonical (yellow) and (b) noncanonical (red) amino
acids into proteins in vivo.


The ribosomal system is not the only evolutionary accomplishment the syn-
thetic chemist might use in pursuit of his ends. The immune system offers
an example of how a biological solution can successfully be brought to exploit
antibodies as enzymatic catalysts. As far as their functions are concerned,
enzymes and antibodies normally are quite different. Enzymes have been
selected for the transition state of a catalyzed reaction over millions of years
[105].Antibodies have been selected for their affinity for the immunogen over
a period ofweeks [135].Ifthe immunogen were a transition state analogue, the
resulting antibodies should catalyze the appropriate reaction. Richard A. Lemer
and Peter G. Schultz with their respective colleagues have designed molecules
1. I; Bringing Biological Solutions to Biological Problems

that could be used to guide the process of clonal expansion and somatic muta-
I 53

tion to generate catalytic antibodies for a variety of reactions [136].Rather than

going into details here, we refer to the authoritative book on catalytic antibodies
11371. The various articles ofthat book make for interesting reading: for the syn-
thetic chemist who wants to design new catalysts as well as for the molecular
biologist who wants to gain structural insight into antibody evolution.

Bringing Biological Solutions to Biological Problems

The composition of this essay followed the matrix

chemical problems biological problems

Biological answers to biological questions are, of course, given by Nature

directly. Man may use the complex systems of Nature with the aim to
correct a fault (as, e.g., was done by Robert Edwards and Patrick Steptoe
[ 1381 in reproductive medicine). Reproductive medicine cannot be discussed
disregarding bioethical aspects [ 1391. The present authors are not competent
to meet the bioethical requirements. For this reason, reproductive medicine is
not further commented on.
Up to now synthetic chemistry has been the dominant part of our reflection.
Now synthetic biology comes in to meet the requirements of the sophisticated
observer who wants to be informed about the newest development. At any
rate, the fundamental question, WHAT IS LIFE? comes up. Under this title,
two essays have been published; one by Erwin Schrodinger [140] in 1944 and
the other by J . B. S. Haldane [141] in 1949. While the former focused on the
physical aspect of the living cell, the latter considered life essentially as a
pattern of chemical processes. A very pragmatic point of view was formulated
in 1994 by Antonio Lazcano 11421 with the statement: “Life is like music, you
can describe it, but not define.”
In a state-of-the-art survey, Biology and the Future o f M a n 11431, of the US
National Academy of Sciences, the chances to realize the dream of a man-made
cell were pondered. The conclusion reached was: “Those who are hopeful about
synthesizing a cell in the foreseeable future have every reason to retain their
optimism.” However, they should be warned against false claims. Synthesis
of life is one such false claim. Living things (i.e., a cell) can be synthesized but
not life itself, and that is what people really mean when they are talking about
synthesizing life.
A question that keeps busy scientists in chemistry as well as in biology
is about where the line separating inanimate from animate matter can be
I drawn.
Chemistty and Biology - Historical and Philosophical Aspects

In the past it has been tried to link the problem to the question of
life’s origin in terms of molecular evolution [144]. Recently, sequencing of
the human and other complete genomes has shed some new light on this
field. The question of what the minimal set of genes would be necessary
for a living organism can be put more concisely in the context of what
is now called synthetic biology [145]. Both approaches, the top-down way of
deactivating more and more genes of an existing species [146]and the bottom-
up way of assembling genes to build an organism with a fully synthetic
genome [147],have not yet reached the goal to explain the transition from the
inanimate to the animate world. On the one hand, results obtained through
different methods to identify the minimal set of genes that constitute a living
organism point to roughly 250 genes [148]. On the other hand, none of
the synthetic constructs obtained so far covers the central functionality of
life, self-construction, metabolism, adaptation, self-repair, reproduction, and
evolution [149].
Nonetheless, the bottom-up route has turned into an engineering approach to
synthetic biology [150].The strategy is to combine predefined DNA modules,
so-called bio-bricks that can be combined to bio-circuits, designed to be
implementations of biological functions [ 1511. In that sense, synthetic biology
is seen as the successor of molecular cloning, in particular, with respect to
safety issues.


To round offthis essay, we point to two issues gaining more and more emphasis
in chemistry. One thing is the problem of shared use of the limited sources of
energy and raw materials. The other thing is the concept of a total synthesis, in
particular for complex natural substances. Both topics underline that organic
chemistry is far from being pure routine applying a comprehensive toolbox
to solve any problem in synthesis [ 1521. Medical therapeutics, agrochemicals,
and high-performance materials must be provided by organic chemistry to
fulfill global needs.

The Fossil Fuel Dilemma o f Present Chemical Industry

For chemical industry, the interdependence of energy source and raw material
supply is typical. This double function of fossil fuel to act as a source of raw
material supply as well as an energy source will have to be terminated in
a not-too-distant future [153]. Being the main source of raw material, fossil
fuel should be maintained as long as possible for the chemical industry. A
final way out to disentangle energy requirement and raw material supply
would be to find new sources for one field or the other. Nuclear energy,
1.7 EPlLOCUE 55

despite political moves to dispense with nuclear power, could play a role
as an alternative to fossil fuel. With petroleum supplies dwindling, there
is increasing interest in selective methods for transforming other carbon
feedstocks into hydrocarbons suitable for transportation fuel. The reductive
oligomerization of CO and H l to produce hydrocarbons (specificallyn-alkanes)
with highly controlled molecular weight (Fischer-Tropsch process [154]) from
the vast reserve of coal, natural gas, oil, or biomass is one such process that
was developed in the 1920s. The Goldman-Brookhart process (tandem alkane
dehydrogenation-olefin metathesis [155]) is of a similar kind, but of recent

Two Lessons From the Wealth o f Published Total Syntheses

The final proof of the structure of a natural product after the latter has also
been synthesized in the chemist’s lab was, for a long time, common procedure
[156]. In a few cases, disagreement raised a few eyebrows. This was the case
for patchouli alcohol and for a molecule called hexacyclinol [157]. Quinine is
an example of the difficulties associated with the notion of a total synthesis.
Shouts [35, 37,1581 and murmurs [llb,159] have been expressed to comment
on the wealth of total syntheses of natural products performed in the second
half of the twentieth century. Synthetic Lesson from Patchouli Alcohol: The Trouble with “the Last
Structural Proof’ [160]
The peculiar case of patchouli alcohol (87) (Scheme 1-31) was told and
commentated by Jack D. Dunitz [IbOa]. Following W. H. Perkin’s jun.
advice [I561 to perform, as a final proof of structure a total synthesis of
a natural product 87 was synthesized [IGOc]. The synthetic product proved
to be identical to sesquiterpene whose structure had been derived from
the results of a long series of chemical experiments lasting more than
50 years and apparently confirmed in 1961 by total synthesis [IGOc]. In
spite of this, X-ray structure determination [IbOa] revealed that the accepted
structure of patchouli alcohol was wrong. A careful reinvestigation showed
that during chemical degradation as well as during synthesis a rearrangement
of the molecular skeleton had taken place. The first reaction step of the
chemical degradation (acetate pyrolysis affording patchoulene 88) and the last
reaction step of the chemical synthesis (hydrolysis of the epoxide 89 obtained
from 88) were accompanied by a rearrangement proceeding in precisely
the reverse direction of the rearrangement in the other case. Taking this
I 1 Chemistry and Biology - Historical and Philosophical Aspects

Degradation 87 Synthesis

a7 88

t i
Scheme 1-31 Synthesis and degradation of Patchouli alcohol.

finding into consideration, a new synthetic approach furnished 87 without any

difficulty [lGOd]. Synthetic Lesson From Quinine 90: The Trouble with Formal Total
Syntheses [161a]
In the period between 1918 and 2001, a series of publications appeared that
changed the claim of the total synthesis of 90 (Scheme 1-32) as a fact into a
myth. It started with a paper of Rabe and Kindler in 1918 [lGlb]on the partial
synthesis of 90 from quinitoxine (91),via quininone (92) (Scheme 1-32a).91 is
a relais compound to 90, since it can easily be made from 90. In 1944 and 1945,
Woodward and Doring published two papers [lGle]where they linked the par-
tial synthesis of Rabe and Kindler to their own synthesis of 91 (Scheme 1-32b),
taking the combination as a total synthesis of 90. Not being convinced of the
view of Woodward and Doring, Stork published a new total synthesis of 90
I 57

92 90 9-epf-quinine

quinidine 9-epr-quinidine

A HO F MeN , Ac - qN, 0

isoquinoline-7dl mixture of


91 90

Scheme 1-32 Synthesis of 90. (a) The Robe-Kindler partial

synthesis of 90 I161 b]. (b) The Woodward-Diin'nglRabe-Kindler
formal total synthesis of 90 [161e]. (c) The Stork total synthesis of
90 [161fl.
I 1 Chemistry and Biology - Historical and Philosophical Aspects



Scheme 1-32 (Continued)

in 2001 [Iblfl. He started from the Taniguchi lactone (94) and proceeded via
desoxyquinine (95) (Scheme 1-32c).According to Stork, a distinction between
a real total synthesis and a formal one is necessary. Accordingly, the work of
Woodward and Doring is an example of a formal total synthesis.


Our own investigations on multicomponent simultaneous procedures were

supported by the German Ministry of Education and Research and carried out by
a team ofpostdoctoral fellows. In addition to these colleagues whose names are
mentioned in the references, Susanne Feiertag, Stefan Kienle, Stefan Raddatz,
Jochen Muller-lbeler,Jochen Muth, Christoph Brucher, Heike BehrensdorJ; Andreas
Kappel, and Marc Pignot have contributed to our understanding of dynamic
variations. Oliver Boden took care of the equipment for the electronic version
of the manuscript. We are indebted to n e o d o r a Ruppenthal for patient and
skillful secretarial help. The greater part of this essay has been translated
from German into English by Dr. Andrew Beard. We are grateful to the
mentioned persons for their assistance and to the indicated institution for its
generous support. Last but not least, we would like to emphasize that it was
Albert Eschenmoser's idea to use p-RNA or analogs for selecting appropriate
candidates from a self-assembly of a dynamic variation.
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Zhuravlev, V.A. Avetisov, The Pathways, and Dreams, J.I. Seeman
definition of life in the context of its (Ed.),American Chemical Society,
origin, Biogeosciences 2006, 3, 281; Washington DC, 1994, p. X X I I ;
(d) D.E. Koshland Jr.,The seven (b) P. Schmalz, Interview mit Gilbert
pillars of life, Science 2002, 295, Stork: Organische - Zukunft und
2215. Gegenwart, Nachr. Chew. Tech. Lab.
150. (a) E. Andrianantoandro, S. Basu, 1987, 35, 349.
D.K. Karig, R. Weiss, Synthetic 160. (a) J.D. Dunitz, X-Ray Analysis and the
biology: new engineering rules for an Structure of Organic Molecules, Cornell
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Biol. 2006, 2, msb4100073; (b) P. Fu, (b) J. Fleming, Selected Organic
A perspective of synthetic biology: Syntheses, Wiley, London, 1973,
assembling building blocks for novel p. 125; (c) G. Buchi, R.E. Erickson,
functions, Biotechnol. /. 2006, 1, 690; N. Wakabayashi, Constitution of
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promise and perils of synthetic 1961, 83,927; (d) G. Buchi, W.D.
biology, Trte New Atlantis 2006, McLeod jr., J. Padilla O., Synthesis of
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151. A registry of standardized modules 1964, 86,4438.
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152. Editorial, Beauties of Synthesis, from quinine, Nature 2001, 21 1, 429;
Nature 2006, 443, 1. (b) P. Rabe, K. Kindler, Uber die
153. K. Weissermel, Energie und Rohstoff partielle Synthese des Chinins, Ber.
entkoppeln, aber wie?, Lecture given dtsch. chem. Ges. 1918, 51, 466;
in Frankfurt am Main, Feb. 22nd, (c)T.S. Kaufman, E.A. Ruveda, The
1980, Hicom GmbH, quest for quinine: Those Who Won the Battles and Those Who Won the
154. K. Weissermel, H.-J. Arpe, Industrial War, Angew. Chem. Internat. Ed.
Organic Chemistry, Fourth Edition, 2005, 44, 854; (d) ].I. Seeman, The
Wiley-VCH, Weinheim, 2003. Woodward-Doeringl Rabe- Kindler
155. A.S. Goldman, A.H. Roy, Z. Ahuja, Total Synthesis of Quinine: Setting
W. Schinski, M. Brookhart, Catalytic the Record Straight, Angew. Chem.
Alkane Metathesis by Tandem Internat. Ed. in press; (e) R.B.
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Using Small Molecules to Explore Biology

Chemical Biology. From Small Molecules to System Biology and Drug Design.
Edited by Stuart L. Schreiber, T a r u n M. Kapoor, and Gunther Wess
Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
Chemical Biology
Edited by Stuart L. Schreiber, Tarun M. Kupoor,and Gunther Wess
CoDvriaht 0 2007 WILEY-VCH Verlaa CmbH & Co KCaA. Weinheim

I 71

Using Natural Products to Unravel Biological Mechanisms

Using Small Molecules to Unravel Biological Mechanisms

Michael A. Lampson and Tarun M . Kapoor


Experimental strategies designed around small molecule inhibitors have been

critical in advancing our understanding ofbiological mechanisms. This chapter
introduces a series of biological questions and illustrates how they have been
addressed by using small molecules to perturb protein function.


Our understanding of biological processes often develops from discovering

or designing ways to perturb the process and observe the effects of the
perturbation. While genetic approaches have been widely used for this
purpose, small molecule inhibitors have several advantages as a means of
perturbing protein function. First, small molecules provide a high degree
of temporal control, generally acting within minutes or even seconds, and
are often reversible, allowing both rapid inhibition and activation of protein
function. The ability to design perturbations on short timescales has proved
particularly valuable in examining dynamic biological processes. Second, dose
can easily be controlled with small molecule inhibitors to allow varying
degrees of inhibition. Third, small molecules can be applied in multiple
biological systems, including different organisms, different cell types, and in
vitro systems. The examples discussed in this chapter illustrate how these
properties of small molecules have been exploited in designing strategies to
dissect biological mechanisms.

Chemical Biology. From Small Molecules to System Biology and Drug Design
Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Gunther Wess
Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
I 2 Using Natural Products to Unravel Biological Mechanisms

Use of Small Molecules to Link a Protein Target to a Cellular Phenotype

Small molecules with dramatic cellular phenotypes have been used, without
knowledge of their protein target, to provide insight into biological processes.
If the effects of a small molecule are well characterized, then identification
of the protein target immediately provides a wealth of information about its
cellular functions because of the known inhibition phenotypes.

2.1 2.1 Colchicine and Tubulin

Cell division is the process by which cells dividetheir contents into two daughter
cells, each ofwhich must receive genetic material identical to that of the mother
cell. Each chromosome is replicated before cell division begins, and a complex
and highly regulated process known as mitosis has evolved to ensure that the
replicated chromosomes are equally partitioned between the two daughter
cells. Progress through mitosis is closely linked to chromosome movements
(Fig. 2.1-1(a)).Chromosomes first move to the center of the spindle, and only
after correct positioning of all chromosomes at metaphase (Fig. 2.1-1(a) iii)
do the sister chromosomes split apart at anaphase (Fig. 2.1-1(a)iv) and move
to opposite sides of the cell before the final division into two daughter cells
(Fig. 2.1-1(a)v, vi). All of these coordinated chromosome movements occur
over the course of approximately one hour. The result is that each daughter
cell receives exactly one copy of each replicated chromosome. Failure of this
process leads to loss or gain of whole chromosomes in the daughter cells, a
condition known as aneuploidy which is strongly associated with developmental
defects and human diseases such as cancer (reviewed in Ref. [I]).
Examination of fixed samples revealed the existence of a fibrous structure,
known as the mitotic spindle, which appears at each mitosis and disappears
after the chromosomes have separated. One of the great challenges in the
study of cell division has been to understand the organization and function
of the mitotic spindle. Use of the small molecule colchicine (Fig. 2.1-1(b))has
contributed to our understanding of the physical properties of the spindle
fibers and how they might drive chromosome movements, as well as their
molecular components.
The fibers that make up the mitotic spindle are optically anisotropic, or
birefringent, with different indices of refraction in different directions (i.e.,
parallel or perpendicular to the fiber axis).Exploiting this property of the fibers,
Inoue developed a sensitive polarized light microscope that allowed him to
directly observe the spindle in living cells [2]. The small molecule colchicine
(Fig. 2.1-1(b))was known to disrupt spindle function, but its mechanism of
action was not known. Using the polarized light microscope, Inoue showed that
the birefringence of the spindle fibers disappeared after colchicine treatment,
indicating loss of the fibers [3]. The time course of this effect ranged from a
few minutes to an hour, depending on the concentration. If colchicine was
removed, the fibers recovered. Small molecule inhibitors of protein synthesis
2.7 Using Small Molecules to Unravel Biological Mechanisms
I 73
Colchicine p'

I 0'
Replicated Sp'indle fiber
chromosome pair

iv V vi

Fig. 2.1-1 (a) Overview o f mitosis. move in opposite directions. (v) The cell
(i) Chromosomes are replicated before divides as the cleavage furrow forms
mitosis. (ii) The spindle forms and between the separated chromosomes.
chromosomes attach to spindle fibers. (vi) Two daughter cells form, each with
(iii) Chromosomes move t o the center ofthe exactly one copy of each chromosome.
spindle at metaphase. (iv) Sister (b) Structures o f t w o small molecules that
chromosomes separate at anaphase and target microtubules: colchicine and taxol.

were used to demonstrate that the fibers recovered by assembly from an

available pool of material [4].Similar results were obtained by changing the
temperature to manipulate the fibers [S]. Together, these findings suggested
that the observed birefringence was due to oriented polymers that were in
equilibrium with free molecules in solution. The equilibrium is shifted toward
the depolymerized state by colchicine or by low temperature, and returns to
its original state after removal of the inhibitor or rewarming.
To demonstrate the potential functional significance of the spindle fiber
dynamics, the same experimental paradigm was used: perturbation of spindle
function combined with observation ofthe fibers in living cells. Treatment with
low concentrations of colchicine caused the fibers to contract slowly rather
than immediately eliminating the birefringence. As the fibers contracted,
chromosomes were pulled toward one pole of the spindle, which was anchored
at the cell surface [ 3 ] .The effect was reversible, as fibers elongated after removal
of colchicine and chromosomes moved away from the pole. This experiment
demonstrated that force could be generated by coupling polymerization and
depolymerization of the fibers to chromosome movement.
In the studies discussed above, colchicine was used to probe spindle func-
tion without knowing its mechanism of action. Tight binding to a intracellular
I target was implied by the low concentration (100 nM) required to arrest cells
2 Using Natural Products to Unravel Biological Mechanisms

in mitosis. A strategy was developed to isolate a colchicine-binding protein.

First, colchicine was labeled with H3 with high specific activity and tested with
a variety of cells, tissues, and organelles [GI. High binding activity was observed
with multiple preparations, including the mitotic spindle, cilia, sperm tails,
and brain tissue, that are enriched in intracellular fibers called microtubules,the
same fibers that Inoue observed in the spindle [7, 81. These results suggested
that the target of colchicine was a subunit of microtubules. Isolated sea urchin
sperm tails were dissolved to extract the colchicine-binding activity, which was
then purified by gel filtration and sedimentation over a sucrose gradient. A
single component with a sedimentation constant of GS was identified. Using
porcine brain as a starting material, the same component was isolated and
shown to bind guanosine triphosphate (GTP) [9, lo]. Because this component
was believed to be the primary constituent of microtubules, the protein was
named tubulin [Ill.
The functions of microtubules in cells depend on the activities of
numerous microtubule-associated proteins (MAPs), including regulators of
polymerization dynamics and molecular motors that move along microtubule
tracks. Identification of MAPS was made difficult by the dynamic nature of
microtubule fibers, particularly the tendency to depolymerize under conditions
used to prepare extracts for biochemical purification. The small molecule taxol
(Fig. 2.1-1(b))was shown to promote microtubule assembly and to stabilize
polymerized microtubules [12, 131, and these properties were exploited to
develop a procedure for purification of MAPS [14]. Taxol was added to brain
or cell extract to polymerize microtubules, which were subsequently isolated
together with bound MAPs. Washing with high salt released MAPS from
the microtubules, which were stabilized with taxol, so that the soluble MAPS
could be separated from the microtubules. One prominent application of this
strategy was the discovery of the founding member of the kinesin family of
microtubule-based motor proteins [15].
The potential of small molecules targeting microtubules as cancer
therapeutics was demonstrated by the vinca alkaloids, such as vincristine
and vinblastine, which have been used in the clinic for 40years. At high
concentrations (10- 100 nM), these compounds depolymerize microtubules,
which eliminates the mitotic spindle. At lower concentrations that are
used clinically, microtubules remain stable but microtubule dynamics are
suppressed. Taxol, which also inhibits microtubule dynamics, is widely used
to treat a variety of cancers (reviewed in Ref. [lG].These drugs induce a mitotic
arrest, which eventually leads to cell death [17]through mechanisms that are
only beginning to be understood [18,19, 201. Cytochalasin and Actin

While colchicine was a valuable tool for examining cellular processes that relied
on microtubules, electron microscopy revealed another filamentous structure,
2. I Using Small Molecules to Unravel Biological Mechanisms
I 75

Fig. 2.1-2 (a) Structure ofcytochalasin B, a small molecule that

targets actin. (b) Force production by the contractile ring in
cytokinesis. A ring o f actin filaments forms at the plasma
membrane and contracts to divide the cell in half.

termed rnicroJlarnents, that was distinct from microtubules. A key step in

understanding the function of microfilaments was to observe a correlation
between the presence of the filaments, their disruption by the small molecule
cytochalasin (Fig. 2.1-2(a)), and the phenotype of cytochalasin treatment in
multiple systems. Although the molecular target of cytochalasin was unknown,
it was shown to inhibit many forms of cellular or intracellular movement, such
as cytoplasmic cleavage in cytokinesis (Fig. 2.1-2(b)),cell motility, membrane
ruffling, and nerve outgrowth [21, 221. In all of these systems, microfilaments
were observed and were shown to be disrupted by cytochalasin. Cells recovered
after removal of cytochalasin as the microfilaments returned to their normal
state. Furthermore, the actions of cytochalasin and colchicine were generally
mutually exclusive, suggesting that the two types of filamentous structures
could function independently in the cell. Microtubule-dependent processes,
which were inhibited by colchicine, were often insensitive to cytochalasin, while
processes inhibited by cytochalasin were generally insensitive to colchicine
[22]. The conclusion from these correlative data was that microfilaments likely
played a fundamental role in the generation of forces at the cellular level:
“the evidence seems overwhelming that microfilaments are the contractile
machinery of nonmuscle cells” [22]. The action of the myosin motor, which
uses energy from adenosine triphosphate (ATP) hydrolysis to slide filaments
made up of polymers of the protein actin, was known to drive contractility in
muscle, but the relevance of this mechanism to other cellular processes had
not been demonstrated.
Using actin filaments purified from muscle, cytochalasin was shown to
decrease the viscosity of actin in solution. This experiment, which established
a direct link between cytochalasin and actin, led to two important conclusions.
First, cytochalasin interacts directly with actin. Second, “an interaction of
l cytochalasin with actin or actin-like proteins in vivo could account for the
2 Using Natural Products to Unravel Biological Mechanisms

ability of cytochalasin to inhibit various forms of cell motility and contraction”

[23].As the molecular target of cytochalasin, actin was implicated as a critical
component of the microfilaments involved in cytochalasin-sensitiveprocesses.

2.1 2 . 3 Small Molecules and Thermal Sensation

Another example of a small molecule with a dramatic cellular phenotype is
capsaicin (Fig. 2.1-3(a)),the natural product that makes chili peppers “hot”.
Its mechanism of action is of particular interest because of the link to
more general pain sensation. A class of neurons that are excited by various
noxious stimuli (chemical, mechanical, or temperature) are also sensitive to
capsaicin [24]. Therefore, capsaicin could be a useful tool in understanding
the basic mechanisms underlying pain sensation. The discovery of a capsaicin

Fig. 2.1-3 (a) Structures ofthe small gated by capsaicin binding, heat, and
molecule capsaicin and menthol. protons. (c) Response of the VR1 receptor
(b) Schematic o f the VR1 receptor, a channel t o capsaicin, temperature, and pH.
nonspecific cation channel. The channel is Adapted from [Ref. 281.
2. J Using Small Molecules to Unravel Biological Mechanisms

receptor, in particular, would provide a molecular handle on this process.

I 77

Studies in cultured neurons showed that capsaicin induced a rapid calcium

influx through activation of a cation channel [25, 261. On the basis of this
knowledge, an expression cloning strategy was devised to identify the receptor
[27]. The underlying logic of this approach was that if nonneuronal cells were
not sensitive to capsaicin simply because they did not express the receptor,
expression of the receptor would lead to a capsaicin-induced increase in
intracellular calcium. A neuronal cDNA library was transfected into human
embryonic kidney (HEK293) cells and screened by calcium imaging in living
cells. The cloned receptor, named VR1 (vanilloid receptor subtype 1) was
shown to be a nonselective cation channel expressed in sensory neurons
(Fig. 2.1-3(b-c)). The sensitivity of VR1 to heat and acid, as well as capsaicin,
indicated its more general physiological importance in detecting noxious
stimuli [28]. At the whole animal level, the role of V R l in detection of noxious
stimuli has been demonstrated by gene disruption studies in mice [29, 301.
A similar expression cloning strategy was used to identify a receptor involved
in transduction of cold sensation. In this case, the natural product used to
induce calcium influxwas menthol (Fig. 2.1-3(a)),which was known to produce
a sensation of cold and even suggested to interact directly with a cold detection
pathway [31].Transient receptor potential (TRPM8), a cation channel from the
same family as VR1, was cloned and shown to be activated by both menthol
and cold [32, 331. Thus, small molecules were used to link our perceptions
of both heat and cold to specific receptors in sensory neurons involved in
thermosensation. Identification of these receptors has opened the door to an
understanding of thermosensation at a molecular level [34].

Small Molecules as Probes for Biological Processes

In strategies developed to use small molecules as probes to understand

biological processes, the effects of the small molecule on the biological
system as a whole are often more important than the specific protein target,
which may not even be known. A number of insightful experiments have
been designed around such perturbations by examining how the system
responds to or recovers from the induced state. Because of the temporal
control available with small molecules and the reversibility of inhibition, these
approaches are particularly powerful with dynamic processes. As initially
shown with colchicine, the mitotic spindle is a highly dynamic structure and
small molecules have played an integral role in understanding its function. Progression through Mitosis

It is clear from observing chromosome movements that cell division occurs
in an ordered sequence of events (Fig. 2.1-1(a)). Chromosomes attach to
spindle microtubule fibers and move to the spindle equator before sister
I chromosomes separate at anaphase and move to opposite sides of the
2 Using Natural Products to Unravel Biological Mechanisms

cell, followed by division into two daughter cells. Successful chromosome

segregation requires that events occur in this order. If anaphase begins
prematurely, before chromosomes have properly attached to the spindle, the
sister chromosomes will not segregate equally, leading to aneuploid daughter
cells. Mechanisms that determine the timing of anaphase onset are therefore
critical for the success of mitosis.
One hypothesis for how anaphase onset might be regulated was through
feedback control. This term refers to a mechanism for controlling progression
past a certain point in the cell cycle,known as a checkpoint, where the completion
of an event generates a signal that allows the next event to begin. Failure to
complete the event causes a cell-cycle arrest. In the context of progression
through mitosis, some critical process, such as spindle assembly, would be
monitored to generate a signal regulating anaphase onset. Consistent with
this hypothesis, colchicine was known to induce a mitotic arrest by disrupting
the spindle. The effect of colchicine did not prove the existence of a feedback
control mechanism, however, because the mitotic arrest could also be explained
by direct inhibition of another microtubule-dependent process required for
anaphase. A prediction of the feedback-control hypothesis is that mutations
in genes required for feedback signaling would allow cells to bypass the
colchicine-induced arrest and progress through mitosis without completing
spindle assembly.
A genetic screen was designed to identify such mutations in budding yeast,
using benomyl, a small molecule inhibitor of microtubule polymerization that
is effective in yeast, to perturb spindle assembly. Benomyl could either be used
at a low dose or washed out, as the effect is reversible, so that cells would survive
the treatment. Cells were arrested in mitosis with high benomyl(70 pg mL-'),
which prevents spindle formation, but proceeded normally through mitosis af-
ter removal of benomyl and continue to grow (Fig. 2.1-4(a))1351. Alternatively,
spindle assembly was slowed with low benomyl (15 pg mL-l), and anaphase
onset was delayed to allow completion of spindle assembly, but cells continued
to grow [36]. In both cases, massive chromosome missegregation and cell
death were expected if cells entered anaphase prematurely in the presence of
benomyl with incomplete or nonexistent spindles. The difference in survival
between cells with functional and defective feedback control was used to select
mutations in genes required for feedback control [35, 361. After creating ran-
dom genetic mutations, cells that failed to grow after benomyl treatment were
selected (Fig. 2.1-4(b)).As in Inoue's studies with colchicine, the reversibility
of the small molecule and the ability to achieve partial inhibition by decreasing
the dose were important components of the benomyl-screening strategies.
The identification of genetic mutations that abolished the benomyl-induced
mitotic arrest provided evidence for a feedback mechanism that delays
anaphase onset until completion of spindle assembly, now often referred
to as the mitotic spindle checkpoint. The names M a d , for mitotic arrest
deficient, and Bub, for budding uninhibited by benomyl, were used for
2.7 Using Small Molecules to Unravel Biological Mechanisms
I 79
Colony grows
Wild-type cell arrests in mitotis without benomyl

8 .

Mutant cell defective in feedback control fails to arrest

Colony dies

with benomyl
Cells dead due to catastrophic
chromosome misegregation

* *
Benomyl Benomyl removed

Fig. 2.1-4 Screening strategy used t o missegregation and eventual cell death.
identify genes required for feedback control (b) Cells were mutagenized, and colonies
o f anaphase onset in budding yeast [35]. were grown from single cells and then
(a) Cells were arrested in mitosis for 20 h transferred t o create two replicate plates.
with benomyl, a small molecule that targets One plate (top) was grown without benomyl.
tubulin and prevents spindle formation. The second plate (bottom) was treated with
After removal o f benomyl, wild-type cells benomyl. Colonies that failed to grow on the
form a spindle and proceed normally second plate, indicating defective feedback
through mitosis. Mutant cells fail to arrest control, were selected from the first plate t o
and enter anaphase without forming a identify the mutated gene.
spindle, causing chromosome

the genes identified in these screens. The Mad and Bub genes, which
are well conserved from yeast to mammals, have provided the foundation
for much of our current understanding of the mitotic spindle checkpoint.
Studies in transgenic mice have confirmed the importance of several of these
genes for faithful chromosome segregation in higher eukaryotes, as reduced
expression increases both aneuploidy and cancer susceptibility. In human
tumors, mutations have been reported in Madl, Mad2, Bubl, and BubRl, a
related vertebrate protein (reviewed in [Ref. 11. Additionally, human germline
mutations in BubR1 have been linked to mosaic variegated aneuploidy, a
condition associated with high risk of cancer [37].
Experiments examining the intracellular localization of Mad2 have suggested
a model for how the feedback control mechanism might operate [38, 391. At
early stages of mitosis, Mad2 localizes to the kinetochore, a structure that forms
on each chromosome and mediates attachment to spindle microtubules. As
I 2 Using Natural Products to Unravel Biological Mechanisms

cells progress through mitosis, however, Mad2 disappears from kinetochores,

and at anaphase onset none of the kinetochores have detectable Mad2. The
loss of Mad2 from kinetochores correlates with microtubule attachment.
Furthermore, when spindle microtubules are depolymerized with the small
molecule nocodazole, Mad2 localizes to all kinetochores. These findings
suggest a mechanistic basis for the feedback-control model. Mad2 binds
kinetochores that lack microtubule attachment as a signal that mitosis
in not complete, which prevents anaphase onset. Microtubule binding
displaces Mad2 from kinetochores, so that when all kinetochores have bound
microtubules, anaphase can begin.
It should be noted that the small molecule benomyl was used in the Mad/Bub
genetic screens not because of its specific protein target but because of the
perturbation of spindle assembly. In principle, the same experiments could
be done by targeting a different component of the spindle. The generality of
the spindle checkpoint has been demonstrated through the use of monastrol,
a small molecule inhibitor of the mitotic kinesin Eg5, which was identified
in a screen for small molecules that arrest cells in mitosis without targeting
tubulin [40].Because Eg5 is required to separate the spindle poles, monastrol
treatment arrests cells in mitosis with monopolar spindles. In the presence of
monastrol, the checkpoint can be overridden by inhibition of Mad2, through
microinjection of inhibitory antibodies [41]. This finding indicates that the
principle of feedback control applies generally to spindle perturbations through
highly conserved mechanisms.
Inhibitors of Eg5 are currently in development as anticancer drugs because,
like taxol and the vinca alkaloids, they arrest cells in mitosis by activating
the spindle checkpoint. The efficacy of these drugs, as demonstrated by
recent studies, requires a prolonged, checkpoint-dependent mitotic arrest [42,
191. Drug resistance is conferred by a compromised spindle checkpoint, for
example, through reduced expression of Mad2. Positioning the Cleavage Plane in Cytokinesis

Monastrol, the small molecule inhibitor of Eg5, has been used in several
studies to address questions in the biology of cell division [41, 43, 441. One
important question is how the position of the cell division (or cleavage)
plane is determined in cytokinesis. The cleavage plane is typically positioned
in the center of the cell so that cellular components are equally divided
between the two daughter cells. Asymmetric divisions do occur, however,
and are particularly important during development, when the location of
the cleavage plane can determine the fate of the daughter cells. Models to
explain the position of the cleavage plane relied on the presence of the bipolar
microtubule array of the mitotic spindle, which would place the division plane
in between the spindle poles.
To test this idea directly, monastrol was used in an experiment designed to
determine if cytokinesis could occur in cells with monopolar spindles [41].To
2. I

Anti-Mad2 antibody
Using Small Molecules to Unravel Biological Mechanisms

Fig. 2.1-5 Assay to examine

I 81

injection cytokinesis in the presence of a

monopolar spindle [41].
Treatment with monastrol, a
small molecule inhibitor ofthe
kinesin Eg5, causes cells to

arrest in mitosis with
monopolar spindles due to
activation of the spindle
checkpoint. Microinjection o f an
antibody against the protein
Mad2 inactivates the checkpoint
b monopolar
so that cellsspindles.
divide with


allow cells to enter anaphase in the presence of monastrol, inhibitory antibodies

against Mad2 were microinjected to override the mitotic spindle checkpoint.
After entering anaphase, the injected cells successfully completed cytokinesis
(Fig. 2.1-5). This experiment demonstrated that a bipolar microtubule array
is not required for cytokinesis. By carefully analyzing microtubule dynamics
during anaphase in the monopolar spindles, a population of microtubules near
the chromosomes was shown to be stabilized at the location where the cleavage
plane formed. These findings suggest a model in which the position of the
cleavage plane is determined by local regulation of microtubule dynamics,
through association with chromosomes. Correcting Errors in Chromosome-spindleAttachments

Accurate chromosome segregation in mitosis requires not only feedback
control of anaphase onset but also regulation of chromosome attachment to
the spindle. Each pair of replicated chromosomes must achieve a particular
orientation in which microtubule fibers attach sister chromosomes to opposite
poles of the spindle. Experiments in yeast showed that inhibition of the
Ipll/Aurora family of kinases stabilized improper attachments [45, 461, but
how the active kinase corrected attachment errors was not known. Because
attachment errors are rarely observed in the presence of active Aurora kinase,
this problem was particularly difficult to address. Inhibition of Aurora kinase,
through experimental approaches such as genetic mutation, could be used
to accumulate attachment errors, but not to examine error correction by the
active kinase. Reversible small molecule Aurora kinase inhibitors present a
I 2 Using Natural Products to Unravel Biological Mechanisms


-b -b

Monastrol Monastrol removed

Hesperadin Hesperadin removed

2.7 Using Small Molecules to Unravel Biological Mechanisms

4 Fig. 2.1-6 Correction o f improper (c) Spindles were fixed after bipolarization
chromosome attachments by activation o f either in the absence (i) or in the presence
Aurora kinase [44]. (a) Structures o f t w o (ii) o f a n Aurora kinase inhibitor.
Aurora kinase inhibitors (AKI), hesperadin Chromosomes are shown in blue and
and AKI-1. (b) Assay schematic. microtubule fibers in green. The arrows
(i) Treatment with the Eg5 inhibitor indicate sister chromosomes that are both
monastrol arrests cells in mitosis with attached t o the same spindle pole.
monopolar spindles, in which sister Projections o f multiple image planes are
chromosomes are often both attached to the shown, with optical sections o f boxed
single spindle pole. (ii) Hesperadin, an regions (1 and 2) t o highlight attachment
Aurora kinase inhibitor, is added as errors. Scale bar 5 pm. (d) After removal o f
monastrol is removed. As the spindle hesperadin, CFP tubulin (top) and
bipolarizes with Aurora kinase inhibited, chromosomes (bottom) were imaged live by
attachment errors fail t o correct so that three-dimensional confocal fluorescence
some sister chromosomes are still attached microcopy and differential interference
t o the same pole o f t h e bipolar spindle. contrast (DIC), respectively. The arrow and
(iii) Removal o f hesperadin activates Aurora arrowhead show two chromosomes that
kinase. Incorrect attachments are move to the spindle pole (marked by a circle
destabilized by disassembling the in DIC images) as the associated
microtubule fibers, pulling the kinetochore-microtubule fibers shorten, and
chromosomes to the pole, while correct then move t o the center ofthe spindle. Time
attachments are stable. (iv) Chromosomes (min:s) after removal of hesperadin. Scale
move from the pole to the center ofthe bar 5 pm. (With permission from Lampson
spindle as correct attachments form. et al. N a t . Cell Biol. 2004, Ref. 44.)

