29th Cyprus Noordwijkerhout Camerino Symposium

Trends in Drug Resarch

Limassol, Cyprus, October 2-7, 2011

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The Book of Abstracts of the Trends in Drug Research 29th Cyprus NoordwijkerhoutCamerino Symposium was published in September 2011 in 300 copies. Cover design: Lightblack Solutions Ltd ISBN 978-9963-9615-2-8
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Committees of the 29th Cyprus-Camerino-Noordwijkerhout Trends in Drug Research Symposium Chairman of the Symposium Prof. A. Makriyannis Local Representatives A. Tsotinis (University of Athens) T. Mavromoustakos (University of Athens) Organizing Committee A. Tsotinis (University of Athens) T. Mavromoustakos (University of Athens) H. Timmerman (VU University, Amsterdam) R. Leurs (VU University, Amsterdam) D. Rotella (Wyeth Res, Chem Sci, Princeton, USA) M. Giannella (University of Camerino) P. Angeli (University of Camerino) D. Papahadjis (National Hellenic Research Foundation) M. Koufaki (National Hellenic Research Foundation) T. Calogeropoulou (National Hellenic Research Foundation) M. Kontoyianni - (University of Edwardsville, Illinois, USA) Scientific Committee (Already Confirmed) D. Sugden (Kings College, U.K.) P. J. Garratt (University College London, U.K.) Alexander Tropsch (University of North Carolina, USA) A. Tsantili-Kakoulidou (University of Athens) P. Koutentis (University of Cyprus) P. Laggner (IBR, Austria) S. Noskov (University of Galgary, Canada) A. Kourounaki (University of Athens) G. Kokotos (University of Athens) P. Marakos (University of Athens) T. Gimisis (University of Athens) A. Geronikaki (University of Thessaloniki) D. Hadjipavlou (University of Thessaloniki) G. Liapakis (University of Crete) T. Tselios (University of Patras) Supporting Organizing Team A. Afantitis, C. Koukoulitsa, G. Melagraki, C. Petrou, P. Afroudakis, V. Belekou, R. Kompogennitaki, A. Tsatsaroni, V. Mouchlis
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Contents
9 The Chairman address to the meeting ............................................................................ 7

The Local Organizers address to the meeting ................................................................ 8 10

11 Minister of Health address to the meeting ..................................................................... 9 15 Scientific Program ..................................................................................................... 11 23 Fragment-based Drug Discovery: a structural perspective .......................................... 18 24 Computational chemistry in Drug Discovery: past, present and future. ...................... 19 25 Biophysics in support of drug discovery. .................................................................... 20 26 .................................................. 21 27 .................................................................................................................... 22

Deuterium exchange mass spectrometry for characterizing protein-ligand interactions: 29 Applications for rational drug design and chemical modeling. ................................... 24
31 X-ray Diffraction Studies on Drug-Membrane Interactions ........................................ 26

Ligand Assisted Protein Structure (LAPS): An Experimental Approach for Target32 Based Drug Design ...................................................................................................... 27
33 Ligand Binding Studies Using NMR Spectroscopy .................................................... 28

A Decision Support System for the Identification of Synthetically Accessible 35 Rheumatoid Arthritis Inhibitors ................................................................................... 30
37 GPCRs: from imaginations to reality; and beyond. ..................................................... 32 38 Advances in GRCR Research ...................................................................................... 33

Structure-based drug discovery for GPCRs ................................................................. 34 39 Recent Crystal Structures of Membrane Bound Proteins Herald a New Era in Drug 40 Discovery ..................................................................................................................... 35 From Benzodioxane to 1,4-Dioxane Scaffold in the Design of 5-HT1A Serotoninergic 41 Full Agonists from 1-Adrenergic Antagonists ........................................................... 36
42 CFTR: A Drugable Target for the Treatment of Cystic Fibrosis. ................................ 37 44 The identification of new HbF inducers for thalassaemia treatment ........................... 39 45 In silico approaches towards the discovery of novel antileishmanial agents............... 40

Structure-based prediction of cytochrome P450 sites of metabolism using induced fit 47 docking and intrinsic reactivity .................................................................................... 42 Novel 5-HT2 Receptor Modulators Demonstrate Antipsychotic Activity In Vivo ..... 43 48 Identification of a New Antimalarial Chemotype ........................................................ 44 49 The Lead Optimization had Pre-Clinical Development of Novel Boron- Containing 50 Small Molecules for the Treatment of Human African Trypanosomiasis ................... 45 Knowledge Recycling in Neglected Diseases Drug Research: PDE Inhibitors as 51 Trypanocidals ............................................................................................................... 46
52 Protect and defend your research intellectual property rights by following "GXP" .... 47

Novel synthetic chemical chaperones as a basis for ALS treatment. .......................... 48 53 Knowledge-driven Multi-Objective Evolutionary Graphs for De Novo Drug Design 51 56
57 Building a Pharmacology Knowledge Space to Profile Drug Safety and Efficiency. . 52 58 Pharmacophores and Their Efficient Use In Lead Discovery and Optimization......... 53

Novel Approaches for the Immunotherapy of Multiple Sclerosis (MS)...................... 55 60 Multiple Antigenic Peptide Systems (MAPs) of the Immunodominant 83-99 Myelin 61 Basic Protein Epitope Implicated in Multiple Sclerosis .............................................. 56 Approaches in the discovery of selective kinase inhibitors for the treatment of cancer. 62 ...................................................................................................................................... 57 QSAR Models that Stand the Test of Time ................................................................. 58 63 Pharmacokinetic optimisation of acidic leads - Missing links and self-inflicted wounds ......................................................................................................................... 59 64 Targeting Diabetes and Obesity: Structure-based Design of Isozyme-selective 65 Inhibitors of Pyruvate Dehydrogenase Kinase. ........................................................... 60 New Small Molecules for Modulating Phosphoinositide-Mediated Signaling: 67 Development and Application. .................................................................................... 62
68 Rational Design for PLA2 Inhibitors as Anti-inflammatory Drugs. ............................ 63 Synthetic Challenges in the Functionalization of [60] Fullerence for the Preparation of Fullerence Derivatives with Antioxidant and anti-HIV Activity ............................................ 70 71 Bioconjugates to Specifically Render Inhibitors Water-Soluble ................................. 66

Licorice Root Triterpenoids as Scaffolds for the Construction of New Medicinal 75 Agents with Target Biological Activity ....................................................................... 68
77 Synthesis and Antiviral Activity of New Glycyrrhizic Acid Analogous ..................... 70

What could be Expected after Anti-TLR4 Antibodies Interaction with Target 79 Receptor? ..................................................................................................................... 72 Preparation and Antimycobacterial Evaluation of Substituted Pyrazinecarboxamides ...................................................................................................................................... 75 82
83 Salicylanilide esters active against MDR tuberculosis ................................................ 76

Synthesis and Biological Evaluation of 4-Substituted 7, 10-Dihydro-6H-[1, 3] 84 diazepino [1, 2-e] purines ............................................................................................ 77
85 New adamantane derivatives with sigma affinity and antiproliferative activity. ........ 78

Inhibition of Monoamine Oxidase by C5-substituted Phthalimide Analogues ........... 79 86 Determination of Residues in the Third Membrane-Spanning Segment of CRF1, 87 which are Accessible in the Ligand Binding-Site Crevice of Receptor....................... 80 Synthesis, Competition Binding and Conformational Studies of Lnear and Cyclc 88 Analogues of C-Terminal Hexapeptide of Neurotensin .............................................. 81 Design of Multi-Targeted Antihyperlipidemic Agents ................................................ 83 90 Isoxazole Substituted Chromans Against Oxidative Stress-Induced Neuronal Damage ...................................................................................................................................... 84 91 Synthesis and Melatoninergic Action of New C4 and C6-Methoxylated N1-OMe N92 acyl tryptamines ........................................................................................................... 85

Synthesis and Melatoninergic Potency of New N-[x-(y-Methoxy-6,1194 dihydroindolo[1,2-b]isoquinolin-11-yl)alkyl]alkanamides ......................................... 87

96 Bioconjugates to Specifically Render Inhibitors Water-Soluble ................................. 89

NMR Spectroscopy and Docking study of complex Ag(tpp)3(asp) to soybean 97 lipoxygenase-1 ............................................................................................................. 90 Use of Dynamic Combinatorial Mass Spectrometry to Identify Potent Prolyl Hydroxylase Domain 2 Inhibitors ................................................................................ 91 98 Neurohormone Melatonin: Solubility and Controlled Release from Cyclodextrin 99 Vehicles........................................................................................................................ 92 Structural Elucidation Conformational Properties and Molecular Docking Study at 100 Bace-1 of Bioactive Molecule BV6 ............................................................................. 93 Docking, Molecular Dynamics and NMR Spectroscopy Study of two new Cytotoxic 101 C2_Substituted Pyrrolo [2,3-f] Quinolines .................................................................. 94 Using Protein Binding Site Comparison to Guide Drug Repositioning; From Human to Trypanosome Phosphodiesterase Inhibitors ............................................................ 96 102
CURRICULUM VITAE .......................................................................................................... 107 SPONSORS ................................................................................................................................. 115

The Chairman address to the meeting

For the past twenty five years, this scientific event has served as a biennial opportunity for researchers and management executives to interact, exchange views and philosophies, preview cutting edge technologies in Drug Discovery and get a glimpse of things to come. The format is the traditional Gordon-style enhanced by the historical ambiance of Cyprus. The place of the conference has been chosen to a seaside five-star Hotel in Limassol, Cyprus. The participants have the opportunity to enjoy swimming in the warm and sandy sea of Cyprus and combine science with relaxation. In addition, the Gala dinner is planned so that the participants can enjoy the traditional foods and beverages of Cyprus. A trip in the middle of the conference is planned where the unique worldwide Roman Mosaics in Pafos will be visited. Also the trip will cover visits in the ancient Greco-Roman theatre of Kourion, goddess of love Aphrodites place of birth Petra of Romios and Colossi Castle. This conference has a twenty-five year history of successfully bringing together world-renowned scientists to discuss the most recent advances and emerging technologies in the field of drug discovery. The format of the conference encourages casual and productive interactions among participants, which are enhanced by the seaside locale and the islands natural beauty.

Alex Makriyannis Chairman of the Symposium

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The Local Organizers address to the meeting

The plenary topics of the 29th Cyprus-Camerino-Norodwijkerhout Trends in Drug Research Medicinal Chemistry EFMC Symposium 2011 focus to aiding medicinal chemists working in academy, research centers and pharmaceutical industry to design and develop new drugs. Today, the advance of technology and methodologies offer new opportunities to tackle major and neglected diseases. In silico rational drug design appears to substitute serendipity and new strategies are applied to the design of new bioactive molecules. These strategies are based on new discoveries in computational chemistry, biochemistry, molecular biology, pharmaceutics, pharmacokinetics, pharmacodynamics and biophysics which offer a tremendous input in this field. In search of developing new medicines, the application of experimental methodologies are also necessary. NMR and MS spectroscopy find new applications in drug discovery. X-ray diffraction is used to resolve the molecular structures of enzymes involved in biological processes and study drug: membrane interactions. A special topic in the scientific program is devoted to advances related to CPCR research as most of the drugs are targeting CPCR-coupled receptors. ADME aspects will also be discussed during the conference. As the majority of the participants are originated from industry, intellectual property issues will also be addressed. Finally, attempts to treat thalassaemia diseases (Thalassa ( ) is Greek for the sea and Haema ( ) is Greek for blood), common in the Mediterranean region, as a result of underproduction of normal globin proteins, will be covered. The local organizers Andreas Tsotinis-Thomas Mavromoustakos

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Minister of Health address to the meeting

It is with great pleasure and interest that I take this opportunity to share my thoughts with you on the issue of Trends in Drug Discovery. There is no better time to revisit this issue which, as time goes by, affects more and more our lives. During the last few years drug discovery has slowed down and is likely to slow down even more. This is due to an increasing requirement on the part of regulators for increased amount of knowledge about all aspects of a drug, before licensing. Such knowledge requires more and more research. Many large Pharmaceutical companies can no longer invest in gaining more knowledge. In addition, in a few years many patents of drugs will expire, leading to loss of revenue by large companies with consequences on the future prospect of drug discovery for novel drugs. Therefore, the concept of Open Innovation has been highlighted as a way to pool knowledge between large companies to tackle common problems in drug discovery. The European Commission is now financing a Public-Private (PP) initiative, known as Innovative Medicines Initiative (IMI), which is the means to engage both the public (Academics and SMEs) and the private sector (large Pharmas) in collaborating to solve some of the bottlenecks in drug discovery. During the Cyprus Presidency of the European Council we will present a European Strategy on this issue as we believe that such PP initiatives are the way forward. Nevertheless, we recognize that certain aspects of drug discovery and testing can be enhanced using more targeted approaches using high throughput omics approaches. Particular emphasis should be given to personalized medicine for instance, a concept that should help in the evaluation of drugs for the future, particularly for oncology patients. I am delighted that this meeting is organized in Cyprus and look forward to taking part at this meeting. Stavros Malas Minister of Health

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SCIENTIFIC PROGRAM

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Scientific Program
Sunday 2.10.2011
17:00-19:00 19:00-20:00 Registration and Poster Set-Up Welcome Drinks Reception

Monday 3.10.2011
8:00-9:00 9:00-9:15 Registration Opening Presentation by the Chairman of the Symposium Prof. A. Makriyannis.