solution to this problem because they can be used to inhibit kinase function
and subsequently removed to activate the kinase. Understanding the function
of Aurora kinases is particularly important because they have been linked to
oncogenesis, and Aurora kinase inhibitors are currently in development as
cancer therapeutics [47, 481.
Several issues needed to be addressed to devise a strategy to address the
question of how attachment errors were corrected. First, kinase inhibition
should be temporally controlled to experimentally isolate the error correction
process, as Aurora kinases have been implicated in multiple mitotic processes.
Second, error correction likely involves some regulation of the dynamics of the
microtubule fibers that attach chromosomes to the spindle. These dynamics
can be analyzed with high temporal and spatial resolution by high-resolution
microscopy in living cells. Finally, the dynamics of individual microtubule
fibers are difficult to analyze if that fiber is obscured by other microtubules in
the spindle. The dynamics can be clearly observed, however, under conditions
in which the improperly attached chromosomes are positioned away from the
spindle body.
All of these issues were addressed through the development of an assay
using several reversible small molecule inhibitors (Fig. 2.1-6) [44]. First,
treatment with the Eg5 inhibitor monastrol arrests cells in mitosis with
monopolar spindles (Fig. 2.1-G(b) i). A particular chromosome attachment
error in which both sisters are attached to the single spindle pole, referred to
as syntelic attachment, is frequent in the monopolar spindles [49]. If monastrol
I is removed, the spindle becomes bipolar, all of the accumulated attachment
2 Using Natural Products to Unravel Biological Mechanisms

errors are corrected, and anaphase proceeds normally. An Aurora kinase

inhibitor was added immediately after removal of monastrol to determine
if Aurora kinase activity is required for correction of the attachment errors.
Because the Aurora kinase inhibitor is added only at this point, its activity was
unperturbed for all the preceding stages of mitosis. To control for possible
off-target activities of the Aurora kinase inhibitors, the assay was performed
with two structurally unrelated inhibitors (Fig. 2.1-G(a)).
Cells expressing GFP (green fluorescent protein) tubulin were used
to examine spindle bipolarization in the presence of an Aurora kinase
inhibitor (Fig. 2.1-G(b-d)).Both chromosome and microtubule dynamics were
analyzed at high resolution by multimode fluorescence and transmitted light
microscopy.The syntelic attachment errors persisted as the spindle bipolarized,
directly demonstrating that Aurora kinase activity is required for correction
of these errors. Notably, some of the improperly attached microtubule fibers
could be clearly observed, unobstructed by other spindle microtubules, as the
chromosomes attached to these fibers were positioned away from the spindle
body. After spindle bipolarization, the Aurora kinase inhibitor was removed to
examine how the active kinase might correct the syntelic attachment errors.
One hypothesis was that attachment errors would correct by chromosome
release from the attached microtubule fiber [50]. Instead the observation was
that improperly attached chromosomes remained attached to the microtubule
fibers and were pulled to the spindle pole as the fibers shortened. Properly
attached chromosomes were not affected, suggesting local regulation of
microtubule dynamics by Aurora kinase activity. After disassembly of the
microtubule fibers, the chromosomes moved to their usual position at the
center of the spindle as correct attachment formed.
Several advantages of small molecule inhibitors, particularly in combination
with high-resolution live-cell microscopy, are demonstrated by this assay. In a
highly dynamic process such as mitosis, many events occur on timescales of
minutes or seconds. Ideally, perturbation of protein function and observation
of the effects of the perturbation would be possible on similar timescales.
Manipulation of protein function through the use of reversible small molecule
inhibitors, together with live-cell imaging, makes this possible. In the assay
described here, inhibitors of both the kinesin Eg5 and Aurora kinases were
effectivelyused as switches to turn enzymes on and off. With this high degree
of temporal control, a mechanism for correcting chromosome attachment
errors could be dissected without perturbing the preceding processes, such as
those involved in spindle assembly. Brefeldin A Principles of Membrane Transport

Our understanding of cell division has benefited greatly from studies with
small molecules, but these tools have also been applied successfully to other
dynamic processes in cell biology. One such process is the transport of lipids
2.7 Using Small Molecules to Unravel Biological Mechanisms

and proteins between distinct membrane-bound compartments, or organelles,

inside the cell. The small molecule Brefeldin A (BFA) was instrumental in
uncovering some of the basic principles of intracellular transport.
A fundamental question in cell biology is how an organelle can maintain
its identity in the presence of constant inward and outward flow of lipids
and proteins. In the secretory pathway, for example, proteins are synthesized
in the endoplasmic reticulum (ER), then transported to the Golgi apparatus
for processing, and finally exit the Golgi in transport intermediates that
fuse with the plasma membrane to release their contents outside the cell
(Fig. 2.1-7(a)). As an indication of the flow of lipids and proteins through
this pathway, bulk ER membrane was estimated to be depleted by transport
to the Golgi with a half-time of 10 min [Sl]. This observation suggested the
existence of a recycling pathway to return membrane to the ER, but the
first direct demonstration of this recycling pathway came from studies with
Brefeldin A (Fig. 2.1-7(b)).Early studies had shown that BFA blocked transport
of proteins out of the ER and caused disassembly of the Golgi [52, 531.
Careful analysis of BFA-treated cells demonstrated that within minutes of BFA
treatment, resident Golgi proteins redistributed to the ER. The redistribution
was shown both by localization of Golgi proteins and biochemically, as resident
ER glycoproteins were processed by the redistributed Golgi enzymes in the
presence of BFA [54, 551. After removal of BFA, the Golgi rapidly reformed
and the usual localization of Golgi proteins was reestablished, again within
minutes. These findings provided direct evidence for a Golgi-ER recycling
pathway and highlighted the dynamic nature of membrane transport between
the two organelles.
Subsequent studies with BFA led to additional insights into some essential
features of membrane traffic from the Golgi. A careful analysis of the timing
of events after BFA treatment showed that a 110-kD peripheral membrane
protein, whose identity was at that point unknown, dissociated from Golgi
membranes as the earliest detectable event (within 30 s) in BFA action and
reassociated after removal of BFA as the Golgi reformed [SG]. Other peripheral
membrane proteins did not dissociate but redistributed to the ER instead, as
had been shown for resident Golgi proteins. These findings suggested that the
110-kD protein played a critical role in the regulation of membrane transport
from the Golgi.
The 110-kD protein was subsequently purified and cloned and shown to be
identical to B-COP, a component of the coat protein 1 (COPI) (or coatamer)
complex, which forms the coat of vesicles budding from the Golgi [57, 581.
This finding, together with the known effects of BFA, led to the hypothesis
that COPI-coated vesicles mediate forward membrane flow from the Golgi.
Inhibition of this process with BFA would allow retrograde flow to dominate,
so that Golgi membranes would be transported back to the ER, as observed.
The hypothesis was tested in a cell-free system in which the budding of
COPI-coated vesicles from Golgi membranes could be reconstituted in vitro
[59].BFA prevented the assembly ofthe COPI coat in this system, as predicted.
I 2 Using Natural Products to Unravel Biological Mechanisms

Fig. 2.1-7 (a) Schematic ofthe secretory ARF CTPase. Exchange o f GDP for GTP on
pathway. Transport vesicles carry membrane ARF triggers ARF-CTP binding t o Colgi
and soluble material from the ER t o the membranes. After ARF-CFP binding, the
Colgi and from the Golgi to the plasma coatamer complex assembles on the
membrane, where the soluble contents are membrane and induces budding o f a
released into the extracellular space. transport vesicle. ARF hydrolyzes CTP after
(b) Structure of the small molecule Brefeldin vesicle budding t o release coatamer and
A. (c) Regulation ofvesicle budding by the ARF-CDP from the membrane.

Together these experiments linked the COPI complex with forward membrane
transport from the Golgi, through the observed effects of BFA on both COPI
coat assembly and the dynamics of ER-Golgi trafficking.
2.7 Using Small Molecules t o Unravel Biological Mechanisms

BFA continued to be instrumental in understanding the regulation of coat

assembly. In a semipermeabilized cell system, GTPy S, a nonhydrolyzable
analog of GTP, was shown to prevent the BFA-induced dissociation of the
110-kD protein (at that point not known to be p-COP) from the Golgi [GO].
This finding suggested that the GTP-GDP (guanosine diphosphate) cycle
was involved in the process inhibited by BFA. A small GTP-binding protein,
adenosine diphosphate ribosylation factor (ARF), was a candidate involved in
this mechanism because it was known to associate with the Golgi and had
been implicated in Golgi transport processes [ G l ] . When the sensitivity of this
protein to BFA was examined, BFA was found to inhibit ARF binding to Golgi
membranes, both in cells and in vitro, while GTPyS prevented this inhibition
[G2]. These results were consistent with the effects of BFA and GTPyS on
,&COP. Furthermore, ARF was shown to be a subunit of the COPI coat
[G3].Together, these findings suggested that the GTP-binding state of ARF
regulates COPI coat formation. To place the events in an ordered biochemical
process, BFA was shown to be required for association of ARF with Golgi
membranes, and ARF was then required for binding of p-COP [G4].
A more detailed biochemical understanding of the mechanism of BFA action
was provided by the finding that an activity associated with Golgi membranes
catalyzes GDP-GTP exchange on ARF and is inhibited by BFA [65, 661.
The interpretation was that BFA acts by preventing nucleotide exchange on
ARF, which prevents ARF binding to membrane, an event required for coat
assembly and vesicle budding. This result suggested a general model for
membrane transport in which ARF proteins regulate assembly of coated
vesicles through changes in the GTP-GDP binding state and therefore control
vesicular trafficking (Fig. 2.1-7(c))[G7].
Much more work has been done with BFA, for example, to understand
its mechanism of action in more detail [G8], but the studies discussed here
illustrate many key features of the small molecule approach. Interest in BFA
was initially stimulated by its dramatic phenotype on a biological process: traffic
of proteins through the secretory pathway. Before the underlying mechanism
was understood in molecular detail, the inhibitor was instrumental in a
series of experiments that revealed some of the key principles of membrane
transport. Though BFA was not directly involved in all of the experiments,
interpretation of many of the findings depended on placing the results in the
context of BFA action. These experiments demonstrated the dynamic nature of
ER-Golgi transport and the role of the COPI coat complex in vesicle formation.
Furthermore, the role of the ARF GTPase in coat assembly led to a model for
regulation of vesicular trafficking.
Several properties of BFA as a small molecule were exploited throughout
these experiments. Reversibility and temporal control were used to understand
the dynamic nature of the events and to place them in an ordered process. In
addition, BFA was used in multiple systems, including various cell types and
in vitro, so that insights from biochemical experiments could be interpreted in
the context of a complex cellular process.
I 2 Using Natural Products to Unravel Biological Mechanisms Catalysis by Ribosomal RNA

Small molecules can be used to address problems at the level of biochemical
reactions as well as larger-scalecellular processes. Puromycin, a small molecule
inhibitor of protein synthesis, has contributed to our understanding of the
catalysis of peptide bond formation. Protein synthesis in a cell takes place on
a large assembly of protein and RNA components called the ribosome. This
structure carries out the complex task of reading the codons of an mRNA
molecule, selecting the appropriate amino acid for each codon, and catalyzing
the formation of a peptide bond between that amino acid and the preceding
one in the polypeptide chain (the peptidyl transferase reaction). It was initially
assumed that ribosomal proteins were responsible for the peptidyl transferase
activity, but experiments in the 1970s suggested that ribosomal RNA might be
directly involved. The discovery of catalytic RNA in the 1980s [69, 701 led to the
hypothesis that ribosomal RNA, rather than protein, might catalyze peptide
bond formation.
An experiment was designed to test this idea on the basis of the logic
that if catalysis is RNA based, it might be possible to remove ribosomal
proteins without loss of peptidyl transferase activity. The assay used to
measure transferase activity had been developed two decades earlier as a model
reaction to study the mechanism of peptide bond formation [71].In this assay,
both ribosomal substrates, the growing polypeptide chain and the incoming
aminoacyl-tRNA,are replaced with simplified molecules: a tRNA fragment,
CAACCA-formyl-methionine, and the small molecule puromycin (Fig. 2.1-8).
The “fragment reaction” requires only the large (50s) ribosomal subunit,
without small subunits or other factors. Peptidyl transferase activity can be
measured as formation of the product f-Met-puromycin, using 35 S-labeled
methionine. Exploiting this model system, catalytic activity was measured
following extraction of ribosomal proteins from the 50s subunit, using
procedure designed to cause minimal perturbation of RNA structure. Ninety-
five percent of the ribosomal protein could be removed by treatment with
SDS (sodium dodecyl sulfate) and proteinase K, followed by phenol extraction,
while maintaining over 80% activity [72]. In contrast, transferase activity was
rapidly lost upon treatment with ribonuclease. While this result could not
formally exclude the possibility that catalysis was carried out by the remaining
5% of ribosomal proteins, it strongly supported the hypothesis that ribosomal
RNA was responsible for peptidyl transferase activity.
In the fragment reaction, the ability of puromycin to mimic the aminoacyl-
tRNA in the peptidyl transferase reaction was exploited to measure catalytic
activity. Puromycin was subsequently used to design a transition-state analog
for the peptidyl transferase reaction, known as the Yams inhibitor, in which
it is linked to the oligonucleotide CCdA by a phosphoramide group [73]. In
a complex with the 50s ribosomal subunit, the Yams inhibitor was used to
define the catalytic site in a high-resolution crystal structure. N o protein was
found within 18 A of this site [74]. This result demonstrated conclusively that
the catalytic activity indeed resides in the ribosomal RNA.
2. I Using Small Molecules to Unravel Biological Mechanisms
I 89

polypeptide chain


polypeptide chain

NHz ReleasedtRNA

", Purornycin

Fig. 2.1-8 (a) Elongation o f a polypeptide peptidyl-tRNA. (b) The small molecule
chain. The amino group ofthe incoming puromycin replaces the arninoacyl-tRNA in
aminoacyl-tRNA joins the carbonyl group o f the polypeptide chain and prevents further
the growing polypeptide chain to replace the elongation.


The experiments described in this chapter illustrate how small molecule

inhibitors have been used to design strategies to address fundamental
biological problems. As our understanding of the biology advances, the use
of small molecules should complement genetic and RNAi-based approaches.
The advantages of small molecule inhibitors have been emphasized here, but
there are also significant limitations that should be considered, particularly
in comparison with genetic approaches. For example, genetics can be used to
target any gene for mutation or deletion without direct effects on any other
gene. Discovery of a new small molecule inhibitor, however, is challenging.
Another limitation is the difficulty of demonstrating specificity of small
molecule inhibitors. Taking a kinase inhibitor as an example, testing the
I 2 Using Natural Products to Unravel Biological Mechanisms

effects on over 500 kinases in the human genome is a substantial undertaking.

Using small molecules in focused assays is one way to address specificity, so
that a narrowly defined biological process is examined and off-target effects are
less likely to be relevant. In combination with this approach, several inhibitors
that target the same protein can be compared. If the inhibitors are chemically
unrelated, they are not expected to have similar off-target activities. Future Directions

Only the availability of inhibitors and the assays that can be designed around
them limit the future use of small molecule inhibitors to address biological
questions. Currently, only a small fraction of the proteome can be targeted
by small molecules. As new inhibitors are identified, small molecule-based
strategies will be applicable to an increasing range of biological problems.
The development of methods to monitor protein function with high temporal
and spatial resolution, particularly in living cells, will also increase the scope
for using small molecules. Recent advances in fluorescence-based probes, for
example, have made it possible to monitor numerous properties of living
cells, including membrane potential, pH, posttranslational modifications,
protease activity, and mediators of intracellular signaling such as Ca2+ and
cyclic adenosine monophosphate (AMP) [75]. These high-resolution readouts,
with the temporal control afforded by small molecule inhibitors, should be
a powerful combination for examining biological mechanisms in living cells.
Methods have also been developed to measure the enzymatic activities of
single protein molecules in vitro. Investigating the effects of small molecule
inhibitors, both at this level and in a more complex cellular context, should
continue to provide insight into protein function.


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52. T . Fujiwara, K. Oda, S. Yokota, 60. J.G. Donaldson,
A. Takatsuki, Y. Ikehara, Brefeldin A J. Lippincott-Schwartz, R.D. Klausner,
causes disassembly of the Golgi Guanine nucleotides modulate the
complex and accumulation of effects of brefeldin A in
secretory proteins in the endoplasmic semipermeable cells: regulation of the
reticulum, J . Biol. Chem. 1988, 263, association of a 170-kD peripheral
18545-18552. membrane protein with the Golgi
53. Y. Misumi, K. Miki, A. Takatsuki, apparatus, J. Cell Biol. 1991b, 112,
G. Tamura, Y. Ikehara, Novel blockade 579-588.
by brefeldin A of intracellular 61. T. Stearns, M.C. Willingham,
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itinerant Golgi proteins to the 62. J.G. Donaldson, R.A. Kahn,
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Chemical Biology
Edited by Stuart L. Schreiber, Tarun M. Kupoor,and Gunther Wess
CoDvriaht 0 2007 WILEY-VCH Verlaa CmbH & Co KCaA. Weinheim

2.2 Using Natural Products to Unravel Cell Biology
I 95

Using Natural Products to Unravel Cell Biology

Jonathan D. Gough and Craig M . Crews


In recent years, a new discipline has emerged from the interface of chemistry
and biology, known as chemical biology. The unique foundation of this field is
the examination of biological questions through the use of chemical probes. An
example of chemical genetics is the use of biologically active natural products
as “inducible alleles” for elucidating protein function. In this chapter, we
discuss a variety of different natural products and their use in understanding
cell biology.


With the sequencing of the human genome, advances in biological research

have grown exponentially. The use of genetic knockouts, RNA interference,
and site-directed mutagenesis to understand the roles of genes and gene
products is now becoming commonplace. Fundamentally, these methods
perturb protein expression at the genetic or transcriptional level. Although
these new tools have significantly improved our understanding of molecular,
cellular, and developmental biology, many questions still remain intractable.
Through the use of chemical genetics, biologically active compounds are now
being used as another means to address difficult biological questions.
Small molecules offer a significant advantage over classical genetic
techniques in that they can serve as “conditional alleles”. For example, a
small molecule that targets a specific protein can be used to “knock out” or
inhibit that protein only at a certain point during the cell cycle or during
an organism’s developmental process. In this approach, small molecules act
as “conditional alleles” that can be used in a temporal manner to induce or
inhibit a specific biological response, thus providing a method to selectively
investigate cell-signaling events within a narrow temporal window. In this
way, chemical genetics has provided the means to answer biological questions
that are difficult to study with standard genetic methods.

Historical Development

Evolution has taught us that biological systems find or create ways to adapt
to exogenous forces or stressors. Natural products are often the result of this

Chemical Biology. From Small Molecules to System Biology and Drug Design.
Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Gunther Wess
Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
l survival mechanism. These often highly potent small molecules encompass
2 Using Natural Products to Unravel Biological Mechanisms

a diverse array of structural variation and biological activities. Historically,

isolated compounds and extracts have been utilized as herbal remedies
or drugs. Initially, pharmaceutical companies utilized natural products as
a source or lead toward new drug candidates. Although most of these
compounds lack the potential for use as drugs, biologists in recent years
have found that natural products are useful for perturbing model cell systems.
As a class of compounds, they offer a unique starting point for investigating
biological systems. Because they are created in a living system, they are often
cell permeable and have specific biological targets. Using structure activity
relationships, via the analysis of analogs, natural products provide a starting
point for the development of new synthetic biological probes and insight into
their mechanism of action.

General Considerations

It is doubtful that Asperillus firnigatus evolved to produce the potent

antiangiogenic natural product fumagillin as a means to inhibit endothelial
cell growth. Nevertheless, secondary metabolites from many natural sources
have unexpected biological activities and have proved useful as cellular probes
or even as drug candidates. While many biologically active natural products
are isolated each year, not all have the potential to be effective biological
tools. Natural products are often isolated based on relatively simple bioassays
such as cell growth inhibition. Those compounds that block cell growth in
a nonselective manner (i.e., DNA intercalcation, ionophore activity, electron-
transport disruption), offer little in the ability to control specific intracellular
signaling processes. Thus, those natural products that most likely serve
as ligands for enzymes offer the most potential use as chemical genetic

Applications and Practical Examples HDAC Inhibitors: Histone Deacetylase Inhibitors

The posttranslational modification of histones provides a code for the
correct regulation of gene expression by affecting chromatin structure
and interaction with regulatory factors. Modifications include acetylation,
deacetylation, phosphorylation, methylation, and ubiquitination [l].Histone
acetyltransferases(HATS)serve to activate gene transcription by acetylating the
E-amine of lysine residues of histone tails. Conversely, histone deacetylases
(HDACs) serve to deacetylate the lysine residues resulting in chromatin
condensation and subsequent transcriptional silencing [2]. Since the discovery
2.2 Using Natural Products t o Unravel Cell Biology

of the first HDAC inhibitors trichostatin A (TSA)1 and trapoxin (TPX) 2 in the
I 97

1990s [3] these, and other similar inhibitors have provided insight into a diverse
array of cell-signaling events: cell cycle arrest, apoptosis, cell differentiation,
angiogenesis, and metastasis inhibition. The general mechanism of action
for many of these natural products entails an aliphatic chain with a metal
chelating moiety that interferes with zinc coordination in the binding pocket
of their targeted HDACs.

3 Trichostatin A
The antifungal natural product TSA, originally isolated from a Streptomyces,was
found to have reversible biological activity at low nanomolar concentrations.
Yoshida and coworkers [4]demonstrated that TSA causes the induction of
Friend leukemia cell differentiation as well as inhibition of the cell cycle of
normal rat fibroblasts in the G I and G2 phases. This initial work revealed that
at low nanomolar concentrations, TSA induces the accumulation of acetylated
histones because of inhibiting HDAC activity within the cell.
TSA has also been shown to induce apoptosis in various tumor cell lines
[5] thereby making HDACs possible targets for cancer treatment. By blocking
HDACs, inhibitors such as TSA affect the level of gene transcription, causing
both the up- and downregulation of many genes ( ~ 2 % of the genome)
[GI. For example, TSA was found to reduce the expression of cyclin B1, a
key cyclin for G2-M transition, but in fact also stimulated expression of
p21C1P/WAF, an inhibitor of cyclin-dependent kinase (CDK)and Cdc2. Through
TSA-mediated HDAC inhibition, the G2-M transition is blocked because of
I 2 Using Natural Products to Unravel Biological Mechanisms

increased transcription of cell cycle regulators, p21C'P/WAF and cyclin B1. This
occurs via the modulation of histone acetylation at these gene promoters [7].
In addition, TSA has proved useful in the elucidation of important nuances
of cell differentiation. Cell cycle inhibitors had shown that inhibition of
proliferation was necessary, but not sufficient, for the differentiation of
neuronal precursor cells into oligodendrocytes [8]. Given the significant level
of chromatin remodeling that accompanies cellular differentiation, Marin-
Husstege and colleagues [9]hypothesized that histone acetylation plays a role
in oligodendrocyte differentiation. Using synchronized primary neonatal rat
cortical progenitors that were induced to differentiate into oligodendrocytes,
the authors showed that there is a temporal window during which histone
deacetylation is correlated with the acquisition of a branched morphology and
myelin gene expression. TSA-treated progenitors were able to exit from the
cell cycle but did not progress into oligodendrocytes. The ability of HDAC
inhibitors to inhibit oligodendrocyte differentiation is cell lineage dependent,
although TSA did not affect the precursor cells' ability to differentiate into
astrocytes. These results suggest that transcriptional repression is a crucial
event during oligodendrocyte lineage progression. Trapoxin
The irreversible HDAC inhibitor TPX was first isolated as a fungal metabolite
that induced morphological reversion of v-sis-transformed NIH 3T3 cells
[lo]. Using the known structure-activity relationship between other HDAC
inhibitors as a guide, a TPX affinity reagent was synthesized and used to
identify its target protein as a HDAC [ll].
TPX was used to elucidate the protein interactions necessary for HDAC
mediated transcriptional repression via the Mad:Max ternary complex [ 121.
Previous studies had suggested that Mad:Max transcriptional repression
was mediated by ternary complex formation with another unknown protein.
Biochemical experiments identified the proteins mSin3A or B as the primary
candidates responsible for this negative transcriptional function. Coexpression
of activated or inactivated MAD (a DNA-binding transcription factor) in the
presence of TPX demonstrated that HDAC activity was necessary for ternary
complex formation. Additionally, these and other experiments showed that
the Mad:Max heterocomplexes repress transcription in a mSin3A-associated
H DAC-dependent manner. Apicidin and Depudecin

Like TPX, the rnicrobially derived HDAC inhibitor depudecin 4 was also
isolated based on its ability to reverse the transformed cellular phenotype
of tumor cells. This diepoxide-containing natural product induced a flat
phenotype in Ki-rus-transformed NIH 3T3 cells and was further characterized
as an HDAC inhibitor by its ability to induce the accumulation of acetylated
histones [13]. Apicidin (APC) 3, a cyclic tetrapeptide HDAC inhibitor with
2.2 Using Natural Products to Unravel Cell Biology

structural similarity to TPX, was shown to possess potent antiproliferative

I 99

activity against various cancer cell lines [14], and like depudecin, displays
potent in uitro and in uivo antiangiogenic activities [15, 161. Thus, given the
ability of HDAC inhibitors to arrest cell proliferation and reverse tumor cell
morphology, HDAC inhibitors have generated much attention as a new class
of antitumor drugs. Cyclin-dependent Kinase Inhibitors

Cyclin-dependent kinases (CDKs) play key roles in regulating cell cycle
progression. Throughout the cell cycle, different CDKs are activated and are
directly responsible for driving the cell from one phase to the next. Individual
CDK activity is regulated by a number of cellular processes: cyclin association,
association with cyclin-dependent inhibitors (CDI),CDK synthesis, proteolysis,
and various posttranslational modifications. Progression through the cell cycle
is controlled by the concentrations of different cyclin proteins, Thus, cyclin
degradation results in the loss of activity from its CDK partner, leading to the
arrest of the cell cycle. The regulation of cell cycle progression is important
for the cells’ ability to deal with external stresses. Therefore, CDKs serve a
checkpoint function, in that the cellular stress can block entry into the next
phase of the cell cycle through the expression of a member of the three major
CDI families, p21C’p’wAF, and ~ 1 6 [17].” ~ ~ ~
The idea of targeting CDKs represents a completely different strategy
for treating tumor cells: finding small molecules that inhibit specific
molecular targets as opposed to drugs that just kill tumor cells. Functionally,
all CDK inhibitors act by competitive inhibition of ATP binding to a
CDK. Whereas disruption of the CDK-cyclin interaction is an attractive
therapeutic strategy given its requirement for CDK activity, the large
protein-protein-binding surface of this interaction makes it a less-than-ideal
target relative to the small, well-defined ATP-binding pocket of CDK.
Accordingly, several antiproliferative natural products target the ATP-binding
site on CDKs. Purine Analogs

The natural products olomoucine 7 and roscovitine 8 are relatively selective
kinase inhibitors that bind CDK1, 2, and 5 but have little effect on CDK4 and
G [18].These selective CDK inhibitory profiles result in cell cycle arrest in
the GI and G2 phases. Both inhibitors act in a dose-dependent and reversible
manner, thus allowing temporal control of CDK activity at different stages of
the cell cycle.
CDK inhibition by these potent natural products results in four major cellular
consequences: (a) inhibition of cell proliferation; (b) induction of apoptosis in
mitotic cells; (c) induction of cellular differentiation; and (d) protection from
apoptosis. Several studies have shown that purine derivatives arrest cells in
I 2 Using Natural Products to Unravel Biological Mechanisms

\ /

5 6 7 8


OH 0

9 10 11

either GI or GZ [19-211 primarily due to CDK2 and CDKl inhibition; however,

the effect on Erkl/2 activity has also been demonstrated [22]. CDK purine
derivative inhibitors also induce apoptosis in mitotic cells when combined
with other drug treatments. For example, roscovitine and olomoucine were
found to synergize with a farnesyltransferase inhibitor [23] to induce apoptosis
of human cancer cell lines. In addition, the combination of the microtubule
stabilizing drug Taxol@with the CDKl inhibitor purvalanol A 9 results in HeLa
cell apoptosis [24]. Treatment with either Taxol@or purvalanol A alone and in
combination (in the reverse order) were ineffectual, demonstrating an ordered
cooperativitybetween the two drugs. Likewise, the induction of differentiation
in murine erythroleukemia cells is triggered by the combined sequential
inhibition of CDK2 (with roscovitine) and CDK6 (via p16"K4a), while the
reverse sequence of inhibition was ineffective [20,25,26].Finally, purine analog
inhibitors of CDKs (5-10) can protect cells from apoptosis via a mechanism yet
undefined. Examples of this phenomenon include the prevention of CAMP-
induced apoptosis in rat leukemia cells [27], etoposide-induced apoptosis
in rat fibroblasts [28], and cell death in human immunodeficiency virus
(H1V)-inducedsyncytia [29]. Flavopiridol
Flavopiridol (FLV) 11 is a sernisynthetic flavinoid derived from rohitukine, an
indigenous plant from India [30]. FLV can induce cell cycle arrest by three
mechanisms: (a) direct inhibition of CDK via binding in the ATP-binding site;
2.2 Using Natural Products to Unravel Cell Biology I 101

(b) inhibition of CDK7/cyclin H consequently leading to loss of CDK activation

[31];and (c) decreased levels of cyclin D1, an oncogene that is overexpressed in
many human neoplasias [32]. Initial studies revealed that FLV arrested cells in
GI or Gz due to CDKl and CDK2 inhibition [33-351. In vitro studies, however,
revealed that FLV inhibits all CDKs thus far examined (IC50 100-300 nM)
[ 35 - 371. Proteasome Inhibitors

Cell homeostasis and proliferation is dependent on both protein synthesis
as well as protein degradation. The proteasome serves as the primary
regulator of intracellular proteolysis. Specifically, the proteasome is a 700 kDa,
multicatalytic protease complex composed of two 19s regulatory particles
flanking the 20s proteolytic cylinder [38], itself consisting of 28 subunits
organized into four rings. The proteasome has three major classes of protease
activities: (a) trypsinlike activity; (b) chymotrypsin-like activity; (c) peptidyl-
glutamyl peptide hydrolyzing (PGPH) activity or caspaselike activity. Each
protease function appears to act independently, thereby degrading most
proteins into six to eight amino acid peptides. Proteins are targeted for
proteolysis via conjugation to 76 amino acid polypeptide ubiquitin (Ub)
catalyzed by a multistep process involving a series of enzymes that: activate
the Ub monomer ( E l ) , recognize the protein targeted for degradation (E3),
and transfer Ub monomers to lysine residues on the targeted protein (E2).
The proteasome has been implicated as a key player in a number of
important cellular processes including apoptosis, cell differentiation, M HC
class I antigen presentation, NF-KB activation, tumor suppression, and cell
division. In particular, the prominent role that the proteasome plays in cellular
proliferation has generated much attention toward the use of proteasome
inhibitors as antitumor chemotherapeutic agents. As more and more cellular
functions are linked to the proteasome, the use of proteasome inhibitors will
be increasingly important in the investigation of various signaling interactions. Lactacystin
Originally characterized as a microbial metabolite that induced neurite
outgrowth in neuroblastoma cells [39, 401, lactacystin 14 was later found
to be a potent inhibitor of cell proliferation [41]. Using a [3H] lactacystin
analog, Fenteany and coworkers [39] demonstrated that lactacystin and
its related clasto-B-lactone covalently bind the N-terminal threonine of the
20s proteasome subunit. Functionally, lactacystin is a relatively nonspecific
protease inhibitor, also showing significant inhibition of peptidyl peptidase I1
and cathepsin A [40].Despite this cross-inhibitory activity, lactacystin has been
used to investigate the role of the Ub proteasome pathway in a diverse array
of systems such as Alzheimer’s disease, breast cancer, neurobiology, kidney
research, and nephrology, to name a few [41-461.
I 2 Using Natural Products to Unravel Biological Mechanisms


14 a,b-Epoxyketones
Selective covalent inhibitors of proteasome have also been developed.
Epoxomicin and eponemycin are members of the cr,B-epoxyketone class
of proteasome inhibitors that were isolated from actinomycete strains and
found to exhibit in vivo antitumor activity against B16 melanoma [47,48]. Early
structure activity studies and structural motifs present in similar molecules
suggested that the terminal epoxyketone moiety was an important aspect of
the functional pharmacophore, possibly via covalent modification of its target
protein. Through synthetic chemistry and biochemical affinity techniques, the
natural products and corresponding biotinylated affinity reagents have been
used to identify the 20s proteasome as the molecular target of epoxomicin 12
and eponemycin 13 [38,491.
X-ray crystallographic analysis demonstrated that the epoxyketone pharma-
cophore of epoxomicin forms a covalent adduct as a morpholino ring [SO] with
the amino terminal threonine of the 20s proteasome. Epoxomicin draws its
specificity from the uniqueness of the proteasomal N-terminal threonine; non-
proteasomal proteases lack an N-terminal nucleophilic residue and thus cannot
form a stable covalent morpholino adduct with the epoxomicin epoxyketone
pharmacophore [50].
These potent and specific proteasome inhibitors have been used to answer
questions in a number of biological fields and systems. For example, protea-
some inhibitors have been used to investigate inflammation, cancer biology
2.2 Using Natural Products to Unravel Cell Biology I 103

and neuroscience. In immune research, chemokines and their receptors play

an important role in host immune surveillance and are important mediators
of HIV pathogenesis and inflammatory response. Chemokines and their re-
ceptors have also been implicated in hematopoiesis, angiogenesis, embryonic
development and breast cancer metastasis. Specifically, they play important
roles in immune and inflammatory responses by regulating the directional
migration and activation of leukocytes. The chemokine receptors CXCR4 and
CCR5 have been shown to act as coreceptors for the entry and infection
of HIV-1 and HIV-2. The proteasome inhibitors lactacystin and epoxomicin
have been used to show that downmodulation mechanisms and chemotaxis
mediated by CCR5 and CXCR4 are dependent upon proteasome activity [51]. TMC-95A
Recently, more selective noncovalent inhibitors of proteasome have been
developed. TMC-95A 15 is a potent and reversible selective inhibitor of the
chymotrypsin-like, trypsinlike, and caspaselike activities ofthe 20s proteasome.
Comparatively, TMC-95A shows no inhibition of calpain, cathepsin, or trypsin.
This selectivity in activity has led to a great deal of current biological interest
in TMC-95A [50, 52,531 including X-ray crystallographic analysis showing that
TMC-95A does not covalently bind the yeast proteasome [54]. ATPase Inhibitors

Vacuolar ATPases (V-ATPases)are a class of enzymes that are found through-
out eukaryotes. Fundamentally, these multisubunit complexes function as
proton pumps, moving hydrogen ions from one side of a membrane to the
other. In so doing, they alter the pH of the distal compartment. Typically,
V-ATPases perform this function on the membrane of cellular vacuoles and
are dependent on ATP for the energy required to carry out their function.
Structurally, eukaryotic V-ATPases are comprised of 13 different polypeptides,
which are defined as comprising two specific functional domains; Vo is the
transmembrane-ion channel domain and V1 is the ATPase or ATP-binding
domain. Small molecule V-ATPase inhibitors are thought to function primarily
through binding to and inhibiting the Vo domain. In recent years, V-ATPase
have become important drug targets because their inhibition leads to highly
specific cytotoxic effects [55]. Bafilomycins and Concanamycins

A series of macrolides, bafilomycins 17 and concanamycins 16 were isolated
in a screen for secondary microbial metabolites having effects similar to those
of the cardiac glycosides ouabain and digitoxin [56].Their V-ATPase inhibitory
effects were not recognized until Bowman and colleagues discovered that
bafilomycins inhibit H+ V-ATPases at nanomolar concentrations [57]. Until
then these compounds had exhibited a wide range ofbiological activities: in vitro
I 2 Using Natural Products to Unravel Biological Mechanisms

inhibition of P-ATPase, antihelminthic activity against Caenorhabditis elegans,

stimulation of y -aminobutyric acid release from rat brain synaptosomes,
selective antifungal activity and inhibition of concanavalin-A-stimulated T-cell


I 0


From a functional standpoint, V-ATPases act as regulators of organelle pH

by pumping protons from the cytoplasm into the lumen. Inhibition of this
regulatory effect results in cytotoxicity. However, because these compounds
bind reversibly, they can be used to perturb a given system for the purpose of
understanding the effect of pH change on other cellular functions or protein
interactions. In addition, as they are reversible, recovery from drug treatment
can also be observed. Examples include inhibition of acidification in pinocytic
vesicles, inhibition of lysosomal acidification and degradation of Epidermal
Growth Factor (EGF)in mammalian cells [55]. Angiogenesis Inhibitors

Angiogenesis is the formation of new blood vessels from preexisting
blood vessels and is required for wound healing and reproduction. In
addition to these homeostatic roles for angiogenesis, the formation of
new blood vessels has been found to be required for the metastasis and
growth of tumors. Since Judah Folkman [58] proposed the link between
angiogenesis and tumor growth/metastases, much effort has focused on the
identificationand developmentof antiangiogenic small molecules as antitumor
chemotherapeutic agents.
Angiogenesis is closely regulated through the complex interactions of
endogenous factors that promote and inhibit the process. In general,
2.2 Using Natural Products to Unravel Cell Biology I 105

angiogenesis proceeds through three steps [59, 601: degradation of the

basement membrane, invasion or migration of cells through the degraded
matrix, and differentiation into mature blood vessels. For endothelial cell
proliferation to occur, the existing blood vessel cells must degrade the
underlying basement membrane and invade the stroma of the neighboring
tissue. Once the barrier has been broken, cells proliferate and migrate
into the underlying tissue. The cells differentiate and form capillary loops.
Subsequently, cell polarity is established and the formation of the lumen
begins. Small molecules that interrupt the various phases of angiogenesis
have been insightful in determining important signaling events that regulate
the various processes involved. Curcuminoids
Curcuminoids, a group of natural products originally isolated from the
Indian spice turmeric, have been known to be potent antioxidant and anti-
inflammatory agents for many years. Curcuminoids reduce tissue factor (TF)
gene expression through the inhibition of the AP-1 and NF-KB transcription
factors and thus lead to the loss of angiogenesis initiation [Gl,621. Fumagillin and TNP-470

Fifteen years ago, an astute observation made during the routine culturing
of endothelial cells led to the identification of a new antiangiogenic natural
product. The natural product fumagillin 18 was isolated from a contaminated
A. &migatus fresenius colony in the Folkman laboratory. Subsequent
derivatization of the parent natural product by Takada Pharmaceuticals yielded
the drug candidate TNP-470 19 that was 50 times more potent than the parent
natural product fumagillin [63]. Using the structure activity relationship as
a guide, a biotinylated affinity reagent was synthesized and used to identify
methionine aminopeptidase 2 (MetAP-2)as the molecular target of fumagillin

I and TNP-470 [G4].X-ray crystal structures of the free and the fumagillin-bound
2 Using Natural Products t o Unravel Biological Mechanisms

MetAP-2 revealed the mechanism of action of this potent natural product;

a covalent bond between the reactive spirocyclic epoxide of furnagillin and
histidine-23 1 of MetAP-2 blocks the active site.
Endothelial cells, unlike fibroblasts, display an impressive sensitivity to
fumagillin and TNP-470 addition. At the molecular level, TNP-470 does
not inhibit early GI mitogenic events such as cellular protein tyrosyl
phosphorylation or the expression of immediate early genes [GS]. However,
TNP-470 was found to induce expression of the CDK inhibitor p21C'P/WAF and
p53 in endothelial cells [GG]. Moreover, the function of both p21C1P/WAF and
p53 were shown to be essential for the endothelial cell cycle GI arrest induced
by TNP-470 and lack of p21C'P/WAF abrogates the inhibitory activity of TNP-470
on corneal angiogenesis in vivo. Thus it was shown that these antiangiogenic
compounds act through p21C'P/WAF induction to GI cell cycle arrest. Immunosuppressant Natural Products

Using the immunosuppressive natural products cyclosporin A (CsA) 20,
rapamycin 22, and FK 506 21, researchers were able to unravel two key


22 21
2.2 Using Natural Products t o Unravel Cell Biology 1 107

signal transduction pathways in T lymphocytes (T cells). T cells respond to

an immune stimulus through the binding of an antigen-presenting cell to the
T-cell receptor (TCR).Binding subsequently initiates a cascade of intracellular
signaling events leading to activation and proliferation of the T cells and other
cell types required for an immune response. Importantly, this process induces
the transcription and thereby production of a range of effector molecules like
interleukin 2 (IL-2);IL-2 is secreted and binds to IL-2 receptors on various cells
including T lymphocytes and stimulates the cells to progress from G I to the
S phase of the cell cycle. This sequential chain of events drives the immune
response. Immunosuppressive natural products have proved useful in the
elucidation of several immune cell signal transduction pathways through the
identification of specific target proteins. Cyclosporin A and FK 506

CsA is a cyclic undecapeptide that was isolated from the fungus Cylindrocarpon
lucidum Booth and Tolypocladium injlatum Gams in 1970 by the Sandoz
Laboratory. Interestingly, CsA has both high potency and selectivity for
inhibition of T-cell activation with low cytotoxicity. The structurally unrelated
polyketide metabolite FK 506, isolated in 1984 by the Fujisawa Pharmaceutical
Company from the fungus Streptomyces tsukubaensis 9996, proved to have
100 times greater immunosuppressive activity than CsA. Although the two
natural products were structurally different and had different potencies, they
exhibit the same phenotypic biological activity; both compounds prevented the
progression from Go to G I during T-cell activation.
CsA and FK 506 have proved to be critical tools in elucidating the signaling
events downstream of the TCR. Both were found to block the same step in
Ca2+-dependentsignaling pathways. Additionally, these natural products were
also found to bind to peptidyl-prolyl cis- trans isomerases, collectively known
as immunophilins. CsA binds cyclophilin [67] and FK 506 binds FKBP 12
[68].Although it appeared that both natural products functioned through the
same mechanism of calcium-dependent gene expression, oddly neither target
protein alone initiated the release of calcium. For the cell cycle inhibition,
both the small molecule and the protein are needed to be present. Using
affinity chromatography with immobilized protein-natural product complexes,
the phosphatase calcineurin was identified as the target of both protein-drug
complexes [69]. In vivo the protein-ligand pairs formed immunosuppressive
complexes that inhibited the calcium-dependent calcineurin phosphatase
The T-cell specific transcription factor, NFAT is held in the cytosol through
the presence of an inhibitory phosphorylated residue. Upon TCR-mediated
calcium release, the calcineurin dephosphorylates NFAT, translocates to
the nucleus. CsA and FK 506 have proved useful in identifying this
pharmaceutically vulnerable step in immune cell signaling [70].
I 2 Using Natural Products to Unravel Biological Mechanisms Rapamycin
The fungal immunosuppressive agent rapamycin was isolated from Strepto-
myces hygroscopicus, originally found in a soil sample from Rapa-Nui, Easter
Island in 1975. Although structurally similar to FK 506, rapamycin demon-
strated markedly different activity. Rapamycin does not affect the progression
from Go to GI, but rather blocks T-cell progression from GI to S phase.
As FK 506 and rapamycin share structural similarities, it was not surprising
that rapamycin also bound FKBP 12. However, binding studies revealed that
the FKBP 12-rapamycin complex does not target calcineurin, as done by the
F K 50G-FKBP 12 complex. Rather, using FKBP 12-rapamycin complex as an
affinity reagent, the lipid kinases target of rapamycin 1 and 2 (TOR1 and
TOR2) were identified [71]; these proteins possess homology to the mam-
malian phosphatidyl inositol-3-kinases, which are involved in the regulation
of cell cycle progression in stimulated cells. Studies have shown that growth
factor addition to cells leads to TOR activation and subsequent increased p70
SG kinase activity [72]. Other Examples of Biologically Active Natural Products Capsaicin
Some of the most commonly and frequently used spices throughout the
world are hot peppers of the Capsicum family, of which capsaicin 23 is the
major pungent ingredient. Because of its analgesic and anti-inflammatory
activities, topical application of capsaicin has been used for the treatment of
a variety of neuropathic pain conditions. Autoradiographic visualization of
a tritiated resiniferatoxin probe in tissues of various species identified the
vanilloid receptor (VR) as a molecular target [73, 741. Additionally, capsaicin
was used as a molecular probe to isolate the first nociceptive receptor, VR1[75].
Characterization of VR1 revealed it to be a member of the Transient Receptor
Potential (TRP)ion channel family and a nonselective cation channel activated
by capsaicin or elevated temperatures.

2.2 Using Natural Products t o Unravel Cell Biology I 109 Parthenolide
Parthenolide 24, the biologically active natural product in the medicinal
herb Feverfew, has been used for 2000 years to treat fevers, headaches, and
inflammation [76]. Initial studies of the anti-inflammatory of parthenolide
activity showed that it was a potent inhibitor of NF-KB nuclear translocation
as well as I K B phosphorylation. Using a biotinylated analog of parthenolide
in affinity chromatography experiments revealed that parthenolide formed a
covalent adduct with IKB Kinase beta (IKK-B) in a specific and dose-dependent
manner [77]. This specific interaction between IKKB and parthenolide was
confirmed by mass spectrometric analysis. Parthenolide was shown to
form a covalent adduct with Cys179 of IKKB, which lies between the two
phosphorylated serines in the kinase activation loop. Moreover, constitutively
activated protein with a Cysl79Ala point mutation was found to be insensitive
to 40 pM parthenolide, indicating that parthenolide inhibits IKKB via Michael
addition by Cys179 in the kinase activation loop [77].

Future Development

Mechanism of action studies of biologically natural products have profited

greatly from the emerging field of chemical biology as chemists and biologists
have worked more closely over the last 15 years. Moreover, these natural
products will continue to be of great use as drug development leads in addition
to their use as tools for understanding intracellular processes.


After a decade, both natural products and cell-based bioassay screening, which
were out of favor, are making a renaissance in the pharmaceutical industry.
Natural products still offer an impressive range of chemical diversity and
have a long track record of providing scaffolds for successful drugs. A greater
appreciation of their potential for the identification of novel hit structures
is propelling a new interest in the use of natural product screens in the
pharmaceutical industry. Likewise, cell-based bioassays are regaining some
of their previous acceptance in the drug development process, primarily
because of the success of novel target deconvolution strategies. New proteomic
technologies are largely behind the belief that the pharmaceutical industry has
the ability to identify the targets of compounds identified in cell-based assays.
Obviously, not all biologically active compounds identified in these screens
will be developed into therapeutic agents. However, this renewed interest in
both natural products and cell-based assays will, in turn, offer many new
2 Using Natural Products to Unravel Biologicd Mechanisms
I opportunities for the development of novel cell biological probes, using the
fruits of these screens.


The authors would like to acknowledge the financial support of the NIH (grant


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Chemical Biology
Edited by Stuart L. Schreiber, Tarun M. Kupoor,and Gunther Wess
CoDvriaht 0 2007 WILEY-VCH Verlaa CmbH & Co KCaA. Weinheim

Engineering Control Over Protein Function Using Chemistry

Revealing Biological Specificity by Engineering Protein- Ligand Interactions

Matthew D. Simon and Kevan M . Shokat


Protein function can be altered in a rapid and graded manner through small
molecule ligand binding in both natural systems and through drug design. In
natural systems evolutionary pressure can lead to accumulation of mutations
that influence ligand binding specificity, thereby altering protein function.
Similarly, in the laboratory, mutations that have well defined effects on a
protein’s ligand specificity can provide a functional handle to elucidate the
protein’s biological role. Here we explore examples of mutations, introduced
in the laboratory or found in nature, that cause significant changes to protein
ligand specificity, with an emphasis on the biological and biochemical lessons
learned from these studies. The examples described here illustrate both the
challenges and the power of engineering protein-ligand interactions in order
to elucidate a protein’s biological role.

3.1 .I

The exquisite specificity observed in biological systems emerges from the

composite specificity of interactions at the molecular level. Understanding the
mapping between molecular interactions and their functional consequences is
the aim of molecular biology. While it is common to characterize biochemical
activities of a protein i n vitro, identifying the biological importance of these
activities in a complex environment such as a cell extract, an intact cell, or
even an entire organism, remains a daunting task. Genetic approaches provide

Chemical Biology. From Small Molecules to System Biology and Drug Design.
Edited bv Stuart L. Schreiber. Tarun M. Kauoor. and Gunther Wess
Copyright 0 2007 WILEY-VCH Verlag G k b H & Co KGaA, Weinheirn
ISBN 978-3-527-31150-7
I powerful means to investigate these biological activities (e.g., observing the
3 Engineering Control Over Protein Function Using Chemistry

phenotype that results from a gene disruption). However, protein engineering

can provide complementary information that connects the biochemical
specificity of a protein to its functional role. Here we discuss examples
where protein-ligand interactions can be engineered to provide a specificity
handle that can in turn be used to link a molecular interaction to a biological
In these experiments a protein is mutated to alter its ligand Specificity.
The resulting engineered protein-ligand interaction is then used to infer
the role of the unmodified protein in the biological system. The success of
this strategy requires that we specifically engineer protein-ligand interactions.
How feasible is it to alter a protein’s ligand specificity in a well-defined manner?
Are mutations that change the ligand specificity of a protein common or rare?
Are mutations that alter the specificity of small-molecule binding more or
less likely to destroy other functions or properties of the protein such as
its catalytic activity, stability, or cellular localization? From all the potential
mutations at a protein-ligand interface, what strategy do we use to identify the
productive mutations that have a desired effect on protein-ligand interactions?
Molecular evolution in nature provides inspiration to help answer these
While the mechanistic details accounting for the success of natural molecular
evolution are distinct from the practical details governing protein engineering,
there are similarities that are worth elaborating. In particular, molecular
evolution in nature demonstrates that a small number of mutations is often
sufficient to cause dramatic changes in the ligand-binding properties of
a protein. Similarly, in protein engineering, a single point mutation is
often enough to provide a specificity handle that allows a protein to be
uniquely sensitive or uniquely resistant to a small-molecule ligand. By keeping
engineered changes to the protein simple, the potential to rationally engineer
proteins is increased, and the chances ofother adverse effects are minimized. In
fact, many engineering strategies based on individual mutations are essentially
indistinguishable from natural strategies found throughout evolution. Here
we discuss several such examples.

The Selection of Resistance Mutations to Small-molecule Agents HIV Protease Inhibition and Substrate Selectivity

Drug resistance mutations are common in patients treated with anti-HIV
compounds such as indinavir and nelfinavir. These drugs act by inhibiting
HIV protease (HIV PR), one of the essential HIV proteins required for viral
growth and infection. These drugs inhibit HIV PR by competing with the
peptide substrate to bind in the active site of HIV PR (see Fig. 3.1-1). The
rapid emergence of inhibitor resistance is caused, in part, by the low fidelity of
3.1 Revealing Biological Specificity by Engineering Protein-Ligand Interactions I 1 1 7

lndinavir NeIfi navir

Fig. 3.1-1 HIV PR bound to a NC-pl peptide substrate [3] (a) and nelfinavir (b) (41.

the HIV-reverse transcriptase. An these experiments a protein is mutated to

alter its ligand specificity. The resulting engineered protien-ligand interaction
is then used to infer the role of the unmodified protein in the biological
system. For example, nelfinavir is a potent inhibitor of the wild-type HIV PR
(Ki = 0.28 nM) However, a double mutant of HIV PR (V82F/I84V) that has
I been observed in patients causes the virus to become refractory to nelfinavir
3 Engineering Control Over Protein Function Using Chemistry

(K,rnut/K,wt = 86) [I].

Given that the inhibitors mimic the protease's natural peptide substrate, it
is perhaps surprising that HIV PR mutants can overcome inhibition without
losing a critical level of enzymatic activity". The most common resistance-
causing mutations are found in close proximity to the substrate-binding
pocket. Given that the inhibitor-binding surface largely overlaps with the
substrate-binding surface, how do these mutations disrupt inhibitor binding
while retaining substrate recognition? Structural analysis reveals that the
inhibitors tend to penetrate deeply into the same pockets that the protease
uses to bind the side chains of its substrates - in fact, the inhibitors tend
to penetrate more deeply into the pockets than the substrates themselves.
Therefore, mutations in the protease can disrupt the deepest inhibitor contacts
while having a smaller effect on substrate binding [2]. Indeed, it appears
that the majority of the characterized inhibitor-resistance mutations work by
disrupting these deep inhibitor contacts, thereby selectively disrupting the
binding of one ligand (the inhibitor) without affecting another ligand (the
While many of the characterized HIV PR mutants do not substantially alter
the protease substrate specificity there are other resistance-causing mutations
that do, the best characterized of which is V82A. When the in vitro substrate
specificity of the V82A mutant (inhibitor resistant) was compared with the
wild-type (inhibitor sensitive) strain, the V82A-containing enzyme was found
to have a statistically significant increase in activity for Val over Ala at the P2
position of the substrate (P2 is the second amino acid N-terminal to the scissile
bond) [S]. So, in this case, mutations in HIV PR were selected, that disrupt
the inhibitor-binding surface, but in doing so, the substrate specificity of the
protease was also affected. So how does the virus accommodate this change in
HIV PR cleaves several substrates during viral development. Among these
sites, cleavage of the nucleocapsid-pl (NC-pl) site is rate limiting to viral
maturation. The occurrence of the V82A mutation in HIV PR correlates with
an alanine-to-valine mutation at the NC-pl cleavage site. In other words, it
appears that, under the pressure of selection caused by the HIV PR inhibitor,
a HIV PR mutant (V82A) was selected with alterations to the inhibitor-
binding site, thereby changing the substrate specificity at P2. Along with the
altered substrate specificity at P2 came compensatory mutations in one of
the substrate sequences (Ala-to-Valat P2). Residue V82 does not make direct
van der Waals contact with the P2 side chain. Rather, incorporation of an
Ala-to-Val mutation at P2 generally increases the quality of fit between the
substrate and the enzyme thereby compensating for loss of substrate-enzyme
contacts at V82 [3]. This structural difference explains why the V82A mutation
1) The V82FII84V mutant HIV PR is func- mutant (kcat/KM = 0.5 mM sc') is com-
tionally active - a vims with these mutants promised relative to the wild-type enzyme
is viable - yet the catalytic efficiency of the ( k c a t / K ~= 30 mM-' s -') 111.
3. I Revealing Biological Specificity by Engineering Protein-Ligand lnteractions 1 119

in the enzyme and the Ala-to-Val mutation in the substrate are found to
There are at least three lessons from HIV PR inhibitor resistance. First,
relatively few mutations are often sufficient to induce inhibitor resistance,
and in many cases a single point mutation is sufficient. Interestingly, several
mutations allow HIV to overcome inhibitor sensitivity demonstrating that
there are numerous solutions to the same engineering problem. While the
mutations are focused in regions that directly contact the inhibitor, as we might
expect, some are sufficiently subtle (e.g., acting through slight rearrangements
of the protein core) that it is hard to imagine predicting similar mutations
while attempting to rationally engineer a protein.
Second, relatively few mutations may be necessary to convergently engineer
a protein and its substrate - in this case natural selection led to a HIV PR
mutation (I82A) that changed its substrate selectivity and a compensatory
change in one of its substrates.
While the first two lessons are encouraging for the purposes of engineering
proteins with altered specificities, the third lesson is largely cautionary:
protein functions can be intimately interconnected. In at least one case,
altering the inhibitor surface of HIV PR affected the substrate specificity
of the mutant proteases. For this reason, engineering projects that intend
to dissect individual functions of a given protein must also take care to
control other unintended changes to the protein function. For example, it is
common that engineering a protein will adversely affect its stability or activity.
This natural example demonstrates the feasibility but also the challenges of
mutating a protein to alter its ligand specificity using only a small number of
mutations. Identification ofthe Target o f Rapamycin

While the emergence of HIV strains resistant to HIV PR inhibitors presents a
serious medical challenge, there are cases where the development resistance
mutations to an inhibitor can be invaluable, particularly when the mode
of action of an inhibitor is unknown. Such was the case with the small-
molecule immunosuppressant rapamycin [GI.The natural product rapamycin
became the subject of intensive study after it was demonstrated to block
helper T-cell activation through an unknown mechanism. Indeed, this is a
common problem with small-molecule agents; although it is straightforward
to isolate compounds that cause interesting phenotypes, identifying the
phenotypically relevant targets of the molecule can be challenging. Similarly,
once a putative target is identified, it can be difficult to establish whether
inhibition of that target is sufficient to cause the biological effect or whether
other targets may also contribute to the observed phenotype. In the case
of rapamycin, several groups conducted research to determine the specific
underlying biochemical interactions that lead to the phenotypic effects of
3 Engineering Control Over Protein Function Using Chemistry



Finding the binding partners of a small molecule is one common approach

in target identification.Attempts to identify the physiologically relevant cellular
targets of rapamycin led to the observation that rapamycin binds tightly to
the abundant peptidylprolyl rotamase, FK506 binding protein (FKBP).While
binding to FKBP appeared to be important for the activity of rapamycin, several
lines of evidence suggested that binding and inhibiting FKBP is not sufficient
to account for the cellular activity of rapamycin. For example, rapamycin is
toxic to yeast, yet strains lacking FKBP are viable; since FKBP is not essential,
its inhibition would not be expected to cause toxicity. Intriguingly, however,
yeast lacking FKBP are insensitive to rapamycin. This and other lines of
evidence [G, 71 lead to the subsequent realization that rapamycin binds FKBP
and that the FKBP-rapamycin complex then targets other cellular factors; it is
these other cellular factors that are responsible for the specific cellular activity
of rapamycin.
Focusing on yeast, a genetic screening was done to identify mutations
that conferred rapamycin resistance [8]. To accomplish this, yeast cells were
mutagenized and rapamycin resistant strains were identified. Some of the
mutations recoveredlocalized to FKBP, as would be expected (see Fig. 3.1-2(b)).
These mutations are recessive, consistent with the role of FKBP as an accessory
protein; even in the presence of the mutant, the wild-type copy is sufficient
to form the active rapamycin-FKBP complex. In addition to the recessive
FKBP mutations, two other proteins were implicated in rapamycin activity,
TOR1 and TOR2 (for target of rapamycin). Unlike the FKBP mutations, it was
found that TOR mutations had dominant effects, suggesting that these TOR
proteins (later identified as protein kinases related to the lipid PI3 kinase),
are the relevant targets of rapamycin responsible for cellular activity (see
Fig. 3.1-2(c)).Indeed, the mutations in the TOR proteins that cause complete
rapamycin resistance have been shown in vitro to block the binding and,
3.7 Revealing Biological Specijcity by Engineering Protein-Ligand Interactions I 121

Fig. 3.1-2 Mechanism of rapamycin lead to loss of inhibition. (c) Dominant

inhibition and resistance. (a) Rapamycin resistance mutations in TOR prevent
inhibits TOR through an FKBP-rapamycin FKBP-rapamycin binding and inhibition.
complex. (b) Resistance mutations in FKBP

therefore, inhibition of TOR by the FKBP-rapamycin complex. Furthermore,

although the initial identification of TOR was performed in yeast, several
studies demonstrated that a human homolog of TOR is responsible for the
immunosuppressive activity of rapamycin. In fact, that the mammalian TOR
deserves its name can be demonstrated using a similar line of experimentation;
the mutation in mammalian TOR analogous to one of those discovered in
yeast (S1975I) confers rapamycin resistance to mammalian cells.
In summary, mutations in a protein that caused small-molecule resistance
were used to map the phenotype induced by the small molecule to its
functionally relevant biochemical targets. Specifically, rapamycin acts by
binding the abundant protein FKBP. The resulting small-molecule protein
complex subsequently binds to and inhibits the TOR proteins leading to the
observed cellular effects of rapamycin. This seemingly complicated mechanism
of action is similar for immunosuppressants FK506 and cyclosporin A
(FK506 binds to FKBP and the complex inhibits the phosphatase calcineurin;
cyclosporin A binds cyclophilin and the resulting complex also inhibits
In the case of rapamycin, it was possible to demonstrate that TOR is the
target using a dominant mutant of TOR that is resistant to FKBP-rapamycin
inhibition. These resistance mutations are the single most definitive means
of demonstrating the phenotypically relevant target of a small molecule.
Unfortunately, the availability of resistance mutations can be limiting; attempts
to engineer such a mutation may adversely affect the function of the protein
3 Engineering Control Over Protein Function Using Chemistry
I (aswas demonstrated by the altered substrate specificity of the V82A mutant of
HIV PR). Similarly, isolating resistance mutations from genome-wide screens
(as was the case with TOR) is only tractable in organisms such as yeast
that are conducive to genetic manipulation. Nonetheless, the use of resistance
mutations to uncover and prove the functionally relevant targets of an inhibitor
is a powerful and definitive experiment. Kinase Inhibitors and Resistance

While inside the laboratory, whole genome screens (enabled by organisms
amenable to genetic manipulation) has made possible the identification of
resistance mutations, outside the laboratory similar screens are inadvertently
taking place in the real lives of cancer patients who are treated with
antineoplastic drugs. The ability to search for increased gene copy number
of known oncogenes and loss of heterozygosity at tumor suppressor loci,
the development of array-based comparative genomic hybridization for
identification of translocation events, and, most relevant here, the ability
to carry out high throughput DNA sequencing of candidate resistance genes
have allowed identification of numerous molecular markers of resistance
to chemotherapeutics. Many resistance loci are associated with increases
in the cancer cell’s ability to pump out the antineoplastic agent, such as
drug efflux pump mutants. These resistance mechanisms are independent
of the targeting agent, causing resistance to cis-platinum, doxorubicin, and
other general antiproliferative agents. Resistance mechanisms to molecularly
targeted therapeutics in contrast provide discreet insights into the mechanism
of action of these new generation antineoplastics.

BAY 43-9006 lmatinib

3.1 Revealing Biological Specificity by Engineering Protein-Ligand Interactions I 123

The prototype molecularly targeted therapeutic agent is imatinib, an

inhibitor of the Bcr-Abl tyrosine kinase. This oncogenic kinase is produced by
translocation of the Bcr locus on chromosome 9 to the c-Abl tyrosine kinase on
chromosome 11, termed the Philadelphia chromosome because of its discovery
in 1960 at the University of Pennsylvania School of Medicine by Peter Nowell
and David Hungerford from the Institute for Cancer Research [9].It was later
demonstrated in 1973 by Janet Rowley that the Philadelphia translocation was
responsible for a specific form of leukemia, chronic myelogenous leukemia
(CML)[lo].In 2001, imatinib was approved for treatment of CML patients, and
produced remarkable results with more than 92% patients achieving 14-month
progression-free survival on imatinib as a monotherapy.
The importance of imatinib in demonstrating the efficacy of a small-
molecule tyrosine kinase inhibitor for cancer therapy is its broad implication
for molecularly targeted therapeutics. First, it discounted the notion that
protein kinases could not be targeted selectively by small molecules that
bind to the ATP-binding site. In particular, the ATP-binding pockets of
different protein kinases were thought to be too similar for small molecules to
discriminate between them, yet imatinib only targets a handful of kinases (the
known targets ofimatinib include Bcr-Abl,c-Abl, PDGFR, and c-Kit).Also, the
high concentration of cellular ATP (>1 mM) was expected to limit the potency
of ATP-competitive drugs, yet imatinib is a potent inhibitor (IC50 < 1 pM). It
was also believed that the side effects associated with inhibition of wild-type
kinases (such as c-Abl) would be prohibitive, yet imatinib causes no overt
toxicity in normal cells while inducing apoptosis in CML leukemia cells.
Second, because of its ability to target Bcr-Abl expressing tumors, patients
could be classified into potential responders based on their Philadelphia
chromosome status. This genomic prescreening for responder populations is
widely viewed as a major avenue for improvement of therapeutic efficacy,
minimization of unnecessary toxicity in nonresponder populations, and
heralds the era of personalized medicine.
A third and more cautionary lesson from imatinib has been the rapid
emergence of imatinib resistance in CML patients. Initially, the advanced
stage CML patients, those in so-called blast crisis stage, who received imatinib
late in disease, showed high rates of resistance. Currently, all CML patients are
given imatinib upon diagnosis, and thus the rate of emergence of resistance is
slower, although still a major challenge to these patients’ long-term survival.
The molecular mechanism behind imatinib resistance mirrors its molecular
mechanism of action. Bcr-Abl gene duplication as well as transcriptional
mechanisms leading to increases in Bcr-Abl transcript levels can lead to
imatinib resistance. Thus, the Bcr-Abl inhibition exerts selective pressure on
CML tumors to increase Bcr-Abl signaling, which is manifest by upregulation
of Bcr-Abl messenger RNA. Another common mechanism of resistance is the
mutation of the Bcr-Abl kinase ATP-binding pocket in which imatinib binds
[Ill. The mutation in the ATP-binding pocket produces a Bcr-Abl protein
kinase, which can carry out ATP-dependent substrate phosphorylation but
I cannot be inhibited by imatinib. Strikingly,the cancer has identified selectivity
3 Engineering Control Over Protein Function Using Chemistry

determinants for imatinib binding, which do not affect ATP binding.

One particular mutation, T3151, is most frequently identified in imatinib
resistant tumors and serves as an illustration of how a single point mutation
can exquisitely control ligand selectivity (see Fig. 3.1-3). The amino acid at

Fig. 3.1-3 The crystal structure of imatinib bound t o Abl kinase [12]. The gatekeeper
residue (T315, colored red) packs tightly against imatinib (PDB: 1 IEP).
3. I Revealing Biological Specificity by Engineering Protein-Ligand Interactions I 125

position 315 of Bcr-Abl makes contact with the exocyclic amine of ATP and,
thus, lines the adenine-binding pocket of the kinase. The ATP-binding pocket
of most protein kinases is larger than necessary for binding ATP, especially
in the vicinity of the exocyclic amine of ATP. Thus, a large hydrophobic
pocket adjacent to adenine is available for small-molecule inhibitor binding.
Importantly, the size of the amino acid residue at position 315 controls access
to this extra pocket, and thus it has been termed the gatekeeper residue. In the
T315I mutant Bcr-Abl kinase, imatinib cannot access the hydrophobic pocket
because the larger isoleucine residue blocks its access. Since the bulkier
isoleuciiie occupies a pocket not used by substrate ATP, the T315I mutant is
still able to efficiently bind ATP and catalyze phosphotransfer reactions.
As the predominance of imatinib resistance mechanisms can be traced to
Bcr-Abl functional upregulation, the clinical resistance offers another proof
of mechanism akin to the genetic screen which identified TOR as the target
of rapamycin discussed in Section In the former case imatinib was
more or less designed to be a Brc-Abl inhibitor, thus its target was known
from the outset of the clinical trial. In the case of rapamycin, a genetic
screen to identify its target(s) was carried out to identify the molecular basis
for its effect on immune suppression. In an amalgam between these two
paradigms for target identification and clinical efficacy, a B-Raf inhibitor
BAY43-9006 displayed disappointing efficacy in clinical trials of myeloma
patients, despite the identification of activating mutations in B-Raf, in this
form of cancer. Luckily, BAY43-9006 was also used in clinical trials of other
cancer types, where it showed surprising efficacy in the treatment of renal
cancer, which is thought to be particularly dependent on vascularization.
Subsequent biochemical studies demonstrated that BAY43-9006, which was
originally thought to be a highly specific B-Raf inhibitor, is a potent inhibitor
of the vascular endothelial growth factor receptor (VEGFR),providing a post
fucto rationale for its efficacy in this VEGFR-dependent cancer type [13].
In another case of small-molecule assisted target identification, the imatinib
response of patients with idiopathic hypereosinophilic syndrome lead to the
identification of a chromosomal rearrangement involving the tyrosine kinase,
and the known imatinib target, PDGFR, as a likely cause of this syndrome
[14]. The link between the PDGFR fusion and hypereosinophilic syndrome
was further strengthened when, after extended imatinib therapy, a relapse in
one patient was observed to correlate with the emergence of a T674I mutation
in PDGFRA. T674 is the gatekeeper residue in PDGFRA.
Similarly, imatinib has been found to be a useful therapy for gastrointestinal
stromal tumors (GIST)which is driven by the c-Kittyrosine kinase, a previously
known “off-target’’ of imatinib when it was being developed as a Bcr-Abl
inhibitor. Again, resistance to imatinib in GIST patients has emerged and c-Kit
ATP-binding site mutations to the gatekeeper residue (T670I) is commonly
found [ 151.
The lessons learned from irnatinib, BAY-43-9006suggest that cancers can
be uniquely dependent on the catalytic activity of a single kinase. Moreover,
I because of the highly conserved nature of the kinase ATP-binding pocket,
3 Engineering Control Over Protein Function Using Chemistry

drug candidates always inhibit multiple family members. In some cases, off-
target effects will lead to new medicines (BAY43-9006).In some other cases
of course, off-target effects will lead to toxic side effects, and will predictably
lead to failures of clinical trials. Moreover, because a single amino acid in
the binding pocket of kinases, the gatekeeper residue, can control inhibitor-
binding specificity, resistance to these drugs has emerged quickly in cancer
patients. A central challenge in all therapeutic areas is to identify key kinase
targets for the treatment of the signaling defects in human diseases.

ExploitingSensitizing Mutations to Engineer Nucleotide Binding Pockets EngineeringUniquely lnhibitable Kinases

One approach for determining the function of every protein kinase in the
genome is to develop a highly selective small-molecule inhibitor of each
kinase. The challenge in achieving high specificity is daunting since over
500 kinases are present in the human genome, containing highly similar
ATP-binding pockets. Our laboratory has addressed this specificity problem by
using protein engineering to target a kinase inhibitor to any kinase of interest.
In fact, this is the inverse of the problem discussed in Section, the
generation of imatinib resistant alleles (T315I) Bcr-Abl. Rather than creation
of an inhibitor resistant allele, the approach to discovery of an inhibitor of
any protein kinase is to create a uniquely sensitive kinase allele, which will be
inhibited by a molecule that does not inhibit any wild-type protein kinase.



This is achieved by mutation of the gatekeeper residue in the wild-type

kinase to a small alanine or glycine residue. Importantly, there are no human,
mouse, worm, fly, or yeast kinases with an alanine or glycine gatekeeper
residue, making the mutant kinase unique. A pyrazolopyrimidine-based
3.1 Revealing Biological Specificity by Engineering Protein-Ligand lnteractions 1 127

Fig. 3.1-4 The structure of kinase inhibitor PP1 bound t o the

ATP-binding pocket o f Hck kinase. The gatekeeper residue (the
surface ofwhich is colored red) packs tightly against the tolyl
substituant of PP1 [16] (PDB: 1QCF).

inhibitor was designed (based on the parent inhibitor PPl), which is only
capable of inhibiting kinases containing a glycine or alanine gatekeeper
residue. Importantly, the kinases with the smallest naturally occurring
gatekeeper residues, serine and threonine, are not inhibited by 1NM-PP1
(Fig. 3.1-4).It is interesting to note that the gatekeeper residue was selected
on the basis of structural models of kinase-ATP crystal structures and docking
models of pyrazolopyrimidine-based inhibitors prior to the discovery of the
gatekeeper mutations in imatinib resistant CML patients. The fact that
gatekeeper mutations can be used to confer inhibitor sensitivity through
rational design and inhibitor resistance through natural selection processes
highlights that this residue is a dominant feature controlling small molecule
access to the ATP-binding pocket without affecting kinase activity. Analog-specific Kinases

The enzymatic function of protein kinases is carried out by phosphorylation
of serine, threonine, or tyrosine residues on target proteins. As an estimated
30% of human proteins are thought to be phosphorylated, identification of
the direct substrates of all human protein kinases is a daunting challenge.
Although a wide range of methods have been developed for isolating the
I 3 Engineering Control Over Protein Function Using Chemistry


ATP N6-Benzyl ATP

phosphoproteome, critical information about the kinase or kinases responsible

for a given phosphorylation event are not provided by phosphoproteomics. To
directly label and identify the targets of each kinase in the genome, kinases
can be engineered to accept surrogate phosphodonors that are not accepted
by any wild-type protein kinases. These N6-substituted ATP analogs, most
commonly N6-benzyl ATP, are accepted by kinases containing an alanine or
glycine gatekeeper residue.
The N6-benzyl ATP accepting oncogenic tyrosine kinase (1338G) v-Src has
been the best characterized analog-specific protein kinase. Several critical de-
sign criteria must be satisfied by an engineered kinase, for it to be useful in
studying kinase-signaling pathways. First and foremost, the substrates phos-
phorylated by the mutant kinase must be identical to those phosphorylated
by the wild-type protein. Three lines of evidence suggest that mutation of the
gatekeeper residue does not alter substrate specificity. First, using combinato-
rial peptide substrates, wild-type Src and (1338G) Src protein kinases exhibit
identical sequence specificity patterns [17]. Second, using a cellular transfor-
mation assay, v-Src and I338G v-Src produce equivalent levels of anchorage
independent cell growth, confirming that the cellular targets phosphorylated
by the mutant are able to fully recapitulate the wild-type kinase-induced phe-
notype [18].Lastly, at the structural level, the crystal structure ofthe mutant Src
(T338G c-Src, see Fig. 3.1-5) shows no rearrangements in the kinase domain
in the phosphoacceptor binding pocket. In fact, the cocrystal structure with
NG-benzylADP shows that the nucleotide binding is unchanged from that of
the ADP/c-Src cocomplex. Thus, available biochemical, genetic, and structural
evidence suggests that the mutation of the gatekeeper residue in the Src kinase
exhibits very limited change to the function of the kinase, while allowing the
use of inhibitors or ATP analogs for the study of Src. Currently, over 30
protein kinases from yeast, mouse, humans, Arabidopsis, and tomato have
been successfully engineered for substrate labeling or inhibitor development. From CTPases to XTPases

Given the convergence between the resistance mutations found in cancer and
the mutations used to engineer orthogonal kinase ligands, it is reasonable
3. I Revealing Biological Specifrcity by Engineering Protein-Ligand Interactions I 129

Fig. 3.1-5 N6-benzylADP is shown bound i n the ATP-binding

pocket o f t h e analog-sensitized Src kinase (PDB: 1 KSW) (Ref.:
Witucki, LA et al., Chem Biol, 2002 19, 25-33).

to consider the gatekeeper residue particularly amenable to engineering.

But the gatekeeper residue is not alone. In fact, the strategy to engineer
orthogonal kinase ligands is the descendant of a similarly successful strategy
to engineer orthogonal nucleotide specificity into the nucleotide binding pocket
of GTPases. This mutation was discovered by Hwang et al. while dissecting
the GTP-binding pocket of EF-Tu, a GTPase essential for ribosome function
in Escherichia coli [19]. Introducing an aspartate to the asparagine mutation
(D138N) disrupted the hydrogen-bonding interactions between GTP and the
GTPase, thus impairing the GTPase activity of the protein. Remarkably,
using XTP as substrate rather than GTP, restored hydrogen bonding (now
reversed, see Fig. 3.1-6) and the activity of the GTPase-turned-XTPase was
nearly identical to the wild-type enzyme. Therefore, this mutation allows the
construction of an orthogonal nucleotide specificity (the XTPase accepts only
XTP; the GTPase only GTP).
This engineered GTPase was particularly useful for dissecting the GTP
requirements of the E. coli ribosome. In vitro translation experiments had
established that two GTPases are necessary for each round of amino acid
addition to a growing polypeptide. EF-G (one of these two GTPases) is
responsible for the translocation of the peptidyl-tRNA from the A site to the
P site of the ribosome. The other GTPase involved in this process is EF-
Tu - the GTPase previously engineered into an XTPase by Hwang et al. The
I 3 Engineering Control Over Protein Function Using Chemistry


Fig. 3.1-6 CTPases contain a conserved aspartate that hydrogen

bonds to the guanine ofCTP. An aspartate t o asparagine mutation
changes the nucleotide specificity from GTP to XTP by altering
these hydrogen bonds.

role of EF-Tu is to ensure proper binding of the appropriate aminoacyl-tRNA

to the ribosome (Fig. 3.1-7). Because the D138N EF-Tu nucleotide specificity
is orthogonal to wild-type EF-G, Weijland and Parmeggiani were able to use
this mutant, radiolabeled nucleotides (either XTP or GTP) to quantitate the
nucleotide consumption of each protein during the translation cycle [20, 211.
From this work it was established that, for every amino acid incorporated into
a growing peptide chain, EF-Tu (D138N) consumes two molecules ofXTP and
EF-G (wt) consumes one molecule of GTP.
At the time when Miller et al. developed the GTPase-to-XTPase mutation in
EF-Tu, they proposed that, because this mutation is in a highly conserved loop
shared by most GTPases, the D138N mutation should be applicable to endow
other GTPases with XTPase activity. This proposal has proven remarkably
accurate; numerous GTPases have been converted into XTPases using this
strategy [22].

Engineeringthe Ligand Selectively of Ion Channels Resistance Mutations in L-type Calcium Channel Signaling

For kinases and GTPases, point mutations can be used to study one member
of a large family by allowing the engineered member to bind to a unique
3. I Revealing Biological Specfcity by Engineering Protein-Ligand interactions I 131

Fig. 3.1-7 The crystal structure o f EF-Tu bound to a nonhydrolyzable CTP analog shows
Asp138 hydrogen bonding t o guanine. (PDB: 1 EXM).

ligand or substrate. An alternative means of isolating the activity of a single

protein in a family is to engineer the protein of interest to be uniquely resistant
to a general inhibitor. This way, the activity of the protein can be unmasked
by inhibiting all the other family members. The function of L-type calcium
channels was dissected in this manner.
Voltage-gatedcalcium channels play an important role in neuronal signaling.
While there are several different types of voltage-gated calcium channels, they
share a common activity: allowing an influx of calcium into the cytoplasm
upon activation. Despite this commonality, calcium influx from different
types of channels is not equivalent; L-type calcium channel specific blockers
diminish calcium dependent CAMP-responseelement binding (CREB) protein
phosphorylation and activation of the MAP kinase pathway while N- and
P/Q-type channel blockers have little-to-no effect. This and other differences
led to the proposal that calcium signal can act locally. For example, L-type
calcium channels may have the means of directing the entering calcium
to affect signaling molecules positioned near the channel. These signaling
molecules may then activate other signaling pathways (such as the MAP
kinase pathway). Testing this hypothesis requires a means of isolating the
role of calcium influx through L-type calcium channels from the role of
calcium influx from other types of voltage-gated calcium channel. This feat
I was accomplished using a mutant L-type calcium channel that is resistant to
3 Engineering Control Over Protein Function Using Chemistry

nimodipine, a dihydropyridine (DHP) antagonist of L-type calcium channel

A dihydropyridine-resistant L-type calcium channel was identified while
trying to map the DHP-binding site [23]. Initially, the binding site was probed
using photoaffinity labels and chimeric channels. These studies implicated
a specific region as responsible for DHP binding. Site-directed mutagenesis
in this region identified several mutations that altered DHP sensitivity. One
mutation, in particular, TlOOGY, was shown to be resistant to antagonism by a
DHP. The agonist binding to the mutant channel was dramatically decreased,
as demonstrated in a radioligand-binding assay. That this effect might be
caused by nonspecific disruption of the channels structure was ruled out
by demonstrating that channel activation and inactivation were not affected
by this mutation. Therefore, biochemical and electrophysiological evidences
suggest that this mutant channel is similar to the wild-type channel with the
exception that the mutant is resistant to DHP antagonists.
In neurons, the TlOOGY mutant channel's activities can be distinguished
from that of the endogenous channel by treating the cells with nimodipine,
thus blocking the wild-type copy and revealing the activity of the transfected
mutant [24]. Upon membrane depolarization in the presence of nimodipine,
the mutant channel rescues the Ca2+ influx and other downstream signaling
pathways including CREB phosphorylation and the stimulation of the MAP

Fig. 3.1-8 The activity of an exogenenous nimodipine, the endogenous, wild-type

L-type calcium channel was dissected using channel (blue) is blocked and the activity of
a mutation that effects nimodipine the mutant channel (green) i s revealed.
resistance (T1006Y). In the presence of
3. I Revealing Biological Specificity by Engineering Protein-Ligund lnteructions 1 133

kinase pathway (Fig. 3.1-8). Thus, the DHP-resistant T1006Y mutant L-type
calcium channel provides the specificity handle necessary to dissect the
activity of L-type calcium signaling. For example, this TlO06Y channel was
instrumental in the identification of a calmodulin-binding site on the C-
terminus of the channel. This binding site provides insight as to how L-type
calcium channel signaling can use local Ca2+ influx to interface specifically
with other cellular signaling pathways. Capsaicin Sensitivity

Similar to the engineering of DHP-resistant mutant calcium channels, there
are natural examples of the emergence of uniquely resistant channels. One
example comes from the small-molecule capsaicin, the component of hot chili
peppers that induces the sensation of burning pain. Capsaicin accomplishes
this effect by binding to and opening the VR1 cation channel found in nerve
endings, including the mouth. That we consider chili peppers “hot” is not
arbitrary - the VR1 channel is also responsible for recognition of noxious
stimuli including heat (>43 “C) and acid [25].