PERSPECTIVES IN STRUCTURE-BASED DRUG DISCOVERY

Chair 9:15-10:00 10:00-10:45 R. E. Hubbard and Sid Topiol Tom Davies - Astex Therapeutics Ltd, U.K. Fragment-based drug discovery: a structural perspective. Andrew Leach-GlaxoSmithKline Research and Development. Current issues in molecular modeling and design Glaxo Research. Coffee Break James Murray-Vernalis, Ltd, U.K. Biophysics in support of drug discovery Vernalis Ltd. Rod Hubbard-University of York, Vernalis Ltd, U.K. What is next for structure-based discovery. M. Kontoyianni - Southern Illinois University Edwardsville. A Structure-Based Approach to Understanding Somatostatin Receptor Subtype Selectivity. Lunch Time - Poster Session

10:45-11:15 11:15-12:00 12:00-12:45 12:45-13:30

13:30-15:00

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15:00-15:45

Dionysios Pantazatos - Rhode Island Hospital Warren Albert Medical School, Brown University, USA Deuterium exchange mass spectrometry for characterization of EGFR protein ligand interactions: Applications for rational drug design and chemical modeling.

X-RAY CRYSTALLOGRAPHY, COMPUTATIONAL AND BIOPHYSICAL METHODS IN DRUG DISCOVERY

Chair 15.45-16.30 G. Kokotos and D. Hadjipavlou M. Rappolt-Institute of Biophysics and Nanosystems Research, Graz Austria X-ray Diffraction Studies on Drug - Membrane Interactions. A. Makriyannis-Center for Drug Discovery, Northeastern University, USA Ligand Assisted Protein Structure (LAPS): An Experimental Approach for Target-Based Drug Design. S. Golic Grdadolnik National Institute of Chemistry, Ljubljana, Slovenia Binding Studies using NMR spectroscopy. A. Afantitis - NovaMechanics, Cyprus. A Decision Support System for the Identification of Synthetically Accessible Rheumatoid Arthritis Inhibitors.

16.30-17.15

17.15-18.00

18.00-18:45

Tuesday 4.10.2011 ADVANCES IN GPCR RESEARCH

Chair 9.15-10.00 10.00-10.45 H. Tiemermann and .R. Leurs H. Timmerman - VU University, Amsterdam GPCRs: from imaginations to reality; and beyond. Nosjean Olivier -Servier Research Institute, France HTS on GPCR made easy - but relevance remains a challenge. Coffee Break
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10.45-11.15

11.15-12.00 12.00-12.45

Miles Congreve -Heptares Therapeutics, UK Structure-based drug discovery for GPCRS. Sid Topiol - 3D-2drug, UK opportunities for molecular modeling from recent GPCR crystal structures. Fabio Del Bello-Scuola di Scienze del Farmaco e dei Prodotti della Salute, Camerino, Italy From benzodioxane to 1,4-dioxane scaffold in the design of 5-HT1A serotoninergic full agonists from a1adrenergic antagonists. Lunch Time-Poster Session

12:45-13:30

13:30-15.15

NEW THERAPEUTIC OPPORTUNITIES IN RARE DISEASES

Chair
15:15-16:00

A. Tsotinis, P.J. Garratt

Hanoch Senderowitz - Bar Ilan University, Ramat Gan, 52900 Israel CFTR: A Dragable arget for the treatment of Cysti Fibrosis. Kleanthous Marina - Institute of Genetics, Cyprus The identification of new HbF inducing agents for thalassaemia treatment. Constantinos Potamitis - National Hellenic Research Foundation, Greece. In silico approaches towards the discovery of novel antileishmanial agents. Andrew Sparkes - Schrodinger, New York, NY 10036, United States Structure-based prediction of cytochrome P450 sites of metabolism using induced fit docking and intrinsic reactivity. Raymond G. Booth - University: Departments of Medicinal Chemistry and Pharmacology, University of Florida. Novel 5-HT2 Receptor Modulators Demonstrate Antipsychotic Activity In Vivo.

16:00-16:30

16:30-16:45

16.45-17.30

17.30-18.15

GALA DINNER 20.30 (optional, ticket holders only)

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Wednesday 5.10.2011 NEGLECTED TROPICAL DISEASES

Chair 8:00-9:00 Dr Christoph Boss Dr Christoph Boss, Actelion Pharmaceuticals Ltd Potent and fast acting novel antimalarials derived from a cell-based screen. Dr Robert Jacobs, SCYNEXIS Inc, USA Lead Optimization and Pre-Clinical Development of Novel Boron-Containing Small Molecules for the Treatment of Human African Trypanosomiasis. Coffee Break K M Orrling, Leiden/Amsterdam Centre of Drug Research Knowledge Recycling in Neglected Diseases Drug Research: PDE Inhibitors as Trypanocidals.

9:00-10:00

10:00-10:15 10:30-11:15

INTELLECTUAL PROPERTY ISSUES

11:15-12:00 David Richard Buckley, (Keynote Speaker) David Buckleyand Associates Pty Ltd Protect and defend your research intellectual property rights by following "GXP".

12:30

EXCURSION to Mosaics of Paphos, Petra of Romios, Ancient Theater of Kourion and Colossi Tower)

Thursday 6.10.2011 ADMET ISSUES -NEURODEGENERATIVE DISEASES

Chair 9:00-9:45 A. Davis and A. Kakoulidou Gruzman Arie - Associate President of Medicinal Chemistry of Israel Novel Synthetic Chemical Chaperones as a New Basis for Amyotropic Lateralsclerosis (Als) Treatment.
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9:45-10:15

C. A. Nicolaou - Computer Science Department, University of Cyprus. "De Novo Drug Design: the promise and potential of multi-objective optimization". Jason Theodosiou- CEO, Aureus Sciences Building a Pharmacology Knowledge Space to Profile Drug Safety and Efficiency. Coffee Break Thierry Langer - Prestwick Chemical SAS Pharmacophores and their efficient use in lead discovery and optimization. T. Tselios - University o Patras, Greece Novel approaches against Multiple Sclerosis. Irene Friligou - Student, Department of Chemistry University of Patras. Multiple Antigenic Peptide Systems (MAPs) of the Immunodominant 83-99 Myelin Basic Protein Epitope Implicated in Multiple Sclerosis. Andreas Goutopoulos EMD Serono Pharmaceutical Company, USA. Approaches in the discovery of selective kinase inhibitors for the treatment of cancer. Lunch Time-Poster Session

10:15-10:45

10:45-11:15 11:15-12:00

12.00-12.30 12.30-12.45

12.45-13:30

13.30-16.00

NEW OPPORTUNITIES IN MAJOR DISEASES

16.00-16.45 16:45-17:30 Davis Andy-Astra Zeneca QSAR Models that Stand the Test of Time. Peter Webborn-Astra Zeneca Pharmacokinetic optimisation of acidic leads - Missing links and self-inflicted wounds. Alexander A. Tokmakov-Kobe University, Japan Targeting Diabetes and Obesity: Structure-based Design of Isozyme-selective Inhibitors of Pyruvate Dehydrogenase Kinase. Round Table - New Trends in Therapeutics
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17:30-18:15

18:15-19:15

Friday 7.10.2011 NEW OPPORTUNITIES IN MAJOR DISEASES

Chair 9.00-9.45 9.45-10.30 10.30-11.00 11.00-11.30 T. Tselios and M. Kontoyianni T. Tselios, M Kontoyianni S. Georgiadis -Kingston University, UK New Binders for G-Quadruplex DNA. Coffee Break V. Mouchlis- University of Athens, Greece Rational design for PLA2 inhibitors as anti-inflammatory drugs. N. Chronakis - University of Cyprus Synthetic Challenges in the Functionalization of [60]Fullerene for the Preparation of Fullerene Derivatives with Antioxidant and anti-HIV Activity. Anna Hirsch - University of Groningen, the Netherlands Bioconjugates to Specifically Render Inhibitors. Closing Remarks by the organizing committee.

11:30-12:00

12:00-12.45 12.45-13:30

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ORAL PRESENTATIONS

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Fragment based Drug Discovery: a structural perspective

Tom Davies, Astex Therapeutics Ltd, Cambridge, UK

The fragment-based approach to drug discovery has received much attention in recent years, and offers several advantages to traditional high-throughput screening methods including improved sampling of chemical space, higher hit rate and the potential for lower attrition during compound development. The use of structural information is of prime importance for the successful exploitation of a fragment hit, and this talk will describe various aspects of structure-guided inhibitor development at Astex. The talk will highlight the insights into fragment molecular recognition afforded by structure, and give examples of the development of potent and efficient inhibitors against selected therapeutic targets. The emphasis will be on discussing our experience of various aspects of FBDD, including fragment-to-lead strategies, and the use of ligand efficiency indices. The wealth of structural information provided by a fragment-based screen also provides an important knowledge-base for understanding favourable interactions within protein active sites, which can help ameliorate current deficiencies in the prediction of binding energetics and protein flexibility. Examples will be presented of how this information is being used at Astex to improve docking and virtual screening results, and to provide information to guide elaboration of fragment hits to potential drugs.

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Computational chemistry in Drug Discovery: past, present and future.

Andrew Leach, GlaxoSmithKline Research and Development.

The goal of this presentation will be to provide some insights into the use and impact of computational chemistry techniques in drug discovery. In the environment of a large Pharmaceutical company many different discovery approaches are employed, each of which creates new challenges and opportunities for the computational scientist. These techniques include hit identification approaches such as highthroughput screening, the use of virtual screening to create focussed sets and fragment-based screening. Projects may subsequently be assisted through the use of protein structure-based design, ligand-based methods and in silico models of physicochemical and ADMET properties. Computation has also played an important role in helping to understand some of the underlying cause of attrition in pharmaceutical development. Examples will be provided to illustrate the impact of computational chemistry in contemporary drug discovery together with an assessment of its strengths and limitations. Some thoughts on future challenges and opportunities will also be presented.

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Biophysics in support of drug discovery.

J. B. Murray,Vernalis (R&D) Ltd, UK

I will discuss the application of biophysical methods to hit identification and lead optimisation. The examples will draw on examples from a range of target classes, including ATPases, protein-protein interactions, kinases and isomerases. The first aspect will focus on the merits and drawbacks of each technique employed in fragment hit identification, such as NMR, SPR, DSF and HCS. The second part of the presentation will focus on the utility of biophysical methods, in particular SPR and ITC, throughout the drug discovery process. This will draw on our experiences at Vernalis and include examples of non-ideal compound behaviour and how this has been overcome. The final section will focus on the prospective use of biophysical methods to guide drug discovery.

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What is next for structure based drug discovery?

R. E. Hubbard, Vernalis (R&D) Ltd, Cambridge & University of York, UK I will begin with a perspective on the development of structure-based discovery methods and their adoption (and adaptation) by the pharmaceutical industry. Broadly speaking, the 1980s saw the introduction of computational design, the 1990s an increased investment in protein crystallography and the 2000s the rise of fragment-based discovery methods. I will briefly chart these developments and discuss how these waves of technology have been assimilated into the overall drug discovery process, with examples from recent drug discovery projects at Vernalis. So - what is next? I will discuss three current areas of research in the structural sciences which I think will have a significant impact on the nature of drug discovery in the 2010s. The first is that structures for some of the major classes of protein target (GPCRs, transporters, ion channels) are becoming available. These are not only having a radical effect on our understanding of the mechanisms underpinning biological function, but also suggest new strategies for therapeutic intervention. The second is that fragment and structure-based discovery has stimulated developments in the various biophysical methods (ITC, SPR, NMR, TSA) that now provide tools with which to ensure optimisation of compounds is driven by hard, rational data. Finally, our increased understanding of mammalian cell biology is revealing intriguing new targets where intrinsically unfolded states and transient multimolecular complexes are suggesting potential new targets.