It has been proposed that capsaicin serves chili peppers by selectively

deterring predators. Birds, productive vectors for seed dispersion, do not
respond to capsaicin. In contrast, mammals are predatory but are deterred by
the capsaicin (with the exception of humans) [26]. The molecular basis of the
differential capsaicin sensitivity between birds and mammals can be traced
to VR1 [27]. The avian homolog of VR1, like its mammalian counterpart, is
responsive to heat but unlike its mammalian counterpart, avian VR1 does not
respond to capsaicin.
The chicken VR1 ortholog (capsaicin insensitive) was compared with the
rat V R l (capsaicin sensitive) and chimeric channels were used to identify
sites on the chicken VR1 sufficient to render rat VR1 capsaicin, insensitive.
When a short stretch of the rat VR1 channel in the third transmembrane
spanning region (presumably at the capsaicin-binding site, although there
are no high resolution structures of the VR1 channel) is substituted with the
chick sequence, the mutant channel is rendered capsaicin insensitive. Using
this chimera as inspiration, it was possible to find individual point mutations
sufficient to render the rat channel capsaicin insensitive while having only a
I modest impact on the channel's response to heat and acid. Interestingly, the
3 Engineering Control Over Protein Function Using Chemistry

best resistance-inducing mutations were the unnatural ones found in neither

receptor; the use of these natural differences serves as an excellent guide but,
as with many of the examples above, it is often necessary to test a panel of
mutations before a productive mutant is found.
Perhaps more remarkable than the ability to use the differences between
chick (insensitive) and rat (sensitive) to construct a mutant insensitive rat
VR1, was the use of the rat receptor to guide the construction of a capsaicin-
sensitive chick receptor. Building the binding pocket required more than a
point mutation; the active construct borrowed 45 amino acids from the rVR1
inserted into the correct position in the cVR1. Essentially, the molecular basis
of this selective deterrence causing birds, but not mammals, to consume chili
peppers is explained by a biochemical change in ligand specificity, induced by
a few amino acids in mammal versus avian VR1.

Conclusion Challenges in Protein Engineering

We have presented several natural and synthetic examples of the alteration
of protein-ligand interactions. Several other examples exist and have been
reviewed elsewhere [28-311. While the utility of altering ligand specificity
is clear, protein engineering remains challenging. Even for the successful
examples presented here, the mutant proteins frequently suffer some level
of compromised function. For example, the space-creating mutation in the
ATP-binding site of Cdkl, an essential yeast kinase involved in the regulation
of cell cycle progression, has a substantial impact on the KM of the kinase for
ATP = 35 pM, KM,,,~ = 320 pM) [32]. In this case, the compromised
KM does not significantly impact the utility of the engineered kinase because
the high cellular concentrations of ATP (>1mM) are substantially above the
KM for both the wild-type kinase and the mutant. This and other similar
concerns can be addressed by using one of the great advantages of convergent
engineering strategies: the activity of the mutant can always be compared to
the activity of the wild type, both with and without ligand (see Table 3.1-1).
Because of these controls, unintended changes to the function of the protein
or the ligand can be dissected. In the case of the analog-sensitized Cdkl, the
mutant compares favorably with the temperature-sensitive mutant that had
previously been used to dissect the function of this kinase. Specifically, this
mutant kinase has been used with INM-PP1 to demonstrate the role of Cdk1
in the G2/M transition [32] and with ~ - ~ * P - l a b e lNG-benzyl
ed ATP to identify
numerous substrates of this kinase [33].Even when the reengineered mutants
do not match the function of the wild type perfectly, they can still serve as
useful tools.
3. I Revealing Biological Spec9city by Engineering Protein-Ligand Interactions I 135

Table 3.1-1 Controls available when using orthogonally

engineered protein-ligand interactions to study the biological
function of a protein

Without ligand With ligand

Wild type Reference state Control for the off-target effects

of the ligand
Mutant Control for the effect of the Experimental condition to probe
mutation the functional consequences of
the protein-ligand interaction

But sometimes the engineered mutations have a substantial impact on the

activity of the protein. For example, while the GTPase-to-XTPase mutation
described in Section has been general for most of the GTPases
studied, attempts to use the Asp-to-Asn mutation to study G-protein coupled
receptor (GPCR) signaling through Go, were initially unsuccessful because
the mutation (D273N) compromises nucleotide binding and GTPase activity
of these G-proteins [34]. In this case, it was possible to rescue the activity of
the mutant G-protein using an additional mutation (Q2SOL) that resides on
the other side of the GTP-binding pocket from D273. The discovery of this
mutation was apparently serendipitous; Q250L mutants are usually GTPase
deficient. Similarly, the space-creating mutations used to study kinases (see
Section occasionally compromise the activity of a kinase severely. In
several cases, it has been possible to identify second-site suppressor mutations
that rescue the activity of the mutant kinase [35]. In light of the natural
examples we have presented above, perhaps this level of feasibility is to be
expected; within the set of single mutants of a given protein there appears
to be significant functional diversity in ligand-binding activities. The best
mutations are sometimes, but not always, easy to rationalize. While using
rational strategies to identie productive mutations undoubtedly enriches
the chances of finding mutants with the desired activities, testing several
mutations is likely necessary. Nonetheless, both the natural and synthetic
examples above illustrate that reengineering a protein’s ligand specificity is a
tractable problem. Engineering Extended Biomolecular Interfaces

While this chapter has focused on the engineering of selectivity for small-
molecule ligands, primarily using single mutations, a similar strategy would
clearly be useful for studying the biological specificity of larger interfaces if
the reagents were available. Toward this end, several studies have attempted
more ambitious engineering projects to redesign large regions of protein
interfaces. For example, computational approaches were instrumental in
developing mutants of maltose-binding protein (and related members of the
I family) with completely reengineered ligand specificities
3 Engineering Control Over Protein Function Using Chemistry

[36] Similarly, many

other computational approaches have made significant progress to aid in the
reengineering of protein interfaces [37]. Alternatively, in vitro selections have
provided a means of enriching desired binders from large libraries of mutants.
For instance, phage display has been used to reengineer both protein-protein
[38] and protein-DNA [39-411 interactions. While reengineering complex
biomolecular interfaces remains difficult, these advances, alone or in
combination, will aid in the development of specifically engineered binding
partners that will provide powerful tools to study the biological importance of
these interactions. Conclusion
Reengineering protein-ligand interactions can provide powerful information
that complements traditional biochemical and genetic approaches. The power
of these engineering approaches will increase as new methods are developed
both in protein engineering and in our ability to genetically manipulate
the organisms we wish to study. These engineering approaches are most
useful in vitro or in organisms where genetic manipulation is tractable,
such as bacteria, yeast, flies, and mice. As pharmacological agents that
target wild-type proteins become increasingly selective, these reagents will
complement chemical genetic tools. Even in these cases, however, engineering
protein-ligand interactions can provide important information about the
specificity of the pharmacological agent, as was discussed earlier for rapamycin.
While the genome is vast, many of its features reoccur (e.g., domains,
cofactors, etc.) in several different signaling contexts. This biochemical
similarity presents a specificity problem on one hand but an engineering
opportunity on the other; introducing specificity handles using carefully
designed mutations can help provide insight into critical connections between
biochemical specificity and biological function.


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Chemical Biology
Edited by Stuart L. Schreiber, Tarun M. Kupoor,and Gunther Wess
Cowriaht 0 2007 WILEY-VCH Verlaq CmbH & Co KCaA, Weinheim

I 3 Engineering Control Over Protein Function Using Chemistry

Controlling Protein Function by Caged Compounds
Andrea Giordano, Sirus Zarbakhsh, and Carsten Schultz


One ofthe major tasks in biological sciences is to dissect complex specimens to

learn more about structures, their functions, and the connections between the
components. These days, science is focusing predominantly on the microscopic
and molecular level and therefore the behavior of each molecule, its fate, its
mobility, and the interaction with other molecules is of interest. TO achieve
this, it is required to generate data with high spatial and temporal resolution.
Most standard methods cannot provide the latter, because they require the
destruction of cells. Even modern techniques like ribonucleic acid interference
(RNAi) or artificial expression of proteins are crude in this respect because
large populations of molecules are affected. It would be most desirable to
interfere with a small subset of molecules in a specific area of a cell or an
organism. Even more advanced would be techniques that permit the onset
of a biochemical reaction or a translocation event at a certain time point
and under the control of the observer. Photoactivatable compounds could
serve these purposes. With a flash of light focused at a particular region of
the specimen, a biologically active compound may be generated or destroyed
within seconds. The caged compound is usually a small molecule that is
able to modulate protein function [l].In the last decade or so, proteins or
peptides themselves are increasingly equipped with photoactivatable groups
generating switchable, biologically active molecules under the direct control
of the experimentalist [2, 31. When applied to proteins, the photolytic removal
would activate or inactivate the molecule spontaneously thus mimicking fast
intracellular changes in enzyme activity. In a few cases, the methodology was
used for other macromolecules like DNA and RNA [4-61. This chapter gives a
brief overview of the various known caging groups suitable for forming caged
proteins, their pros and cons, and the methods of introducing the groups
chemically. Chiefly, the current knowledge of applying cages to proteins
and the questions answered by using caged proteins are described. During
the preparation of this manuscript, a splendid book describing most of our
knowledge on caged compounds and proteins was published [7].

Photoactivatable Groups and Their Applications Nitrobenzyl and Nitrophenyl Groups

In 1962, Barltrop et al. reported the release of glycine from its nitrobenzyl
carbamate upon photolysis IS]. Today, the o-nitrobenzyl group and its

Chemical Biology. From Small Molecules to System Biology and Drug Design.
Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Giinther Wess
Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
3.2 Controlling Protein Function by Caged Compounds I 141

derivatives are the most prevalent photocleavable caging groups in use.

Formally, the reaction is a photochemically induced isomerization of
o-nitrobenzyl alcohol into o-nitrosobenzaldehyde, thereby releasing the
substituent as the free acid (Scheme 3.2-1).Esters, carbamates, and carbonates
are converted into an acetal derivative that spontaneously collapses into the
aldehyde and the released fragment. If the leaving group is a carbamate or
a carbonate the latter undergoes spontaneous decarboxylation and yields free
amines or alcohols, respectively.
The groups are usually uncharged, of average lipophilicity, and fairly small;
all features that are desirable for cell applications. Nitrobenzyl groups as
well as other caged groups were successfully employed, especially to mask
charged groups like acids, phosphates, and amines (as carbamates) [g]. For
compounds like CAMP the corresponding nitrobenzyl ester or coumaryl esters
were rendered uncharged by the masking groups and the compounds were,
therefore, able to penetrate cell membranes [I1, 121. After photolysis, however,
the released charged compounds were again impermeable and hence trapped
inside cells. This prodrug-like approach combines two crucial features of
biochemical tools: cell permeability and photoactivation. This combination of
properties could also be of major interest in peptide-based tools in the future.
The unsubstituted 2-nitrobenzyl (NB) group (Fig. 3.2-1A) has several
shortcomings that limit its application. First, the wavelength that is required
for deprotection (260 nm) is too short for optical equipment and is known
to damage living cells [13]. Second, the N B caging group is not suitable to
examine fast reactions because there is a lag of a few milliseconds between
the photolysis and the release of the bioactive molecule [14, 151. Third, the
photoproduct 2-nitrosobenzaldehyde may react with the released compound or
other components, leading to cell damage [16].These three factors (photolysis
wavelength, kinetics, and product) are most relevant for all cages used in living
A more suitable photolysis by-product is released from the 1-(2-nitro-
pheny1)ethyl (NPE) group (Fig. 3.2-1B) [16], which is also removed by
short UV light (265 nm). It generates the less reactive nitrosoacetophe-
none and therefore exhibits less toxicity. Also, NPE’s photolysis rates
are significantly higher at 260nm than those for N B (10000 versus
850 s-l). Even better are a-carboxy-2-nitrobenzyl (CNB) groups (Fig. 3.2-1C,
17000 s-l) [17]. However, the NPE group is chiral, a property that is
often undesirable due to the formation of diastereomers with chiral
biomolecules. The diastereomers might have different biological and pho-
tochemical properties and separation is usually difficult on a preparative
NPE-caged ATP was used to probe the kinetics of muscle contraction, but
its release rate was modest and, more importantly, the caged compound was
not completely inactive [18, 191. Sometimes, the increased lipophilicity of the
cage is undesirable. To prevent the interaction of NPE-caged carbamoylcholin
with the nicotin acetylcholine receptor before photolysis a negatively charged
I 3 Engineerhg Control Over Protein Function Using Chemistry

"&" \ /

a: I ko


3.2 Controlling Protein Function by Caged Compounds I 143


&x &x @x W N H 2 H3CO

\ \ \ \


Fig. 3.2-1 Structures o f nitrobenzyl groups used for light-induced

deprotection. X represents a leaving group, either in the reagent
used to introduce the cage or for the photochemical release.

carboxylate group was attached to the cage (CNB, Fig. 3.2-1C), eliminating
the problem 1171. In addition, this CNB group showed faster release kinetics
than the N B group [17]. CNB has also been successfully used to cage glycine
derivatives [20]. However, additional charges are not always beneficial. CAMP-
dependent protein kinase A (PKA) was made to react with CNB bromide to
yield a caged version of the enzyme [21]. The caging group was introduced at
Cys199 and inactivated PKA. Unfortunately, the caged protein was unable to
undergo significant photoactivation. In contrast, simple o-nitrobenzyl bromide-
modified PKA not only exhibited a substantial loss in kinase activity but also
showed a 20-30 fold reactivation of the catalytic activity upon exposure to UV
light (for more detailed information on caged PKA, see below).
A particular form of CNB is (2-nitropheny1)glycine (Npg). This artificial
amino acid (Npg, Fig. 3.2-1D) was successfully incorporated into ion channels
like the nicotinic acetylcholine receptor [22] by nonsense suppression, a
technique developed by Peter Schultz and coworkers [23-261. Irradiation (4
h, > 360 nm) of proteins containing Npg led to peptide backbone cleavage in
Xenopus oocytes [22].
Like the nitrobenzyl group, NPE and CNB groups absorb only weakly at
wavelengths greater than 340 nm, thus limiting applications in the suitable
range of 350-400 nm. Wavelengths under 300 nm are inconvenient because
of considerable absorption by proteins and nucleic acids as well as by any kind
of glass, including microscope lenses.
This was overcome when electron-donating groups were added to the
aromatic moiety. The 4,5-dimethoxy-2-nitrobenzyl (DMNB) (Fig. 3.2-1E) cage
(2-nitroveratryl) was introduced in 1970 by Patchornik and Woodward as
I a “nitrogen” protecting group [27]. The substituents on the aromatic ring
3 Engineering Control Over Protein Function Using Chemistry

were located to give a major absorption band at 350nm. This relatively

long wavelength is attractive, because absorbance of radiation by proteins
and nucleic acids is significantly reduced. Until today, DMNB is still one
of the few photolabile protecting groups working at lower energy levels (up
to 420 nm). Marriott employed DMNB chlorocarbamate (Fig. 3.2-2A) to cage
G-actin at LysGl [28].This modification blocked the polymerization of G-actin
to F-actin. Additionally,he prepared a cysteine-caged myosin using the DMNB
bromide [29]. The DMNB chlorocarbamate and bromide (Fig. 3.2-2A and B)
are both commercially available and are the most commonly used reagents
to introduce the DMNB group. Nitrophenyl-substituted Michael acceptor
systems (Fig. 3.2-2C) have also been employed to cage proteins, for instance
B-galactosidase,probably by reaction with a cysteine residue [30].
Katritzky et al. examined the effect of the electronic nature of nitrobenzyl
groups and two different types of linkage groups, ether and carbonate,
upon photolysis [31]. The 4-monomethoxy substituted nitrobenzyl group
(Fig. 3.2-1F) had a more electron-rich benzylic carbon atom than that of
the 4,s-dimethoxy substituted nitrobenzyl compounds, because, according
to the authors, the methoxy substituent in the meta position was electron
withdrawing with respect to the benzyl carbon atom. On the basis of
quantitative stucture-activity relationship calculations it was expected that
monomethoxy-substituted nitrobenzyl molecules would decompose faster
than their dimethoxy analogs under photolysis conditions [311. Dimethoxy
substitution of caged nitrobenzyl phenylephrine increased the maximum
absorption wavelength and also increased the rate of photolysis relative to
the unsubstituted nitrobenzyl phenylephrine analog, showing that electron-
donating benzyl substituents promoted photolytic cleavage of 2-nitrobenzyl
phenolic ethers. Furthermore, it was shown that molecules with ether linkages
decompose faster than molecules with a carbonate linkage. The faster kinetics
of release of DMNB compared to the corresponding NPE-caged versions were
demonstrated for caged cyclic nucleotides [32].
The 1-(4,5-dirnethoxy-2-nitrophenyl)ethyl (DMNPE) (Fig. 3.2-1G) group
which combines both the modifications of DMNB and NPE groups failed
to show fast release kinetics with ATP or amino acids [32, 331. As mentioned
above, another major problem is the formation of diastereomers due to the

Fig. 3.2-2 Structures of commonly used DMNB reagents.

3.2 Controlling Protein Function by Caged Compounds I 145

stereocenter at the benzylic carbon atom. As expected, the DMNPE group is

removed with UV light > 350 nm, which is less harmful to cells. Furthermore,
the photo-by-product is again a nitrosoacetophenone that is less reactive than
the corresponding aldehyde released by photolysis of commonly implemented
o-nitrobenzyl caging groups. Therefore, depending on the application, the use
of the DMNPE group might be beneficial, especially when the formation of
diastereomers is not causing problems. The isomeric 2-ethyl form [34] as
well as the related 2-propyl variety [35] were also examined as cage groups.
The photorelease happened via B-elimination. Because of favorable quantum
yields, these groups may be some of the most promising caging groups in
future applications.
Some of the isomeric nitroaromatic groups were tried as photocages for
phosphates in the 1960s. The 3,s-dinitrophenyl (DNP) (Fig. 3.2-1H) caged
inorganic phosphate was converted by irradiation at 300-360 nm ( E ~ ~ ~
about 3000 M-lcm-l) with a reasonable quantum efficiency (0.67) and
released phosphate at > l o 4 s-' at pH 7. However, the only successful
example that employs the DNP group was the photoreleasing phosphate
in crystals of glucogen phosphorylase b, thereby permitting to monitor
its catalytic cycle by Laue X-ray diffraction [36]. DNP-caged ATP was at
least 100-fold less photosensitive than DNP phosphate, clearly a setback for
applications involving compounds with a chromophore. Recently, N-methyl-
N-(2-nitrophenyl)carbamoyl chloride (MNPCC) was introduced to specifically
mask the catalytic serine in butyrylcholinesterase (BChE). Reactivation was
achieved by irradiation at 365 nm [37].
A very recent addition to the nitrobenzyl-based photocleavable protecting
groups are the 1-(2-nitrophenyl)-2,2,2-trifluoroethyl
(NPT) (Fig. 3.2-1K)and the
~-(~,5-dimethoxy-(2-nitrophenyl)-~,~,~-trifluoroethyl (DMNPT) (Fig. 3.2-1L)
groups [38]. However, these groups are not stable under the harsh reaction
conditions of the Williamson synthesis. Therefore, it was required to attach the
NPT and DMNPT groups to various alcohols via Mitsunobu coupling. Primary
alcohols reacted with good yields while secondary alcohols gave only poor cou-
pling. An advantage ofthe NPT and DMNPT groups is the high quantum yields
(0.4-0.7). Unfortunately, besides the slow fragmentation kinetics observed for
decaging alcohols [38]this caging group exhibited very poor hydrolytic stability
for carboxylic esters (M. Goeldner, personal communication).
An interesting nitrobenzyl-based photocage is the 2,2'-dinitrobenzhydryl
(DNB) group [27]. Here, the benzylic methylene group is substituted with
another o-nitrophenyl group. This group, which was used to cage amino acids,
does not lead to diastereomers due to its symmetry. The related bis(2-nitro-
4,5-dimethoxyphenyl)methylgroup was used to cage ion chelators [39,40].
A novel cage variety is the 2-(dimethylamino)-5-nitrophenyl (DMNP) group.
With its major absorption band at 400 nm, fast release kinetics, and a decent
extinction coefficient (9000 M-'cm-') this group appears to be promising for
in vivo applications [41].
I 3 Engineering Control Over Protein Function Using Chemistry

Is it possible to use several of these photoactivatable groups in one molecule

for orthogonal deprotection by wavelength-selective cleavage? First attempts
with various nitrobenzyl group derivatives were only partially successful mainly
because of energy transfer between the chromophores [42,43]. Other Caging Groups

A significant number of photoremovable protecting groups that are not
derivatives of nitrobenzyl group cages have been devised by organic chemists
for applications in peptide and nucleotide syntheses. These groups and their
respective uses have been extensively reviewed before [9,44].We will therefore
describe only those groups that were useful to cage peptides and proteins,
in detail. However, several caging groups used to date for small molecules
or as photoremovable protecting groups for synthetic purposes may be very
useful for applications with proteins in the future. Unfortunately, many of
these require photolysis with short wavelength ultraviolet light (<300 nm)
and would be impractical for biological systems. Some, however, are cleaved
at higher wavelengths and do not cause the photodestruction of amino acids
such as tryptophan and tyrosine. These are, in particular, phenacyl esters. They
were used to mask phosphates [45,46]and peptides [47] and generated mostly
phenylacetic acid derivatives after photocleavage due to an intramolecular
rearrangement reaction [48, 491. Sheehan introduced substituted benzoin
esters as a protecting group for the carboxyl group, over 30years ago [SO].
Later, this moiety was reinvestigated as replacement for the NPE group
to protect phosphates. Promising results were achieved with cu-benzoyl-3,S-
dimethoxybenzyl phosphate due to a high quantum yield (0.78 at 347 nm/0.64
at 366 nm) and fast photolysis rates (>10’ s-I) [51,52]. A water-soluble diacetic
acid derivative was also introduced 1531. A very elegant application of a benzoin
group is the formation of a peptidic loop by cyclization via a bifunctional
chromophore that keeps the peptide in a partially unfolded state. Photolysis of
the benzoin broke the cyclic structure thereby permitting the peptide to fold,
which was followed by CD spectroscopy [54].
Other groups like the sisyl (tris(trimethylsily1)-silyl)group are probably too
lipophilic to be used in an aqueous environment and might interfere with
protein conformation or solubility [55].This problem has been anticipated for
coumarin-based cages. While coumarins were successfully used for caging
y-aminobutyric acid (GABA) derivatives [56] and for two photon photolysis
of glutamate in brain slices [57], the (7-methoxy-coumarin-4-y1)methyl esters
of CAMP and cGMP were poorly soluble [58, 591. More recently, however,
substituted coumarylmethyl ester (7-diethylaminocoumarin-4-y1)methyl ester
(DEACM), (7-carboxymethoxycoumarin-4-y1)methyl ester (CMCM), and [6,7-
bis(carboxymethoxy)coumarin-4-yl]methyl ester (BCMCM) were developed to
cage cyclic nucleotide monophosphates. The CMCM and BCMCM groups in-
creased the hydrophilicity and solved the solubility problem [59].The DEACM
protecting group on the other hand, exhibited remarkable photochemical
3.2 Controlling Protein Function by Caged Compounds I 147

properties [60]. The caged cyclic nucleotides could be efficiently released at

nondamaging wavelengths (405 nm). All caged compounds were released
very quickly and show very high rates of photocleavage. 7-Hydroxycoumarinyl
methyl esters of CAMP were also sufficiently soluble to allow for biological
applications [61]. Hence, coumarin-based groups have a high potential for
successful applications in proteins. Other groups worth investigating are aryl-
azides [62],nitroindilines [63, 641, as well as N-acyl-2-thionothiazolidines [65]
and 5-azido-l,3,4-oxadiazoles [66].Most of these groups suffer from laborious
preparation procedures or have just not been investigated for applications with
large molecules. Exceptions are cinnamate-based caging groups.

32 . 2 . 3 Vi nylogenic Photocleavable Croups

The cinnamate cage was used in one ofthe earliest examples ofa caged enzyme.
In contrast to other caging groups, the cinnamate cage relied on E + Z pho-
toisomerization (Scheme 3.2-2). Porter and coworkers showed that a number
of serine proteinases could be inactivated with p-Amidinophenyl-o-hydroxy-
methylcinnamate, which forms a stable acyl enzyme intermediate upon release
of the pamidinophenol leaving group [67,68]. After photoisomerization to the
Z derivative, the aromatic hydroxy group was sufficientlyclose to the ester ofthe
acylated enzyme to permit reesterification (Scheme 3.2-2). This sterically favor-
able arrangement allowed the regeneration ofthe free serine hydroxy group and
gave the decaged protein. Limitations reside in the extensive overlap between
enzyme and inhibitor absorbance spectra. The intensity of the light source had
to be substantial. At the same time long irradiations degraded the enzyme.
Other photocleavable protecting groups that take advantage of E + Z
photoisomerization are the vinylsilanes (Fig. 3.2-3) [69, 701. Unfortunately,
these compounds require harsh, short wavelength light (254 nm) for
photoconversion. The introduction of a methylenedioxy group (Fig. 3.2-3B)
failed to shift the absorption to higher wavelengths, but the naphthalene
derivative (Fig. 3.2-3C)was effectively photolyzed at 350 nm in methanol. Attaching Photoactivatable Croups

The introduction of a cage usually requires a nucleophilic group at the molecule
of interest. The relevant groups in proteins and peptides are amino, thiol, or

w o , eH n z y m e


Scheme 3.2-2 Decaging of a proteinase via an intramolecular reesterification.

I 3 Engineering Control Over Protein Function Using Chemistry


Fig. 3.2-3 Vinylsilanes as photocleavable protecting groups require E +2


alcohol groups. Amino groups are readily reacted with chloroformate deriva-
tives (Scheme 3.2-3).In fact, the most commonly used nitrobenzyl derivative
(DMNB-OCOC~)is commercially available. Other reagents are prepared by
reaction of the alcohol with phosgene or alternatively with carbonyldiimida-
zole (CDI) [42]. Caging reactions proceed under mild conditions in aqueous
solution at slightly basic pH (9-10) [28]. An alternative is p-nitrophenyl car-
bonate esters. The leaving group permitted the formation of a carbamate
directly from the hydrochloric acid salt of glutamate in the presence of
4-(dimethylarnino)pyridine(DMAP) at room temperature (Scheme 3.2-3) [57].
Thiol groups are preferentially reacted with aryl methylhalogenides, for
instance, bromo nitrobenzyl derivatives (Scheme 3.2-4). The conditions are
extremely mild (Tris buffer pH 7.2) and reactions were reported to be finished
within an hour [71].When the reactive caging group is equipped with a suitable
amino acid docking sequence, a specific cysteine can be labeled, even with a
300-fold excess of the reagent [21]. Another photoactivatable caging reagent
that covalently binds to thiols in proteins is the a-haloacetophenone group.
Its aromatic character is recognized particularly well by phosphotyrosine
phosphatases (PTP) [72,73].Accordingly,haloacetophenone groups are potent
photoreleasable inhibitors of PTPs in vitro. No details about the labeling
procedure have been published so far.
It is of special interest to label serine and threonine residues, due to their role
as acceptors for posttranslational modifications, namely, for phosphorylation.

Scheme 3.2-3 Introduction of caging groups to amino residues.

3.2 Controlling Protein Function by Caged Compounds I 149

Tris buffer
- PH 7.2 R
R-SH +

Scheme 3.2-4 Introduction ofcaging groups to thiol residues. X is O H or halogen

To achieve the necessary alkylation, much harsher conditions are required.

Unfortunately, the strongly basic conditions of the Williamson ether synthesis
are unsuitable for halogenated o-nitrobenzyl reagents [74]. A more suitable
leaving group than the halogene is the well-known trichloracetamide group
(Scheme 3.2-5). However, the successful reaction requires strongly acidic
conditions (CF3S03H)and is used for protected amino acids rather than entire
peptides [75].
A milder method that is suitable for caging hydroxyl groups in proteins is the
reesterification with p-amidino esters of arylcinnamates. With the help of the
leaving group, deactivation of thrombin was achieved within 8 h at pH 7.4 [68].
The phosphorylated varieties are as important for functional studies of
peptides and proteins as the hydroxyl groups. Since the nucleophilicity of a
phosphate is only moderate, thiophosphates are frequently used as targets
for caging reactions. The same conditions that work for labeling cysteines
are applied for thiophosphates [71].Alternatively, 4hydroxyphenacyl bromide
(HP-Br) is employed to label a thiophosphothreonine in protein kinase A
under very mild conditions (1mM reagent, pH 7.3) [76]. For peptides, caged
phosphates can be conveniently introduced during solid phase synthesis via
phosphorous ( I I I) reagents [77 -801.
The above-mentioned coumarin cages were introduced to CAMPor cGMP
via the corresponding diazoalkanes [60]. The introduction of cages via diazo
compounds has great versatility and was used for numerous applications,
in particular, for caging small biologically active phosphate esters like ATP
and myo-inositol 1,4,5-trisphosphate (InsP3) [lo, 811. Usually, the carbonyl


R-OH + Cl3C"Q
' CH2C12 Rm*ocH3


Scheme 3.2-5 A method that does not require base to form ethers o f hydroxy amino acids.
B r A T s N H - N h - B r d O H T s EtSN ~~d
\ \

Scheme 3.2-6 Two commonly used synthetic routes to diazo compounds. Ts - tosyl.

derivative of the caging group was reacted with hydrazine, followed by oxidation
to the diazo compound in the presence of MnOz (Scheme 3.2-G(a))[lo,81,821.
After the removal of MnOz by filtration and several washes, the diazo reagent
was used mostly without further purification. In an alternative method, a tosyl
hydrazone was formed. Treatment with base then gave the diazo compound
(Scheme 3.2-G(b))[GO, 611.

Caged Peptides and Proteins

The synthesis of caged peptides is accompanied by a series of obstacles.

That is the reason for the formerly small amount of caged peptides available
compared to other low-molecular-weight caged species. Proteins contain a
variety of nucleophilic sites and therefore the major problem is the site-specific
modification of a protein with an exogenous caging agent. Furthermore,
the absence of an appropriate nucleophilic residue at or near the desired
site of modification can be a problem. Finally, unlike low-molecular-weight
compounds, proteins and most peptides are generally not membrane-
permeant. The most obvious way to prepare a caged protein seems to be
the addition of a photoactivable group to a residue that is essential for protein
function. The problem is that the chemistry required needs to deal with entire
proteins and that the residue of interest is not usually unique within the
protein. Nevertheless, several approaches addressed the direct introduction
of cage groups on proteins, either on several residues simultaneously or
specifically on a single amino acid side chain. Multiresidue Protein Caging

Preparation of caged proteins by introduction of an o-nitrobenzyl group
directed toward specific residues dates back to the mid-1990s. In a pilot
study, bovine serum albumin (BSA) was randomly labeled with up to 15
3.2 Controlling Protein Function by Caged Compounds 1 151

o-nitrobenzyl groups at Lys residues using either 2-nitrobenzyl alcohol or

1-(2-nitrophenyl)ethanolin the presence of diphosgene or l,l’-CDI, which
yielded up to 90% of caged protein [83]. Notably, the secondary alcohol
coupled with diphosgene, but not with 1,l’-CDI. Exposure of NB-labeled
BSA to UV light led to the release of about 60% of the coupled cages. The
incomplete photolysis was probably due to the propensity of the photoproduct
nitrosobenzaldehyde to either back-react with the protein or to dimerize to
azobenzene-2,2’-dicarboxylicacid, which was suggested to act as an internal
filter lowering the efficiency of photolysis [84]. NPE-labeled BSA, on the other
hand, readily furnished up to 95% of the native protein after UV treatment
(365 nm) with a time-dependent release of about 1/3 of coupled residues
after 1-2 min and about 213 of that after 5 min of exposure. Performing
the same caging strategy and using antibodies as models for both receptor
and ligands, these authors successfully modulated affinity of antibody-binding
sites for antigen, antigen binding sites for antibodies, and antibody Fc binding
sites for protein A using a NPE-coated human IgG before and after UV
treatment [85].
With the aim of studying the regulation of the G-actin monomer
pool and the assembly of F-actin filaments in living cells, Marriott
described both preparation and properties of G-actin conjugates [28, 861.
Using the lysine-directed 4,5-dimethoxy-2-nitrobenzyl chloroformate (DMNB-
OCOCl) and an optimized water-based chemistry protocol that avoided
overlabeling of the target protein (and thus, circumventing problems of
denaturation/insolubility/low yields of photoactivation), caged monomeric
G-actin was prepared in 30-60% yield, with an average of four DMNB
groups per monomer. Such LysG1-caged G-actin showed to be unable to
polymerize to F-actin in vitro, confirming that residue Lys6l was forming part
of an actin-actin interface in F-actin. Upon photo-deprotection with UV light
(320-400 nm) for 12 min, polymerized F-actin was obtained in 60-95% yield.
More recently, Lys-targeted protein caging with DMNB-OCOCl was
performed on the G-actin binding protein thymosin 8 4 (TB4) [87]. TB4 is
thought to be involved in the regulation of the large intracellular G-actin
pool. Native TB4 is known to inhibit actin polymerization in vitro by
binding to G-actin via a conserved nine-residue segment (LKKTETQEK,
residues 17-25) [88]. In the cited study, DMNB-labeled TB4 was shown
to be unable to bind to G-actin in vitro as a result of the unaffected rate
of polymerization compared to control actin. Subsequently, DMNB-labeled
TB4 was introduced by bead loading in locomoting fish epithelial keratocytes
and was photoactivated locally in the cell wings (871. Upon UV irradiation
(365 nm), very specific changes in the global locomotory pattern of keratocytes
were observed in vivo, with noticeable turning of cells. These observations may
be explained by local perturbation in actin filament dynamics brought by the
spontaneous increase of active, decaged TB4 concentration in the region of
3 Engineering Control Over Protein Function Using Chemistry

Single Residue Protein Caging

A second labeling strategy aimed at the preparation of caged protein conjugates
is based on the targeted modification of essential cysteine residues using
photolabile alkyl halides [86], such as 2-bromo-2-(2-nitrophenyl)aceticacid
(CNB-Br),NB-Br, or DMNB-Br. Proteins to be caged at Cys residues can be
engineered from other proteins by cysteine-scanning mutagenesis: the useful
mutant will be the one that is inactive only after labeling with a thiol-reactive
caged reagent. Because only a single cage group is removed from a cysteine-
targeted caged protein, the photoactivation yield is usually higher compared to
DMNB-caged proteins [89].The main disadvantage of this approach may be
the necessity of generating and screening a large collection of mutants.
The synthesis and utilization of the water-soluble CNB-Br as a Cys-targeted
caging reagent was reported by Bayley and coworkers [go]. Staphylococcal
a-hemolysine ( a H L ) is a toxic polypeptide lacking cysteine residues. The
protein self-assembles to form a heptameric pore in cell membranes. A single
cysteine mutant R104C maintained this feature, while pore-forming activity
toward rabbit erythrocytes was lost upon derivatization of CyslO4 with CNB-
Br (100 10 mM Dithiothreitoe in aqueous buffer at pH 8.5, yield ca 80%).
Toxicity ofthe R104C mutant was regenerated by photoactivation with UV light
(300 nm, 30 min, yield ca 60%) and subsequent exposure to rabbit erythrocytes
(Fig. 3.2-4).
Marriott and Heidecker reported a Cys-caged heavy meromyosin (HMM)
using DMNB-Br and evaluated the capacity of photoactivated HMM to couple
the energy of calcium/actin-activated ATP hydrolysis to the movement of
F-actin filaments in an in vitro motility assay [29, 861. It was known from
labeling studies with the thiol-reactive fluorophore tetramethylrhodamine

0 20 40 60
Time (min)

Fig. 3.2-4 Hemolytic activity of decaged R104C a-hemolysine

(black circles) toward rabbit red blood cells (rRBC) measured by
monitoring light scattering at 595 nm versus a nonilluminated
sample (white circles). With permission from Ref. [go].
3.2 Controlling Protein Function by Caged Compounds I 153

iodoacetate (IA-TMR) directed against Cys707 that this residue was crucial
for sliding of F-actin filaments in the in vitro motility assay. Therefore, it
was reasoned that Cys707-caged HMM could show a similar behavior, which
eventually could be reverted upon photoactivation. HMM was reacted with
DMNB-Br in aqueous buffer at pH 7.4. Two cage groups per HMM molecule
(or one cage per ATPase domain of HMM) were incorporated in the reported
protocol. Although the calcium/ATPase activity of purified caged HMM was
increased fivefold compared to unlabeled HMM, caged HMM failed to produce
appreciable sliding of F-actin filaments, unless irradiated with pulsed (500 ms)
340-400 nm UV light, conditions that produced sliding of 90% of F-actin
filaments in the in vitro motility assay with a velocity of up to 4 pm s-l, a value
comparable to unmodified HMM [%I.
Protein kinases constitute a large family of enzymes (>500) whose activity
includes the transfer of the y -phosphoryl group of ATP to serine, threonine,
and tyrosine residues in a wide range of protein substrates, giving rise to
a large collection of phosphorylation-based signal transduction pathways. A
well-defined spatially and temporally activatable kinase is of invaluable utility
in elucidating many aspects of signal transduction phenomena in living cells,
under both physiological and pathological conditions.
One of the best-studied kinases is protein kinase A. An interesting
comparison of the behavior of three different caged catalytic subunits of
PKA was reported by Bayley and colleagues [91]. Working with a single
cysteine mutant (C343S) of the murine catalytic subunit of PKA, the unique
Cys residue 199 was masked with the thiol-reactive cage groups NB-Br, CNB-
Br, and DMNB-Br. Cys199 is placed in close proximity to the critical Thr197
in the “activation loop” of the enzyme [92]. The caged protein showed, as
expected, a significant inactivation when kinase activity was tested in vitro with
the artificial substrate Kemptide (LRRASLG).Interestingly, only the NB-caged
enzyme showed, among the three, low values of residual activity after caging
(3-5%) and satisfactory activity after photolysis (pH 6.0,80- 100%)with respect
to the unmodified enzyme. Moreover, the quantum yield of photolysis was an
impressive 0.84. The ‘‘lesson’’from this work, using the authors’ phrasing,
is that given a particular target protein a variety of photoremovable protecting
groups have to be tested since a reagent that works well with one protein (for
instance, the CNB-caged aHL described earlier) may not work well with others.
Cofilin is a kinase-regulated, F-actin binding protein whose activation state
is regulated by phosphorylation at Ser3 through the LIM-domain-containing
kinase (LIM kinase). Unphosphorylated cofilin monomers bind cooperatively
to F-actin in vitro leading to depolymerization of actin filaments [93], while
phosphorylation by LIM kinase inactivates these features of the cofilin function
(Fig. 3.2-5).Lawrence and coworkers [94]observed that the cysteine mutant S3C
cofilin is constitutively active because it is unable to undergo phosphorylation
by LIM kinase, while a CNB-caged S3C cofilin is unable to depolymerize
actin filaments in vitro. This shows the importance of Ser3 for cofilin activity.
Accordingly, S3C cofilin activity was restored up to 80% upon irradiation and
I 3 Engineering Control Over Protein Function Using Chemistry

Fig. 3.2-5 Activity o f cofilin initiated by local decaging. A 2-s laser pulse aimed at the area
indicated in F gave local protrusion within 1 t o 3 rnin. With permission from Ref. [95].

depolymerization of rhodamine-labeled actin filaments was assessed via an in

vitro light microscopy assay. Subsequently, these investigators could elegantly
extend the role of cofilin in vivo by microinjecting caged CNB-S3C cofilin (up
to 20 pM) into MTLn3 carcinoma cells and by exposing cell territories to UV
irradiation [95].
Cell-wide photoactivation increased free barbed ends, F-actin content, and
cellular locomotion, while highly localized activation generated lamellipodia
and determined direction of cell locomotion. Showing all the intrinsic power
of caged proteins in biological investigations in vivo, this study expanded the
effective role of cofilin in contrast to motility models in vitro, where cofilin was
predicted to only depolymerize F-actin.
Protein phosphorylation on tyrosine residues is an important posttransla-
tional modification playing a vital part both in physiological processes, such
as transmembrane signaling, and in pathological processes, for instance, in
cancer and immune dysfunctions [96].The levels of tyrosine phosphorylation
are regulated by the opposing action of protein Tyr kinases (PTKs),which cat-
alyze the formation of phosphotyrosine residues (pY) on target proteins, and
phosphotyrosine phosphatases (PTPs), which hydrolyze pY. PTPs of various
origins share a common domain of about 250 residues containing the unique
“signature motif’ (I/V)-HCxAGxxR(S/T) in which the catalytic phosphatase
cysteine is located [97]. Being generally less well characterized than protein
kinases, the precise role of PTPs in physiological and pathological conditions
still remains to be investigated in more detail.
Recently, a-halogenated acetophenones (phenacyl groups) have been
reported as a novel, membrane-permeant, non o-nitrobenzyl-based class of
caging reagents. They are capable of covalent, photoreversible (350 nm)
inhibition of PTPs at the catalytic cysteine (Scheme 3.2-7) [72,73].The different
3.2 Controlling Protein Function by Caged Compounds I 155

a-bromo and a-chloro acetophenone derivatives were employed i n vitro to cage

the catalytic cysteine ofvarious prototypical phosphatases such as PTPlB, SHP-
1, and the catalytic domain of SHP-1, SHP-1 (ASH2). Recovery of enzyme
activity after irradiation at 350 nm (15 min) was in some cases obtained to a
maximum of 80% of the original value.
In the last years, reports have demonstrated the possibility of producing
caged proteins by targeting specific amino acid residues that are different from
lysine or cysteine.
After having described a catalytic Ca subunit of PKA caged at Cys199,
Bayley along with Zou and others presented a Ca caged at the active
threonine (Thr197) using the above-mentioned 4-hydroxyphenacyl photore-
movable protecting group [76]. The advantage of such a caging group
with respect to the classical o-nitrobenzyl derivatives was the rapid photo-
deprotection (k % 107-10s s ~ and ~ the
) lack of reactivity of the photolysis
product 4-hydroxyphenyl acetic acid [47, 981. The phenacyl methodology was
also employed to prepare caged thiophosphoryl peptides (see also below)
[76, 991: Ca catalytic subunit was first expressed as a recombinant mutant
protein (H6-T197C199A/C343S) in Escherichia coli. Exclusive thiophosphory-
lation of Thr197 was performed with the phosphoinositide-dependent kinase
(PDK-1) in the presence of ATP(y)S. Confirmation of thiophosphorylation
was assessed by Western blotting and gel-shift electrophoresis. Finally, pu-
rified thiophosphorylated Ca was caged with 4-HP-BR (Scheme 3.2-8) giving
rise to the modified protein HP-PsT197Ca showing an 18-fold reduction-
of specific kinase activity i n vitro toward Kemptide. Activation by photolysis
was performed with UV light (312 nm) at pH 7.3 with an 85-90% yield in
photoactivation, a quantum yield of 0.21, and a 15-fold increase in activity.
These are promising values for future in vivo studies.
Photoregulation of the catalytic activity of natural and recombinant human
BChE was described in 2003 [37].This enzyme is closely related to acetylcholine


s o

hV / +$OH
/ + Cys-protein
+ /e i $

X = CI, Br ; R = H, CH,, CH,COOH

Scheme 3.2-7 Cysteine-containing proteins like phosphatases are caged in the active site
with phenacyl bromides or chlorides.
I 3 Engineering Control Over Protein Function Using Chemistry

Tlg7Ca b Ti 97Ca
PDK-1 kinase hv
0 0
Br -s-p=o S-P-OH

OH 0


Scheme 3.2-8 Caging ofthe catalytic subunit Ca of PKA was

achieved by thiophosphorylation and subsequent alkylation o f the
thiophosphate by 4-hydroxyphenacyl bromide (HP-Br).

esterase (AChE),the serine hydrolase that terminates cholinergic transmission

by hydrolysis of the neurotransmitter acetylcholine. Despite the fact that its
endogenous substrate has not been identified yet, this enzyme plays a key role
in detoxification by degrading esters such as succinylcholine and cocaine. In
the reported study, BChE was treated with a novel photoremovable alcohol-
protecting group, MNPCC targeted at the catalytic serine residue ofthe enzyme.
MNPCC seemed to act as a pseudoirreversibleinhibitor and the X-ray structure
of the MNPCC:BChE conjugate showed a nonambiguous carbamylation of
the catalytic residue as the only modification on the protein [37].Reactivation
of the caged enzyme was obtained at 365 nm (20 min, pH 7.4) and exhibited
an efficiency larger than 80%, as was determined by the Ellman test. The
same group previously intended to explore the efficient photoregulation in
crystals of the MNPCC:BChE conjugate was used to further determine the
mechanistic properties of BChE by time-resolvedX-ray crystallography under
cryophotolytic conditions [loo].

Caged Proteins by Introduction o f Photoactive Residues via Site Directed,
Unnatural Amino Acid Mutagenesis

Photochemical control of processes such as protein folding, protein-protein

or protein-ligand interactions may be achieved via an alternative procedure by
which the photochemical trigger - that is, the caged amino acid - is directly
incorporated into the native protein sequence as an unnatural residue.
The elegant and sophisticated - yet laborious - biosynthetic methodology
introduced by Peter Schultz made a wider exploration of protein functions
possible by de facto expanding the natural genetic code [23-251.
Introduction of an unnatural amino acid follows a series of defined steps that
are summarized here briefly: (a)the codon for the amino acid to be replaced
3.2 Contro/hg Protein Function by Caged Compounds 1 157

is substituted with a nonsense codon (like the amber stop codon UAG)
via standard site-directed mutagenesis, (b) a specific “nonsense suppressor”
tRNA able to recognize this codon is prepared and acylated with the desired
unnatural amino acid, (c) addition of the mutagenized gene or mRNA and the
aminoacylated suppressor tRNA to an in vitro extract or biosynthetic apparatus
generates a mutant protein containing the unnatural amino acid at the desired
Thus, the generation of the specific suppressor tRNA, its acylation with the
unnatural residue, and the synthesis of sufficient amount of mutagenized
protein are the key steps of the entire methodology, more recently expanded
in some technical aspects from its original design [101-103].
With this technique, caged amino acids have been successfully introduced
into various protein sequences as unnatural residues. Enzymatic catalysis
before and after photoirradiation has been explored by means of caged residues
replacing the natural ones in critical positions. Schultz and coworkers described
a mutant phage T4 lysozyme (T4L)containing an aspartyl /3-nitrobenzyl ester
in place of the wild-type Asp20 in the active site of the enzyme [104]. This
residue, along with Glull, is responsible for the catalytic activity [105]. The
caged protein, produced in 37% yield, showed no activity in vitro. Conversely,
activity was restored to a 32% level compared to the wild-type enzyme after
irradiation at 315 nm (Hg-Xe arc lamp 1000 W). In another experiment these
investigators managed to photochemically initiate protein splicing from the
Thermococccus litoralis DNA Vent polymerase by introducing the 2-nitrobenzyl
ether of serine in the place of the conserved Ser1082 [106].
NB- or DMNB-caged aspartates were instrumental in controlling the
dimerization of HIV-1 protease [107].This enzyme exists as a 22-kDa monomer
that self-assembles into the active dimeric aspartyl protease. The active site is
placed at the interface of the homodimer and consists of Asp25 and Asp125,
both necessary for the proteolytic activity [108, 1091. Introduction of a NB-Asp
into position 25 led to minimal proteolytic activity, while its recovery after UV
irradiation (500 W mercury-xenon lamp, 10 min, 0 “C,pH 6.0) was about 97%
as revealed by a fluorescence-based protease assay [110]. The introduction of
the caged aspartate did not prevent dimerization, suggesting that H bonding
involving the wild-type residue is not a prerequisite for monomer association
of HIV-1 protease. Instead, it was believed that it affected the stability of the
dimer [107].
A similar behavior was shown by the H133A mutant of BamHI endonuclease
having incorporated a caged Lys132 [lll].Lys132 along with Glu167, Glu170,
and His133 participates in the salt-bridge network at the dimer interface of the
active wild-type enzyme [112, 1131. Site-directed introduction of DMNB-OCO-
Lys132 (yield 55%) in the H133A mutant did not prevent dimer formation
but abolished enzyme activity almost completely. Photoirradiation (365 nm,
20min, 0°C) led to a recovery of both activity and specificity toward a
substrate DNA (ADNA). A different behavior was shown for the H133A
BamHI mutant incorporating DMNB-Glul67 or DMNB-Glul70 which did not
158 3 Engineering Control Over Protein Function Using Chemistry
I exhibit recovery of activity after photoactivation, suggesting misfolding of the
protein subsequent to the introduction of these caged residues. A site-directed
incorporation of a phenylazo-Phe residue (azoAla) at the same position 132
was also performed (incorporation efficiency of 52%) [114]. Dimer formation
and enzyme activity was achieved by inducing trans-cis photoisomerization
of the azobenzene moiety. The substihtion K132azoAla produced a mutant
enzyme with drastically reduced activity (measured by cleavage efficiency of
a DNA substrate), while after irradiation and trans-cis isomerization almost
full activity was recovered compared to the wild-type enzyme. Thus, in its
trans conformation, the bulkiness of the azoAla residue prevented a correct
association of monomers, while the more compact size of the cis isomer did
not preclude the proper assembly into the active form. Gradual gain of activity
was observed within 5 min of photoirradiation (366 nm, 0°C) without further
increase in a global 20 min exposure time.
Several proteins are naturally produced as inactive proenzymes and acquire
full activity only when cleaved at a specific position by another enzyme.
Caspase-3, a cysteine protease, is a key component of the apoptosis signaling
pathway. Its inactive form procaspase-3 is cleaved at position Ser176 by caspase-
8 in the “death receptor-induced’’ apoptosis pathway, eventually forming the
active tetramer. Majima and coworkers artificially reproduced the activation
mechanism of procaspase-3 by photoinducing the cleavage of the backbone
in a mutant protein containing a Npg residue specifically introduced at
position 176 [115]. The incorporation efficiency of Npg by using an i n vitro
transcription/translation system was only 15%. Nevertheless, photoactivation
(366 nm, O’C, up to 10 min exposure time) of Npg-caspase-3 was followed
within 1 min by a clear activation of enzymatic activity as quantified by the
change in fluorescence of the peptidic substrate Z-DEVD-rhodamine 110.
All these studies were performed i n vitro. Some i n vivo experiments with
caged proteins engineered by nonsense suppression were successful, especially
on the acetylcholine receptor.
In the mouse muscle nicotinic receptor (nAChR), NB-tyrosine was
incorporated at positions 93 and 198 of the (Y subunit. These are conserved
residues crucial for acetylcholine binding. The mutagenized mRNA and the
relative nonsense suppressor tRNA charged with the NB-Tyrwere injected into
Xenopus oocytes. The channel was successfully expressed and incorporated into
the egg membrane [ 1161. In the following voltage-clamp study, a train of about
20 near-UV laser pulses (300-350 nm) was able to increase acetylcholine-
induced conductance across the membrane with about 5% of decaged Tyr
residues in any one flash.
A qualitatively similar result was achieved in another elegant experiment
where the same ion channel was mutagenized by direct incorporation of
NB-Cys or NB-Tyr replacing a conserved leucine residue in the y subunit that
is known to be involved in channel gating [117].As stated by these authors, the
work represented the first successful incorporation of caged amino acids into a
transmembrane segment of a membrane protein. Interestingly, the presence
3.2 Controlling Protein function by Caged Compounds 1 159

of the bulky nitrobenzyl group did not disturb both assembly and trafficking
of the receptor, but likely distorted its conformation leading to an alteration of
the conductance. This condition was reverted by photoactivation performed
with 1-ms pulses of UV light. The different and characteristic kinetics of
channel activation after flash photolysis for tyrosine and cysteine for the
respective caged receptors were determined. Oocytes expressing the mutant
acetylcholine receptor wVall32Npg showed acetylcholine-induced conductance
similar to the wild type! but upon photoinduced cleavage of the backbone in
the localized region of the w subunit about 90% of the current was lost. Thus,
in addition to playing a key role in the correct assembly of the various subunits,
this conserved portion proved to be essential for receptor function [22].
The work of this group clearly showed the importance and usefulness of
caged proteins as tools for the elucidation of protein function in living cells
[118- 1201.

Small Caged Molecules Used to Control Protein Activity

An alternative method to modifying the protein of interest is to control its

function by an inhibiting or activating ligand. Since these ligands can be
small peptides or other small molecules, a caging group is usually introduced
by preparative chemistry. After decaging, interaction between ligand and
protein is permitted, the protein is either silenced or activated. For life cell
applications, the small molecule ligand has to be membrane-permeant or needs
to be introduced by physical methods like microinjection or electroporation.
Among the many caged ligands reported so far are various cyclic and noncyclic
nucleotides [19, 59, 82, 121, 1221, nitric oxide [123], lipids [go, 124-1261,
carbohydrates [80, 127, 1281, inositol polyphosphates [81, 129-1311, ion
chelators [40,132, 1331, amino acids [57, 134, 1351, receptor agonists [136,
1371, and many others [138]. Because the synthesis and application of these
small molecules has been thoroughly reviewed before [l,7, 44, 1391, we will
not discuss them in detail. Caged Peptides

Some of the most potent modulators of protein function are peptides. To
introduce a cage at the correct position, essential residues need to be known.
Alternatively, libraries of potential binding peptides have to be prepared and
tested. There are only a handful of amino acid residues suitable for introducing
a caged group. Typical side chains are those of the basic and acidic amino
acids and the nucleophilic thiol group of cysteine. In addition, phosphorylation
usually takes place at the alcohol groups of serine, threonine, or tyrosine and
caging groups on these residues render the phosphorylation site inaccessible
until the cage is removed. Solid phase peptide synthesis (SPPS) also permits
I the introduction of phosphorylated residues equipped with a cage group
3 Engineering Control Over Protein Function Using Chemistry

attached to the phosphate. From a synthetic standpoint, there are two ways
of preparing caged phosphopeptides: by using an already assembled caged
phosphoamino acid or by introducing the caged phosphate after cleavage of
the mature peptide from the resin. Phosphopeptides will bind to proteins
usually interacting with phosphoproteins as soon as the cage is removed. With
the help of membrane-penetrating peptide sequences, “peptide interference”
is now on its way into biology labs. Caged Basic Residues

Caged lysine in form of N‘-o-nitrobenzyloxycarbonyllysine was reported as
a building block suitable for Fmoc-SPPS. It was used for the preparation
of caged AIPs, autocamtide-2 related inhibitory peptides [2, 1401. AIP
(KKALRRQEAVDAL) is a highly specific inhibitor of calcium/calmodulin-
dependent protein kinase I1 (CaMKII). The first two lysine residues play
an important role for its activity [141]. As expected, caged AIPs showed
significantly reduced inhibitory activity in vitro toward CaMKII (IC50 =
1.2 x M) and gave instantaneous recovery of activity after irradiation
(IC50 = 3.6 x lo-’ M, as for natural AIP). Interestingly, the photolysis by-
product nitrosobenzaldehyde did not interfere with the behavior of the
photoactivated peptides. Caged Tyrosine Residues

One of the first caged peptides contained a NB-caged tyrosine that was
introduced via Fmoc-SPPS [142]. Fmoc-Tyr(NB) was used to prepare caged
neuropeptide Y (NPY) and caged angiotensin I1 (AII) peptide [142]. NPY
is a 36-amino acid peptide containing Tyr residues at both the N- and the
C-termini. It localizes in both the central and peripheral nervous system and
is potentially involved in various physiological roles, including blood pressure
regulation, anxiety, circadian rhythms, and feeding behavior. Structure/activity
relationship studies indicated that both the N- and the C-terminal fragments
of NPY are essential for the activation of Y 1 receptors [143]. Introduction of
one caged Tyr at the naturally occurring Tyr positions at the N/C-termini of
NPY led to a decrease of about 1 order of magnitude after activation of the
Y1 receptors in SK-N-MCcells, with additional reduction when two caged Tyr
were incorporated at both termini of NPY. Restoration of activity assessed by
the binding assay performed after UV irradiation demonstrated the successful
role ofthe nitrobenzyl group as a cage for Tyr residues and for the NPY peptide
Interestingly, no differences in activity toward A11 receptors in human
neuroblastoma SMS-KAN cells were found between caged and unmodified
A11 peptides, indicating that the Tyr residue in this eight-amino acid peptide
is not involved in binding to the receptor [142].
3.2 Controlling Protein Function by Caged Compounds I 161

The 20-amino acid residue peptide RS-20, whose sequence derives from
smooth muscle myosin light chain kinase (M LCK),is a well-known calmodulin
binding peptide [144]. Both, RS-20 and LMS-1, a 13-residue peptide derived
from the autoinhibitory domain of MLCK, have the capability of inhibiting
MLCK phosphorylation activity, normally directed toward the molecular motor,
actin binding protein myosin 11, which is involved in physiological phenomena
like cell polarization and locomotion [145, 1461.
The interaction of RS-20 with its target protein calmodulin has been
extensively studied and hydrophobic residues Trp5 and Leu18 were shown to
be critical for binding [147, 1481. Tyr9 in LMS-1 peptide is in turn crucial for
the inhibitory effect as is predicted from mutagenesis studies on MLCK [149].
Walker and others expanded the study on these molecules, both in vitro and in
vivo, using a caged version ofboth peptides (Scheme 3.2-9)[150].Trp5 in RS-20
was replaced with a masked tyrosine bearing a CNB cage on the phenolic group.
The carboxylic group of the cage mimicked the negative charge of a glutamate,
a mutation known to have a negative effect on binding. Accordingly, the caged
RS-20 peptide was largely unable to bind to calmodulin, as assessed in vitro
by a quantitative calmodulin-dependent MLCK assay. The photoproduct 5Y-
RS-20 generated after 10-min irradiation at 300-400 nm showed an apparent
50-fold increase in its affinity toward calmodulin. A similarly Tyr9-caged
LMS-1 proved to be an effective switchable inhibitor of MLCK in vitro, being
indistinguishable from authentic LMS-1 in its inhibitory potency. The effect
of local photoactivation of the two caged peptides was finally assessed in
vivo in fast-moving Newt eosinophil cells [151]. Peptides were introduced by
microinjection in an estimated concentration of 20-100 pM. Photoactivation


L I 1

- hv
peptides +

r~ 1

Scheme 3.2-9 The calmodulin binding peptide RS-20 and LMS-1,

a peptide that inhibits myosin phosphorylation, caged at different
tyrosines. Both peptides were successfully used in eosinophils
after microinjection [151].
I was performed locally by pulsed near-UV laser light (series of 10 pulses with
3 Engineering Control Over Protein Function Using Chemistry

a 5 ms duration at 20 ms intervals) with concomitant microscopic observation

of cells. Photorelease of active peptides was followed, within a few seconds,
by acute paralysis of cell movement, ceased flow of cytoplasmic granules
and inhibition of forward motion of the leading lamellipodia. These results
suggested that calcium/calmodulin regulation of MLCK activity is a major
signaling pathway underlying locomotion in eosinophil cells in vivo, and that
the myosin I1 motor target of MLCK activity is strongly involved in these
motility functions. Caged Cysteine and Thiophosphoryl Residues

As mentioned above, Pan and Bayley reported a generally applicable approach
for caging cysteine-containing peptides or thiophosphorylated peptides on
serine residues in aqueous solution using o-nitrobenzyl bromides such
as NB-Br, CNB-Br, and DMNB-Br [71]. Kemptide (LRRASLG), C-kemptide
(LRRACLG), and CS-kemptide dimer (LRRACLGLRRASLG) were used as
model peptides in this study. Both, Kemptide and CS-Kemptide dimer, were
successfully thiophosphorylated on Ser residue using ATP(y)S and PKA
catalytic subunit. Thiophosphorylated kemptide peptide was subsequently
treated with the three different cages, respectively. At pH 7.2, only NB-Br
and DMNB-Br cages were found to react satisfactorily with the thiophosphate
group, producing the corresponding caged peptides in 95% yield. CNB-Br was
found to be close to unreactive (10% yield at pH 4.0), hence the synthesis to
this caged peptide was no longer pursued.
Photoactivation of NB- and DMNB-thiophosphoryl-caged Kemptide at
290-380nm was obtained with a yield of 70 and 55% and with quantum
yields of 0.23 and 0.06, respectively (Scheme 3.2-10).
Selective caging was examined on the CS-Kemptide dimer. The goal was
to selectively introduce a cage at a thiophosphoryl-Ser residue over a cysteine

SH op02s-

NB-Er. pH 4.0

O z N q s

0 SH 0

Scheme 3.2-10 The selective introduction of cages to thiophosphates versus cysteines is

p H dependent.
3.2 Controlling Protein Function by Caged Compounds I 163

residue. At pH 4.0 NB-Br (2 mM in 100 mM sodium acetate) showed good

selectivity for the alkylation of thiophosphate, while at pH 7.2 both Cys
and thiophosphoryl residues reacted with NB-Br as was determined by
Cysteine-targeted caging of C-Kemptide was performed with all three
photolabile groups mentioned above at pH 7.2 with a consistent yield of
caged product (95%), while photolysis with UV light (h,,, = 312 nm) gave
yields varying from 62 to 70% and quantum yields from 0.15 to 0.62 at pH 5.8,
with a slight decrease in performance at pH 7.2.
Finally, NB-caged thiophosphoryl kemptide was used to test the activity of
phage h protein phosphatase (h-PPase)before and after photoactivation. The
thiophosphate group of NB-caged thiophosphoryl kemptide was fully protected
against h-PPase activity, whereas the correspondent group in the unmodified
peptide was hydrolyzed to an extent of 90% when incubated at 30°C for
3 h. After UV treatment for 40 min, the uncaged thiophosphoryl kemptide
underwent thiophosphate hydrolysis to about 70%.
A similar strategy was employed to produce caged thiophosphotyrosyl
peptides [99]. The sequence EPQYEEIPILG was thiophosphorylated on the
tyrosine residue by action of hematopoietic cell kinase (Hck) in the presence
of Co" ions (the authors explain how thiophosphorylation on Tyr with
ATP(y)Sand tyrosine kinases failed in conditions that normally work well with
standard ATP) and afterwards attached the thiophosphate group again with
both NB-Br and 4-HP-Br, respectively. The peptides EPQYp,(HP)EEIPILG
and EPQYp,(NB)EEIPILG were obtained in 90 and 75% yield, respectively,
regardless of the pH of the reaction buffer (range 5.8 to 8.0). Irradiation
of the EPQYp,(HP)EEIPILG peptide at 312 nm afforded the photoproduct
EPQYP,EEIPILG with 50-70% yield. Quantum yields were 0.65 and 0.56 at
pH 5.8 and 7.3, respectively. The same treatment of EPQYp,(NB)EEIPILG
gave EPQYp,EEIPILG in 50 to 60% yield, with quantum yields 0.37 and 0.25
at pH 5.8 and 7.3, respectively. It was verified that the caged peptides were
no longer able to bind to an SH2 domain in vitro, while this feature was
completely restored upon photolysis (Scheme 3.2-11).Despite the promising
characteristics of the above described thiophosphorylated peptides (especially
the HP-caged one), to the best of our knowledge, no study has yet been reported
in vivo.
By means of caged peptides, Lawrence and coworkers successfully prepared
a caged protein kinase A in two different ways, (a) via a peptidic affinity label
[21] and (b) via a caged inhibitor [152].The peptidic affinity label was designed
to target Cys199 in the active loop of the enzyme, interacting with PKA active
site only in its caged form, while transforming itself into a low affinity ligand
upon photoactivation. This peptide was synthesized by SPPS (see Fig. 3.2-6)
and coupled at the C-terminus to the a-carboxyl group of a CNB cage via a
diethylamine linker. The caged ligand was subsequently coupled to the thiol
group of Cys199, finally affording the caged enzyme.
I 3 Engineering Control Over Protein Function Using Chemistry

Kck kinase, Co"
NB-Br (75%)
hv 312nm I

hv 312nm HP-Br
(50-70%) (90%) 02N7$
NB-cagedpeptide (inactive)

HP-cagedpeptide (inactive)


Scheme 3.2-11 Tyrosine residues equipped with various caging groups rendered
peptides inactive with respect to SH2-domain binding.

Fig. 3.2-6 Protein kinase A

labeling approach. Underlined

letters represent amino acids in
the one-letter code.

This caged PKA showed less than 2% of the activity displayed by the native
protein, while UV irradiation (300-400 nm, up to 15 min) restored about 50%
of the activation of the unmodified enzyme in vitro. Following these in vitro
observations, 3-7 pM solutions of caged PKA were microinjected in living rat
embryo fibroblasts (REF)-10-fold dilution was estimated after injection - and
irradiated with near-UV light (300-400 nm, up to 15 min). In these cells,
photoactivation of PKA led to disruption of actin stress fibers, membrane
rufling, and change of cell shape from flat to rounded, in accordance with
the phenotype observed when unmodified, active catalytic PKA subunit was
injected into the same cells. Microinjected cells that were not exposed to UV
irradiation retained their stress fibers and flat morphology, indicating that the
PKA-inducedpathway had not been activated [21].
PKI is a heat-stable protein first described in 1982 as a potent inhibitor
of PKA [153]. On the basis of a short binding sequence, a potent inhibitor
peptide with the sequence GRTGRRNAI was identified. The underlined
Arg residue played an essential role for the inhibitory behavior of this
3.2 Controlling Protein Function by Caged Compounds 1 165

peptide [154].Consequently, a peptide containing an L-ornithine replacing the

arginine residue was prepared. The latter was guanidinylated to obtain a caged
arginine, the first example described of this kind [ 1521. The guanidinylating
reagent resulted from the synthesis of DMNB-OCOCIand S-methylisothiourea
(Scheme 3.2-12). The caged peptide was shown to be a SO-fold poorer
inhibitor of PKA in vitro (K;= 20 pM) compared to the uncaged counterpart
(K;= 420 nM).
When REFS were exposed to the membrane-permeant PKA activator,
8-(4-~hlorophenylthio)-cAMP (CPT-CAMP),they underwent the same mor-
phological transformation as described above (disruption of actin stress fibers
leading to cell shape changes). In contrast, cells microinjected with the caged
peptide (5 pM estimated intracellular concentration) and exposed to UV irradi-
ation were unable to respond to the CPT-CAMPstimulus, demonstrating that
the CPT-CAMPactivation of the PKA pathway had been efficiently blocked in
vivo by the decaged peptide [152]. Caged Phosphorylation Sites and Caged Phosphopeptides

Recently, a Ser-caged,photoactivatable fluorescent peptide probe that monitors
protein kinase C (PKC) activity was described [75].As expected, the Ser-caged
peptide failed to serve as an effective PKC substrate in vitro,but upon light-
induced deprotection (300-400 nm, h,,,360 nm, 90 s), the serine became
phosphorylated and enzyme activity was recorded as a convincing change in
the fluorescent properties of the probe. Photoconversion was estimated to
occur with 60% yield and a quantum yield of 0.06.
With this probe, the investigators also studied the light-induced sampling
of PKC activity in HeLa cells in vivo. Exposure of cells to phorbol ester (TPA)
normally induces PKC activity. HeLa cells microinjected with the caged probe
at an estimated concentration of 20 pM failed to display a fluorescent response
to TPA, while a robust response was recorded as a result of a concomitant TPA
treatment and UV irradiation (365 nm at 1 J cm-2).

Scheme 3.2-12 A peptide caged at an arginine residue was

prepared by attaching a DMNB-coupled S-methylisothiourea
reagent t o ornithine [152].R represent further amino acids.
I 3 Engineering Control Over Protein Function Using Chemistry

The phosphorylated varieties with a cage attached to the phosphate

are as desirable as caged serine or threonine. Imperiali and colleagues
have lately introduced an elegant and general method for the synthesis
of peptides containing 2-nitrophenylethyl-caged phosphoserine, phospho-
threonine, and phosphotyrosine by integrating an interassembly approach
into Fmoc-SPPS [78]. The recently reported method for the synthesis of
the phosphocaged Fmoc-building blocks - namely, N-a-Fmoc-phospho(1-
nitrophenylethyl-2-cyanoethyl)-~-serine, -threonine and -tyrosine - is superior
to the introduction of cages to the growing peptide on resin. Especially, the ox-
idation step required in phosphorous(111) chemistry was potentially hazardous
toward oxidation-sensitive residues C-terminal of the caged amino acid [79].
A caged phosphoserine octapeptide equipped with the environmentally
sensitive fluorophore 6-(2-dimethylaminonaphthoyl)alanine (DANA) [155]was
used in vitro to probe the phosphorylation-dependent binding to 14-3-3
proteins [156], a highly conserved family of proteins that plays a role as an
intermediate in the cell cycle regulation through phosphorylation-dependent
protein-protein interactions [157].The caged phosphopeptide was unable to
bind to the target 14-3-3protein as opposed to the photoproduct after irradiation
at 365 nm. This could be monitored by the shift of fluorescence of the DANA
amino acid from heml = 522 n m (unbound peptide) to hem2= 501 n m (bound
The investigators have more recently described the use of such caged
phosphoserine-containing phosphopeptides to perform a UV-induced, “chem-
ical” knock-down of the entire 14-3-3 protein family thereby observing
the effects on cell cycle progression in vivo [158]. A derivative caged at
the phosphoserine position of a good 14-3-3-binding motif sequence like
MARRLYRpSLPAKK [159]was prepared by SPPS. The efficiency of the pho-
toactivationwas first tested in vitro under conditions mimicking irradiation of
cultured cells (365 nm, 90 s, 2.8 J m-’ irradiation dose).The uncaged phospho-
peptide was obtained in about 80% yield, quantum yield of 0.43 and was able to
compete with cellular proteins for 14-3-3binding in vitro, as demonstrated by
competitive binding assays performed in U20S cell lysates (Scheme 3.2-13).
The caged phosphopeptide was subsequently supplied to living U20S cells by
connecting it to the cell-permeable Penetratin sequence [161]via a disulphide
bond between N-terminal cysteine residues. After internalization and release
from vector peptide by spontaneous hydrolysis of the disulfide bridge, effects
of uncaged phosphopeptide disturbance on 14-3-3 binding to natural target
proteins were studied under several conditions.
For instance, synchronized U20S cells that received the peptides in an early
G2 phase and were subjected to UV treatment (365 nm, 90 s) showed (a) an
increased cell death ratio compared to controls, (b) an uncontrolled premature
entry into M phase accompanied by mitotic catastrophe, and (c) a striking
reduction in the stable G1 cell population, suggesting that 14-3-3 proteins
normally regulate the onset and timing of mitosis in cycling cells and maintain
stable interphase arrest in noncycling cells. The role of 14-3-3 proteins in
3.2 Controlling Protein Function by Caged Compounds I 167



522 nm

O f

h = 501 nm
\ /N\

Scheme 3.2-13 An octapeptide equipped with the

environmentally sensitive dye DANA. Only after decaging, binding
t o 14-3-3 domains is possible and is measured by a shift in
fluorescence due t o the change in the lipophilicity ofthe
environment [160].

the S-phase checkpoint response to DNA damage is speculative, since cells

incubated with caged peptides and simultaneously exposed to both UV-A
and UV-B (respectively 365 and 302 nm, 90 s) to induce uncaging and DNA
damage were unable to sustain S-phase arrest compared to controls, resulting
in ca SO% early apoptotic cell death.
To prepare larger phosphoproteins with cages on the phosphate moiety,
it was necessary to combine the synthesis of caged phosphopeptides [78,
791 with expressed protein ligation [162, 1631. The ligation of a recombinant
Smad2-MH2 thioester with the doubly NPE-caged C-terminal phosphopeptide
yielded a recombinant protein that formed a heterodimer with the cytosolic
retention factor Sara (Smad anchor for receptor activation). Decaging permitted
the release of Sara and subsequently the formation of active homotrimers.
Decaging was also followed in digitonin-permealized HeLa cells by monitoring
nuclear entry of Srnad2-MH2 after illumination [162]. This methodology was
extended using a cage in the backbone of the MH2 peptide. Photorelease of
the bulky N-terminus permitted homotrimerization. This was made visible by
adding fluorescein next to the phosphorylation sites and a dabcyl quencher to
the N-terminus. Photoinduced homotrimerization was therefore accompanied
by a strong increase of the fluorescein fluorescence [164].
I 3 Engineering Control Over Protein Function Using Chernistv

Fig. 3.2-7 A chemotactic tripeptide caged
at the N-formyl group.
Me0 H

0 Other Caged Residues

Some N-formylated peptides are known to promote chemotaxis in mammalian
leukocytes, acting specifically via the formyl peptide receptor (FPR) located
on the plasma membrane of neutrophils [165].Among them, the most active
peptide is the tripeptide N-formyl-&)Met-&)Leu-(L)Phe.Caged versions of
such a peptide have been synthesized employing either nitroveratrylaldehyde
or nitropiperonaldehyde as photoremovable protecting groups at the N-formyl
moiety (Fig. 3.2-7) [lGG]. Although the described caged peptides exhibited a
drop of activity by 3-4 orders of magnitude in a rat basophilic leukemia RBL-
2H3 cell line, a study concerning photoactivation in vivo and related effects on
chemotaxis has not yet been reported.


Caged compounds including caged proteins are extremely useful tools to

study biochemical processes inside and outside of living cells. The respective
molecules have been employed in a large variety of areas. However, the overall
number of research groups benefiting from the technology is still fairly small.
It would be desirable if novel caging groups, caged molecules, and ready-to-use
decaging equipment would be more easily accessible. We will definitely see
more of the exciting applications in the future. For this, as in more and more
areas in biology, the close collaboration of chemists and biologists will be


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Chemical Biology
Edited by Stuart L. Schreiber, Tarun M. Kupoor,and Gunther Wess
Cowriaht 0 2007 WILEY-VCH Verlaq CmbH & Co KCaA, Weinheim

I 3 Engineering Control Over Protein Function Using Chemistry

EngineeringControl Over Protein Function; Transcription Control by Small

John T.Koh


Ligand-inducible transcription factors, whether derived from heterologously

expressed prokaryotic regulatory proteins or reengineered eukaryotic receptors,
continue to play an invaluable role in studying gene function. Through the
study of reengineering ligand-binding specificities of nuclear receptors and
other transcription factors, new tools for exploring emerging extranuclear roles
for these receptors can be generated. Developing new strategies for selective,
functionally orthogonal, ligand-receptor pairs can be applied more broadly in
chemical biology in the form of chemical inducers of dimerization (CIDs), or
analog-specific enzymes. Similar design principles may also be applied to the
functional rescue of disease-associated mutant proteins that have defects in
binding small molecules.
The impact that ligand-inducible transcription factors have had on the study
of biology over the past decade highlights the importance of developing new
methods to precisely manipulate and study complex biological systems at the
molecular level. The availability of multiple ligand-dependent transcription
factors further increases the level of complexity and sophistication with which
we can probe complex biological phenomena. In the future new systems such
as light-directed transcription control may play a powerful role in dissecting
the roles of genes that act through their unique spatiotemporal patterns in
tissue. These efforts will similarly require continued development of new tools
based on the marriage of both chemical and biological methods.


This chapter reviews strategies for manipulating or engineering de novo

proteins that can regulate gene expression in response to small molecules.
Methods that allow us to control the expression of genes in a spatially and
temporally defined manner provide powerful tools for the study of gene
function. The study of naturally occurring ligand-inducible transcriptional
regulators affords insights into the strategies that nature uses to remotely
regulate protein function, thus providing a basis with which to control
and study the actions of virtually any gene product through the remote
regulation of its expression. Ligand-receptor engineering can be used to
create new transcriptional regulators, to provide the means to selectively

Chemical Biology. From Small Molecules to System Biology and Drug Design
Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Gunther Wess
Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
3.3 Engineerjng Control Over Protein Function; Transcription Control by Small Molecules I 175

activate one of many cellular pathways responsive to the same ligand, and may
further provide new strategies to rescue disease-associated mutants of ligand-
dependent proteins. In addition, new methods to control gene expression with
light can be used to spatially and temporally pattern genes in tissues.

The Role of Ligand-dependent Transcriptional Regulators

Many naturally occurring inducible transcriptional regulators have been used

in heterologous systems for controlling protein expression. However, more
recently a number of research groups have used a combination of chem-
ical and genetic approaches to reengineer the specificity of transcriptional
regulators [l-31. Emerging methods may allow one to convert otherwise
nonligand-responsive proteins into ligand-responsive systems. Several new
technologies offer unprecedented control over gene expression using nucleic
acids such as antisense, ribozyme interference, and RNAi [4-6]. New methods
to control mRNA translation in a ligand-dependent manner offer a new dimen-
sion of transcriptional control [7,8].These methods can be used in conjunction
with ligand-dependent expression systems to provide spatial and temporal con-
trol of genes. In addition to strictly “on/ofl” responses, dose-dependent or
“rheostatic” control expression can provide exquisite control of gene functions
dependent on specific stoichiometries or spatiotemporal patterning. Ligand-dependent Transcriptional Regulators Derived from Natural

Practical applications of ligand-regulated transcription factors need to function
in a highly specific manner that should ideally only effect the expression of the
gene of interest. Some of the most common ligand-inducible transcriptional
regulators are derived from naturally occurring proteins. For example, the lac
repressor binds to operons (i.e., genetic binding sites) in the promotor region
preceding genes, and blocks transcription (Fig. 3.3-1). The lac repressor forms
a homotetramer that spans two operon sites and blocks association of the
transcription machinery through a combination of direct occlusion of DNA-
binding sites and perturbation of DNA topology [9, 101. Binding of the small-
molecule lactose or the stable synthetic analog isopropylthiogalactropyranoside
(IPTG) occurs near a dimerization interface, induces a conformational change
that disrupts oligomerization and DNA binding, thus exposing the full
promotor sequence and allowing for gene expression. The lac operon is highly
inducible and widely used for controlling protein production in eukaryotes.
This is particularly important when expression of the target protein, either
toxic to the cell or otherwise, adversely affects growth.
Several prokaryotic repressors can also be used in eukaryotes. Most notable
is the tetracycline (TET) inducible expression system, which has had a
I 3 Engineering Control Over Protein Function Using Chemistry

Fig. 3.3-1 Prokaryotic repressors can be exploited t o control

eukaryotic expression. (a) Repressor used to turn off transcription,
(b) repressor-activator chimera used to “turn-on” gene
expression. AD - activation domain.

tremendous impact on the study of eultaryotic protein function [ll, 121.