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A Structure Based Approach to Understanding Somatostatin Receptor Subtype Selectivity

Zhaomin Liu, A. Michael Crider, and Maria Kontoyianni Departments of Chemistry and, Pharmaceutical Sciences, Southern Illinois University, Edwardsville, Il 62026 In a recent review, it was concluded that somatostatin receptor subtypes 4 and 5 would be high-impact templates for homology modeling if their 3D structures are resolved.[1] Specifically, the somatostatin receptors include five subtypes (SSTR 1-5) and mediate the inhibitory effects of somatostatin on secretion and proliferation depending upon receptor subtype and tissue localization. To better understand the binding modes of the structurally diverse agonists of SSTR4 we generated a homology m odel of the receptor using the newest active state 2 adrenoreceptor crystal structures.[2] We subsequently docked a variety of agonists into the model-built structure, using experimental restraints, and were able to explain observed activity profiles. For further validation, a virtual screening experiment was performed with three docking algorithms (eHiTS[3, 4], Glide[5-7] and LigandFit[8]) and seven scoring functions. We employed a compound library comprised of 990 random molecules, also seeded with 16 known agonists, in order to explore the feasibility of the model and its ability to retrieve known actives. Results will be presented. References: 1. Mobarec, J.C., Sanchez, R., and Filizola, M. (2009). Modern homology modeling of G-protein coupled receptors: which structural template to use? J Med Chem 52, 5207-5216. 2. Rasmussen, S.G., Choi, H.J., Fung, J.J., Pardon, E., Casarosa, P., Chae, P.S., Devree, B.T., Rosenbaum, D.M., Thian, F.S., Kobilka, T.S., Schnapp, A., Konetzki, I., Sunahara, R.K., Gellman, S.H., Pautsch, A., Steyaert, J., Weis, W.I., and Kobilka, B.K. (2011) Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 469, 175-180. 3. Zsoldos, Z., Reid, D., Simon, A., Sadjad, S.B., and Johnson, A.P. (2007). eHiTS: a new fast, exhaustive flexible ligand docking system. J Mol Graph Model 26, 198-212. 4. Zsoldos, Z., Reid, D., Simon, A., Sadjad, B.S., and Johnson, A.P. (2006). eHiTS: an innovative approach to the docking and scoring function problems. Curr Protein Pept Sci 7, 421-435. 5. Halgren, T.A., Murphy, R.B., Friesner, R.A., Beard, H.S., Frye, L.L., Pollard, W.T., and Banks, J.L. (2004). Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J Med Chem 47, 1750-1759. 6. Friesner, R.A., Banks, J.L., Murphy, R.B., Halgren, T.A., Klicic, J.J., Mainz, D.T., Repasky, M.P., Knoll, E.H., Shelley, M., Perry, J.K., Shaw, D.E., Francis, P., and Shenkin, P.S. (2004). Glide: a new approach for rapid, accurate
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docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 47, 1739-1749. 7. Friesner, R.A., Murphy, R.B., Repasky, M.P., Frye, L.L., Greenwood, J.R., Halgren, T.A., Sanschagrin, P.C., and Mainz, D.T. (2006). Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem 49, 6177-6196. 8. Venkatachalam, C.M., Jiang, X., Oldfield, T., and Waldman, M. (2003). LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites. J Mol Graph Model 21, 289-307.

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Deuterium exchange mass spectrometry for characterizing protein ligand interactions: Applications for rational drug design and chemical modeling.
Dionysios Pantazatos, COBRE CCRD Rhode Island Hospital, Warren Alpert Medical School, Brown University

X-ray crystallography is the predominant and most accurate method for determining protein structure and has proven to be an invaluable approach for understanding the molecular facets of ligand binding. The caveat is that it represents a static structure forced to adopt a crystalline state and it does not provide solution-based information of structural stability and conformation. Characterizing changes in protein structure, conformation, and dynamics may not only provide insight for understanding the molecular mechanism(s) of protein activation or inhibition induced by ligand binding but also the mechanism by which a small molecule may modulate protein function by altering protein structure. Over the past decade, alternative methods for probing structural dynamics and conformation have been developed that take advantage of the sensitivity and throughput of mass spectrometry. One of the most increasing and widely used methods utilizes isotopic labeling of backbone amides via hydrogen/deuterium exchange[1]. This method is also referred to as deuterium exchange mass spectrometry (DXMS). DXMS is a powerful and sensitive method for QSAR studies. It is has proven effective for identifying ligand interaction sites[2], regions of solvent exposure, and characterizing drug ligand interactions[3, 4]. DXMS can be performed in the presence or absence of ligand in order to identify ligand binding sites and regions of allosteric change and dynamics induced upon inhibitor binding. DXMS provides valuable information on the degree of solvent accessibility and structural stability at the local environment of the amide. This method has previously been utilized for characterizing of the interaction of clinically relevant molecules with their therapeutic target[4]. The talk will provide an overview of DXMS methodology, two applications of DXMS for 1). Characterizing conformational changes of matrix metalloproteinase 9 (MMP9) induced by the small molecule inhibitor doxycyline. 2). Determining of the structural facets that mediate coagulation Factor VIII activity induced by membrane binding with insights for the development of novel anticoagulant strategies. 3) Understanding EGFR kinase inhibition and activation with proposed methods for facilitating drug screening.
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1.

Tsutsui, Y. and P.L. Wintrode, Hydrogen/deuterium exchange-mass spectrometry: a powerful tool for probing protein structure, dynamics and interactions. Curr Med Chem, 2007. 14(22): p. 2344-58. Garcia, R.A., D. Pantazatos, and F.J. Villarreal, Hydrogen/deuterium exchange mass spectrometry for investigating protein-ligand interactions. Assay Drug Dev Technol, 2004. 2(1): p. 81-91. Garcia, R.A., et al., Molecular interactions between matrilysin and the matrix metalloproteinase inhibitor doxycycline investigated by deuterium exchange mass spectrometry. Mol Pharmacol, 2005. 67(4): p. 1128-36. Hamuro, Y., et al., Hydrogen/deuterium-exchange (H/D-Ex) of, PPARgamma LBD in the presence of various modulators. Protein Sci, 2006. 15(8): p. 1883-92.

2.

3.

4.

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X ray Diffraction Studies on Drug Membrane Interactions

Michael Rappolt, Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences Biomimetic membrane systems are indispensable in basic lipid research and applied bio-nanotechnology. Here, especially synchrotron-based Xray scattering techniques offer a very versatile approach to probe their detailed structural, dynamical and also mechanical characteristics. After a brief overview of the latest experimental and methodological highlights, the presentation of prominent examples from the field of pharmaceutical research will be given. This includes studies on interactions of antihypertensive drugs (angiotensin II receptor blockers) with lipid bilayers (Fig. 1), membrane-mediated effects on ion channels induced by the anesthetic drug ketamine, time-resolved structural elucidations of light activated vesicles for on demand release of ocular drugs, and recent activities in the formulation of new drug delivery systems such as cubosomes and hexosomes.

Figure1: Action of the Antihypertensive drug valsartan on lipid bilayers in the gel-phase (A to B) and in lamellar fluid phase (C to D). The displayed membrane models have been derived from synchrotron small angle X-ray scattering experiments (scheme taken from Potamitis et al. (2011) Biochim. Biophys. Acta 1808: 1753-1763).

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Ligand Assisted Protein Structure (LAPS): An Experimental Approach for Target Based Drug Design
Alexandros Makriyannis, Ph.D., George D. Behrakis Chair in Pharmaceutical Biotechnology. Director, Center for Drug Discovery, Northeastern University

The endocannabinoid biochemical system provides very promising opportunities for therapeutic intervention. Currently, the CB1 and CB2 cannabinoid receptors, as well as the two critical enzymes involved in endocannabinoid deactivation, fatty acid amide hydrolase (FAAH) and monoacyglycerol lipase (MGL), are being seriously explored as therapeutic targets. Our laboratory has been developing biochemical and biophysical methods to obtain structural and functional information on the interaction of ligands with the target proteins. Such information is used in the design and development of novel medications. I shall discuss Ligand Assisted Protein Structure (LAPS), a recently developed approach combining molecular biology and mass spectroscopy-based focused proteomics as well as H/D exchange LC/MS/MS. Recently, we have introduced the use of multinuclear NMR to obtain more detailed information on the structural and pharmacokinetic profiles on ligand molecules of interest. LAPS is currently being used to study ligand-protein interactions with both GPCRs and enzymes.

(Supported by NIH grants DA3801, DA7215, DA00493)

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Ligand Binding Studies Using NMR Spectroscopy

Simona Goli Grdadolnik
Laboratory of Biomolecular Structure, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia, EN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia, e-mail: simona.grdadolnik@ki.si

NMR spectroscopy is a capable physical tool for identification and characterization of the ligand-receptor interactions for a broad range of ligand binding affinities. NMR studies provide structural and dynamics insight into ligand binding modes and can afford important findings for design of new drug leads that complement conclusions from SAR study and crystal structure studies. It is generally accepted that conformational dynamics might strongly affect the stability of the ligand-protein complex. Dynamic insight into the observed differences in the biological activity of novel ligands may significantly contribute to the rational design of more active derivatives. With the recent development of cryogenic probe technology NMR spectroscopy has become also a high-throughput screening method, which is especially powerful for identification of binding of low-affinity, low-molecularmass fragments and offers excellent experimental basis for fragment based drug-discovery.

The capabilities and limitations of NMR methods for the investigation of ligand-receptor interactions will be presented. Our case-studies of NMR-assisted discovery of novel inhibitors of attractive antimicrobial targets will be used to explain the application of ligand-based and
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protein-based NMR methods [1-3]. Special attention will be given to the advantage of NMR spectroscopy for the studies of dynamic processes in molecular complexes. The influence of conformational dynamics on the stability of ligand-receptor interactions and on the inhibitory activities of novel ligands will be presented.
REFERENCES [1]. Sim i , M., Hodo ek, M., Humljan, J., Kristan, K., Urleb, U., Goli Grdadolnik, S., J. Med. Chem., 52, 2899-2908, 2009. [2]. Tomai , T., Zidar, N., Kova , A., Turk, S., Sim i , M., Blanot, D., MllerPremru, M., Filipi , M., Goli Grdadolnik, S., Zega, A., Anderluh, M., Gobec, S., Kikelj, D., Peterlin Mai , L., ChemMedChem., 5, 286-295, 2010. [3]. Mavromoustakos, T., Durdagi, S., Koukoulitsa, C., Sim i , M., Papadopoulos, G. M., Hodo ek, M., Goli Grdadolnik, S., Curr. Med. Chem., 18, in press, 2011.

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A Decision Support System for the Identification of Synthetically Accessible Rheumatoid Arthritis Inhibitors
Antreas Afantitisa, Georgia Melagrakia, George Kolliasb. aDepartment of ChemoInformatics, NovaMechanics Ltd, Nicosia, Cyprus,b Biomedical Sciences Research Center "Alexander Fleming", Athens, Greece Rheumatoid arthritis (RA) is a devastating, chronic inflammatory disorder that affects approximately 1% of the worldwide population, and is characterized by persistent active inflammation with concurrent tissue destruction. Recent reports from several major pharmaceutical companies indicate that there is a significant interest for the identification of small-molecule antagonists of rheumatoid arthritis (RA). Small-molecule inhibitors could provide a less-expensive, orally administered alternative to parenteral biologic agents [1]. Current market for RA medicines is estimated at 7 billion annually; this is expected to approach 9 billion by 2011. In this work an in silico exploration of the relationship between the structural characteristics of novel coumarin and chromene derivatives and their TNF- inhibition activity has been conducted [2]. Among a pool of 250 descriptors the most important features responsible for the TNF- inhibition activity were identified. Different machine learning (Adaboost, Randomtree, J48 and RBF) and variable selection (Ranker and BestFirst) methodologies have been explored individually and in combination. Consensus predictions based on the majority vote of predictions coming from each methodology independently were found to be the most accurate and reliable model (ligand based). One of the common core structures identified in the study has been used for data mining databases of commercially available chemicals to identify more similar molecules in terms of structural, pharmacophore, shape similarity and scaffold relatedness. Several promising synthetically accessible small molecules have been identified with our database mining methods which are based on principles of chemical similarity to a probe (lead compound) and/or predictions from in silico models and/or chemical space. In addition docking studies have been performed by applying structure based virtual screening techniques. The proposed combination of both ligand and structure based virtual screening techniques utilize multiple sources of information rather than just structural or ligand assay data. The combined approach uses structure-based and ligand-based methods to identify compounds with features important for the TNF- inhibition and to ensure that the shape, size and energetic interaction potential of the putative ligands complement that of the target protein respectively [3]. The series of
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compounds identified in silico by the proposed workflow, will be evaluated both in vitro and in vivo. REFERENCES [1]. Palladino M et. al. Nature Reviews Drug Discovery 2, 736-746, 2003 [2]. Cheng, J-F et. al. Bioorganic & Medicinal Chemistry 14, 2411-2415, 2004. [3]. Melagraki, G & Afantitis, A. Current Medicinal Chemistry 18 26122619, 2011

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GPCRs: from imaginations to reality; and beyond.