Similar to LacR, DNA binding of TET is also conformationally controlled by
the association of a small molecule, tetracycline (tet). Tet binding triggers
dissociation of the repressor dimer and loss of DNA binding (Fig. 3.3-l(a)).
These ligand-dependent repressors have been converted to ligand-dependent
activators through fusion of LacR or TET to the potent HSV (herpes simplex
virus) transactivation domain (VPlG). These systems provide tight control
over genes of interest placed behind minimal promoters having three-
flanking operator binding sequences (Fig. 3.3-1(b)).The LacR-VP1G chimera
has approximately 1000-fold inducibility but is slow to respond to the addition
of IPTG [lo]. In the original TET system, cells continuously treated with tet
repress gene expression. When tet is removed gene expression is activated.
The need to continuously treat cells with tet was a significant drawback
as it was unclear what effects long term exposure to tet could have on a
specific system. Bujard et al. were able to reengineer the TET so that it only
bound DNA in the presence of doxycycline (dox). Fusion of this modified
form of TET to the VPlG transactivation domain formed a dox-responsive
transactivator, tTA or “Tet-On,” that tightly and rapidly upregulates the
transcription 105-foldwhen dox is added [12]. These pioneering studies have
lead to the development of a number of ligand-inducible activators based on
prokaryotic proteins.
Ligand-dependent transcriptional regulators have been derived from
prokaryotic repressors that bind DNA in response to small-molecule ligands
to commercially available antibiotics of the macrolide and streptogramin
families [13,14].Because the protein binds DNA only when liganded, chimeras
generated from the fusion of these repressors to transactivation domains can
serve as potent ligand-dependent transcriptional activators.
3.3 Engineering Control Over Protein Function; Transcription Control by Small Molecules I 177


OH -.


Fig. 3.3-2 Prokaryotic regulators of transcription have been

adapted for use as eukaryotic transcriptional regulators:
(a) macrolide, (b) streptogramin, (c) quorum signaling
p-0x0-hexanoylhomoserine lactone. Exploiting Prokaryotic Ligand-dependentActivators

Another class of small-molecule transcription factors is the quorum-
sensing receptors that often respond to surprisingly simple small molecules
(Fig. 3.3-2(c)).These naturally occurring small-molecule dependent transcrip-
tional regulators have been pursued as a means to control prokaryotic genes
only recently and therefore their discussion here is brief [lS]. Nonetheless,
these naturally occurring prokaryotic transcriptional regulators hold promise
as an important new source of ligand-dependent transcriptional regulators of
eukaryotic genes. An interesting example is that of the acetaldehyde respon-
sive protein that controls expression in response to gaseous molecule and can
therefore enforce transcription control in a whole animal transgenic model
through its air supply [lG]. Reprogramming Eukaryotic Transcriptional Regulators

A critical requirement for any transcriptional regulator to be used in
the study of gene function is the strict selectivity of the ligand-receptor
pair to activate only the gene of interest. Several groups have developed
methods to “reprogram” the ligand-binding specificity and gene target-
ing specificity of transcriptional regulators. The need to change both
ligand-binding specificity as well as DNA-binding specificity greatly limits
the possible receptors that can be directly reengineered to provide con-
trol over transgene expression. For example, G-protein coupled receptors
(GPCRs) are an important class of signaling receptors that regulate gene
expression in response to small molecules. However, GPCRs regulate ex-
pression through signaling pathways that involve the intermediary actions
of multiple proteins. In this case, the ligand binding and DNA recognition
events are separated on many different proteins, making their “reprogram-
ming” difficult.
Nuclear and steroid hormone receptors (NHRs),in contrast to GPCRs, are
ligand-inducible transcription factors, which when liganded directly bind to
I hormone response elements in eukaryotic genes and upregulate transcription
3 Engineering Control Over Protein Function Using Chemistry

through a ligand-dependent transactivation domain [ 17, 181. When in their

unliganded forms, most steroid hormone receptors are not bound to DNA,
but are instead sequestered by heat-shock proteins (hsps). Steroid hormone
binding causes dissociation from hsps, dimerization, and DNA binding. In
contrast, the unliganded forms of the “nuclear” receptors such as thyroid
hormone, retinoic acid, peroxisome proliferator-activated receptor (PPAR),
and vitamin D receptor (VDR) are generally bound to DNA as heterodimers
with RXR retinoid X receptor and bind corepressor proteins that actively
repress gene expression (Fig. 3 . 3 - 3 ) .
The ligand-dependent transactivation domain and the DNA-binding do-
mains of these receptors function relatively independent of each other,
allowing one to create functional chimeras that redirect the actions of
specific hormones to new genes through alternate DNA-binding domains.
For example, an early study by Greene and Chambon demonstrated that
by exchanging the glucocorticoid receptor (GR) ligand-binding domain for
that of the estrogen receptor (ER), glucocorticoid-responsive genes could
be rendered responsive to estradiol (E2) [19]. A number of other functional
chimeras have been constructed by exchanging DNA-binding domains from
other NHRs including thyroid hormone receptor (TR)/retinoid X receptor
chimeras [20], retinoic acid/VDR chimeras (211, and TR/GR chimeras [22].
Functional chimeras have also been generated using non-NH R DNA-binding

Fig. 3.3-3 General mechanism of their unliganded forms. (b) Nuclear

nuclear/steroid hormone receptor action. receptors can bind to DNA in the absence of
(a) Steroid hormone receptors are generally ligand and can associate with transcriptional
sequestered by heat-shock proteins (hsp) in repressors.
3.3 Engineering Control Over Protein Function; Transcription Control by Small Molecules I 179

domains such as the progesterone receptor (PR) Gal4 DNA-binding do-

main chimera, developed by Wang and O’Malley [23]. Several studies have
shown that DNA-binding domains can be reengineered or evolved to bind
to new DNA-binding sequences [24, 251. Therefore, NHRs are an attrac-
tive scaffold from which to develop new, selective transcriptional regulators
as they in principle can be modified to regulate almost any transgene of
interest. However, application of these systems is still limited by the pres-
ence of other endogenous receptors that are also responsive to the same
The use of heterologous NHRs is one way to selectively control only
the targeted gene of interest. The ecdysone receptor (EcR) is unique to
insects and crustacea and therefore has been widely used to selectively
regulate mammalian genes [26, 271. Inducible gene expression in mammals or
mammalian cell culture can be achieved with EcR, although highly inducible
expression generally requires coexpression of RXR. It is unclear if over
expression of RXR influences expression of other NHR responsive genes.
Nonetheless, the EcR has become an important heterologous regulator of
mammalian gene expression. The need for additional and multiple ligand-
inducible transcription factors has prompted several groups to develop new
transcriptional regulators by reengineering the ligand-binding domains of
existing NHRs.

Engineering New Ligand Specificities into NHRs

The reengineering of NHR ligand-binding domains to selectively respond

to synthetic ligands has proved to be an important and challenging area
in ligand-receptor engineering. Since the original studies of Kirsh and
Holbrook directed toward reengineering substrate specificity of enzymes,
ligand-receptor engineering has become an important tool for studying
complex biomolecular systems [28, 291. Schreiber was perhaps the first to use
a combination of mutagenesis and synthesis to generate selective probes for
biological function in the form of chemical inducers of dimerization (CIDs;
covered elsewhere in this volume) 130-321. The basic design principle used in
these studies was the use of “bumps and holes” to alter the interface between
ligand and protein in a complementary manner [31]. The bump refers to a
molecular appendage on the ligand that would cause a steric clash if it were to
try to bind to the wild-type receptor. However the “bumped ligand” could bind
to a receptor that is appropriately modified through mutagenesis to contain
a compensatory “hole.” The “bump and hole” approach to ligand-receptor
engineering has been applied to a number of protein-ligandlenzyme substrate
systems. One ofthe most successful systems is the ATP analog-selective-kinase
systems by Shokat et al. [33].
I 3 Engineering Control Over Protein Function Using Chemistry

The Requirement of “Functional Orthogonality”

The application of “bump and hole” engineering toward the generation

of selective transcriptional regulators has been limited, largely because
“hole-modified’ proteins often retain substantial aflinity for their natural
ligand [34]. For some applications, such as the selective labeling of kinase
substrates by radiolabeled ATP analogs that are only recognized by modified
kinases, competing reactions by the natural (nonlabeled ATP) substrate for
the kinase is not strictly required [35].
However, a selective transcriptional regulator used to study gene function
would have to be function independent of any endogenous receptors. Absolute
selectivity over all concentrations of ligand is rarely observed. In practice, it is
sufficient for a modified ligand-receptor pair to be “functionally orthogonal”
such that the modified receptor is nonresponsive to endogenous concentrations
of the natural ligand and that the modified ligand is unable to activate the
natural receptor at concentrations used to modulate the modified receptor [33,
341. It is important to recognize that while high potency is generally desirable,
the ligand-analog need not bind the modified receptor with the same affinity
as the natural ligand-receptor pair so long as it has high selectivity.

Overcoming Receptor Plasticity

The greatest challenge presented by engineered nuclear receptors is the

significant structural flexibility of the ligand-binding domain. NH R ligand-
binding domains undergo substantial structural reorganization upon hormone
binding. The hormone generally provides a hydrophobic nucleus around which
the ligand-binding domain repacks its core. The structural changes to the
receptor’s core cause changes to the receptor surface resulting in coactivator
recruitment and changes in receptor dimerization. It is therefore not surprising
that the estrogen receptor binds many ligands that are substantially larger than
E2 and would otherwise appear to be too large to fit within the binding
pocket observed in the E2-ER crystal structure (Fig. 3.3-4) [36]. These studies
imply that identifying “bumped” hormones that will not bind wild-type NHRs
could be more challenging than ligand-receptor engineering with more rigid
Through targeted site-directed mutagenesis, Corey et al. searched a library
of failed drug candidates, “near drugs,” for their ability to selectively activate
the 9-cis retinoic acid receptor, RAR [37, 381. These mutants were carefully
selected not to have significant activity with the natural hormone 9-cis retinoic
acid. Although mutants that improved activity of these ligands with the mutant
receptor were identified, these ligands that largely contained only hydrophobic
groups, aside from the requisite carboxylate, were generally less than 10-fold
3.3 Engineering Control Over Protein Function; TranscriptionControl by Small Molecules I 181


Estrddio (E?) RU-58668 ICI-IX2.780

Hanson: 17u phenylvinyl estradiol Katrenellenbogen; 4-ally1

Fig. 3.3-4 The estrogen receptor has sufficient flexibility to accommodate a diverse array
of ligands that interact with ER a t low or sub-nanomolar potencies.

selective for the mutant over the wild-type receptor. These studies highlight
the remarkable ability of the wild-type receptor to accommodate ligands that
differ in hydrophobic shape even when modeling might suggest that these
ligands should not be accommodated by the ligand-binding site. In general,
protein plasticity limits the use of “bump and hole” engineering of flexible
Our group has therefore focused on exploring methods to manipulate polar
groups to impart specificity to engineered ligandlreceptor pairs, following
the general notion that polar interactions impart specificity to molecular
recognition events because mismatched polar interactions cannot be easily
avoided by simple side-chain reorganization. In an early work on the retinoic
acid receptor, hormone-binding selectivity was changed by modifying a key
arginine residue, (Arg278) that forms a salt bridge to the carboxylate of
bound retinoic acid [39].Although a neutral ethylamide analog of retinoic acid
displayed some mutant versus wild-type selectivity, this analog was notably
less potent than the wild-type retinoic acid- RAR (retinoic acid receptor)
pair and showed only partial selectivity. A more dramatic attempt to impart
selectivity through the manipulation of polar interactions was the reversal of
a ligand-receptor salt bridge by creating a guanidine functionalized retinoid,
which showed selective but weak activity for the charge-complementing mutant
RARy (S289G/R278E).The weaker cellular activity of this ligand-receptor pair
is not entirely unexpected in the light of studies by Warshel suggesting that salt-
bridge interactions are stabilized protein dipoles that would be destabilizing
if the salt bridge were reversed [40, 411. In general, charged or neutral polar
I groups found in the interior of proteins are stabilized by multiple polar
3 Engineering Control Over Protein Function Using Chemistry

interactions from the protein in the form of ion pairs, hydrogen bonds, and
local or macrodipoles. Adding, removing, or rearranging polar groups found in
the interior of protein-ligand complexes is generally disfavored as it leaves the
associated polar groups unsatisfied. The solution to this problem of selectivity
is not immediately obvious but in at least some cases can be solved.
The Koh and the Katzenellenbogen groups simultaneously explored estro-
gen analogs that could complement the same Glu353 + Ala or Ser mutation
in the estrogen receptor [42-441. Glu353 forms an intramolecular salt bridge
with Arg274 and both residues form key hydrogen bonds to the 3-hydroxyl
of E2 (Fig. 3.3-5(a)).Mutations to Glu353 greatly reduce the receptor’s affinity
for the natural ligand E2. While a number of estrogen analogs bearing neutral
functional groups in place of the 3-hydroxyl of E2 could activate the Glu353
mutants with high affinity, in almost all cases, these analogs activated the
wild-type ERs with equal or greater potency. A few low-potency ligands ( t 2 %
wild-type potency) show receptor selectivities as high as 34-fold (mutantlwild
type) (Fig. 3.3-G(a))[42]. By comparison, carboxylate-functionalized estrogen
analogs designed to restore (intermolecularly) the lost protein salt bridge
with Arg274 form high affinity/potency complexes with the mutant receptor
(Fig. 3.3-5(b)).These complexes are not of higher affinity than the analogs
having neutral appendages, suggesting that the favorable energetics of form-
ing a salt bridge with Arg274 is offset by the substantial cost of desolvating
the ligand-associated carboxylate [44].However, carboxylate-functionalizedlig-
ands of appropriate size and shape provided a significant gain in selectivity,
which can be as high as 95- to 400-fold in favor of the mutant over the wild-type

Fig. 3.3-5 Accessible surface model of functionally orthogonal

ER/ES8 pair. (a) Wild-type ER-E2 receptor based on structure
modeled by Brzozowski et al. [45].(b) Modeled structure o f
3.3 Engineering Control Over Protein Function; Transcription Control by Small Molecules I 183

RTP = I .S RTP = 15 RTP = 17 KTP = 0.9 RTP = 2
RS = 34 RS = 1.3 R5= I1 KS = 9.2 RS = 1.6

RTP = 0.8 RTP = 3.0 RTP = 38

RS = 22 RS = 95 RS = 56

Fig. 3.3-6 Complements for ERa(E353A). structure provide high selectivity without
(a) Neutral modifications tend t o provide significant loss in affinity. RTP - relative
only modest mutant versus wild-type transcription potency; RS - receptor
selectivity. (b) Acidic analogs of appropriate selectivity (ECSowild type/ECSomutant).

ERs (Fig. 3.3-6(b)).This greater selectivity is imparted as a result of weaker

binding of the carboxylate-functionalized ligands to the wild type, presumably
as a result of mismatched polar interactions at the ligand-receptor interface.
We termed the process of exchanging polar groups across the lig-
and-receptor interface as “polar group exchange”. [43] In essence, the same
key functional groups are present in more or less the same positions in the
wild-type and the engineered ligand-receptor complexes but differ only in their
covalent connectivity ofa key polar group. In the present example, the carboxy-
late group is presumed to be in more or less the same position but covalently
linked to the ligand than to the receptor. This minimizes the impact of altering
polar groups within the interior of the protein by preserving the orientation
of key dipolar interactions. The most selective system reported is ERB(E305A)
with the synthetic ligand ES8. This mutant is no longer activatedby endogenous
concentrations of E2, but can be fully activated by concentrations of ES8 that do
not activate the wild-type ERs. This system therefore comprises a functionally
orthogonal ligand-receptor pair that, in principle, can be used to regulate gene
expression independent of endogenous estrogen responsive receptors.

Nuclear Receptor Engineering by Selection

Miller and Whelan were perhaps the first to recognize the potential of screening
or selecting NHR mutants from receptor libraries to identify ERs with modified
ligand specificities [46,47].Using error prone PCR, they generated populations
of mutant ERs in yeast that decreased responsiveness to E2 but has increased
responsiveness to the synthetic diphenyl indene-ol GRl32706X. Despite their
I elegant plan, the selected mutants had good potencies but relatively modest
3 Engineering Control Over Protein Function Using Chemistry

selectivities, exhibiting only a 10- to 25-fold improvement in the potency

of GR13270GX with the mutant when compared to wild type. One of the
limitations of the Miller and Whelan study was that their modified ER
regulated the expression of p-galactosidase, which was laboriously followed
colorimetrically. Doyle has recently succeeded in using a true selection method
to screen codon randomized libraries of RXR that were activated by the
synthetic compound LG335 [2]. A key component to their strategy was to
utilize a fusion of the nuclear receptor coactivator ACTR linked to the potent
Gal4 activation domain (ACTR-GAD).This provided tight control of ADE2
expression to conditionally control survival of the P JG9-4Aauxitroph on media
lacking Trp and Leu. The mutant RXR(I2G8V/A272V/I310L/F313M) was 300
times more responsive toward LG335 than wild-type RXR in mammalian cell
culture. This particular ligand-receptor pair has only 30% of the wild-type
efficacy but nonetheless represents a significant advance in the strategies used
to develop functionally orthogonal transcriptional regulators. This general
strategy could be easily extended to other NHRs.

Ligand-dependent Recombinases

Other NHR reengineering strategies do not require engineered ligand bound

complex to be transcriptionally active but can exploit the ligand-dependent
association of steroid receptors to hsps. Pioneering work by Chambon’s group
demonstrated that site-specific recombinases can be placed under the control
of nuclear receptor ligand-binding domains [48-SO]. The chimeric fusion
protein composed of the site-specific recombinase Cre with the ER ligand-
binding domain is only active in the presence of an ER ligand such as E2 or the
antagonist tamoxifen (Fig. 3.3-7). The unliganded ER ligand-binding domain
is associated with hsp90 and interferes with the formation of the tetrameric
Cre complex, which mediates recombination. Ligand-dependent recombinases
provide a powerful tool for the gene expression because flanking a gene of
interest with Cre recognition sites can be used to permanently turn on or turn
off its expression. Because recombination causes a permanent change to the
cellular genome, all the progeny of a cell that has undergone recombination
will propagate the same genomic change. Conditional recombinases used in
conjunction with cell-type specific promotors can therefore be powerful tools
for following cell lineages in vivo [511.
Since the development of the original Cre-ER system, mutagenesis and
screening strategies have identified modified ER ligand-binding domains that
have reduced responsiveness to E2 but can mediate tamoxifen-dependent
recombination [48]. It is important to make the distinction that these
modified ligand-receptor pairs do not necessarily form transcriptionally active
complexes. Since the first report of the Cre-ER system, several new systems
3.3 Engineering Control Over Protein Function; Transcription Control by Small Molecules I 185


edRE-ER a
ER ligand


Fig. 3.3-7 Site-specific recombinases can presence o f an ER ligand. Recombination

be used t o control gene expression. can be used t o switch on or off genes by
(a) Homologous recombination by Cre is placing them downstream of promoter
performed a t specific LoxP sites. (b) The sequences.
chimeric Cre-ER i s only active in the

have been reported that make use of Cre or the site-specific recombinase Flp
including Cre, Cre-PR (progesterone receptor fusion), Cre-GR (glucocorticoid
receptor fusion), and EcR-Flp [Sl-531.
Although some of these ligand-dependent recombinases have been
reengineered to selectively respond to synthetic receptor antagonists such
as Tamoxifen responsive Cre-ER or RU486 responsive Cre-PR, the need to
treat cells for up to several days with these potent receptor antagonists may
have unwanted side effects, particularly, when used in in vivo developmental
models [SO, 531. This suggests that functionally orthogonal ligands may still
have an important role to play, providing the next generation of highly selective
ligand-dependent recombinases. Chemical Biology o f NHRs and the Potential o f Engineered Nuclear

A rapidly emerging area in nuclear receptor biology is the “nongenomic” or
“extranuclear” actions of NHRs [54]. Several lines of evidence suggest that
nuclear receptors may activate signaling complexes outside of the nucleus that
only indirectly affect gene transcription. For example, the rapid nongenomic
I actions ofvitamin D receptor (VDR)have been known for many years. Vitamin
3 Engineering Control Over Protein Function Using Chemistry

D analogs that selectively activate the nongenomic actions of vitamin D have

played an invaluable role in the study of its nonnuclear actions [55-571.
Nongenomic activities of thyroid hormone [58],glucocorticoids, androgens,
and mineralcorticoids have also been identified [54, 591. Currently, the most
well characterized of these systems involves estrogen and the estrogen recep-
tor. In addition to identifying that the GPCR GPR30 is an estrogen responsive
receptor [60-621, several studies have also confirmed that the estrogen receptor
can also act outside the nucleus in complex with scaffolding proteins such as
MNAR to activate Src kinase or in palmitoylated form in association with cave-
olins to activate PI3 kinase (Fig. 3.3-8) [63-661. In this case, the nuclear receptor
is found to play multiple extranuclear roles in regulating cellular signaling
pathways. Analog selective hormone receptors may yet play an important role
in dissecting the multiple signaling pathways activated by steroid hormones.

Complementation/Rescue o f Genetic Disease

The development of analog-specific forms of nuclear/steroid hormone

receptors has prompted us to investigate many naturally occurring mutations
found in nuclear receptors associated with genetic disease. Mutations to

Fig. 3.3-8 Estradiol i s involved in many activation of Src kinase, c - palmitoylated

different signaling pathways some ofwhich ER can localize t o caveolins in an estrogen
involve the same ligand-receptor pair. dependent manner, d - CPCR signaling by
a - classic nuclear activation of estradiol. Pathways a, b, and c may
transcription, b - MNAR scaffolded potentially involve E R a and ERP.
3.3 Engineering Control Over Protein Function; Transcription Control by Small Molecules I 187

nuclear receptors are associated with a family ofhuman genetic diseases, which
include VDR mutations associated with rickets, TR mutations associated with
resistance to thyroid hormone, mineralcorticoid resistance, PPAR mutations
associated with certain forms of severe insulin independent diabetes,
and androgen receptor mutations associated with androgen insensitivity
syndrome [67-691. Additionally, mutations to the androgen, estrogen, and TRs
are associated with the pathology of prostate, breast, and thyroid cancers [70].
A significant subset of these disease-associated mutations is located at the
receptor-hormone interface suggesting that appropriately designed hormone
analogs may be able to “complement” or “rescue” the function of these
receptors. Unlike current gene therapy strategies that use nucleic acid analogs,
hormone analogs typically have good druglike properties (i.e., bioavailability,
biostability) suggesting that hormone receptor complements may represent a
new strategy toward developing new treatments for genetic disease.
The possibility of using hormone analogs to rescue nuclear receptor
mutations was perhaps first explored by DeGroot et al. who demonstrated that
some synthetic hormone analogs were more potent than triiodothyronine (T3)
in mutant forms of TR, associated with resistance to thyroid hormone [71].
More recently, Feldman and Peleg similarly screened vitamin D3 analogs
that partially complement VDR mutants associated with vitamin D resistant
rickets [72], and Chatterjee et al. have identified PPAR agonists that can
restore activity to PPAR mutants associated with severe insulin independent
diabetes [73]. The first example of a molecule being designed as a rescuing
function to a mutant protein associated with a genetic disease was the
development of the thyroid hormone analog HY1, which was designed
to complement the RTH (thyroid hormone resistance) associated mutant
TRB(R320C)[74].This study represented a significant advance over the earlier
studies by DeGroot, in that the complementing analog was selective for the
mutant form of TRB over the TRcr subtype. In more recent work, new thyroid
hormone analogs have been developed that restore efficacy and potency to three
ofthe most common RTH-associated mutants Arg320 -+ Cys, Arg320 + His,
Arg316 + His (Fig. 3.3-9) [75, 761. All of the compounds used to rescue these
mutations affect the carboxylate-binding cluster of arginines, and are based on
the same general complementation strategy involving more neutral hydrogen
bonding groups in place of the ligand’s carboxylate. This suggests that once
general rules for designing complementing analogs are established, the process
of identifying new compounds may be reasonably efficient.
It is important to distinguish these “functional rescue” studies from sev-
eral other important studies showing that small molecules can stabilize or
chaperone folding of mutant proteins such as mutant p53 associated with
cancer [77, 781, mutant forms of V2R associated with nephrogenic diabetes
insipidus [79, SO], mutant forms of opsin associated with retinitis pigmen-
tosa [81],and B-glucosidase mutants associated with gaucher disease [82, 831.
By contrast, nuclear receptor mutants are often well-folded,stable proteins that
I 3 Engineering Control Over Protein Function Using Chemistry


TRfl(R320C) EC,=7.0 nM TRp(R320H) EC= , 0.46 nM
rnuffrx selectivity = 5.5 rnuffu selectivity = 1.O

H KG-8 H
TRp(R320C) EC& 7 nM TR[$(R316H)EC=, 12.6 nM
rnuffn selectivity = 12 muffu selectivtty = 4

Fig. 3.3-9 Analogs that rescue function t o TRP mutants

TRP(R320C), TRB(R320H), and TRP(R316H) associated with
resistance t o thyroid hormone. Receptor selectivity of ligand,
mutlcr, defined as (ECso with TRcr)/(EC50 with mutant TRB).

have lost ligand-dependent transactivation function that can be complemented

by appropriate ligand design.
The challenge to designing compounds that rescue mutations associated
with genetic diseases is that there are generally very few individuals with
any specific mutation. This poses an even greater challenge to chemists to
efficiently design compounds that can complement any specific mutation
in a receptor-binding pocket. We evaluated the ability of computer-aided
design to discover molecular complements for the rickets associated mutation
VDR(R274L),which is more than 1000 times less responsive to the natural
hormone 1,25-dihydroxyvitamin D3. We used a virtual screening strategy to
evaluate a focused library of analogs of the synthetic VDR agonist LG190155
(Fig. 3.3-10) [84]. Although the bound structure of LG190155 with wild-type
VDR was not available, half of the analogs selected by virtual screening were
able to restore more than GO% activity at 200 nM. When tested in cell based
assays, the best analogs were able to restore almost fully the potency and
efficacy to this otherwise unresponsive mutant. Computer-aided design was
similarly successful at identifying seco-steroid analogs that could complement
this same mutant (Fig. 3.3-10) [85]. These findings suggest that for at least
some mutants, computer-aided molecular design can be used to efficiently
design compounds that rescue genetic mutations.

De Novo Design of Ligand-binding Pockets

In addition to reengineering existing ligand-binding pockets, it is also possible

to generate de novo ligand-binding sites into proteins. A notable early
example shown by Matthews was the formation of de novo benzene- and
guanidine-binding sites by making Phe + Ala or Arg + Ala mutations into
3.3 Engineering Control Over Protein Function; Transcription Control by Small Molecules I 189

1,25dihydroxyvitaminD, ss-Ill
Wild-type VDR; EC,=2.0 nM VDR(R274L); EC=, 7.0 nM
VDR(R274L); EC, 2000 nM

LG190155 0
Wild-type VDR; EC,= 110.0 nM ss-Ill
VDR(R274L); EC, = 85 nM VDR(R274L); EC=, 3.3

Fig. 3.3-10 Molecular rescue of rickets associated mutant VDR(R274L) by designed

synthetic analogs of known agonists.

lysozyme [86].Although these de novo binding sites have only weak affinity for
these solvent substrates, they clearly demonstrated that new small-molecule
binding sites could be created into proteins. Barbas and Schultz have been
able to use this strategy to create zinc finger domains that bind only in the
presence of isoindole derivatives [87]. By fusing these inducible zinc finger
domains to transactivation domains, the isoindoles can be used to remotely
regulate gene transcription. Currently, the affinity of these de novo designed
cavities for their ligands are of only modest potency. However, combined with
recent advances in computational methods to de novo design ligand-binding
cavities [88-911, this general strategy provides a potentially powerful approach
to creating ligand-inducible transcriptional regulators.

Light-activatedGene Expression from Small Molecules

A new and exciting area in ligand-induced transcriptional regulators is

the development of photoresponsive transcriptional regulators, which utilize
photocaged small molecules. Just as ligand-inducibletranscriptional regulators
have revolutionized our study of protein function, light-activated transcription
(or translation) systems may prove to be a powerful tool for studying the
function of genes that elicit their effects only through their expression in precise
three-dimensional patterns, gradients, or arrays. This includes morphogens,
which are important guidance cues for neurogenesis, vascular genesis, and
limb development as well as other critical steps during development [92, 931.
Spatial gene patterning may also potentially play a role in creating artificial
I 3 Engineering Control Over Protein Function Using Chemistry

By photocaging nuclear receptor agonists, Koh et al. were able to show

that transcription could be controlled in an exposure-dependent manner [94].
Currently, photocaged agonists for the estrogen, thyroid, retinoic acid, and
VDRs have been used to place nuclear receptor mediated transcription under
the control of light [94-961. Using a photocaged agonist of the ecdysone
receptor, Lawrence et al. have demonstrated that even though photoreleased
agonists are freely diffusing, spatially discrete patterns of expressed genes
can be made on the micron scale in cultured cells [97]. The photoregulation
of gene expression by uncaging small molecules presents many challenges.
Small-molecule triggers for transcription have the advantage of being easily
delivered into cells by passive diffusion. Therefore, a multicellular system or
organism is only light sensitive after the addition of the caged compound.
Conversely, a cultured cell monolayer can be again rendered light-insensitive
minutes after the caged compound is removed from the media.
Ligand diffusion can affect the resolution at which genes can be patterned,
as the photoreleased activator can diffuse into neighboring cells. When the
patterned feature sizes are small, the region of activation will be confined
through the effects of ligand dilution upon bulk diffusion. In other words, the
concentration of released hormone activator will be too dilute to activate cells
that are remote to the site of activation. Photocaged antagonists may provide a
means to selectively turn off gene expression in a small region of cells within
a larger tissue [96].
The photorelease of nuclear receptor agonists in a subpopulation of cells
within a tissue presents another challenge, as the diffusion of ligand back
out of the cell will limit the duration of transcription response. For some
ligand-receptor pairs, the duration of reporter gene response may be limited
to less than a few hours, whereas for other ligand-receptor pairs, reporter gene
expression can last for several hours and as long as 1.5 days [95].The duration
of reporter gene response is much longer than the half-life of free-ligand
within the cell because many ligand-receptor complexes have very slow off-
rates. However, ligand-receptor pairs with apparently slow off-rates, can have
a relatively limited duration of response as NHR transcription complexes are
generally disassembled by chaperones and are targets of ubiquitin ligases and
proteasomes [98- 1011.The effects ofphotoreleased antagonists to turn off gene
expression can be similarly limited by ligand off-rates and receptor proteolysis.
Even when a covalent-binding antagonist that has a very long ligand-receptor
half-life is used, gene expression is recovered over several hours as new protein
is resynthesized by the cell [9G]. The long duration response observed, for at
least some ligand-receptor pairs, suggests that photocaged agonists can be
used to generate unique spatiotemporal patterns of gene expression.
The use of small molecules to activate gene expression should be compared
to methods used to photocage proteins or nucleic acids [102-1061. In gen-
eral, photocaged biopolymers are difficult to deliver into cells or organisms,
whereas caged small molecules can in principle be added in vitro or in vivobut
require the use of transfected cells of transgenic animals. Tsien et al. elegantly
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gene product. The use of caged nucleic acids to photoregulate gene expression
was first demonstrated by Hasselton et al. in mouse models [103-1051. The
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Chemical Biology
Edited by Stuart L. Schreiber, Tarun M. Kupoor,and Gunther Wess
CoDvriaht 0 2007 WILEY-VCH Verlaa CmbH & Co KCaA. Weinheim


Controlling Protein- Protein Interactions

Chemical Complementation: Bringing the Power o f Genetics to Chemistry

Pamela Peralta-Yahya and Virginia W. Cornish


Genetics in many ways is the underpinning of modern cell biology, having

provided a straightforward experimental approach to identify the proteins
involved in a given biological pathway. As practised, however, genetics leaves
us with a picture of the cell composed largely of proteins. The roles of other
molecules, such as phosphoinositides or siRNAs, have long been overlooked.
With growing interest in developing a complete description of a living cell
and with the backdrop of the genome sequencing projects, the question would
seem to be how to extend the ease of genetics to these other classes of
molecules. With a complete palette, it would then be possible to fully harness
the powerful synthetic and functional capabilities of the cell for chemistry
beyond that naturally carried out by the cell (Fig. 4.1-1).Here we consider a
particular genetic assay, the yeast two-hybrid assay, in light of these challenges.


The two-hybrid assay, which detects protein-protein interactions as reconsti-

tution of a transcriptional activator, provides a general, high-throughput assay
for cloning any protein on the basis of its interaction with another protein.
Introduced only in 1989, the two-hybrid assay has proven so robust that today
roughly half of the known protein-protein interactions are determined in part
using the two-hybrid assay. In this, chapter we look at more recent efforts to
extend this powerful genetic assay to read-out the other important molecules in

Chemical Biology. From Small Molecules to System Biology and Drug Design
Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Gunther Wess
Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
I 4 Controlling Protein-Protein Interactions

Fig. 4.1-1 Chemical Complementation combines the power of

genetic assays and small molecule chemistry to understand small
molecule function and develop new chemistry inside the cell.

the cell, such as nucleic acids and small molecules. We also consider the pos-
sibilities for exploiting the two-hybrid assay for chemical discovery-extending
the power of genetics to chemistry not naturally carried out in the cell.
The two-hybrid assay works by detecting protein-protein interactions as
reconstitution of a transcriptional activator, a natural eukaryotic transcription
factor, and as activation of a reporter gene. One protein is fused to the
DNA-binding domain (DBD) of the transcriptional activator, and the other
protein is fused to the activation domain (AD).If the two proteins bind to one
another, they effectively dimerize and hence reconstitute the transcriptional
activator (Fig. 4.1-2). In practice, this assay is used not just to test a single
protein-protein interaction, but to test all of the proteins expressed in a given
organism or cell line for binding to the protein of interest. A library of AD-
fusion proteins, encoding all ca lo4 different proteins, is transformed en masse
into an appropriate two-hybrid selection strain containing the DBD-protein
fusion of interest. Only cells expressing an AD-protein fusion that binds
to the DBD-protein fusion will then survive under the appropriate reporter
gene selection conditions. The assay is general because the transcription-
based selection works for any protein-protein interaction. Therefore, while
4. I Chemical Complementation: Bringing the Power ofGenetics to Chemistv I 201

Fig. 4.1-2 In the yeast two-hybrid system, activator recruits the transcriptional
dimerization of fusion proteins machinery t o the promoter region of the
X-DNA-binding domain and Y-activation reporter gene, initiating its transcriptional
domain reconstitutes the transcriptional activation.
activator. The reconstituted transcriptional

traditional genetic assays rely on pathway-specific cell survival selections or

phenotypic screens, to which not all pathways or proteins in a pathway are
amenable, the two-hybrid assay can be applied to any given protein-protein
interaction, since the transcription-based read-out is independent of the
particular pathway being studied. The assay is high-throughput because
standard molecular biology techniques allow large libraries (ca 105-107
in yeast) to be tested simultaneously, where only the cells expressing an
interacting protein pair survive.
The other strength of the two-hybrid assay is the ease with which it can
be carried out using modern methods in molecular biology. At the end of
a two-hybrid selection, the interacting proteins can be read-out simply by
extracting the DNA encoding the AD-fusion proteins from the surviving cells
and by sequencing the DNA, As a proof of the power of this approach, the
two-hybrid assay is now essential to any effort to clone proteins along a given
biological pathway. Moreover, the fortuitous development of the two-hybrid
assay concurrent with genome sequence projects, enables the construction of
exact cDNA-ADlibraries based on this data, thus facilitating protein identity to
be readily extracted from a random DNA library. The high-throughput nature
of the two-hybrid assay even allows protein interaction studies to be carried out
on a genome-wide scale. For example, analyzing all ca 6000 proteins expressed
in yeast for binding to one another by testing all GOO0 DNA-binding protein
fusions to their 6000 AD counterparts.
As with the field of genetics as a whole, the two-hybrid assay is biased
toward proteins. As variations of this assay, which can detect DNA, RNA,
and small molecule binding, are now developed, it is exciting to imagine
I the potential for basic science discovery for the roles of these molecules in
4 Controlling Protein-Protein Interactions

the cell. Furthermore, these so-called n-hybrid assays extend these powerful
transcription-based genetic assays to chemistry not naturally carried out in
the cell. This extension should allow these genetic assays to be used not only
for the discovery of biological pathways but also for new chemistry, including
drug discovery and the directed evolution of molecules with new functional


Since the conception of the two-hybrid assay to detect protein-protein

interactions in vivo at the end of the 1980s, key modifications to this assay
have expanded its scope to detect DNA-, RNA-, and small molecule-protein
interactions in so-called n-hybrid assays. More recently, “n-hybrid” assays
have also been used to detect enzyme catalysis, where enzyme activity is
linked to cell survival via transcription of a reporter gene. Here we look at the
initial publications that moved the two-hybrid assay into each of these new
directions. Protein-Protein Interactions

In 1989 Fields and Song introduced the “Yeast Two-Hybrid Assay”
which provides a straightforward method for detecting protein-protein
interactions in uivo [l].Until the development of the two-hybrid methodology,
protein-binding interactions had been detected using traditional biochemical
techniques such as coimmunoprecipitation, affinity chromatography, and
photoaffinity labeling [2]. There are three significant advantages to this in vivo
assay that led almost immediately to its widespread use: first, it is technically
straightforward and can be carried out rapidly; second, the sequence of the
two interacting proteins can be read off directly from the DNA sequence of the
plasmids encoding them; and third, it does not depend on the identity of the
interacting proteins and so is general.
The two-hybrid assay was based on the observation that eukaryotic
transcriptional activators can be dissected into two functionally independent
domains, a DBD and a transcription AD, and that hybrid transcriptional
activators can be generated by mixing and matching these two domains [3].
It appears that the DBD only needs to bring the AD into the proximity of the
transcription start site, suggesting that the linkage between the DNA-binding
and the AD can be manipulated without disrupting activity. Thus, the linkage
in the two-hybrid assay is the noncovalent bond between the two interacting
As outlined in Fig. 4.1-3(a),the yeast two-hybrid system consists of two
protein chimeras, and a reporter gene downstream from the binding site for
4. J Chemical Complementation:Bringing the Power ofGenetics to Chemistry I 203

I >

I >
I DNA binding site I I Reporter gene I I DNA binding site I I Reporter gene I


I DNA bindinq site I I DNA binding site I

Fig. 4.1-3 Different yeast n-hybrid systems given DBD. (c) The three-hybrid system that
that have been developed t o study can detect RNA-protein interactions has
protein-protein, protein-DNA, one more component than the yeast
protein-RNA, and protein-small molecule two-hybrid system: a hybrid RNA molecule.
interactions. (a) In the original version o f One half ofthe hybrid RNA is a known RNA
the yeast two-hybrid system, transcriptional (R) that binds to the MS2 coat protein
activation o f the reporter gene i s (MS2) with high affinity and serves as an
reconstituted by recruitment o f the anchor. The other half i s RNA X, whose
activation domain (AD) to the promoter interaction with protein Y is being tested.
region through direct interaction o f protein (d) Another version o f the yeast three-hybrid
X and Y, since protein X is fused t o a system can be used t o detect small
DNA-binding domain (DBD) and protein Y molecule-protein interactions. Ligand L1
i s fused to the AD. (b) In the one-hybrid that interacts with protein X is covalently
system, the AD is fused directly t o the DBD. linked to ligand L2. Thus, i f L2 interacts with
This system can be used to assay either Y, transcriptional activation of the reporter
DBDs that can bind t o a specific DNA gene will be reconstituted.
sequence or the in vivo binding site for a

the transcriptional activator. If the two proteins of interest (X and Y) interact,

they effectively dimerize the DNA-binding protein chimera (DBD-X)and the
transcription activation protein chimera (AD-Y). Dimerization of the DBD and
the transcription AD helps to recruit the transcription machinery to a promoter
adjacent to the binding site for the transcriptional activator, thereby activating
transcription of the reporter gene.
The assay was demonstrated initially by using two yeast proteins known to
be physically associated in vivo [l].The yeast S N F l protein, a serine-threonine
protein kinase, was fused to the GAL4 DBD, and the SNFl activator protein
SNF4 was fused to the GAL4 transcription AD. A GAL4 binding sequence was
placed upstream of a /?-galactosidasereporter gene (lacz).Plasmids encoding
I the protein fusions and the reporter gene were introduced into the yeast.
4 Contro//ing Protein-Protein Interactions

Positive protein-protein interactions lead to the increase in B-galactosidase

activity inside the cell, which can be tested in a colorimetric assay using
5-bromo-4-chloro-3-indolylB-D-galactosidase (X-gal)that turns the cells blue,
or by direct measurement of enzyme activity using chlorophenol red B-D-
galactopyranoside as a substrate. Control experiments established that neither
the DBD and AD domains on their own nor the individual protein chimeras
induced B-galactosidase synthesis above background levels. B-Galactosidase
synthesis levels were increased 200-fold when the DBD-SNF1 and SNFCAD
fusion proteins were introduced together. By comparison, the direct DBD-AD
fusion protein activated B-galactosidase synthesis levels 4000-fold.
It was quickly realized that the strength of the two-hybrid assay would lie
not in its ability to detect a single protein-protein interaction but rather to
screen an entire genome to detect novel protein-protein interactions [4-91. For
example, Murray and coworkers, as a first step toward testing their hypothesis
that the cyclin-dependent kinase (CDK) Cdc20 is involved in the spindle
assembly checkpoint in budding yeast, used the yeast two-hybrid assay to
determine if any of the proteins known to be involved in the spindle checkpoint
physically interact with Cdc20 [lo]. In this experiment, haploid strains
containing DBD-MAD (mitotic arrest defective) fusions were crossed with
haploid strains containing AD-Cdc2O fusions. Protein-protein interactions in
the resulting diploids lead to transcription activation of the lacZ reporter gene.
As controls, haploid strains containing SNF1-AD and SNF4-DBD fusions
were also mated and tested for B-galactosidase activity. The yeast two-hybrid
system detected three new protein partners for Cdc2O: MAD1, MAD2, and
MAD3. In this experiment, the yeast two-hybrid assay was the key in rapidly
and effectivelyidentifying the new protein-protein interactions. Identification
of these interactions using more traditional biochemical methods, such as
coimmunoprecipitation,would have been cumbersome and time consuming
since those methods require prior isolation of large quantities of all possible
interacting proteins before running the assays. By facilitating the discovery
of cascades of interacting proteins - in this case, the spindle assembly
checkpoint - the yeast two-hybrid assay helps researchers put together entire
biochemical pathways and to begin understanding how these proteins function
together inside a cell. DNA-Protein Interactions

Early on it was appreciated that, just as the yeast two-hybrid assay could be
used to detect protein-protein interactions, transcriptional activators could
be used directly, in a “one-hybrid” assay, to detect DNA-protein interactions
(Fig. 4.1-3(b))[ll,121. DNA-binding proteins that bind to a given target DNA
sequence could be isolated from cDNA libraries encoding all the proteins
expressed in a given organism or specific cell type. Alternatively, the optimal
or naturally occurring recognition sequences for a given regulatory protein
4. I Chemical Complementation: Bringing the Power ofGenetics to Chemistry I 205

could be determined. With such an approach, Wang and Reed isolated a

complementary DNA for the transcriptional activator, Olf-1, believed to be the
critical switch for the coordinated expression of olfactory-specific genes [ 131.
To achieve this, they fused an olfactory cDNA library, consisting of 3.6 million
clones, to the GAL4 transcription AD. The reporter plasmid consisted of three
tandem Olf-1 binding sites upstream of a low activity promoter directing the
transcriptional activation of the H I S 3 gene. The reporter plasmid requires the
AD-cDNA fusion protein to bind to the Olf-1 sites and activate the transcription
of the HIS3 gene. Therefore, only cells expressing the AD-cDNA fusion are
able to grow on medium lacking histidine. RNA-Protein Interactions

Selecting for RNA-protein interactions is less straightforward because RNA-
protein fusions cannot be generated directly in vivo and because routine
biochemical assays that turn RNA-binding events into an amplifiable signal
are not available. These difficulties were circumvented by adding a third
component to the two-hybrid system to generate a “three-hybrid” assay
(Fig. 4.1-3(c)) [14, 151. The third component is a hybrid RNA molecule, in
which one half is a well-studied RNA molecule that binds to a known protein
with high affinity and the other half is the RNA molecule of interest whose
protein-bindingpartner is in question. In total, the three-hybrid system consists
of two protein chimeras, one RNA chimera, and a reporter gene. The hybrid
RNA molecule bridges the DNA-binding and AD-fusion proteins and activates
transcription of a reporter gene.
In a proof of principle experiment, Wickens and coworkers showed that
the RNA three-hybrid system could detect the interactions between two well-
studied protein-RNA pairs: the iron regulatory protein (IRPl) to the iron
response element (IRE) RNA sequence, and the HIV transactivator (TAT)
protein to the HIV transactivation response (TAR) element RNA sequence
[16]. First, they constructed a bifunctional RNA containing a RNA sequence
known to bind the coat protein MS2 and the RNA sequence of either IRE
or TAR. Next, they fused the DNA-binding domain to the coat protein MS2,
and the AD to either the IRPl or TAT proteins. The two protein fusions and
the bifunctional RNA were introduced in a yeast strain containing a reporter
construct that directs activation of both a lacZ reporter gene and a H I S 3
reporter gene upon RNA-protein interaction. These reporter genes allow the
authors to carry the assay as a colorimetric screen using the lacZ reporter gene
and as a selection where only cells containing an interacting RNA-protein
pair survive on medium lacking histidine. Furthermore, using 3-amino-1,2,3-
triazole (3-AT),a competitive growth inhibitor of the enzyme encoded by the
HIS3 gene, Wickens and coworkers were able to select only cells with elevated
expression levels of the H I S 3 gene, reducing the number of false positives in
the HIS3 growth selection.
I 4 Controlling Protein-Protein Interactions Small molecule-Protein Interactions

Just as a dimeric RNA molecule can be introduced to mediate the interaction
between the DNA-binding and ADS, so can a dimeric small molecule [17].
In fact, well before their use in a small molecule three-hybrid assay, dimeric
small molecules were used as “chemical inducers of dimerization” (CIDs)
to artificially oligomerize fusion proteins in vivo [18]. In the yeast three-
hybrid system, the union of two protein fusions and a CID reconstitute
the transcription of a reporter gene (Fig. 4.1-3(d)).In 1996, Licitra and Liu
built what they called a yeast three-hybrid assay [19]. This assay consists of
two fusion proteins and a heterodimeric small molecule CID that brings
these fusion proteins together to activate the transcription of a reporter gene
(Fig. 4.1-3(d)).
Licitra and Liu employed two fusion proteins: the glucocorticoid receptor
(GR)fused to the DBD LexA, and FK 506-binding protein (FKBP12) fused to
the transcription AD B42 [19].A heterodimeric dexamethasone (Dex)-FK506
molecule that binds to GR and FKBP12, respectively, bridges the two fusion
proteins and activates the transcription of a lacZ reporter gene. Further,
using the GR-LexA fusion protein and the Dex-FK506 molecule in their yeast
three-hybrid assay, Licitra and Liu were able to isolate the FKBP isoform
with the highest affinity for FK506 (FKBP12) from a Jurkat cDNA library.
This experiment opened the yeast three-hybrid system as a tool for drug
discovery. Catalysis
In all the previous applications, the n-hybrid assay is used to detect a binding
event, whether it is protein, DNA, RNA, or small molecule binding. Our
laboratory and others have been interested in the idea that this powerful
genetic assay could be brought to bear on a broader variety of questions.
Several different approaches have now been devised for linking enzyme
catalysis to reporter gene transcription using the n-hybridassay. Our laboratory
introduced “Chemical Complementation”, which detects enzyme catalysis of
bond formation or cleavage reactions on the basis of covalent coupling of two
small molecule ligands in vivo (Fig. 4.1-4) [20]. In this assay, the enzyme is
introduced as a fourth component to the small molecule yeast three-hybrid
system, and the linker in the small molecule CID acts as the substrate for the
enzyme. Bond formation is detected as synthesis of the CID and hence the
activation of an essential reporter gene; bond cleavage is detected as cleavage
of the CID and hence the repression of a toxic reporter gene. In theory, this
approach should be readily extended to new chemistry, simply by synthesizing
small molecule heterodimers with different chemical linkers as the enzyme
substrates. Inspired by traditional genetics, our hope is to make a general
complementation assay that would link enzyme catalysis of a broad range of
chemical reactions to cell survival-extending genetic selections to chemistry
beyond that naturally carried out in the cell.
4. I Chemical Comp/ementation: Bringing the Power ofGenetics t o Chemistry I 207


I DNA binding site I I Reporter gene I
Fig. 4.1-4 Chemical Complementation. A either cleavage or formation of the bond
reaction-independent complementation between the two small molecules can be
assay for enzyme catalysis based on the detected as a change in transcription o f the
yeast three-hybrid assay. A heterodimeric reporter gene. The assay can be applied t o
small molecule bridges a DNA-binding new chemical reactions simply by
domain-receptor fusion protein and an synthesizing small molecules with different
activation domain-receptor fusion protein, substrates as linkers and adding an enzyme
activating transcription o f a downstream as a fourth component t o the system.
reporter gene in vivo. Enzyme catalysis o f

In our initial report, we chose cephalosporin hydrolysis by the Enter-

obacter cloacae P99 p-lactamase (P99) as a well-studied enzyme catalyzed
cleavage reaction around which to develop Chemical Complementation [20].
Cephalosporins are B-lactam antibiotics, and p-lactamases are the bacterial
resistance enzymes that hydrolyze and inactivate these antibiotics. The P99
B-lactamase is well-characterized biochemically and structurally, and the syn-
thesis of cephalosporins is well established. First, we designed a small molecule
CID cephalosporin substrate, incorporating the CID ligands at the C 3’ and
C7 positions of the cephem core. Using a lacZ reporter gene, we showed
that Chemical Complementation could be used to detect B-lactamase activity
using this dexamethasone-methotrexate (Dex-Mtx)heterodimer with a cephem
linker (Dex-Cephem-Mtx). In the absence of enzyme, the Dex-Cephem-Mtx
CID dimerizes the appropriate DBD- and AD-fusion protein activating tran-
scription of a lacZ reporter gene. Expression of the P99 p-lactamase then
presumably leads to cleavage of the Dex-Cephem-Mtx CID, disrupting tran-
scription activation. We also showed that the system could distinguish the
wild-type (wt) enzyme from the inactive P99:SG4A variant, in which the critical
4 Controlling Protein-Protein fnteractions
I active site serine nucleophile has been mutated to an alanine, via a lacZ
screen. These experiments established the feasibility of detecting enzyme
catalysis using the yeast n-hybrid assay.
Benkovic and coworkers took a related approach in an assay they called
Quest (Querying for Enzymes using the Three-hybrid system), which detects
catalysis by coupling substrate turnover to transcription of a reporter gene
[21]. Here, the CID that dimerizes the transcriptional activator is a homodimer
of the substrate. Enzyme catalysis of free substrate to product is detected as
displacement of homodimeric CID substrate from the transcriptional activator
fusion proteins. Although this approach has the advantage ofusing unmodified
substrate, a new CID-protein pair has to be developed for each new reaction.
In a more biological approach, Peterson and coworkers have developed a
two-hybrid-based system to detect protein tyrosine kinase (PTK) activity [22].
This assay relies on the PTK-dependent phosphorylation of a tyrosine residue
present in a peptide that has been fused to the DBD. The phosphorylated
tyrosine is then bound by the phosphotyrosine-binding protein fused to the
AD, leading to transcriptional activation of the reporter gene. While limited
to peptide substrates, this approach has the advantage that it does not require
chemical synthesis, making it more accessible to biologists.

General Considerations

Whether being applied as in the original two-hybrid assay to detect

protein-protein interactions or in the related n-hybrid assays to detect
protein-DNA, RNA, or small molecule interactions, the basic components
of the n-hybrid assay remain the same. Thus, while we focus in this section on
the small molecule three-hybrid assay because it is in this that our laboratory
specializes, this section could also be used as a technical introduction to any of
the other n-hybrid systems. The real strength of the n-hybrid assays lies in how
straightfonvard they are to implement in the laboratory with basic knowledge
of Escherichia coli and Saccharomyces cerevisiae molecular biology. Moreover, the
commercial availability of the components of the two-hybrid system permits
any laboratory to rapidly implement the system. Finally, laboratories without
prior experience working with S. cerevisiae should not be deterred from carrying
out n-hybrid assays, as molecular biology techniques for this organism are
similar to those for E. coli. The Chemical Inducer o f Dimerization (CID)

The effectiveness ofany three-hybrid system depends critically on the CID used
to dimerize the transcriptional activator in vivo [23,24]. The subject of CIDs has
been considered fully in the previous chapter by Clackson, so here we focus
on the issues we have found particularly important for the use of CIDs in the
4. I Chemical Complementation: Bringing the Power ofGenetics to Chemistry I 209

three-hybrid assay. Our presentation of these considerations is based largely on

our own work with the yeast three-hybrid system and the CID ligand/receptor
pairs Dex/GR, FKS06/FK506 binding protein 12 (FK506/FKBP12), a syn-
thetic analog of FK50G SLF/FK506 binding protein 1 2 (SLF/FKBP12),
methotrexate/dihydrofolate reductase (Mtx/DHFR), 06-benzylguanine/06-
alkylated guanine-DNA alkyltransferase (BG/AGT),estrone/estrogen receptor
(ES/ER), and biotin/streptavidin (biotin/SA) (Fig. 4.1-5) [19, 23-28].





Estrone Biotin

Fig. 4.1-5 Small molecules used t o create chemical inducers of dimerization (CIDs) for
the yeast three-hybrid system.
I 4 Controlling Protein-Protein Interactions

First and foremost, a successful three-hybrid system seems to require a

high-affinity (low nanomolar KD) CID pair [29]. Using the most sensitive
reporter genes commercially available for the Brent LexA yeast three-hybrid
system, we found that FK506-Dex, Mtx-Dex, Mtx-Mtx, and Mtx-SLF could all
activate transcription, but Dex-Dex and Dex-SLF could not [25]. Second, the
directionality of the system is important for a strong transcription read-out. We
reported that the Dex-Mtx yeast three-hybrid system showed higher levels of
transcription activation when DHFR was fused to the DBD than when fused to
the AD [30]. Third, as with any CID application, the ligandlreceptor pair must
be considered in the context of the host cell line. For example, the Dex/GR
interaction is dependent on associated heat shock proteins. Thus, the KD of
this interaction is significantly higher in S. cerevisiae, in which there are only
homologous heat shock proteins, than in the native mammalian background.
Also, this CID pair cannot be used in E. coli, in which there are no such
homologous heat shock proteins. Finally, there are also more subtle effects.
For example, for reasons we do not understand, only the E. coli DHFR, not the
murine homolog, is functional in the Dex-Mtx yeast three-hybrid system [30]. The Genetic Assay

For a laboratory new to the three-hybrid assay, we recommend beginning with
the yeast two-hybrid system, which is based on reconstitution of a eukaryotic
transcriptional activator protein. Not only is this assay straightforward to
practice but also all the necessary strains and plasmids are commercially
available. As discussed below, however, there are potential advantages to
working in E. coli or using a nontranscription-based assay. Several E. coli-
based transcription assays and general protein complementation assays (PCA)
have now been developed as two-hybrid assays. Notably, while the E. coli
transcription assays have proven amenable to the introduction of small
molecule CIDs, the PCAs have not. The Yeast n-Hybrid System

There are two key versions of the yeast two-hybrid system. The GAL4 system
originally introduced by Fields and Song uses the DBD and the AD of the
yeast GAL4 gene [ l ] . The LexA system introduced by Brent and coworkers
uses the E. coli DBD LexA and the E. coli B42 AD [31]. Over time, these
two systems have benefited from a number of improvements. Convenient
DBD and AD vectors were developed to carry diverse bacterial drug-resistance
markers, yeast origins of replication, and yeast auxotrophic markers. These
technical improvements facilitate the testing of large pools of protein variants
(ca lo6) using growth selections. In addition to the basic activator system,
reverse and split-hybrid systems were developed to detect the disruption of
protein-protein interactions, and a transcriptional repressor-based system has
been reported [32, 331. Today components for these systems are commercially
4. I Chemical Complementation: Bringing the Power ofGenetics t o Chemistry I 211

available, including Stratagene and Clontech, which market the Gal4 system,
Origene, for the LexA system, and Invitrogen, which offers versions of both
systems. All of the basic features of the two-hybrid system have been covered
already in several excellent reviews and the chapters on methods.
In our laboratory we have used the Brent two-hybrid system to build our
Dex-Mtx yeast three-hybrid system. We favor the Brent system, which uses
LexA, an E. coli transcription factor, and B42, an artificial activator isolated
from E. coli genomic DNA. Both LexA and B42 are orthogonal to standard
yeast genetic tools and nontoxic to the yeast cell, yet the artificial LexA-B42
transcriptional activator is on par with the strongest transcriptional activators
endogenous to S. cerevisiae [31].Moreover, the LexA system permits the use of
the tightly regulated GAL1 promoter to drive the expression of the LexA DBD
and B42 AD-protein fusions by varying the ratio of galactose and glucose in the
growth medium. As reported by Lin et al., we use pMW103, a multicopy 2~
plasmid with a HIS3 maker, to encode the LexA DBD fusions and pMW102,
a multicopy 2,u plasmid with a TRPl marker, to encode the B42 AD fusions.
Rather than the original EGY48 LEU2 selection strain, we chose the FY251
strain (MATa trplA63 his3A200 ura3-52 leuZAlGal+), which provides an
additional selective marker for greater flexibility. The LEU2 or URA3 markers
can then be used either for the transcription activation growth selection or
introduction of additional plasmids. In this initial publication, we then used the
lacZ reporter plasmid pMW112, which encodes the lacZ gene under control of
eight tandem LexA operators. Thus, small molecule CID-induced transcription
activation could be detected using standard lacZ transcription assays either on
plates or in liquid culture [25]. Further optimization of the yeast three-hybrid
system in our lab led us to conclude that integration of either the AD or DBD
into the yeast chromosome stabilizes the transcription read-out of the reporter
gene without loosing transcriptional strength, effectively reducing the number
of false positives in the detection of novel ligand-receptor interactions [34]. E. coli Transcription Activation Assays

Widespread use of the yeast two-hybrid system led several groups to develop
alternate transcription-based assays. While the yeast two-hybrid assay is quite
powerful, a bacterial equivalent would increase by several orders of magnitude
the number of proteins that could be tested, as the transformation efficiency
and doubling rate of E. coli are significantly greater than those of S. cerevisiae.
There may also be applications where it is advantageous to test a eukaryotic
protein in a prokaryotic environment, in which many pathways are not
conserved. The yeast two-hybrid assay cannot, however, be transferred directly
to bacteria since the components of the transcription machinery and the
mechanism of transcriptional activation differ significantly between bacteria
and yeast.
The first bacterial repressor assay was developed in 1990 by Sauer and
coworkers, who adapted a bacterial h transcriptional repressor system to
I read-out the GCN4-leucine zipper fusion [ 3 5 ] .The transcriptional repressor
4 Controlling Protein-Protein fnteractions

h d controls the lytic/lysogenic pathway in bacteriophage h. As a dimer,

hcI is bound to the h operator and prevents the expression of genes
involved in the lytic pathway, allowing integration of the h DNA into the
bacterial chromosome. Taking advantage of the hcI dimerization requirement,
Sauer and coworkers fused the DNA-binding domain of two hcI to a
GCN4 leucine zipper dimerization motive to restore a functional hybrid
Seven years later, Hochschild and coworkers designed a bacterial two-
hybrid activation system based on the transcription mechanism of E. coli
RNA polymerase (RNAP) [ 3 6 ] .This assay is based on their observation that
binding of the C-terminus of the a subunit of the RNAP (a-CTD) to an
upstream element leads to transcription activation of a downstream gene. To
create a bacterial two-hybrid system, the authors replaced the a-CTD with
the C-terminus of the transcriptional repressor hc1 (hcI-CTD), generating a
ahcI chimera. Binding of the transcriptional repressor hcI to the h operon,
leads to recruitment of RNAP via the ahcI chimera, which in turn directs
transcription activation of a reporter gene downstream of the h operon. By
simply replacing the ahcI chimera with arbitrary protein-protein interactions,
they created a bacterial two-hybrid activation system. This technology was
successfully applied to detect two interacting yeast proteins, Gal4 and Galll,
fused to hcI and a-NTD (N-terminus of the alpha subunit of the RNAP)
respectively (Fig. 4.1-6).
Our development of a successful yeast three-hybrid system and the
advantages promised by an analogous system in bacteria, led us to construct
a bacterial three-hybrid system from the RNAP two-hybrid system developed
by Hochschild and coworkers [ 3 7 ] . We chose to adapt this assay because it
is a transcriptional activation system, and reconstitution of transcriptional
activation should be largely conformation independent. The key to converting
this two-hybrid assay into a three-hybrid system was the design of a dimeric
ligand that could bridge hcI and a-NTD through the receptors of the ligand.
For the bridging small molecule, we chose to prepare a heterodimer of Mtx and

Fig. 4.1-6 The bacterial two-hybrid system and Y. Binding ofthe Acl repressor t o the A
developed by Hochschild and coworkers. operon followed by dirnerization o f X and Y
The Acl repressor and the a-subunit o f recruits RNAP leading t o transcription
RNAP are fused t o two arbitrary proteins, X activation o f a downstream reporter gene.
4. I Chemical Complementation: Bringing the Power ofGenetics t o Chemistry I 213

a synthetic analogue of FK506 (SLF).We call this heterodimer Mtx-SLF. We

did not pursue building a bacterial three-hybrid system based on the Mtx-Dex
heterodimer previously used in our yeast three-hybrid system because the
Dex/GR interactions require heat shock proteins that are absent in E. coli. The
heterodimer Mtx-SLF gives a strong transcription read-out in the E. coli RNAP
three-hybrid system, providing a robust platform €or high-throughput assays
based on protein-small molecule interactions. Protein Complementation Assay

All of the above assays are based on transcription of a reporter gene. A
different method for studying protein-protein interactions is the use of a
PCA. Here an enzyme with a phenotype detectable via either a screen or
a selection is divided into two nonfunctional fragments that are fused to
proteins to be tested for dimerization. If the tested proteins dimerize, the two
enzyme fragments are brought into close proximity leading to reconstitution
of enzyme activity (Fig. 4.1-7) [38, 391. Since PCAs are independent ofthe cell’s
transcription machinery, they can be used to detect protein interactions
in any cell type or cell compartment in vivo or in vitro. Furthermore,
PCAs can potentially quantify protein-protein interactions since there is a
simple relationship between protein dimerization and reconstituted enzyme
activity. PCAs have been developed using a variety of proteins including
B-galactosidase, B-lactamase, DHFR, GFP (green fluorescent protein), and
YFP (yellowfluorescent protein) 140-421.
For example, in a proof of principle paper, Michnick and coworkers showed
that mDHFR can be split into two fragments that show no detectable

Fig. 4.1-7 Protein complementation reconstituted enzyme activity on their own

assays. A protein that carries out a (blue and green), but can effectively
detectable function is separated into two reconstitute enzyme activity when fused t o
fragments that show no detectable two interacting proteins, X and Y.
I reconstituted enzyme activity on their own but can effectively reconstitute
4 Controlling Protein-Protein Interactions

enzyme activity when fused to two interacting proteins. Bacteria expressing

a functionally reassembled mDHFR can easily be selected since mDHFR
activity is essential for growth of E. coli in the presence of trimethoprim,
which selectively inhibits bacterial DH FR but not its eukaryotic counterpart
mDHFR. Further, the mDHFR PCA works as a selection system in eukaryotic
cells deficient in endogenous DHFR activity [43]. In a remarkable application
of this system, Michnick and coworkers were able to detect a protein-protein
interaction, locate the interaction to a specific cell compartment, and place
the interaction in a signal transduction pathway by doing a single assay based
on the DHFR PCA in mammalian cells deficient of DHFR [44].Specifically,
they examined protein interactions in the well-studied signal transduction
pathway of receptor tyrosine kinase, which mediates control of initiation of
translation in eukaryotes. From 35 interactions tested, the DHFR PCA selection
identified 14 interacting partners that were localized to specific intracellular
compartments using fluorescein-Mtx,a fluorophore in which the Mtx portion
binds to the reconstituted DHFR with nanomolar affinity. The position of
the protein interaction in the signal transduction pathway was determined
by using three small molecule inhibitors known to act at key points of the
In view ofthe advantages PCAs would bring to the detection ofprotein-small
molecule interactions, our laboratory has made some efforts to develop a small
molecule PCA three-hybrid assay, though without success [45]. Specifically,we
tested both the Mtx-SLF adenylate cyclase PCA and the Mtx-SLF b-lactamase
PCA in E. coli (E. Althoff, V. Cornish, unpublished results). In addition,
we tested a Dex-Mtx GFP PCA also in E. coli in collaboration with Regan
and coworkers (E. Althoff, V. Cornish, T. Magliery, L. Regan, unpublished
results). From both, a simple thermodynamic consideration and these results,
we hypothesize that without the high degree of cooperativity found in
the transcription-based assays, the PCAs cannot detect a three-component
interaction. Problem Choice

The two-hybrid assay was originally used simply for cloning proteins based on
their interaction with other proteins in a given biological pathway. However,
the more recent development of one- and three-hybrid assays opens the door
to studying DNA, RNA, and small molecule interactions, and even catalysis.
Though developed as a genetic assay for cloning, there is no reason that the
n-hybrid assays cannot be used for a broad range of applications, including
drug discovery, directed evolution, and enzymology.
It is interesting to consider how well suited the two-hybrid assay is for
its original conception - the discovery of new proteins on the basis of their
binding to other known proteins - particularly as this assay begins to be carried
out on a genome-wide scale. An important paper that bears on this question,
4.1 Chemical Complementation: Bringing the Power ofGenetics to Chemistry 1 215

in our opinion, comes from Golemis and Brent, in which they estimated that
the KD cutoff for the yeast two-hybrid assay is ca 1 p M [4G].Assuming that
the proteins are being expressed at ca 1 p M concentrations, the two-hybrid
assay can only detect relatively high-affinity interactions (ca K D = 1 pM).
Thus, while the two-hybrid assay is quite successful at identifying new
interactions, it is probably not appropriate to assume that a high-throughput
two-hybrid assay gives a snapshot of all interactions. In fairness, however,
it should be pointed out that traditional affinity chromatography approaches
are even further impaired because they rely on the natural abundance of
any given protein in the cell. Extending this analysis to drug discovery
using the small molecule three-hybrid assay, it is our opinion that the three-
hybrid assay was long underutilized because the original systems had low
sensitivity owing to the CID anchor. Recently, we have shown that our
Mtx three-hybrid system has a KD cutoff of ca 100nM [29].Consistent
with this idea, GPC Biotech reported last year the use of the Mtx three-
hybrid system for identification of protein targets of CDK inhibitors [47].
Interestingly, Hochschild and coworkers have shown that they can build
additional sensitivity into their bacterial two-hybrid assay by adding cooperative
interactions [48].
The n-hybrid assay can also be used for directed evolution. For example,
Pabo and coworkers have adapted a bacterial one-hybrid assay to evolve zinc-
finger variants with defined DNA-binding specificities [49].Starting with a
three zinc-finger protein that has nanomolar affinity for its DNA-binding
site, the authors replaced the binding site for the third zinc finger with a
new DNA sequence and then randomized the third finger to evolve a zinc-
finger variant with increased affinity for the target sequence. Impressively,
the evolved zinc finger showed DNA affinity within 10-fold of the wt protein,
KD = 0.01 nM, and a 10- to 100-fold preference for the modified over the
wt DNA sequence. Given the low K D cutoff and the fact that the n-hybrid
assay is governed by equilibrium binding, there are two likely limitations to
using this assay for directed evolution. First, the assay cannot effectively detect
initial, weak binders. Second, the assay is limited in its ability to distinguish
evolved variants on the basis of improvements in KD since energy differences
of only a few kilocalories per mole determine whether a molecule is bound
at equilibrium. In theory, however, these limitations could be overcome by
varying the concentration of the n-hybrid components or, again, by building in
a series of tunable, cooperative interactions. Pabo and coworkers, then, choose
their problem well. They began with a zinc-finger protein with two out of three
zinc fingers intact. This initial binding affinity enabled them to select good
binders in a single round of selection, rather than trying to improve binding
affinity through multiple rounds of selection. A similar analysis suggests that
the n-hybrid assays may be ideally suited to catalysis applications since large
differences in catalytic activity are needed to significantly affect the half-life of
product formation.
I 4 Controlling Protein-Protein lnteractions


Although introduced only in 1989, the yeast two-hybrid assay has emerged as
an integral tool for biology research. Two-hybrid screens now appear regularly
in the biology literature. Genome-widetwo-hybrid screens are even the focus of
major research publications. Somewhat surprisingly then, there have been few
applications of the related n-hybrid technologies to detect protein interactions
with DNA, RNA, and small molecules, or applications beyond cloning. Here
we look at more recent applications of n-hybrid assays with an eye for asking
whether this discrepancy results from the relative power of these different
n-hybrid assays or rather the biases of current research. Protein-Protein lnteractions

Traditional genetic assays and more recently the yeast two-hybrid assay have
been primarily used to identify natural protein-protein interactions. Two-
hybrid screens are now fully integrated into the biologist’s toolbox and
appear routinely in the published literature. Almost half of the published
protein-protein interactions to date have been detected, at least in part, using
the yeast two-hybrid assay [SO]. Beyond these simple cloning applications,
the two-hybrid assay would seem perfectly suited for genomics. For example,
automation techniques were used to identify all possible protein-protein
interactions in S. cerevisiae [51]. Every open-reading frame encoding a protein,
ca GOOO in S. cerevisiae, was fused both to a DNA-binding domain and an AD,
and the two fusion libraries were screened against one another. The major
challenge in this project was how to transform all combinations of the GO00
DBD and GOOO AD fusions into yeast and then how to assay so many cells. Since
a library of lo7 is at the limit of the transformation efficiency of yeast, it is in
theory achievable. Uetz and coworkers compared two approaches. In the first
approach, they explicitly mated haploid mating type (MATa) cells containing
192 DBD fusions with haploid MATa cells containing the GOOO AD fusions
in a spatially addressable format, such as microtiter plate, and assayed each
well using a HIS3 growth selection. In the second one, MATa cells containing
the GOOO DBD fusions were mated with MATa cells containing the GOOO AD
fusions, and only diploids that survived in a LEU2 growth selection were
arrayed and analyzed individually. Interestingly, there were significantly more
“hits” in the first spatially addressable format, underscoring the importance of
parameterizing new methods for high-throughput screening and the problem
of distinguishing false positives and negatives in genomics. This example
highlights how well suited the n-hybrid assays are for extracting some of the
information provided by recent genome sequencing efforts.
While the two-hybrid method has been extensively used to detect natural
protein-protein interactions, it should also be well suited for protein evolution.
Brent and coworkers demonstrated that the two-hybrid assay can be used to
4. J Chemical Complementation: Bringing the Power ofGenetics to Chemistry I 217

Table4.1-1 The sequences and binding affinities of 14 different

aptamers for binding to Cdk2 isolated in a yeast two-hybrid system

Aptamer KO (n M) Amino acid sequence


Pep2 *
Peplo *

a ND - not determined

identify peptide aptamers that inhibit Cdk2 from a library of random peptide
sequences (Table 4.1-1) [52]. The 20-residue peptide library was displayed in
the active site loop of E. coli thioredoxin (TrxA).The TrxA loop library was
fused to the AD, and Cdk2 was fused to the DBD. In a single round of assay,
6 x lo6 TrxA-AD transformants, a very small percentage of the 20mers
possible, were tested for binding to LexA-Cdk2. From this assay, they isolated
66 colonies that activated transcription of both a LEU2 and a lacZ reporter
gene. Remarkably, these colonies converged on 14 different peptide sequences
that bound Cdk2 with high affinity. Using surface plasmon resonance, the
peptide aptamers were shown to bind Cdk2 with KDs of 30-120 nM. In kinase
inhibition assays, the peptide aptamers had ICsos for the CdkZ/cyclin E kinase
complex of 1- 100 nM. What is particularly impressive about this experiment is
that nanomolar affinity ligands are being isolated in a single round of selection
from a library only on the order of 106-108. Similar results have been obtained
using peptide aptamers in a traditional genetic selection [53].
Given the success of this and related “aptamer” selections, it is somewhat
surprising that these “aptamer” scaffolds are not more widely used.
There are several potential advantages to directed evolution over traditional
monoclonal antibody technology for generating selective binding proteins.
Optimistically, six months are required from the start of immunization,
through immortalization, and finally screening to generate a monoclonal
antibody. On the other hand, if several peptide aptamer libraries were
maintained for routine use, the libraries could be screened against a new target,
false positives could be sorted out, and biochemical assays could validate a
target in less than a month and at considerably less expense. Moreover, protein
I scaffolds other than antibodies may prove more robust for use as reagents and
4 Controlling Protein-Protein lnteractions

therapeutic applications. Perhaps because monoclonal antibody technology

has become so robust over the years, the momentum does not seem to be
there to seriously explore replacing this technology with directed evolution. It
is also interesting to compare these “aptamer” scaffolds to chemical genetic
approaches for generating inhibitors for a broad array of biological targets. DNA-Protein Interactions

Just as the yeast two-hybrid assay can be used to detect protein-protein inter-
actions, transcriptional activators can be used directly to detect protein-DNA
interactions. In truth, this type of experiment was done before the one-hybrid
assay was conceptualized as such. For example, as early as 1983 a His6 +
Pro Mnt variant was generated that preferentially binds a mutant Mnt oper-
ator using a transcription-based selection [54]. A plasmid encoding Mnt was
mutagenized both by irradiation with UV light and by passage through a
mutator strain. The mutant plasmids were then introduced into E. coli and
selected against binding to the wt operator and for binding to the mutant
operator. Because there are a variety of convenient reporter genes, the E. coli
was engineered to link DNA recognition to cell survival in both the negative
(selection against binding to the wt operator) and the positive (selection for
binding to the mutant operator) directions. Binding to the wt Mnt operator was
selected against by placing a tet resistance (tetR)gene under negative control
of the wt Mnt operator. If a Mnt mutant bound the wt operator, it would
block synthesis of the tetR gene, and the E. coli cells would die in the presence
of tetracycline. Then Mnt variants with altered DNA-binding specificity were
selected for on the basis of immunity to infection by a P22 phage containing
a mutant Mnt operator. The mutant Mnt operator controlled synthesis of the
proteins responsible for lysing the bacterial host. If a Mnt variant could bind
to this mutant operator, it would turn off the lytic machinery, and the bacteria
would survive phage infection. Four independent colonies were isolated from
the two selections. Again, only a single round of selection was required for each
step. All four colonies encoded the same His6 + Pro mutation, two by a CAC
+ CCC and two by a CAC + CCT mutation. Not only did these mutants bind
to the mutant operator but they also did not bind efficiently to the wt operator.
More recently, Pabo and coworkers adapted a bacterial two-hybrid assay into
a bacterial one-hybrid system to evolve zinc-finger variants with defined DNA-
binding specificities [49]. In this assay, three tandem zinc fingers function as
the DBD of this one-hybrid system and are fused to Gall1 protein, known
to dimerize with Ga14, which is fused to the RNA polymerase. Binding of
the three tandem zinc fingers to a specific DNA sequence upstream of the
reporter gene, mobilizes the RNAP to the promoter region of the reporter gene
and initiates transcription thereof (Fig. 4.1-8).This assay allows testing f 1 0 8
protein variants per round of selection. However, if all three zinc fingers were to
be randomized simultaneously it would create 8 x protein variants (using
4. I Chemical Complementation: Brhging the Power ofGenetics to Chemistry I 219

1 round of -
s e T I d g I

2F3 F

DNAbindiny 18fe Reporter ene

Fig. 4.1-8 Development ofzinc fingers the cy-subunit o f RNAP. I f ZF3 bound t o the
specific for a specific DNA sequence using a first site with high affinity, the RNAP
one-hybrid assay adapted from a bacterial complex would be recruited, activating
two-hybrid system. Zinc fingers (ZF) 1, 2, transcription o f a HIS3 reporter gene.
and 3 from the Zif268 protein were fused to Significantly, in just one round o f assay,
the Call 1 protein. The Gal4 protein, which several proteins were identified that bound
binds Gall 1 with high affinity, was fused to specifically to the target DNA sequence.