Henk Timmerman, VU University, Amsterdam More than 100 years ago the term receptor appeared for the first time in the scientific literature; famous names as Ehrlich and Langley come to mind. During the next 50 years no much progress was made, though Clark introduced the well-known occupation model for receptor activation and blockade; the lock and key metaphor became generally accepted. With the transition of pharmacology from in vivo approaches only into a molecular science, with the book of Arins Molecular Pharmacology as a milestone, much progress became possible. The introduction of simple in vitro techniques by Arins and as simple mathematics of the pharmacon receptor interactions led to easy determinations of affinities of the ligands. Moreover the definition of intrinsic activity, partial agonists, spare receptors led to a much better understanding of receptors in general. Receptor subtypes emerged and selective ligands were introduced. It is highly remarkable that the new pharmacology dealt not at all with the chemical nature of receptors. In the late 1960ies Arins admitted that when he was talking on receptors I know I am talking about something I do not know anything about. New technologies were needed to make progress in receptor research. First of all analytical chemistry, which made it possible to find factors which we know as second messengers by now. During a short while adenylcyclase, responsible for the formation of cyclic-AMP, was even considered to be a receptor. Much progress was made when molecular pharmacology became applied in pharmacology research. Receptor proteins were defined, as well as several factors involved in signal transfer processes. Major contributions in understanding receptors, their structures and the mechanism involved in activation processes came from computational approaches and from crystallography. Currently open issues are the meaning, the role of receptors receptor dimers, of heteromers etc. In this lecture attentions will be given to historical aspects and especially to moments at which it was felt that the imaginations had become reality. But also to the fact that realty is often much more complex than we at first sight might think it to be.
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Advances in GRCR Research

HTS on GPCR made easy - but relevance remains a challenge. Servier Research Institute - Olivier Nosjean G protein-coupled receptors (GPCRs) are among the oldest targets in drug discovery, and as such have been the focus of intense technology innovation to make their manipulation easy in the laboratory. Their very peculiar quaternary structure depends mainly on the surrounding cellular membrane environment, be it the phospholipid bilayer itself or associated proteins. This specific feature, shared by other membrane targets such as ion channels, restrained GPCRs from gaining instant benefit from the technology advances that proved to be so powerful in the area of soluble proteins, namely biophysics and structural biology. Although some important progress is on-going in this respect for GPCRs, the main technological advances that helped the screening laboratories to address GPCRs have been made at the cellular level. A quick review of these technologies will show us that HTS on GPCRs has, indeed, been made very easy. Besides, academic researches as well as efforts made in the drug discovery area have shown that GPCRs are highly flexible - both in structural and in functional respects and that their action is regulated by a large number of protein partners, making the ligand to receptor to cellular response part of a much more complex mechanism of action than previously considered. Although the all is now possible picture is both exciting and scaring at the same time, we will see that the drug discovery process can benefit from these advanced concepts, provided that a reasonable strategy is followed, and that sufficient time is dedicated to mechanistic studies.

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Structure based drug discovery for GPCRs

Miles Congreve Head of Chemistry Heptares Therapeutics Limited Heptares has developed a technology that facilitates the study of Gprotein-coupled receptors (GPCRs) by dramatically stabilizing these important proteins outside of the cell membrane. The new stabilized human receptors (StaRs) are much more robust than the corresponding wild type proteins; they are amenable to crystallography, biophysical/fragment screening and for raising monoclonal antibodies. The process whereby the StaRs are first engineered will be explained. The utility of this new technology for structure-based drug design and biophysical screening of GPCR targets will be presented. The Heptares virtual and fragment screening hit identification approaches will be outlined with some example results against receptor targets illustrated. A short case study of drug discovery for the adenosine A2A receptor will be described. Adenosine A2A is a validated therapeutic target for treatment of Parkinsons disease and also of interest for other CNS indications. Leads with good ligand efficiency and drug-like properties have been identified from virtual screening and have been progressed to a candidate drug in less than 18 months.

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Recent Crystal Structures of Membrane Bound Proteins Herald a New Era in Drug Discovery
Sid Topiol, Chief Scientific Officer, 3D-2Drug, LLC

Structure-Based Drug Design (SBDD) has become a powerful tool in drug discovery. This has been made possible through developments in computational technologies and crystal structure determinations. The most widespread, successful application of these approaches has been in their deployment to study enzymes and other soluble proteins targets. This has made possible X-ray structure determinations and SBDD approaches for these targets, such as kinases, secretases, and phosphodiesterases. Many disease targets are, however, membranebound proteins. For these proteins, X-ray structures have been harder to obtain due to difficulties in isolating and purifying the high quantities needed for crystallization. In the past few years, there has been significant progress in this area including, most notably, G-protein coupled receptors (GPCRs), the largest category of protein targets for drugs. These 7-transmembrane (7TM) proteins first started to succumb to X-ray structure determinations for light-activated Rhodopsin and, more recently, for ligand-bound class I GPCRs such as the 1- and 2adrenergic receptors, the adenosine A2a receptor, and a now growing list of others. For the class III GPCRs, the endogenous ligand-binding domain (LBD) resides in the extracellular region outside of the 7TM domain. For the metabatropic glutamate receptors (mGluRs) of class III, X-ray structures have been determined as well. Another category of drug targets, transporters, have also experienced advancements as exemplified by the X-ray structure determinations of the leucine transporter (LeuT), a bacterial homolog of the human endogenous amine transporters such as the serotonin, dopamine, and norepinepherine transporters. Ion transporters represent yet another example of such protein targets. The role of X-ray structures of these targets for drug discovery will be illustrated. For class I GPCRs, we will discuss their use in identifying active compounds. We analyze the relative merits of targeting the extracellular versus 7TM domains of mGluRs for drug discovery from a structural perspective.
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From Benzodioxane to 1,4 Dioxane Scaffold in the Design of 5 HT1A Serotoninergic Full Agonists from 1 Adrenergic Antagonists
Fabio Del Bello, Yogita Aniket, Alessandro Bonifazi, Mario Giannella, Alessandro Piergentili, Maria Pigini, Wilma Quaglia, Scuola di Scienze del Farmaco e dei Prodotti della Salute, Universit degli Studi di Camerino,Via S. Agostino 1, 62032 Camerino, Italy.

The quite planar 1,4-benzodioxane structure of the non-subtype selective 1-adrenergic antagonist WB 4101 (1) [1] has successfully been replaced by the less conformationally constrained 1,4-dioxane nucleus substituted in its positions 5 or 6 with one or two pendant phenyl rings. Indeed, the compounds characterized by N-(6,6-diphenyl-1,4dioxan2ylmethyl)-2-phenoxyethanamine structure, where R is 2,6-OCH3 (2), 2OCH3 (3), or H (4), showed full 5-HT1A receptor agonism, selective 1Dadrenergic antagonism or significant cytotoxic activity, respectively [2]. Considering that a high degree of homology exits between 5-HT1A and 1-adrenergic receptors [3] and WB 4101 itself displays 5-HT1A affinity, the improved 5-HT1A/ 1-adrenergic profile of compound 2 appeared noteworthy. Therefore, to identify stereochemical requirements for the selective recognition of the above mentioned biological targets, the derivatives 2-4 have been resolved into the corresponding enantiomers. In addition, novel ligands, bearing one or two phenyl groups in positions 2 or 3 of the 1,4-dioxane scaffold, have been prepared and studied.

REFERENCES [1]. [2]. Mottram, D. R.; Kapur, H. J. Pharm. Pharmacol., 27, 295-296, 1975. Quaglia, W.; Piergentili, A.; Del Bello, F.; Farande, Y.; Giannella, M.; Pigini, M.; Rafaiani, G.; Carrieri, A.; Amantini, C.; Lucciarini, R.; Santoni, G.; Poggesi, E.; Leonardi, A. J. Med. Chem., 51, 6359-6370, 2008. Trumpp-Kallmeyer, S.; Hoflack, J.; Bruinvels, A.; Hibert, M. J. Med. Chem., 35, 3448-3462, 1992.

[3].

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CFTR: A Drugable Target for the Treatment of Cystic Fibrosis.

Hanoch Senderowitz, Department of Chemistry, Bar Ilan University, Ramat Gan, 52900 Israel

Cystic Fibrosis (CF) is the most common, genetic lethal disease among Caucasians, affecting ~75,000 people worldwide. The median survival age of CF patients is 37 years and the only currently available treatments to the disease are symptomatic. CF is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a Cl- channel whose malfunctioning disrupts the liquid-salt balance in the lungs ultimately leading to chronic infection of the respiratory tract, pulmonary failure and death. CFTR is therefore a highly relevant target for the treatment of CF. Unfortunately, the development of CFTR-based therapeutics has been limited due to several factors including the challenging nature of the required pharmaceutical intervention (i.e., correcting a malfunctioning protein as opposed to more traditional agonists/antagonists approaches), the lack of a high resolution structure of CFTR and the paucity of ligands known to exert their effect by directly binding to it. Over the last few years we have applied a multi-pronged approach to the development of CFTR-based therapies to CF whereby we focus on specific CFTR domains, domain-domain interfaces and the full length protein as targets for computer aided drug design. Thus, we have modeled the 3D structure of CFTR using a combination of ab intio and homology modeling tools. The resulting models were validated and used to identify putative binding sites for CFTR modulators which were subjected to in silico screening campaigns. Each such campaign began with a database of ~4M commercially available, drug-like compounds that were filtered and clustered based on binding site properties and characteristics of known CFTR modulators to create several, ~150K compounds, site-focused libraries. These libraries were docked and scored in the different binding sites and the ~200 best scoring compounds were selected as virtual hits and tested, in vitro, for

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biological activity. Multiple active hits were identified and submitted to lead optimization. More recently we have shifted our attention to a specific CFTR domain, namely, the first Nucleotide Binding Domain (NBD1) which is also the site of the most prevalent CFTR mutation, 5F508. Recent experimental data suggest that: (1) The main defect in 5 F508-NBD1 is its ground state thermal instability and (2) While the ground states of wt- and ofF5 F508NBD1 are similar differences exist in their relative dynamic excursions from this state. These experimental findings are supported by recent MD simulations. Take together these data suggest that the impaired nature of 5 F508-NBD1 could be repaired upon binding of chemical modulators to specific sites within this domain. F508-NBD1 is therefore becoming a focal point for virtual screening campaigns.

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The identification of new HbF inducers for thalassaemia treatment

Marina Kleanthous and Marios Phylactides, the Cyprus Institute of Neurology and Genetics

Thalassaemia is one of the most common inherited blood disorders in the world. Patients develop a profound anaemia that if untreated by regular blood transfusion leads to death in the first decade of life. A well-adjusted transfusion regimen and associated medication help extend the life of thalassaemics, who nevertheless still have a reduced life expectancy and quality of life compared to healthy individuals. At present, allogenic bone marrow transplantation from a matching donor represents the only curative treatment, but is theoretically available to no more than 30% of those affected. Moreover, the treatment carries many risks, such as adverse reaction to the transplant (graft-versus-host disease, GvHD), and in practice is only taken up by a small percentage of eligible patients. Therefore other approaches for thalassaemia treatment are investigated, such as the pharmacological reactivation of -globin genes for the production of foetal haemoglobin (HbF), as it can substitute for the nonfunctional or absent adult haemoglobin, and such as gene therapy (GT) using autologous bone marrow transplantation of genetically corrected haematopoietic stem cells (HSC). The only HbF-inducing drug currently approved for treatment of patients is hydroxyurea (HU), but owing to the highly variable response of thalassaemics its use is restricted to sufferers of sickle cell disease (SCD). Indeed, less than 60% of thalassaemics respond to this drug, while prolonged treatment has cytotoxic effects. In what has become a highly active research field, our group at the CING is investigating the activity of putative HbF-inducing agents from several classes of chemicals, which exert their effect on -globin expression by diverse mechanisms. The compounds investigated include, amongst others, histone deacetylase (HDAC) inhibitors, which alter chromatin structure, metabolites and hormones. The tolerability of these agents and their effect on globin expression are assessed in murine in vitro and in vivo models and in primary human cells.

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In silico approaches towards the discovery of novel antileishmanial agents

Constantinos Potamitis1, Panagiotis Zoumpoulakis1, Theodora Calogeropoulou1, Giorgio Maccari2, Maurizio Botta2. 1Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 11635, Athens, Greece. 2 Department of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Siena, via Aldo Moro 2, 53100, Siena, Italy.

Protozoan parasites of the trypanosomatidae family are the causative agents of various forms of the neglected disease Leishmaniasis (Leishmania species). Trypanothinone Synthetase (TryS) is the sole enzyme responsible for the biosynthesis of trypanothione in the human pathogenic parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. Since Trypanothione is absent from humans but essential for the survival of the parasites, the enzymes related to Trypanothione biosynthesis are targets for the development of new antileishmanial agents. An homology model of Leishmania major-TryS has been produced, based on the crystal structures of Leishmania major Trypanothione Synthetase Amidase (TSA)1 and E. coli Glutathionylspermidine Synthetase (GSPS). Based on the optimized docked complex of the TryS with the native ligand glutathione, a pharmacophore model was generated (Figure 1). Pharmacophore-based in silico screening of commercially available compound libraries in combination with molecular docking studies has led to the identification of putative inhibitor candidates. Among the most important pharmacophores of the derived hits is a negatively charged moiety in most cases sulfonic group- which approaches the coordination sphere of the Mg2+ ion and H-bond acceptors i.e. hydroxyl group forming hydrogen bonds with crucial amino acids of the pocket. he above findings led to the design of new molecules using different, rigid scaffolds with improved docking scores.

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Figure 1: Generated pharmacophore model for the complex of glutathione-TryS along with the lipophilic surface of the binding pocket. Reference 1. Fyfe, P.K.; Oza S.L.; Fairlamb A.H.; Hunter W. N. Leishmania Trypanothione Synthetase-Amidase Structute Reveals a Basis for Regulation of Conflicting Synthetic and Hydrolytic Activities. J. Biol. Chem. 2008, 283, 17672-17680.