24 codons at six amino acids per three zinc finger = (246)3),which cannot
be covered by this high-throughput method. Thus, the authors are limited to
randomizing one finger at a time, while keeping the other two unchanged. We
believe that conserving the high affinity of two zinc fingers for the DNA may be
important for the success of Pabo and coworkers’ directed evolution, because
starting a directed evolution with a high-affinity protein for DNA ensures the
evolution of proteins within the dynamic range of the n-hybrid system. For this
zinc-finger evolution, they created a library of ca 10’ variants, and identified
a total of nine sequences that bound specifically to three target DNAs with a
preference of 10-to100-fold for the modified over the wt DNA.
Comparing their results for the zinc-finger evolution using the bacterial
hybrid system with earlier results obtained in a similar zinc-finger evolution
study using phage display, Pabo and coworkers conclude that the affinity and
specificity of the selected zinc fingers is superior to those obtained in earlier
phage display studies. Moreover, the bacterial hybrid system is a more rapid
alternative to phage display because it permits isolation of functional fingers
in a single selection step instead of using multiple rounds of enrichments.
Speaking to the power of this approach, Sangamo uses a modified one-hybrid
assay for its selection of artificial DNA-binding proteins for commercial appli-
cations [55, 561. The success found here raises the question of other binding
interactions. One could speculate that the success here depends on starting
with two known zinc fingers with high affinity for their DNA target, except that
the protein “aptamer” scaffold selections described in the previous section
have begun with scaffolds with no measurable affinity for their protein target. RNA-Protein Interactions

Before the development of the RNA three-hybrid system, identification of
protein-RNA interactions was limited to in vitro methods such as pull-down
assays using radiolabeled RNA. The introduction of the RNA three-hybrid
system has allowed not only the detection of well-studied protein-RNA
I pairs, but also the identification of novel protein-RNA
4 Controlling Protein-Protein Interactions

interactions. An
impressive application ofthis system is the cloning of a regulatory protein from
Caenorhabditis elegans that binds to the 3’ untranslated region of the FEM-3
(fern-33’UTR)and mediates the sperm/oocyte switch in hermaphrodites [57].
In this assay, a bifunctional RNA plasmid possessing fern-33’UTRand the RNA
ligand for the MS2 coat protein was introduced into a yeast strain expressing
a DBD-MS2 upstream of the HIS3 and lac2 reporter genes. Into this strain,
a complementary DNA-AD library was introduced. Cells containing a positive
protein-RNA interaction were selected first for HIS3 and lacZ activation
followed by screening for the presence of the bifunctional RNA plasmid. The
RNA plasmid from successful candidates was lost by reverse selection and
the cells were tested again for lacZ activation to reduce the number of false
positives. Cells that failed to activate lacZ after plasmid loss were tested for
fern-33’UTR binding specificity by reintroduction of the bifunctional RNA
plasmids. The protein encoded in the only cDNA-AD that satisfied all selection
and screening criteria was found to have 93% homology at the nucleotide level
with two genes encoded in the C. elegans genome. Further testings confirmed
these genes to be regulators of the sperm/oocyte switch in hermaphrodite
C. elegans. The specificity with which the RNA three-hybrid assay selected
just one protein from thousands for the selected protein-RNA interaction
illustrates the power of this assay for finding novel protein-RNA interactions
[lG].The recent discovery, for example, of RNAi highlights the need not to forget
about molecules other than proteins when carrying genetic assays [58, 591. Small Molecule-Protein Interactions

While several small molecule three-hybrid systems have now been reported, it
was only in 2004 that such a system was used successfully for drug discovery
research. Specifically, Becker and coworkers reported that the Mtx yeast three-
hybrid system developed in our laboratory could be used to clone novel protein
targets of CDK inhibitors (Table 4.1-2) [47].The CIDs used in this study took
advantage of the low picomolar affinity of Mtx for DHFR [25]. Three known
CDK inhibitors, roscovitine, purvalanol B, and indenopyrazole, were linked to
Mtx and introduced into a yeast strain expressing a DBD-DHFR protein fusion
upstream of the HIS3 reporter gene and a library of kinase cDNAs linked to
a transcription AD. With this system they isolated, besides the known CDK
targets, 29 new kinase targets, 22 of which were either confirmed by in vitro
binding or enzyme inhibition assays. We speculate that the success here was
from the use of the high-affinity Mtx/DHFR anchor, which, as we recently
showed, gives a KD cutoff of ca 100 nM in the yeast three-hybrid assay. Catalysis
The widespread utility and robust transcription read-out of the n-hybrid system
motivated several laboratories to develop general methods to detect enzyme
4. I Chemical Complementation: Bringing the Power ofGenetics to Chemistry I 221

Table 4.1-2 Summary of biochemical analysis o f purvalanol

B-Protein interactions. Binding o f proteins t o immobilized
purvalanol B but not t o CDK-inactive-N6-methylated purvalanol B
was evaluated by immunoblotting or liquid chromatography-mass
spectrometry (for endogenous Jurkat proteins). Enzyme assays
were performed with purified enzymes and percentage inhibition
o f kinase activity observed with 1 pM purvalanol B

catalysis in vivo around the small molecule three-hybrid system. Several

proofs of principle papers have been published in the last few years, and
now the key test of these systems is whether they can be readily applied to
new chemistry. Toward that end, our laboratory recently demonstrated that
Chemical Complementation could be used to detect glycosidic bond formation
using a glycosynthase [GO].
We chose glycosidic bond formation because despite the fundamental role
of carbohydrates in biological processes and their potential use as therapeutics,
carbohydrates still remain difficult to synthesize. Specifically, this system was
developed using the E197A mutant of Cel7B from Humicola insolens, which
I had previously been shown to be an efficient“glycosynthase” using an a-fluoro
4 Controlling Protein-Protein Interactions

donor substrate. Here, enzymatic activity is detected as formation of a bond

between a Mtx-disaccharide-fluoridedonor (Mtx-Lac-F)and a dexamethasone-
disaccharide acceptor (Dex-Cel), which dimerize DBD-eDHFR and AD-GR
activating transcription of a LEU2 reporter gene that permits survival under
appropriate selective conditions. The growth advantage conferred by the
glycosynthase activity was used to select the Ce17B:E197A glycosynthase from
a pool of inactive variants (Cel7B).A mock library containing 100: 1 inactive
variants to glycosynthase underwent 400-fold enrichment in glycosynthase
after a single round of selection. Encouraged by this result, we carry out the
directed evolution of the glycosidase Cel7B to improve its glycosynthase activity
using a Glu197 saturation library. From a library of lo5 mutants, Ce17BE197S
was selected, which showed a fivefold improvement glycosynthase activity over
the known Ce17B:E197A glycosynthase (Table 4.1-3).
As intended, no further modifications to Chemical Complementation were
needed to extend this assay to detect glycosynthase activity. All that was
required to detect glycosynthase activity was to add the Dex and Mtx saccharide
substrates. This result shows the generality of Chemical Complementation,
and the ease with which it can be applied to new chemical reactions. Moreover,
it shows that Chemical Complementation can detect not only bond cleavage
but also bond formation reactions. Although, the size of the Glu197 saturation
library selected here was quite small, with only 32 members at the DNA level,
the transformation efficiency of S. cerevisiae, however, allows much larger
libraries, in the order of lo5-10’.

Future Development

The yeast two-hybrid assay no doubt will continue to be a mainstay technique

for the discovery of new protein-protein interactions. As biological pathways

Table 4.1-3 Clycosynthase activities and protein purification

yields for Cel7B variants

E197A E197S N196D/E197A

Specific activity (mol [F])/(min-’ mol [&I) 8 f2 40 f 5 7&1

Protein purification yield [nmol IF1] 6.1 4.6 7.3
Glycosynthase activity for tetrasaccharide synthesis from a-lactosyl fluoride and
p-nitrophenyl p-cellobioside (PNPC) was measured for the Humicola insolens Cel7B
variants in sodium phosphate buffer, pH 7.0, at room temperature. Specific activities
were determined by measuring the fluoride ion release rate by a fluoride ion selective
electrode. The protein purification yields are the yield of purified protein as determined
by western analysis from total cell culture.
References I 2 2 3

are being studied increasingly at the systems level, the two-hybrid assay has
the potential to be quite useful for analyzing total protein dynamics in living
cells. As seen in the PCA work by Michnick and coworkers, it is here that
technical improvements will prove important for the two-hybrid assay.
But it is the n-hybrid assays that have the potential to extend the power
of genetics to molecules other than proteins, such as nucleic acids and
small molecules. Despite this enormous potential, use of these other n-hybrid
assays pales in comparison to that of the two-hybrid assay. As we argue in
this chapter, a consideration of the published literature suggests that this
discrepancy is not the result of some inherent technical limitation to the
n-hybrid assays, but rather likely reflects the bias of current practice. Thus,
it is here that we believe there is most potential for the future development
of the n-hybrid assay and indeed genetics as a whole. Technically, the n-
hybrid assays probably still can be further developed for different classes
of molecules or posttranslational modifications. But already in their present
form these assays seem to have tremendous potential for biological discovery,
uncovering new functions for the many classes of molecules that make up
the cell.
These advances also expand our ability to engineer the cell to harness
its synthetic and functional capabilities for chemical discovery. Just as
protein engineering impacted both basic research and the biotechnology
and pharmaceutical industries in the last 25 years, so should cell engineering
in this century. Such systems engineering likely will require a much more
quantitative understanding of cellular processes, and accordingly the n-hybrid
assays will have to be characterized and rebuilt on this level, allowing, for
example, the K D cutoff of the assay to be dialed-in. Using this genetic assay
in entirely new ways should then open the door for new chemistry, with the
potential to match the complexity of cell function.


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Chemical Biology
Edited by Stuart L. Schreiber, Tarun M. Kupoor,and Gunther Wess
CoDvriaht 0 2007 WILEY-VCH Verlaa CmbH & Co KCaA. Weinheim

4.2 Contro//ing frote;n-frote;n Interactions I 227

Controlling Protein-Protein interactions Using Chemical inducers and
Disrupters of Dimerization

Tim Clackson


Transient interactions between proteins are a common mechanism of

information transfer in biological systems. Chemical inducers of dimerization
allow these interactions to be brought under specific, real-time chemical
control, and have become established tools for cell biology research. This
chapter reviews the diverse types of ligands and cognate binding proteins
that can be used to control protein-protein associations, and discusses
the applications of the technology, both in basic research and in potential
therapeutic settings.


Many cellular processes are triggered by the induced interaction of signaling

proteins [I, 21. Examples include the clustering of cell surface receptors
by extracellular growth factors and the subsequent stepwise recruitment
and activation of intracellular signaling proteins. Indeed, many signaling
cascades proceed almost entirely through such interactions, from the initial
extracellular receptor engagement through signaling to the nucleus, proximity-
driven activation of gene transcription, and subsequent effector steps such as
regulated protein secretion.
A chemical inducer of dimerization, or “dimerizer”, is a cell-permeant
organic molecule with two separate motifs each of which bind with high
affinity to a specific protein module. In principle, any cellular process that
is activated (or inactivated) by protein-protein interactions can be brought
under dimerizer control by fusing the protein(s) of interest to the binding
domain(s) recognized by the dimerizer. Addition of the dimerizer then non-
covalently links the chimeric signaling proteins, activating the cellular event
that it controls (Fig. 4.2-l(a)).
This conceptually simple approach, described more than 10 years ago [ 3 ] ,
has proved broadly applicable and has been widely adopted not only in the
chemical biology community but also across biological research in general. It
has also spawned several related technologies, such as systems for “reverse
dimerization”. This chapter will review the various protein-ligand systems
that have been designed, and describe examples of their use, both in research
and drug discovery.

Chemical Biology. From Small Molecules to System Biology and Drug Design
Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Gunther Wess
Copyright 0 2007 WlLEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31150-7
4 Controlling Protein-Protein Interactions

Fig. 4.2-1 Schemes showing the principle cells. (b) Heterodimerization. In this
of chemically induced dimerization o f example, one fusion protein is membrane
proteins. (a) Homodimerization. in this tethered; the other is expressed as a soluble
example, fusion proteins are tethered t o the cytosolic protein and is recruited to the cell
cell membrane through fusion to a peptide membrane upon addition ofdimerizer.
sequence that becomes myristoylated inside

Development o f Chemical Dimerization Technology

The concept of chemically induced dimerization was introduced by Schreiber

and Crabtree and their colleagues in 1983 [ 3 ] .The inspiration for their work
came from the mechanism of the natural product immunosuppressive drug
FKSOG, which binds simultaneously to FK50G binding protein 12 (FKBP12
or FKBP), a ubiquitous peptidyl-prolyl cis-trans isomerase, and the signaling
phosphatase calcineurin, inhibiting the latter’s phosphatase activity and hence
blocking signaling. This suggested a general way to bring any protein-protein
interaction under small molecule control. Bifunctional organic molecules
could be designed, with two protein-binding moieties. Target proteins for
these molecules could be appended to the signaling domains of interest at
the genetic level to create chimeric proteins. Addition of the bifunctional
organic molecule to cells expressing the chimeric proteins would induce
dimerization of the engineered proteins, mimicking the natural activation
process (Fig. 4.2-l(a)).
4.2 Controlling Protein-Protein lnteractions I 229

In the initial paper, Spencer et al. used the FK506-FKBP interaction itselfto
provide building blocks for the dimerizer system. They generated a dimerizer
by linking two molecules of FK506 to create FK1012, a molecule that can bind
two FKBP domains simultaneously (but not calcineurin). They then created
a suitable variant of their target protein, the T-cell receptor zeta chain, by
appending three copies of FKBP. Addition of FK1012 to cells expressing the
engineered protein led to clustering of the protein and activation of authentic
downstream cellular events.
FK1012 is a homodimerizer, with two identical binding motifs. It was
quickly recognized that induced heterodimerization should also be feasible, by
fusing the two proteins of interest to different protein-binding domains
that are targeted by a suitable nonsymmetrical dimerizer (Fig. 4.2-l(b))
[4-61. Dimerizers used for such approaches have included, for example,
dimers of FK506 and cyclosporine (FK-CsA) [4]. However, it is most
straightforward to simply use the bifunctional natural products directly.
Rapamycin, an immunosuppressive drug related to FK506, functions by
binding simultaneously to FKBP and the protein kinase FRAP/mTOR [7]and
can be used to heterodimerize proteins fused to these protein modules [5, 61.
The ability to induce a protein-protein interaction inside cells provided a
general way to generate inducible alleles of signaling and other proteins - one
that can be activated in real time, in contrast to classical genetic approaches [8].
This suggested a series of important applications, ranging from mechanistic
analysis of protein function to understanding the consequences of activating
signaling in whole cells and even transgenic animals. Initial hopes have been
more than fulfilled, and several hundred papers have now been published that
describe diverse uses of the technology [9].

Dimerization Systems

A major focus, following the initial reports, was on refining the tools used to
achieve chemical dimerization - in particular, the dimerizers themselves.
Important aims were to improve chemical feasibility, specificity, and
pharmacological properties, the latter to permit studies in experimental
animals. This section will describe the options that have evolved for
different types of induced dimerization. The focus will be on the FKBP-
based technologies and applications developed by the author’s group and its
collaborators, although other systems will also be mentioned. Homodimerization
A series of FK1012 variants has been described with different linkers and, in
some cases, facile syntheses using FK506 as a starting point (Fig. 4.2-2) [lo].
All of these can be used to effect dimerization between FKBP fusion proteins.
I 4 Controlling Protein-Protein Interactions

FK1012 Linker X

OMe Me0 H2 ii3


Fig. 4.2-2 First-generation homodimerization agents FK1012 and

AP1510. These molecules are able to induce homodimers between
wild-type FKBP fusion proteins. The variant FK1012s differ only in
the linker region.

We sought to develop fully synthetic, lower-molecular-weightreplacements

for FK1012, to allow full exploration of structure-activityrelationship (SAR)
and optimization of pharmaceutical properties. These efforts led to the design
of A m 1 0 (Fig. 4.2-2), which comprises two synthetic FKBP-binding ligands
joined by a short spacer [ll].Although AP1510 binds less tightly to FKBP
than FK1012, it is more potent in most applications, perhaps due to a greater
conformational rigidity.
FK1012s or AP1510 can be used to induce discrete homodimers between
molecules ofan FKBP fusion protein when that protein contains a single FKBP
domain. Higher-order clustering can, in principle, be achieved by including
two or more FKBP domains, although the geometry and stoichiometry of the
resulting complexes are difficult to control.
In addition to FKBP-based systems, homodimerization has also been
achieved using the naturally dimeric natural product coumermycin, which
can dimerize proteins fused to Escherichia coli DNA gyrase [12]. Heterodimerization
Although early heterodimerization studies used molecules such as FK-CsA,
the most common approach is the use of rapamycin, which naturally functions
4.2 C o n t r d i n g Protein-Protein lnteractions 1 231

as a heterodimerizer [7]. One protein is fused to FKBP, and the other to the
-100 amino acid domain of FRAP/mTOR which binds to the FKBP-rapamycin
complex, termed FRB (for FKBP-rapamycin binding domain) [13]. FKBP and
FRB have no detectable affinity for one another in the absence of rapamycin,
yet the drug binds simultaneously to both proteins with high affinity. Thus,
addition of rapamycin to cells expressing FKBP and FRB fusion proteins leads
to strictly drug-dependent heterodimerization.
Because of its inherent directionality, heterodimerization is often a more
precise tool than homodimerization and can be used in many configurations.
For example, a protein can be inducibly recruited to the plasma membrane
by fusing it to one of the drug-binding domains, and fusing the other
to a myristoylation motif (see Fig. 4.2-l(b)) [4]. A major application of
heterodimerization is in the control of transcription (see Section [5, 61.
In addition to the rapamycin system, other heterodimerization systems
have been described, including dimers of methotrexate and dexamethasone
to target dihydrofolate reductase and glucocorticoid receptor fusion proteins,
respectively [14, 151, and dimers of estrogen analogs and biotin analogs to
target fusions to estrogen receptors and streptavidin [16]. Refining Ligand-Protein Pairs: “Bumps and Holes”

Although the ligand-protein interfaces provided by nature are good starting
points for building dimerization systems, there is room for improvement.
In particular, it is desirable to maximize the selectivity of the ligands for
their target fusion proteins compared to endogenous proteins, to ensure
that the ligands have no effect on natural cellular physiology. In the case of
FKBP-based homodimerization, the ligands might interfere with the natural
function of FKBP as a modulator of transmembrane signaling proteins
(although this is unlikely given the high intracellular FKBP levels). There
is also the possibility that dimerizer potency could be blunted by sequestration
of the drug into the extensive cellular FKBP pool. In the case of rapamycin-
based heterodimerization, adding rapamycin to cells inhibits endogenous
mTOR/FRAP activity, inducing antiproliferative effects.
The solution devised for these problems has become known as “bumps
and holes”, and takes advantage of the fact that the sequences of the drug-
binding domains are available for genetic modification, since they are being
expressed heterologously in the cell (Fig. 4.2-3). In this approach, the ligand
is modified to introduce a steric clash (a “bump”) that abolishes binding to
the target protein. Then, using structure-guided or screening approaches, one
or more compensating mutations are identified in the drug-binding domain
that restore the ability to bind the modified ligand (a “hole”). The bumped
dimerizer is now able to bind only to the modified drug-binding domain of the
chimeric protein and not to endogenous proteins.
In addition to affording highly specific protein-ligand pairs, this interface-
engineering approach has also provided insights into the structural and
I 4 C o n t r o h g Protein-Protein interactions

Fig. 4.2-3 Engineering specificity into FKBP system. Bumped “rapalogs” are able to
dimerizing agents using “bumps and induce heterodimers between FKBP fusion
holes”. (a) Homodimerization system. proteins and FRB fusion proteins engineered
Bumped homodimers are able t o induce with a specific “hole”. The compounds can
dimers between FKBP fusion proteins still bind to endogenous FKBP, but have
engineered with appropriate “holes”, while reduced or eliminated antiproliferative
evading endogenous FKBP. activity because this complex cannot bind
(b) Rapamycin-based heterodimerization effectively t o endogenous FRAP/mTOR.
4.2 C o n t r o h g Protein-Protein interactions 1 233

thermodynamic plasticity of small molecule-protein interfaces [ 17, 181. The

approach has echoes in many other areas of chemical biology, in particular
the pioneering work of Shokat and coworkers in engineering allele-selective
kinase inhibitors and substrates (see Chapter 3.1). Bumped Hornodirnerizers

Highly potent and selective hornodimerizers have been designed by engineer-
ing the interface between AP1510 and FKBP. X-ray crystallographic analysis
suggested that alkyl substitution of a specific carbonyl group on the FKBP lig-
and would destroy binding and that loss-of-size mutations at FKBP residue F36
should restore affinity (Fig. 4.2-3(a)).Subsequent studies resulted in AP1903,
a bumped dimerizer with very high affinity (& 0.1 nM) and 1000-foldselec-
tivity for the FKBP mutant F36V compared to the wild-type protein (Fig. 4.2-4)
[ 191. Related dimerizers with different linkers but equivalent potencies have
also been described (such as AP20187; see Fig. 4.2-4).
These dimerizers, in general, have proved to be much more potent than their
unbumped cousins and suitable for in vivo studies in a range of experimental
animals. Numerous studies have reported the use of FKBP-F36V fusion
proteins and AP20187 to control cellular processes [9],and AP1903 itself has
completed a phase I clinical trial in healthy human volunteers, where it was
found to be safe and well tolerated [20]. Bumped Heterodirnerizers: “Rapalogs”

“Bumped” raparnycin systems have been developed by chemically modifying
the FRB-binding portion of rapamycin, to generate “rapalogs” with reduced

~ Dtrnerizer x Linker Y


Fig. 4.2-4 Bumped homodimerizers. These compounds are designed to bind potently
and specifically to the F36V mutant of FKBP.
I 4 Controlling Protein-Protein hteractions

or eliminated FRB binding and, hence, biological activity. Compensating mu-

tations in FRB have then been identified using structure-guided mutagenesis
and screening/selection, which can then be introduced into target protein FRB
fusions (Fig. 4.2-3(b)).
Several rapamycin bump-hole solutions have been described (Fig. 4.2-5).
In one, bulky substitutions at the Cl6 methoxy group of rapamycin were used
to abrogate binding to wild-type FRB. In a structure-guided screen, mutation
of FRB residue Thr2098 (which abuts Cl6) to Leu was found to allow binding
of a wide range of Cl6-substituted rapalogs (Ref. 21 and our unpublished
work) (Fig. 4.2-5). In fact, the T2098L substitution is a versatile “tag” that
functionally accommodates numerous rapamycin analogs, modified at C 16
and/or other positions, as well as rapamycin itself. As a result it is routinely
incorporated into all our FRB fusion protein constructs and has been used
with C16-bumped rapalogs in numerous in vitro and in vivo studies.
Another system uses C20-methallyl rapamycin (Ma-rap; Fig. 4.2-5), which
is unable to bind wild-type FRB and is therefore devoid of FRAP/mTOR
inhibitory activity [22]. Ma-rap was found in a screen to bind very specifically
to a triple mutant of FRB known as PLF [22]. Using the PLF mutant of FRB,
Ma-rap can be used to achieve highly selective heterodimerization of proteins

AP rapalogs Rapalog R16 R32
Rapamycin OMe II

OMe /I
Me0 AP22594 0


AP1861 II
Me0 ~


AP21967 I


AP23102 HN,koa I1

Fig. 4.2-5 Bumped rapalogs used as rapamycin), in which the triene portion of
heterodimerizers. The rapalogs listed in the rapamycin is modified as shown, is active in
panel are all active in dimerization systems dimerizeration systems incorporating the
incorporating the T2098L mutation in FRB specific FRB triple mutation PLF
fusion proteins. Ma-rap (CZO-methallyl (K2095P/T2098L/W2101 F) [22].
I 4 Controlling Protein-Protein lnteractions

Fig. 4.2-6 Schemes for controlling transcription using chemically induced dimerization.
(a) Control using homodimerizers. (b) Control using heterodimerizers (rapalogs).

of FKBP binds to itself in a manner that can be reversed using an FKBP

ligand [27]. The phenomenon was initially noted in a two-hybrid assay
and subsequently confirmed by biophysical studies on the purified protein.
Although the monomer-monomer affinity is relatively weak (& 30 yM),
the interaction is specific, and concatenated F36M domains form discrete
aggregates by virtue of multivalent binding. Interactions can be completely
disrupted by addition of a monomeric “bumped” ligand analogous to one
half of AP1903 (see Fig. 4.2-4),suggesting that the F3GM mutation, similar to
F36V, introduces a “hole” in the protein surface. This result also implies that
the proteins interact through their ligand-binding sites - a finding confirmed
crystallographically (see next section).
This system can be used to reversibly aggregate any protein to which multiple
F36M domains are attached. For example, intracellular expression of a fusion
between four F36M domains and green fluorescent protein (GFP) results in
large fluorescent intracellular aggregates that disperse within minutes upon
adding monovalent ligand [27]. Removal of ligand leads to rapid re-formation
of aggregates.
4.2 C o f l t r o h g Protein-Protein Interactions 1 237

Fig. 4.2-7 Comparison of conventional and proteins. (b) Reverse dimerization system
"reverse" FKBP dimerization systems. using monomeric ligand (AP21998) and
(a) induced dimerization using bumped F36M fusion Proteins.
homodimerizer AP20187 and F36V fusion Structural Basis of Induced Dimerization

One attraction of using inducible dimerization is that the interacting molecules
are understood in great detail. The high-resolution X-ray structures of
all three FKBP-based complexes in the dimerized state are available - the
AP1903 homodimerization system (our unpublished work), rapamycin
heterodimerization system [7], and the F36M reverse dimerization system
[27] (Fig. 4.2-8). These structures have been invaluable for engineering and
optimizing the drug-protein interfaces. In addition, they provide important
guidance on the orientations in which the binding proteins can be fused
to heterologous proteins of interest, in order to induce dimerization of the
appropriate geometry.


With protein-protein interactions being pervasive throughout biology,

chemically controlled dimerization has proved to be a remarkably versatile
technology, and more than 300 papers have described use of the approach
[9]. These applications can be broadly separated into two classes. The first
is the use of dimerization technologies in basic and applied biological
research, to understand the functions of proteins or pathways, and to create
I 4 Controlling Protein-Protein Interactions

Fig. 4.2-8 X-ray crystal structures of (b) Structure o f raparnycin in complex with
dimerized complexes. In each case, protein wild-type FKBP green and the FRB domain
N-termini are marked in blue and C-termini of FRAP/rnTOR gray (Protein Data Bank
in red. (a) Structure ofAP1903 in complex (PDB) ID: 4FAP) [7]. (c) Structure ofthe
with two molecules o f FKBP-F36V (our homodimeric complex o f the
unpublished data). The two proteins are self-associating FKBP mutant F36M
brought close to each other in a “parallel” (PDB ID: 1 EYM) [27]. The two molecules
configuration, and intramolecular interact through their ligand-binding sites in
drug-drug interactions are extensive. an “antiparallel” configuration.

inducible animal models of disease. The second is the direct use of the
technologies in potential therapeutic applications, generally in the context of
cell or gene therapies. Examples of both will be reviewed in the following
4.2 Contro//ing Protein-Protein interactions 1 239 Analysis o f Protein Function

A very common and powerful application is creating an inducible allele of a
protein in order to dissect its function. Typically, the protein of interest is fused
to a dimerization domain, cells expressing the fusion protein are exposed to
dimerizer, and the consequences are assessed by any appropriate technique,
such as assaying downstream signaling or profiling mRNA expression. The
key advantages of chemically induced dimerization are that activation can be
restricted to one particular protein and can be initiated and then monitored in
“real time” by addition of drug. This allows very specific questions to be asked
about the function of a protein or of the pathway that it controls.
Over 100 proteins have been successfully brought under dimerizer control
in this way 191. In many cases, these are signaling proteins such as cell
surface receptors, intracellular protein kinases, and signaling proteases such as
caspases. Often, the experimental goal is simply to test whether dimerization is
sufficient to activate the protein. For example, such studies support an induced
proximity model for activation of Raf-1 [12], caspase 8 [28], and leukemia-
associated fusion proteins [29]. However, more complex questions can be
asked, particularly through combined use of homo- and heterodimerization.
Dimerizable alleles of the epidermal growth factor (EGF) receptor family have
beenused to show that EGFRl homodimers, EGFR2 (HER2)homodimers, and
EGFR1-EGFR2 heterodimers all have different effects on breast tumor cell
proliferation and invasion in three-dimensional culture models [30]. By using
dimerizable alleles, the roles of each complex could be probed independently
and without the complicating effects of the natural receptor ligands.
More broadly, dimerization can be used to gain control over a specific
molecular process or even cellular event that can be induced by proximity:
examples include cell adhesion and rolling [31],DNA looping [32], recombinase
enzymatic activation [33], RNA splicing [34], protein splicing [35], and
glycosylation [3G]. These inducible alleles allow the process in question to
be dissected, but often also provide tools that have applications in their
own right: for example, the use of inducible recombinase activity to achieve
temporal control of gene deletion [33]. Animal Models of Disease

Because the inducing compounds are suitable for use i n vivo, and are generally
orthogonal to mammalian biology, studies can also be performed in a whole-
animal context. A common approach is to generate transgenic mice in which
expression of the fusion protein is restricted to a tissue of interest. These mice
allow study of protein or pathway function i n vivo, but can also provide an
inducible model of any disease that is associated with activation (or inhibition).
For example, transgenic mice expressing inducible versions of either fibroblast
growth factor receptor 1 (FGFR1) or FGFR2 specifically in the prostate have
been used to show that only the former receptor can induce the neoplasia and
hyperplasia typical of early prostate cancer [37] (Fig. 4.2-9).These mice could
I 4 Controlling Protein-Protein Interactions

Fig. 4.2-9 Use of dimerization technology prostate tissue. Administration of dimerizer

t o probe the roles of FGF receptor subtypes (AP20187) induced prostate neoplasia and
in prostate cancer development. Transgenic hyperplasia only in the FGFRl mice,
mice were prepared in which implicating this receptor subtype in early
dimerizer-inducible alleles of FGFRl or Prostate cancer development.
FCFRZ were expressed exclusively in

also be used to test potential drugs for the ability to block the induced FGFRl
signal and its consequences.
A general approach to creating animal models of degenerative diseases is to
induce apoptosis specifically in target tissues or organs. This can be achieved
through tissue-specific expression of inducible alleles of the Fas receptor or
through any number of downstream caspases. Mice in which hepatocytes can
be inducibly ablated represent a valuable model for liver diseases [38], and
mice expressing inducible caspase in macrophages are a valuable resource for
probing the roles of these cells [39]. Regulated Cell Therapies

A powerful use of dimerizer technology is in controlling the proliferation,
differentiation, and/or survival of genetically engineered cells [40]. Cell
therapies have broad potential to treat diseases but suffer from limitations,
including the inability to manipulate the cells once introduced into the body.
Blau and coworkers have used dimerizer-activated alleles of cytokine receptors
to acquire control over cell proliferation. Cells modified with a gene of
interest are also engineered with this “cell growth switch”; administration of
dimerizer then stimulates proliferation only of modified cells, in vitro or in vivo
(Fig. 4.2-10). This approach has been successfully demonstrated in small [41]
and large animal studies [42]and offers a way to expand very rare modified cell
populations into a therapeutic range. Other signaling proteins can be used to
achieve different outcomes - for example, dimerizing CD40 induces a potent
4.2 C o n t r o h g Protein-Protein lnteractions I 241

Fig. 4.2-10 Application o f a receptor. Although transduced cells are rare,

dirnerization-based “cell growth switch” to following infusion in vivo they can be
achieve expansion of genetically modified selectively expanded by administering
cells. Hernopoietic cells are transduced with dimerizer (AP20187), which induces their
a retrovirus encoding a therapeutic gene proliferation and differentiation. Expansion
along with a fusion between FKBp.F36V and can akO be carried O u t in Cell CultUre.
the signaling domain o f rnpl, a cytokine

immunomodulatory response in cells and could be used as part of a cellular

cancer vaccine [43].
The opposite approach to inducing proliferation is to induce cell death,
using conditional alleles of Fas or caspases. A Fas “death switch” has been
used to eliminate engineered T cells infused into animals [44],as a model
for depleting the T cells that cause graft-versus-host disease following bone
marrow transplantation [45].More potent caspase-based switches can also be
used [46] and, in principle, could be installed into any therapeutic cell to provide
a “fail-safe” mechanism for cell destruction should adverse events ensue. Regulated Transcription and Regulated Gene Therapies

Use of dimerizers to control transcription of engineered target genes represents
an alternative to technologies such as the tetracycline-inducible (“Tet”) system
I that rely on allosteric activation [47].A key advantage of dimerizer approaches
4 Controlling Protein-Protein Interactions

is the very low background transcription in the absence of dimerizer, most

likely because the AD is physically separated from DNA prior to activation (see
Fig. 4.2-6) [25].This feature has been exploited to achieve inducible expression
of proteins that are highly toxic, such as diphtheria toxin [21],or highly potent,
such as activators of viral replication [48].The modular nature of the dimerizer
system also facilitates control of endogenous (as opposed to introduced) genes,
achieved by fusing FKBP modules to a DBD engineered to recognize the
appropriate natural promoter [49].
There is considerable interest in the use of dimerizer-controlled gene ex-
pression in regulated gene therapies. Extensive work has gone into optimizing
the rapamycin-inducible system for potential clinical use, including iden-
tifying rapalogs with optimal pharmacology, and developing “humanized”
DNA-binding and activation domains, so that all protein components of the
system are of human original to minimize immunogenicity in a clinical set-
ting (reviewed in Refs 25, 47). The rapamycin system has been successfully
incorporated into most gene therapy vector contexts, including adenovirus and
adeno-associated virus (AAV) [SO], onco-retrovirus, lentivirus, herpes simplex
virus, and naked DNA (reviewed in Ref. 25). Tightly controlled erythropoietin
(Epo) production in response to rapamycin has been demonstrated in nonhu-
man primates for over 6 years following a single intramuscular administration

Fig. 4.2-11 Use of dimerizer-controlled indicated doses (mg/kg, intravenously

transcription to achieve long-term regulated triangles) induced discrete and reversible
expression of a therapeutic gene in a increases in serum Epo levels (black
nonhuman primate. At time zero, the symbols, left axis) and commensurate
animal received a single intramuscular elevations in hematocrit (open symbols,
injection of adeno-associated viral vectors right axis). Inducibility has persisted for over
encoding primate erythropoietin (Epo) 6 years t o date and the study is ongoing.
under the control o f the rapamycin- This figure was originally published in Blood
regulated dimerization system. Subsequent [51]. 0The American Society of
administrations o f rapamycin at the Hematology.
4.2 Controlling Protein-Protein lnteractions I 243
ofAAV vectors (Fig. 4.2-11)[51].Rapamycin- or rapalog-controlled gene expres-
sion has also been demonstrated in animal models after gene delivery to the
liver [52], eye [53],and brain [54].These studies support the concept ofbringing
therapeutic protein production under dimerizer control in the clinical setting. Three-hybridApproaches
Another use of dimerizer-controlled transcription is in three-hybrid assays
[14, 151. In these applications, the “third hybrid” is the dimerizer, and gene
activation serves merely as an assay to report on the interaction between a
dimerizer and the two fusion proteins, rather than as the end in itself. Three-
hybrid assays can be used to identify target proteins for a given small molecule
(by incorporating the molecule into a dimerizer and screening against a cDNA
library fused to an AD; see Chapter 18.2), or to identify small molecules that
bind a given target (by cloning the target as an AD fusion protein and screening
against a library of dimerizers in which one monomer is diversified). More
recently, they have been applied to directed evolution of the catalytic properties
of proteins using “chemical complementation” (see Chapter 4.1). Regulated Secretion Using “Reverse Dimerization” System

The reverse dimerization system (Section has been used to develop an
approach for the regulated pulsatile secretion of proteins [55].The aim of this
work was to mimic the natural, rapid release of proteins such as insulin using a
regulated gene therapy strategy. Since control at the transcriptional level takes
place on the timescale of days, it is necessary to directly regulate the secretion
process. To achieve this, the protein ofinterest is expressed as a secreted protein
fused to tandem copies of the FKBP-F36M domain, resulting in the formation
of aggregates in the endoplasmic reticulum (ER) that are too large to exit to the
Golgi (Fig. 4.2-12). Addition of a monomeric ligand breaks up the aggregates,
allowing the proteins to proceed to the Golgi, where they are processed by the
endogenous protease furin, releasing the authentic protein for secretion.
Using this system, rapid pulses of insulin secretion could be iteratively
induced by adding ligand to cells in uitro (Fig. 4.2-12(c)).Furthermore, in a
mouse model of insulin-dependent diabetes, induced release of insulin could
transiently reverse hyperglycemia [55].More recently, we have incorporated the
system into an AAV vector and demonstrated long-term inducible secretion
following gene transfer into mice (our unpublished studies). These findings
suggest that regulated secretion could be useful for regulating the expression
of proteins that require delivery in rapid pulses.
The ability to reversibly induce large protein aggregates has also provided a
useful tool in basic research on the mechanisms of intracellular transport - for
example, allowing demonstration, for the first time, of the existence of
“megavesicles” that traffic between the ER (endoplasmatic reticulumn) and
Golgi [56].
4 Controlling Protein-Protein Interactions
4.2 C o n t r d h g Protein-Protein interactions I 245

4 Fig. 4.2-12 Use ofthe reverse dimerization Cells expressing an insulin-F36M fusion
system t o control protein secretion in protein were exposed t o AP21998 for three
mammalian cells. (a) Scheme for inducible 1-h periods as indicated, and medium was
secretion. (b) Chemical structure o f collected every hour and assayed for insulin
monomeric ligand AP21998. (c) Pulsatile levels [55].
release o f insulin from engineered cells.

Future Development

Inducible dimerization technologies are now firmly established as research

tools. The components of the various systems are largely developed, although
refinements will likely continue in some areas - for example, the optimization
of protein-ligand pairings, particularly rapamycin analogs. A worthwhile goal
now within reach is the simultaneous regulation of multiple pathways or
proteins using dimerizers and binding proteins that are completely orthogonal
to one another [24].
Some of the most powerful research applications of the technology are only
now starting to be explored - a consequence of the time necessary to establish
transgenic mouse lines expressing appropriate fusion proteins. The next few
years will likely see many more reports using such mice to dissect the roles of
individual proteins and pathways in normal physiology and in disease.
Similarly, although the feasibility and promise of therapeutic uses of
dimerizer technology has been well established in animal models, translation
into the clinic has been slow owing to the general issues and complexities
associated with gene and cell therapies. As these issues are resolved, dimerizer
technology may have a key role to play in conferring control and safety on such
Looking further ahead, interesting extensions of the dimerizer concept are
emerging. These include attempts to enhance the potency of drugs by linking
them to another small molecule, such as an FKBP ligand, that can recruit an
endogenous protein and improve overall binding affinity [57]. The ultimate
extrapolation of chemical dimerization would be dimerizers that bind directly
to native target proteins, as opposed to engineered fusion proteins. Attempts
to build fully synthetic transcriptional activators that directly bind both DNA
and transcriptional regulators are a step in this direction [58],and compounds
that directly dimerize and activate cytokine receptors may, in time, become a
therapeutic alternative to recombinant proteins such as Epo [59].


Chemically controlled dimerization represents a clear and successful example

of how chemical biology approaches can “cross over” into mainstream biology
I and become established as powerful and generally accepted research tools. The
4 Controlling Protein-Protein Interactions

technology has contributed significant new insights into numerous biological

processes and, in turn, has inspired new directions in chemical biology
research. Both of these benefits are likely to continue as the technology
becomes more broadly utilized.


I thank Len Rozamus, Xiaotian Zhu, Vic Rivera, and Renate Hellmiss
for preparing the figures. I am indebted to my many ARIAD colleagues
and collaborators, past and present, who have contributed to our work on
dimerization technology. Particular thanks are due to Vic Rivera for numerous
discussions over many years. Kits for the regulated dimerization of proteins
may be requested through ARIAD’s website at


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Chemical Biology
Edited by Stuart L. Schreiber, Tarun M. Kupoor,and Gunther Wess
Cowriaht 0 2007 WILEY-VCH Verlaq CmbH & Co KCaA, Weinheim

I 4 Contro//;ng Prote;n-Protein interactions

Protein Secondary Structure Mimetics as Modulators o f Protein-Protein and
Protein- Ligand Interactions

Hang Yin and Andrew D. Hamilton


The development of low-molecular-weight agents that modulate pro-

tein-protein interactions has been regarded as a difficult goal due to the
relatively large and featureless protein interfacial surfaces involved [l-31. Con-
ventional methods for identifylng inhibitors of protein-protein interactions
generally involve the preparation and screening of large chemical libraries to
discover lead compounds [4]. Despite significant advances in high-throughput
methods, screening a large number of compounds cannot guarantee the
delivery of potential drug candidates with necessary potency and selectivity.
Structure-based design is an area of great current interest and represents a
much-considered alternative to conventional methods. In this chapter, we will
review some representative studies ofusing synthetic agents that mimic protein
secondary structures in drug discovery, in particular, to target protein-protein