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Structure based prediction of cytochrome P450 sites of metabolism using induced fit docking and intrinsic reactivity
Woody Sherman, Tyler Day, John Shelley, Mee Shelley, David Rinaldo, Andrew Sparkes, Ramy Farid. Schrodinger, New York, NY 10036, United States

Structure-based methods have great potential for predicting sites of oxidation by specific isoforms of cytochrome P450 (CYP). However, there are two primary factors that hinder the success of traditional rigid-receptor docking approaches: (a) sites of oxidation do not correlate well with the most stable binding mode of a substrate, and (b) in order to accommodate structurally diverse substrates, CYP active sites are highly flexible. We describe a new method for predicting sites of oxidation based on analysis of ensembles of structures generated by Induced Fit Docking combined with empirically derived rules that define the intrinsic reactivity of a given site. The method is shown to be successful in predicting sites of oxidation for a dataset of known CYP 2C9, 2D6, and 3A4 substrates.

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Novel 5 HT2 Receptor Modulators Demonstrate Antipsychotic Activity In Vivo

Raymond G. Booth, Organization/University: Departments of Medicinal Chemistry and Pharmacology, University of Florida. Clinton E. Canal, Drake Morgan.

Serotonin 5-HT2A, 5-HT2B, and 5-HT2C GPCRs signal primarily through G q to activate phospholipase C and formation of inositol phosphates and diacylglycerol second messengers. The human 5-HT2C receptor is found exclusively in the central nervous system and there is compelling evidence in humans and lab animals that 5-HT2C receptor activation is pharmacotherapeutic for severe neuropsychiatric disorders such as schizophrenia, as well as, obsessive-compulsive behavioral disorders such as over-eating and psychostimulant drug abuse. Meanwhile, 5HT2A receptor activation produces hallucinogenic effects an d 5-HT2B activation leads to heart valve damage and pulmonary hypertension, thus, there is zero tolerance for drug activation of these 5HT2 subtypes. A 5-HT2C agonist drug that does not also activate 5HT2A and/or 5-HT2B receptors, so far, has not been developed. Drug discovery targeting the 5-HT2C receptor is challenging because its shares 75% transmembrane sequence identity with 5-HT2A/2B. The highly conserved transmembrane domains and same second messenger signaling complicates development of ligands that activate 5-HT2C but not 5-HT2A or 5-HT2B receptors. Our medicinal chemistry program incorporating site-directed mutagenesis and molecular modeling studies reveals important differences in molecular determinants for activation of the 5-HT2 GPCR family members. We have developed several novel N,N-dimethyl-4-(4-substitutedphenyl)-1,2,3,4-tetrahydro-2-napthalenamine (phenylaminotetralin, PAT) derivatives that are agonists at human 5-HT2C receptors, but, inverse agonists at 5-HT2A/2B receptors expressed in clonal cells. Offtarget screening indicates no significant affinity of PATs at 440 kinases and several-dozen GPCRs, ion channels, transporters, and signaling/metabolizing enzymes. Accordingly, preclinical studies in laboratory animals were conducted to assess psychotherapeutic activity and safety for lead analogs administered orally or via intraperitoneal injection. Using 2 different rodent models of schizophrenia, potent and efficacious antipsychotic activity, without weight-gain, was demonstrated for several PATs. No adverse affects were apparent in treated animals. Medicinal chemistry, molecular modeling, and in vitro/in vivo pharmacology data will be presented at the Symposium. Research Funding: United States National Institutes of Drug Abuse and Mental Health (RO1 DA023928, DA030989, MH081193).
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Identification of a New Antimalarial Chemotype

2) Ralf Brunner 1) Bibia Heidmann, 1) Hamed Aissaoui, 1) Romain Siegrist, 1) Olivier Corminboeuf, 1) Christoph Binkert, 1) Solange Meyer, 1) Stephane Delahaye, 1) Amelie LeBihan, , 2) Sergio Wittlin, 2) Reto Brun and 1) Christoph Boss. 1) Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, CH-4123 Allschwil, Switzerland and. 2) Swiss Tropical and Public Health Institute (Swiss TPH), Socinstrasse 57, CH-4051 Basel, Switzerland

Malaria, caused by protozoan parasites of the genus Plasmodium remains a major public health problem and the increasing number of resistant strains underscores the need for new drugs with novel modes of action. More than 240 million cases of malaria occur every year and the number of fatalities is estimated at over 800000. The disease accounts for 20% of all childhood deaths in Africa [1]. The two main issues which prevent efficient malaria control in the most strongly affected regions are: too expensive medications and increasingly drug-resistant parasite strains. The need for new and affordable drugs is therefore urgent and indisputable. We will describe a representative of a novel class of antimalarial agents which potently inhibits in vitro erythrocytic Plasmodium falciparum growth of all tested strains with IC50 values in the low single-digit nanomolar range, irrespective of their resistance properties. The compound exhibits oral bioavailability in mice and shows pharmacological efficacy in two mouse models of malaria. The mode of action seems to be different from registered antimalarials. First SAR investigations as well as our efforts on the broader characterization of the compound will be disclosed [2].
[1] Word Health Organization, 2010. Malaria Factsheet Nr. 94. World Health Organization, Geneva. [2] C. Binkert, C. Boss, O. Corminboeuf, C. Grisostomi, S. Meyer, WO2007/046075 (Actelion Pharmaceuticals Ltd).

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The Lead Optimization had Pre Clinical Development of Novel Boron Containing Small Molecules for the Treatment of Human African Trypanosomiasis
Robert Jacobs1, Bakela Nare1, Steve Wring1, Cy Bacchi2, Reto Brun3, Jacob Plattner4, Beth Beaudet1, Tana Bowling1, Daitao Chen1, Yvonne Freund4, Eric Gaukel1, Matthew Jenks1, Marcel Kaiser3, Luke Mercer1, Andy Noe1, Matt Orr1, Robin Parham,1 Ryan Randolph1, Cindy Rewerts1, Jessica Sligar1, Nigel Yarlett2, Robert Don5. 1SCYNEXIS, Inc., Research Triangle Park, NC, USA, 2Pace University, New York, NY, USA, 3Swiss Tropical Institute, Basel, Switzerland, 4Anacor Pharmaceuticals, Inc., Palo Alto, CA, USA, 5Drugs for Neglected Disease initiative, Geneva, Switzerland

Human African trypanosomiasis (HAT) represents a significant public health problem in sub-Saharan Africa affecting hundreds of thousands of individuals. An urgent need exists for the discovery and development of new, safe, and effective drugs to treat HAT, as existing therapies have poor safety profiles, difficult treatment regimens, limited effectiveness, and a high cost of goods. From a collaborative effort between SCYNEXIS, Anacor Pharmaceuticals, Pace University, and DNDi, we summarize optimization of a novel class of small boroncontaining compounds, the oxaboroles. We have identified SCYX-7158 as an effective, safe, and orally active treatment for HAT in a mouse model of stage 2 (CNS) diseases. SCYX-7158 is active in vitro against relevant strains of Trypanosoma brucei, including T. b. rhodesiense and T. b. gambiense (IC50 values 0.180.98 M), and is efficacious in both stage 1 and stage 2 murine HAT models. Physicochemical and in vitro ADME properties of SCYX-7158 are consistent with the compound being orally available, metabolically stable, readily CNS permeable and with low risk for drugdrug interactions. In the stage 2 mouse model, SCYX-7158 is effective orally at doses as low as 12.5 mg/kg (QD per 7 days). In vivo pharmacokinetic characterization of SCYX-7158 shows that the compound is highly bioavailable in rodents and non-human primates, has low intravenous plasma clearance, a 24-hr elimination half-life, and a volume of distribution that indicates good tissue distribution. Most importantly, in rodents brain exposure of SCYX-7158 is high, with Cmax higher than 10 ug/mL and AUC0-24hr higher than 100 g*hr/mL following a 25 mg/kg oral dose. Furthermore, SCYX-7158 readily distributes into the CSF to achieve therapeutically-relevant concentrations in this compartment. Based on these properties, which promise lower rates of recrudescence than with current standard of care, SCYX-7158 has been selected as a preclinical candidate for treatment of stage 2 HAT.
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Knowledge Recycling in Neglected Diseases Drug Research: PDE Inhibitors as Trypanocidals

K M Orrling1, C Jansen1, A Shanmugham1, T Seebeck2, H Tenor3, G J Sterk4, I de Esch1, R Leurs1. 1Leiden/Amsterdam Centre of Drug Research (LACDR), Division of Medicinal Chemistry, VU University Amsterdam, The Netherlands, 2Institute of Cell Biology, University of Bern, Switzerland, 3Nycomed, Konstanz, Germany, 4Mercachem, Nijmegen, The Netherlands. Email: K.M.Orrling@vu.nl

African sleeping sickness causes significant morbidity and mortality and seriously affects society in the poorest areas of the world. The current therapies suffer from severe side effects, inconvenient administration and high costs.1 In the search for an urgently needed new treatment, cyclic nucleotide phosphodiesterases (PDEs) have emerged as attractive molecular targets. For example, both genetic knock-down and chemical inhibition of PDE activity resulted in halted proliferation and eventually elimination of Trypanosoma brucei, the causative agent of African sleeping sickness.2 The vast knowledge and generated expertise within the field of human PDEs are believed to provide a shortcut to efficient inhibitors of parasitic PDEs. The catalytic domains of the human PDEs and parasitic PDEs show a high degree of homology, as well as parasite-specific features (Figure 1).3 This has allowed several medicinal chemical approaches to be applied, e.g. ligand-based drug design, structurebased drug design and fragment-based drug design, in the quest for a new efficient and safe trypanocidal drug.

No L pocket

L pocket

Figure 1( left) The vdW surface of the active site of hPDE4B (magenta) cocrystallized with rolipram and ( right) the homology model TbrPDEB1 (cyan) with rolipram superimposed and the arrow pointing at the parasitespecific L-pocket. This project is facilitated by Dutch Top Institute Pharma and involves eight consortium members and research labs in four European countries. (1) World Health Organisation, Global Burden of Disease: 2004 Update. (2) Oberholzer et al. FASEB J, 2007, 720-731. (3) Wang et al. Mol Microbiol, 2007, 1029-1036.

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Protect and defend your research intellectual property rights by following "GXP"
David R Buckley, David Buckleyand Associates Pty Ltd and Associates Pty Ltd

After your Eureka moment you will find many greedy eyes scouring your scientific research efforts in a perfectly legal attempt to take away your intellectual property (IP) and your livelihood. Protecting your research integrity and defending ownership of such research efforts is critical to you achieving your just rewards, whether they are monetary or not. In different jurisdiction, first to discover is sometimes more important than first to patent. Protecting research IP is important right from the moment of design of experiments and documenting results. It is important to have a strict discipline on this documentation, whether it is electronic or hardcopy, and to protect your hard-won IP you must have knowledge of GXP. That is Good Research Practices, Good Clinical Trial Practices, Good Documentation Practices, etc. You must protect from the risk of tampering, fraud, and inadmissibility in court. But most R&D organizations often overlook the fact that by just discovering their Eurekas, they haven't fully implemented the protections they need to prove ownership, authenticity and "time-ofcreation" (first to invent). This paper will outline some GXPs necessary for research organizations need to implement to prove the IP was created at a specific point in time and hasn't been altered since.

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Novel synthetic chemical chaperones as a basis for ALS treatment.

Omer Green and Arie Gruzman, Organization/University: Bar Ilan University

Novel synthetic chemical chaperones as a basis for ALS treatment. Working Hypothesis: ALS is fatal neurodegenerative disease characterized by the selective death of motor neurons in the cortex, brainstem, and spinal cord. The majority of ALS is acquired spontaneously (SALS), with inherited disease accounting for only 10 15% of all cases. Although several different mutations have been implicated in familial ALS (FALS), mutations in the gene encoding SOD1 are the most common cause of inherited motor neuron disease. Recent studies provide compelling evidence that the misfolding of SOD1 into a disease-specific and toxic conformer underlies both sporadic and inherited ALS. Artificial chaperones that include polyols, trimethyl Noxide (TMAO), phenylbutiric acids and different amino acid derivatives, have been linked to ability to reverse the mislocalization and aggregation of proteins associated with different human diseases. The most limiting factor using of chemical chaperones as drugs is their very high active concentration (nM). Under our proposed research plan, we will synthesize and use chemical chaperons to prevent misfolding, accelerate refolding and redirect proteins to the degradation pathways. Our preliminary results have demonstrated that this method is suitable as a strategy for the treatment of both sporadic and familial ALS. In our search for small molecules that can prevent the formation of toxic misfolding and aggregates, we have synthesized chemical chaperons that primarily targeted cell organelles and areas where WT and mutated SOD1 (among other proteins) are aggregated (ER, lysosomes and mitochondria). Refolding by chemical chaperons will enable lysosome proteolytic enzymes and proteosome system to cleave the misfolded proteins properly. This activity will prevent formation of toxic for motor neuron aggregates. As an ER targeting motive we used mannose-6-phosphate. Lysosome targeting motives were represented by four moieties: 1-aminoisoquinoline, 1-dodecylimidazole,
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2, 4, 5-trimethylpyridine and harmine. Triphenylphosphine was selected as a targeting motive for mitochondria. Some of these molecules have high permeability properties and selective ability to accumulate in targeted organelles. This strategy has never been applied for the development of anti-ALS drugs. Studies on motor neurons (NSC34) in oxidative stress conditions, using our novel compounds in nM concentrations, demonstrate decrease formation of mutant and WT SOD1 aggregates, reduce ER stress and prolong cell survival. We propose to use our new strategy for the development of disease modifying agents in familial and sporadic ALS patients. Research aims: 1. To synthesize novel chemical chaperons with targeting moieties to selective cellorganelles (ER, mitochondria and lysosomes) with nM biological active concentration. These compounds will have different cleavable and non cleavable linkers between targeting moieties and chaperone-like domain that allowed regulating pharmacokinetic and pharmacodinamic characteristics of the active molecules. Moreover, any design of an antiALS drug must consider blood-brain barrier (BBB) permeability of the potential candidates. Therefore, we plan to synthesize molecules that will match already known physicochemical parameters that increase the probability for BBB penetration. We will try to increase oral bioavailability of drug candidates. For this purpose we will synthetase molecules with reduced molecular flexibility, low polar surface area and low total hydrogen bound count. 2. To test biological effect of novel chemical chaperons in NSC-34 cells transfected by human WT an d G93ASOD1 by estimation following parameters: mutant and WT SOD1 degradation, formation of aggregates, level of ER stress and prolong survival in oxidative stress conditions. 3. To validate mechanism of action of test compounds. For this aim we plan to use in vitro aggregation assay for mutant and WT SOD1 proteins. For validation of intracellular targeting we plan synthesis of fluorinated version of active chemical chaperons with following 19FNMR analysis of targeted subcellular fractions. 4. To test the effect of active in vitro compounds in ALS mice models. We plane to treat (i.v./p.o.) human SOD1 G93A transgenic mice with our compounds and test mice motor functions,
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motor neurons survival, tissue morphology changes and life span,. 5. To optimize pharmacokinetic parameters of active in vivo compounds by adding to the lead compound additional motives to improve its bioavailability, oral activity, biodegradation, transmembrane permeability, potency and efficacy. Methods: For in vitro experiments we will use stable transfected with human wtSOD1 or SOD1G93A mouse NSC-34 hybrid cell line. In vitro aggregation of SOD1 will be produced using metal catalyzed oxidation system. Ultracentrifugation will be used for subcellular fractionation. Mutant SOD1 degradation, ER stress and decrease formation of SOD1 contain aggregates will be estimated by western blot. For the purification of the compounds, we will use a HPLC system. 1H, 13C and 19F NMR spectra of compounds will be obtained with a Bruker 300 MHz apparatus. Mass spectrometry of the molecules will be measured on a Thermo Quest Finnigan LCQDuo. Male transgenic mice expressing the human mutant SOD1G93A will be used for the in vivo studies. The effect of tested compounds will estimated by measurement of motor performance, delay of weight loss, attenuation of motor neuron loss and survival of G93A mice as compared to the untreated control group. We will perform immunohistology studies, to follow the SOD1 aggregations. Preliminary results: We have synthesized and tested in vitro several such compounds. Some of them exhibit biological effect in vitro in nM concentrations range. These compounds inhibited aggregation of oxidized pure human SOD1 in vitro, following prevention of formation of SDS resistant aggregates that included high molecular weight mutant SOD1 in NSC-34 cells. Moreover, decreasing phosphorylation level of CHOP, BiP and ATF4 by tested compounds indicated that they reduced tunicamycin induced ER stress response in SOD1G93A transfected NCS-34 cells. Finally, we generated H2O2 production in NSC-34 cells by adding glucose oxidase to the medium to induce permanent oxidative stress. In these conditions, our novel chemical chaperons significantly protected cells against oxidative stress induced apoptosis.

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Knowledge driven Multi Objective Evolutionary Graphs for De Novo Drug Design
C. A. Nicolaou1, 2 C. Kannas2 C. S. Pattichis2 1 CaSToRC, The Cyprus Institute, P.O.Box 27456, CY-1645 Nicosia, Cyprus3 Computer Science Department, University of Cyprus P.O.Box.20537, CY-1678 Nicosia, Cyprus

De novo design (DND) involves searching an immense space of feasible, drug-like molecules to select those with the highest chances of becoming drugs. Traditionally, DND has focused on designing molecules satisfying a single objective, such as increased potency, and ignored the presence of additional constraints that impact the quality of the compounds produced, including pharmacokinetics, toxicity and synthetic feasibility. Recent improvements in DND methodology addressing such known limitations of existing approaches have caused renewed interest as shown by an increased number of publications and reports [1]. Recent improvements focus primarily on the synthesizability of the proposed compounds [1] and the incorporation of multiple objectives to the design process [2]. Substantial research has been performed to identify suitable algorithmic methods for compound design and optimization, for proposing appropriate building blocks for the virtual synthesis of compounds and for measuring the performance of the designed compounds against multiple objectives. In this presentation we briefly review the algorithmic methods used by recent DND methods paying special attention to the optimization method used and the handling of multiple objectives. We then describe the Multi-objective Evolutionary Graph Algorithm (MEGA) [3] and introduce recent work that enables the method to exploit problem specific knowledge during the search process. A description of the key elements of the specific implementation of the algorithm is given, including the methods for obtaining molecular building blocks, evolving the chemical graphs, scoring the designed molecules and exploiting domain specific knowledge. Following, we present results from the application of MEGA to design molecules that satisfy multiple objectives as part of a specific drug discovery project. The results show that the method is capable of producing a diverse set of chemical designs that can serve as idea-generators to medicinal chemistry teams.
References: 1. Schneider, G.; Hartenfeller, M.; Reutlinger, M.; Tanrikulu, Y.; Proschak, E.; Petra Schneider, P. Voyages to the (un)known: adaptive design of bioactive compounds Trends in Biotechnology. 2009, 27(1), 18-26. 2. Nicolaou, C. A.; Brown, N.; Pattichis, C. Molecular optimization using computational multi-objective methods. Curr. Opin. Drug Discov. Dev. 2007, 10(3), 316-24. 3. Nicolaou C. A., Apostolakis. J, Pattichis C. S. De Novo Drug Design Using Multi-Objective Evolutionary Graphs.J.Chem. Inf. Model, 2009, 49(2),295307.

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Building a Pharmacology Knowledge Space to Profile Drug Safety and Efficiency.

Jason Theodosiou, CEO, Aureus Sciences As the attrition of new drug approvals is increasing, the pharmaceutical industry is exploring new ways to improve the efficacy and safety of new drug candidates and reduce the cost and the risks of the R&D process. The use of new technologies to profile and predict biological properties of new drug candidates becomes even more important. These technologies are using existing scientific knowledge in order to provide accurate profiles and predictions and contribute to increasing the success rate of research projects. During the last 10 years, Aureus Sciences has built an extensive modern pharmacology knowledge space providing access to detailed experimental data used in various systems and applications. This presentation will describe this knowledge space as well as examples of applications.

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Pharmacophores and Their Efficient Use In Lead Discovery and Optimization

Prof. Thierry Langer, Prestwick Chemical SAS, Blvd. Gonthier d'Andernach, 67400 Strasbourg-Illkirch, France

In silico or virtual screening has gained considerable impact for the efficient discovery of novel bioactive compounds in modern pharmaceutical research. The concept of chemical feature-based pharmacophore models has been established as state-of-the-art technique for characterizing the interactions between a macromolecule and a ligand. The results of numerous case studies have been published, clearly indicating the merits of this approach for efficient hit discovery [1]. While in ligand-based drug design, feature-based pharmacophore creation from a set of bioactive molecules is a frequently chosen approach; structure-based pharmacophores are still lacking the reputation to be an alternative or at least a supplement to docking techniques. Nevertheless, screening using 3D pharmacophores as filters bears the advantage of being faster than docking. Additionally, it transparently provides the user with relevant information that is used by the screening algorithms to characterize the ligand-macromolecule interaction. At Inte:Ligand GmbH, LigandScout [2] has been developed, as a rapid and efficient tool for automatic interpretation of ligand-protein interactions and subsequent transformation of this information into 3D chemical feature-based pharmacophore models As an extension of this approach, we have introduced parallel pharmacophore-based screening as an innovative in silico method to predict the potential biological activities of compounds by screening them with a multitude of pharmacophore models.[3] Using LigandScout, the entire Protein Databank has been processed, and a pharmacophore database of structure-based pharmacophore models for all targets of potential interest for drug development has been generated, in addition to ligand-based models for targets that lack information about their 3D structure.
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We present an overview of this technology together with the results of an application example employing a set of antiviral compounds that were submitted to in silico activity profiling using a subset of the Inte:Ligand pharmacophore database. The results of the screening experiments show a clear trend towards correct prediction of activity profiles. In addition, using our approach one is able to obtain information about binding of the ligands under investigation also to 'anti'-targets, such as enzymes of the cytochrome P450 family[4], or to the hERG channel. Thus, off-target activity can be determined easily, giving support to the medicinal chemists in their hit-to-lead and lead optimization studies.
References [1] T. Langer, Mol. Inform. 29, 470-475 (2010) [2] G. Wolber, T. Langer, J. Chem. Inf. Model. 45, 160-169 (2005) [3] T. M. Steindl, et al., J. Chem. Inf. Model. 46, 2146-2157 (2006) [4] D. Schuster, et al., Curr. Drug Discov. Technol. 3, 1-48 (2006)

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Novel Approaches for the Immunotherapy of Multiple Sclerosis (MS)

Theodore Tselios, Organization/University: Department of Chemistry, University of Patras, Greece

Multiple Sclerosis (MS) is a chronic, progressive disease of the central nervous system (CNS), characterised by focal inflammation. MS is generally considered to be an autoimmune disease involving CD4+, CD8+ T and B cells. This is strongly supported by the association of the Major Histocompatibility Complex (MHC II) with the disease and the ability of Th1 and Th17 (T helpers) CD4+ T lymphocytes to lead the disease to an animal model for MS called Experimental Autoimmune Encephalomyelitis (EAE). Candidate autoantigens for MS include constituents of the myelin sheath, such as Myelin Basic Protein (MBP), Proteolipid Protein (PLP) and Myelin Oligodendrocyt e Glycoprotein (MOG). Modern therapeutical approaches against MS involve the design and synthesis of peptide analogues of disease-associated myelin epitopes in order to induce tolerance in CNS specific T cells. Mutated linear or conformational restricted cyclic peptide analogues, derived from proteins of the myelin sheath, have been designed and synthesized in order to inhibit EAE through the alteration of immune response from Th1 to Th2. The peptide analogues have been rationally designed using a combination of Nuclear Magnetic Resonance (NMR) and Molecular Dynamic (MD) calculations. Moreover, a number of peptides were conjugated to mannan (polymannose) in its reduced or oxidized form via Schiff bases. Mannan has successfully been used as a carrier to target T cell antigens to the mannose receptor of macrophages and dendritic cells leading to the induction of Th1 or Th2 immune responses in cancer immunotherapy protocols. The ability of mannan to induce different T cell cyto kine profiles depending on the mode of conjugation is of great importance to our research. The synthesized analogues were evaluated in vivo and in vitro using the EAE animal model and blood derived from patients with MS respectively.

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Multiple Antigenic Peptide Systems (MAPs) of the Immunodominant 83 99 Myelin Basic Protein Epitope Implicated in Multiple Sclerosis
Dr. Irene Friligou, University of Patras, Greece

The multiple antigenic peptide system (MAP) is a distinct type of dendrimers and it is based on a core of branched lysines on which multiple dendritic peptides are attached [1]. Such multivalent peptides have been demonstrated to show enhanced immunogenicity. MAPs with two, four, eight, or sixteen copies of synthetic peptide antigens can be produced by utilizing oligolysine cores with one to four sequential levels of lysine residues [2]. Here, we report two methodologies for both divergent (direct) and convergent (indirect) microwave-assisted synthesis of a MAP with two branches carrying the immunodominant 83-99 Myelin Basic Protein Epitope (MBP83-99) which is implicated in Multiple Sclerosis (MS). In the divergent approach, the MAP was synthesized on a solid support using the CEM Liberty automated microwave peptide synthesizer, while the branch of the MAP was introduced by coupling a Fmoc-Lys(Mtt)-OH residue on the core. Also, in order to prevent steric hindrance, -Ala was used as a spacer between the core and the two peptide epitopes. In the convergent synthesis, the core of the MAP and the peptide epitope were synthesized independently on a solid support using CEM Liberty [3], and coupled in a later step using CEM ExplorerTM, to give the final MAP. [1] Tam, J.P. (1988) Proc. Natl. Acad. Sci. USA, 85, 5409. [2] Crespo, L. et al. (2005) Chem. Rev., 105, 1663. [3] Friligou, I. et al. (2010) Amino Acids, doi 10.1007/s00726-010-0753-6.

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Approaches in the discovery of selective kinase inhibitors for the treatment of cancer.
Andreas Goutopoulos, Serono Pharmaceutical Company, USA.

The Raf/MEK/ERK cascade is a major survival signaling pathway regulating cell proliferation, differentiation and apoptosis. This pathway is often up-regulated in a variety of malignancies including melanoma, pancreatic, and colorectal cancer because of pathwayactivating mutations (ie: Ras, Raf, EGFR), resulting in increased proliferation and tumor progression. Two different approaches towards the discovery of selective, type III, MEK inhibitors will be presented. The first approach stemmed from a conventional HTS campaign and the second was a fragment-based approach. Both approaches resulted in allosteric inhibitors of MEK from varying scaffolds and with different biochemical and DMPK properties. Highlights of the lead optimization and candidate selection will be discussed, with particular focus on the characterization of the compound that is currently investigated in oncology clinical trials.

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QSAR Models that Stand the Test of Time

Andy Davis- Astra Zeneca Quantitative structure-activity models have been core computational chemistry tools since their popularization by Hansch and Fujita in the early 1960s. For many years they were limited in their applicability to congeneric series and small datasets. Research in the 1990s highlighted the potential to tune-down or tune-out ADME (absorption, distribution, metabolism, elimination) linked development attrition by prudent ADME screening in Discovery. The growing ADME datasets, and the fact that many of these endpoints were more bulk-property controlled, rather than structure-class specific, provided fruitful opportunities for computational chemistry to use QSAR tools to build global cross project ADME QSAR models. These have been very successful in building-in good ADME properties into molecules at the compound design-stage. More recently a similar focus on toxicology has provided tox-based QSAR models, allowing us to add T to the acronym to broaden the study to ADMET models. But QSARs are empirical models that only offer the potential to encode information provided within the description of the training set. Predictions can only confidently be made when the prediction molecule is within the domain of applicability of the original model. But molecular design evolves within a project, as data feedback guides compound design, and therefore the project chemical space tends to grow away from the original QSAR model. This poses a problem for QSAR models. We will describe our own exploration of the relationship between prediction quality and time, for a number of representative ADMET endpoints in Discovery projects. We will describe how we have identified robust machine learning techniques, and used these together with informatics automation to build, and update global cross-project and local series-specific ADMET QSAR models that can stand the test of time.

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Pharmacokinetic optimisation of acidic leads Missing links and self inflicted wounds
Peter Webborn The physicochemical/pharmacokinetic properties of acidic compounds present particular challenges for the optimisation of acidic leads. Poorly understood clearance mechanisms, transporter effects, toxic metabolites, low volumes of distribution can all contribute complex PK and PKPD. The susceptibility of acidic compounds to transporter mediated uptake and/or efflux; make this class of compounds particularly prone to nonmetabolic clearance mechanisms [1]. Hepatic uptake transporters add a further level of complexity and in certain circumstances may be the rate determining step in drug clearance [2]. Plasma protein binding is high for even moderately lipophilic acidic drugs, although the impact of this can be misinterpreted [3]. Description of acyl glucuronide metabolites as toxic compounds in an FDA guidance document [4], again potentially complicates the development of acidic compounds, but consideration of the pharmacokinetic properties may mitigate this risk [5]. This presentation explores these issues, possible misconceptions and ways forward. 1. International Transporter Consortium, Nat Rev Drug Discov. 2010 Mar;9(3):215-36 Membrane transporters in drug development. 2. Yamazaki M, Akiyama S, Nishigaki R, Sugiyama Y. Pharm Res. 1996 Oct;13(10):1559-64. Uptake is the rate-limiting step in the overall hepatic elimination of pravastatin at steady-state in rats. 3. Smith DA, Di L, Kerns EH. Nat Rev Drug Discov. 2010 Dec;9(12):929-39. The effect of plasma protein binding on in vivo efficacy: misconceptions in drug discovery. 4. FDA Guidance for Industry Safety testing of metabolites 2008
5. Regan SJ, et al., Biopharm. Drug Dispos. 31: 367395 (2010) Acyl

Glucuronides: The Good, The Bad and The Ugly

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Targeting Diabetes and Obesity: Structure based Design of Isozyme selective Inhibitors of Pyruvate Dehydrogenase Kinase.
Alexander A. Tokmakov Research Center for Environmental Genomics, Kobe University, Japan Targeting Diabetes and Obesity: Structure-based Design of Isozymeselective Inhibitors of Pyruvate Dehydrogenase Kinase Alexander A. Tokmakov Research Center for Environmental Genomics and Graduate School of Science, Kobe University, Rokko dai 1-1, Nada, Kobe 6578501, Japan tokmak@phoenix.kobe-u.ac.jp Regulation of glucose metabolism in cells and hence glucose homeostasis in the organism depends on activity of the pyruvate dehydrogenase complex (PDC), which catalyzes oxidative decarboxylation of pyruvate and provides acetyl-CoA and NADH for the tricarboxylic acid cycle and biosynthetic processes. In turn, PDC activity is regulated by interconversion between the active (nonphosphorylated) and inactive (phosphorylated) forms. This interconversion is catalyzed by highly specific pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs). Four genetically and biochemically distinct isozymes of the PDK family (PDK1, 2, 3 and 4) have been identified in mammals. They display different tissue distributions, phosphorylation site specificities, and binding affinities for the inner lipoyl domain (L2) of the PDC E2 subunit. Among them, PDK4 was found to be selectively upregulated in response to starvation and physical exercise. In addition, aberrant function of PDK4 has been implicated in type 2 diabetes and obesity. Thus, PDK4 is recognized as a potential therpeutic target for treatment of these diseases. Structure determination of individual PDK isozymes is important for understanding isoform- and tissue-specific regulation of PDK activity and for development of isozyme-selective inhibitors. Structures of several PDK-famiy proteins have been deposited in the Protein Data Bank. They provide a basis for rational design of isozyme-selective inhibitors of PDKs. The detailed comparison of these structures reveals important differences in the conformation of the nucleotide-binding site of PDKs. These differences can be exploited for development of novel isoform-selective PDK
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inhibitors targeting the ATP-binding site. Recently, inhibitor-bound structures of several PDK family proteins have been reported [1, 2]. The inhibitors used in these studies (Figure 1) can be considered as potential lead compounds for development of therapeutic drugs to treat type 2 diabetes and obesity. Figure 1. Chemical structures of PDK inhibitors. (A) AZD7545, (B) radicicol and (C) P4A (4-[4-(4-methoxy-phenyl)-5methyl-1H-pyrazol-3-yl] benzene- 1,3-diol).
REFERENCES

1. Kato, M., Li, J., Chuang, J.L., Chuang, D.T. Structure, 15, 992-1004, 2007. 2. Kukimoto Niino, M., Tokmakov, A., Terada, T., Ohbayashi, N., Fujimoto, T., Gomi, S., Shiromizu, I., Kawamoto, M., Matsusue, T., Shirouzu, M., Yokoyama, S. Acta Crystallogr Sect D, 67, in press, 2011.

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New Small Molecules for Modulating PhosphoinositideMediated Signaling: Development and Application.
Savvas Georgiades, University of Cyprus

Phosphatidylinositols are integral components of key signaling pathways, and their various phosphorylation states act as specific signals for keeping these pathways at check. Any deregulation of phosphorylation levels can form a basis for disease (e.g. cancer, diabetes). Small molecules can provide useful chemical biological tools for modulating aspects of these signaling pathways at will, thus affording new possibilities for development of treatments. Examples of such molecules from our recent synthetic experience will be presented, including psammaplysene A (a natural product) that regulates the cellular localization of FOXO transcript ion factors downstream of phosphoinositide signaling in cancer cells, a Zn(II)-hydroxamic complex that increases Akt kinase activation and provides promise for diabetes research, and a modular boronic acid molecule that directly alters phosphoinositides equilibrium.

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Rational Design for PLA2 Inhibitors as Anti inflammatory Drugs.

Varnavas D. Mouchlis,a,b Victoria Magrioti,a Efrosini Barbayianni,a Thomas Mavromoustakos,a Michael H. Gelb,c George Kokotos.a of Organic Chemistry, Department of Chemistry, University of Athens, Department of Cheminformatics, NovaMechanics Ltd, Nicosia, Cyprus, cDepartment of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA
aLaboratory

The phospholipase A2 (PLA2) superfamily consists of different groups of enzymes which are characterized by their ability to catalyze the hydrolysis of the sn-2 ester bond of phospholipid molecules. The products of PLA2s activity play several important roles in a variety of physiological processes. There are four main types of PLA2s: the secreted PLA2s (sPLA2s), the cytosolic PLA2s (cPLA2s), the calciumindependent PLA2s (iPLA2s), and the lipoprotein-associated PLA2s (LpPLA2s) [1]. Various potent and selective PLA2 inhibitors have been reported up to date and have provided outstanding support in understanding the mechanism of action and elucidating the function of these enzymes [2]. In an effort to develop new PLA2 inhibitors, our team has employed computer-aided drug design, organic synthesis and in vitro evaluation [3]. Our attention has been focused on GIIA sPLA2 and GIVA cPLA2, which are mainly involved in inflammatory diseases. The combination of molecular docking along with 3D-QSAR allows to study GIIA indole inhibitors [4]. By combining organic synthesis, in vitro evaluation and molecular docking, we have studied long chain 2oxoamide inhibitors based on -amino acids [5]. The study of the binding of 2-oxoamide inhibitors in the active site of GIVA cPLA2 by combining molecular docking along with molecular dynamics is in progress.

The active site of GIIA sPLA2 (PDB ID: 1DB4). The calcium ion and the catalytic dyad His47/Asp91 participate into the catalytic mechanism of the enzyme.

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REFERENCES

[1]. [2]. [3].

[4]. [5].

Burke, J. E.; Dennis, E. A., Cardiovasc. Drugs Ther. 23, 49-59, 2009. Magrioti, V.; Kokotos, G., Expert Opin. Ther. Pat. 20, 1-18, 2010. Mouchlis, V. D.; Barbayianni, E.; Mavromoustakos, T. M.; Kokotos, G., Curr. Med. Chem. 18, 2566-82, 2011. Mouchlis, V. D.; Mavromoustakos, T. M.; Kokotos, G., J. Chem. Inf. Model. 50, 1589-1601, 2010. Mouchlis, V. D.; Magrioti, V.; Barbayianni, E.; Cermak, N.; Oslund, R. C.; Mavromoustakos, T. M.; Gelb, M. H.; Kokotos, G., Bioorg. Med. Chem. 19, 735743, 2011.

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Synthetic Challenges in the Functionalization of [60]Fullerene for the Preparation of Fullerene Derivatives with Antioxidant and anti HIV Activity
Nikos Chronakis, Department of Chemistry, University of Cyprus, P. O. Box 20537, 1678 Nicosia, Cyprus. nchronak@ucy.ac.cy.

Over the past decade, surface-modified, water soluble fullerene derivatives (Figure, e,e,e and trans-3,trans-3,trans-3 trisadducts) have been shown to exhibit strong antioxidant activity against reactive oxygen species (ROS) in vitro and to protect cells and tissues from oxidative injury and cell death in vivo. Progress in developing fullerenes as drug candidates has been hampered by three development issues: 1) lack of methods for scalable synthesis, 2) inability to produce highly-purified, single-species regioisomers compatible with pharmaceutical applications, and 3) inadequate understanding of structure-function relationships with respect to various surface modifications (e.g., anionic versus cationic versus charge-neutral polarity). To address these challenges, we have designed and synthesized novel water-soluble fullerenes that can be purified as single isomers and which we believe can be manufactured to scale at reasonable cost [1].

O HO

O OH HO

O OH O HN N HN O O N H O H N O

NH NH2 HN NH NH2

OH O O OH HO

OH O O

HO O O OH HO

OH O O HN

N H HO NH2

e,e,e

trans-3,trans-3,trans-3

HN

Furthermore, we have become interested in the design and synthesis of new fullerene derivatives that exert improved inhibitory activity with the active site of the HIV-1 PR [2]. The complementary spatial relationship between [60]fullerene and the active site of the HIV-1 PR enzyme has led to the suggestion that fullerene-based derivatives could have potential use as effective HIV-1 PR inhibitors. Combining the results derived from molecular docking simulations with the principles of molecular recognition, we have designed the fullerene derivative 1 (Figure) which is a potential HIV-1 RP inhibitor with the highest predicted binding energy compared with standard HIV drugs, like amprenavir.
REFERENCES [1]. Witte, P., Beuerle, F., Hartnagel, U., Lebovitz, R., Savouchkina, A., Sali, S., Guldi, D.,Chronakis, N.,Hirsch, A., Org. Biomol. Chem., 5, 3599-3613, 2007. [2]. Durdagi, S., Mavromoustakos, T., Chronakis, N., Papadopoulos, M. G., Bioorg. Med. Chem., 16, 9957-9974, 2008.

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Bioconjugates to Specifically Render Inhibitors Water Soluble

Anna K. H. Hirsch, Organization/University: University of Groningen T. Masini, S. Wieczorek, H. G. Brner.

Water solubility is a recurring problem in chemical biology. In medicinal chemistry, an increase in affinity is often accompanied by a concomitant increase in hydrophobicity. The resulting inhibitors often suffer from low water solubility, precluding in vitro studies. A novel approach that renders inhibitors water-soluble is presented: a polymer peptide conjugate is designed, in which the polymer moiety affords water solubility, while the peptide sequence interacts specifically with the insoluble inhibitor,[1] leading to a soluble inhibitorbioconjugate complex.[2] This concept was applied to render small-molecule inhibitors of the kinase IspE,[3] an attractive antimalarial target, watersoluble. This strategy avoids compromising the inhibitor structure to afford water solubility, opening up a much wider part of chemical space.

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POSTER PRESENTATIONS

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Licorice Root Triterpenoids as Scaffolds for the Construction of New Medicinal Agents with Target Biological Activity
Lidia Baltina, Anton Budaev, Marina Khudobko, Lilia Mikhailova Institute of Organic Chemistry Ufa Scientific Centre of the Russian Academy of Sciences, prospect Oktyabrya ,71, Ufa, 450054, Russian Federation;E-mail: baltina@anrb.ru

Synthesis of novel biologically active compounds of medicinal use based on the low molecular plants metabolites is one of the important trends in the modern organic and medicinal chemistry. Pentacyclic triterpenoids are the wide distributed plants metabolites possessing different kinds of biological activities. Development of the triterpenoids chemistry last twenty years led to the opening of some unique biologically active substances to be of the interest for medicine as medicinal preparations for the treatment of viral, cancer and methabolic diseases. Licorice root are widely used medicinal plant known in medicine more than 5000 years. The major triterpenic acids of this roots are Glycyrrhizic and Glycyrrhetic acids (1) (GLA) which are known as anti-inflammatory, anti-ulcerogenic, anti-allergic, antioxidant, antitumor and antiviral agents. GLA is a perspective natural scaffold for the construction of novel antidiabetic agents due its possibility to inhibit 11 -hydroxysteroiddehydrohenase type 1 (HSD1), enzyme responsible for the exchange of glucocorticoids [1]. The present work is devoted to the target chemical modifications of some licorice root triterpenoids (14) to obtain novel derivatives and modified analogs for the structureactivity relationships study.

COOH O

COOH

HO (1) COOH

HO (2) COOH

HO (3)

HO (4)

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GLA (1) was isolated from Glycyrrhiza uralensis Fisher roots collected in Siberian regions of Russia, and oxidative and skeletal transformations of its derivatives (2-4) were carried out with the production of novel oxygen and nitrogen containing derivatives and analogs with modified rings A and C. Methods of synthesis of new derivatives of triterpenoids containing additional oxo- and hydroxyl functional groups were developed by the use of ozone, metachloroperbenzoic acid, dimethyldioxirane and airs oxygen as oxidants. The changes in ring A of the given group of triterpenoids were made with the transformation into A-nor, 1,2-en, 2-hydroxy, -carboxy, halogen and cyan-3-oxo-derivatives. Synthesis of novel conjugates of licorice triterpenoids and their 3-oxo-derivatives with aminoacides and dipeptides was carried out. New compounds are studing as antiviral, antitumor, anti-inflammatory and antidiabetic agents. This work was supported by the grants of RFBR (projects 08-03-00366, 11-0300452), SC-3756.2010 and SK 14.740.11.0367.
REFERENCES

[1]. Tolstikov G.A., Baltina L.A., Grankina V.P., Kondratenko R.M., Tolstikova T.G. Licorice: biodiversity, chemistry, and application in medicine, II, V, Academic Publishing House Geo, Novosibirsk, 2007.

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Synthesis and Antiviral Activity of New Glycyrrhizic Acid Analogous

Baltina, aOlga Stolyarova, aLidia Baltina*, bTatyana Ilina, bOlga Plyasunova, cRaffaello Pompei aInstitute of Organic Chemistry Ufa Scientific Centre of the Russian Academy of Sciences, prospect Oktyabrya, 71, Ufa, 450054, Russian Federation; E-mail: baltina@anrb.ru bState Scientific Centre Virology and Biotecknology Vektor, Koltsovo, Novosibirsk region, Russian Federation cUniversita di Cagliari, Sezione di Microbiologia Applicata, Via G.T.Porcell, 4-09124 Gagliari, Italy.
aLia

Search of novel antiviral agents for the treatment of different viral infections is one of the important tasks of the modern medicinal chemistry and drug discovery due to the wide distribution of HIV, viral hepatitis B and C, and the appearance of new socially dangerous viral infections such as avian flu, swine flu and imerging infections causing by influenza A viruses. Glycyrrhizic acid (GA) is the main biologically active triterpene saponin of licorice root (Glycyrrhiza glabra L., Gl. uralensis Fisher)possessing multi-medicament properties including anti-inflammatory, anti-ulcer, anti-allergic, anti-dote, antioxidant, anti-tumor and antiviral activity. Chemical modification of GA is a perspective rout to construct new antiviral agents. Herein we describe the synthesis of novel GA analogues with modified aglycon, and its antiviral activity (anti-HIV-1 and anti-influenza A) in vitro. Reduction of GA by using an excess of NaBH4 in 2-propanol-water mixture under boiling leads to the formation of crude glycoside 1 with 95-100% yields after the acidification of the reaction mixture with 5% hydrochloride acid. The pure 3 -[2-O- -D-glucopyranosyl- -Dglucopyranosyl)-oxy]-olean-9(11),12(13)-dien-30-oic acid 1 was isolated by column chromatography on silica gel using of chloroform-methanolwater mixtures with 45-50% yields.

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COOR 12 13

11 9 COOR O OH HO COOR O OH HO OH O (I) O

Cytotoxicity and anti-HIV-1 activity of compound 1 were studied on the traditional model of primary HIV infected lymphoid MT-4 cells by using HIV-1/EVK strain. Cytotoxicity of glycoside 1 was estimated as the concentration of compound causing 50% of cells death (CD50). AntiHIV-1 activity of compound 1 was estimated by the measurement of the virus specific protein 24. It was found that glycoside 1 is nontoxic for cells (CD50 24000 g/ml) and in 12.3 times less cytotoxic as compared to GA. Tested compound 1 possesses an expressed anti-HIV-1 activity in MT-4 cells and effectively inhibits the accumulation of HIV-1 p24 protein. HIV-1 RT RNA-dependent DNA polymerase activity was determined using 0.2 units/ml of poly(rA)-oligo(dT)12-18, 2 nM RT, 5 M [3H] TTP and it was found that tested glycoside inhibited HIV-1 RT RNA-dependent DNA polymerase activity. The 50% inhibitory concentration (IC50) relative to the drug-free control was 3.10.4 mM. GA is not active as an inhibitor of RT HIV-1. Cytotoxic and antiinfluenza A viral activity was performed on MDCK cells for the Influenza A virus (strain WSN). The maximum non toxic dose (MNTD50), that is the maximum dose of compound inhibiting the cell multiplication of less than 50%, was calculated by the regression analysis. The antiviral activity was determined as the cytopathic effect in the presence of the compound by the viral plaque method. Glycoside 1 showed MNTD of 250 g/ml and an IC50 on Influenza A virus (strain WSN) of 8 g/ml, which resulted in protection index (PI) of 31.2. In the same conditions GA demonstrated MNTD of 2000 g/ml and IC50 of 125, with a PI of 16.

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What could be Expected after Anti TLR4 Antibodies Interaction with Target Receptor?
D.S. Kabanov and I.R. Prokhorenko, Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia kabanovd1@rambler.ru

TLR4 is a toll-like receptor expressed by monocytic as well as neutrophilic cells and recognizing microbial molecules such as lipopolysaccharides (LPS) of Gram-negative bacteria [1]. It is well known that minimal LPS concentrations prime neutrophils for enhanced release of reactive oxygen species (ROS; respiratory burst) in response to very weak secondary stimulus, such as the chemotactic factor formyl-methionyl-leucyl-phenylalanine (fMLP) [2]. LPS binding protein (LBP), an acute-phase plasma protein, binds to LPS, leading to efficient LPS interaction with the cell surface receptor CD14 [3]. The complex of LBPLPSCD14 or LPSCD14 then interacts with TLR4, by which transmembrane signals are generated to affect cellular function [4]. Although TLR4 is considered to be the primary receptor for LPS [5], his role in neutrophil priming by S- or Re-LPS structures is not clear at present. So, in our study we addressed the question of whether neutralization of TLR4 by mouse anti -human TLR4 monoclonal antibodies (Ab) HTA125 (Serotec), might interfere with priming of human neutrophils by S- or Re-LPS from Escherichia coli (E. coli). Human neutrophils were isolated from heparinized blood of healthy volunteers by standard procedure [6]. To assess the functional role of TLR4, neutrophils were incubated with or without HTA125 or isotype control IgG2a for 30 min before stimulation with S- or Re-LPSE.coli. Then, samples containing 2 105 cells, 2% autologous serum, glucose and luminol in Ca2+-PBS buffer (pH 7.3), were placed in chemiluminometer\'s chambers (37C). Next the cells were primed by S- or Re-LPSE.coli (100 ng/ml) for 30 min (37C). ROS production was triggered by addition of fMLP (1 M). The chemiluminescence (CL) reaction was monitored continuously for 7 min. Results were expressed as CL units, which were calculated as the area under the curve of
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millivolts versus time (in minutes). Neutrophils which were not treated by HTA125 were used as the control. The obtained results on ROS release from human neutrophils (n = 6) during the first 50 sec after fMLP addition are summarized in the table: The effects of anti-TLR4 mAb on fMLP-induced chemiluminescence of LPS-primed and unprimed neutrophils Effect of LPS priming on ROS release Effect of anti-TLR4 mAb + LPS on ROS release Re-LPS S-LPS Control Control + TLR4 mAb S-LPS Re-LPS 121 20 173% 108 20 154% 70 11 100% 73 14 100% 141 29 193% 161 28 221% From obtained results we concluded that the HTA125 increase the ROS generation by LPS-primed neutrophils in response to fMLP. The Re-LPS structure caused the most neutrophil priming effect than that did S-LPS structure in comparison with the controls. The obtained results are in a good agreement with the date presented by Blomkalns et al (2011) indicating that while R-LPSE. coli K12LCD25-induced interleukin-8 release by human monocytes was TLR4-dependent (i.e., blocked by HTA125 (eBioscience, San Diego, CA)), superoxide formation was largely TLR4-independent [7]. They suggested that activation of the NADPH oxidase by LPS is mediated through alternative receptors in human monocytes [7]. However the same clone of anti-TLR4 Ab HTA125 (eBioscience) inhibited respiratory burst of neutrophils upon simultaneously addition of Ab and LPSE. coli O111:B4 to the cells [8]. To explore the possibility that HTA125 influence on CD11b up-regulation we estimated the neutrophil CD11b surfac e expression after the cell incubation with HTA125. Our results indicated that the increase of CD11b surface expression during experimental procedure did not attribute to HTA125. Thus we suppose that the HTA125 interaction with TLR4 might cause particular conformational changes in target receptor organization which in turn induced effects similar to those of LPS priming. Also, we hypothesize that unexpected response of HTA125-treated neutrophils to LPS and fMLP might be explained by extensive LPS loading to another neutrophil LPS receptors when LPS-binding site of TLR4 is blocked. Further studies are warranted to elucidate the mechanism of HTA125induced amplification of ROS release from LPS-primed neutrophils in response to fMLP. REFERENCES: [1]. Beutler B., and Poltorak A., Eur. Cytokine Netw., 11, 143152, 2000. [2]. Yan R.S., Al-Hertani W., Byers
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D., Bortolussi R., Infect. Immun., 70, 40684074, 2002. [3]. Wright S.D., Ramos R.A., Tobias P.S., Ulevitch R.J., Mathison J.C., Science, 249, 1431 1433, 1999. [4]. Henneke P., and Golenbock D.T., Nat. Immunol., 2, 828 830, 2001. [5]. Medzhitov R., and Janeway C. Jr., Trends. Microbiol., 8, 452456, 2000. [6]. Haslett Ch., Guthrie L.A., Kopaniak M.M., Johnston R.B., Henson P.M., Am. J. Pathol., 119, 101110, 1985. [7]. Blomkalns A.L., Stoll L.L., Shaheen W., Romig-Martin S.A., Dickson E.W., Weintraub N.L., Denning G.M., J. Inflammation, 8, 414, 2011. [8]. Stadlbauer V., Mookerjee R.P., Wright G.A.K., Davies N.A., Jrgens G., Hallstrm S., Jalan R., Am. J. Physiol. Gastrointest. Liver Physiol., 296, 1522, 2009.

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Preparation and Antimycobacterial Evaluation of Substituted Pyrazinecarboxamides

Martin Dolezal, Cynthia Carillo, Jan Zitko, Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Krlov, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic; e-mails: martin.dolezal@faf.cuni.cz.

Tuberculosis (TB), the worlds leading infectious disease, caused by Mycobacterium tuberculosis, remains a poverty-related disease mostly in developing countries. Today, nine million individuals still develop TB annually, of whom three million die. The situation is worsening because of the increasing incidences of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB [1].
O O CH2 N N R
1

NH R2

N N R
1

N H

R2

R1 = H, Cl, alkyl; R2 = H, alkyl, Cl, CF3, NO2, etc. 1 2

Design, synthesis approach, results of in vitro antimycobacterial evaluation, and study of structure-activity relationships of various pyrazinoic acid derivatives are presented. This communication deals with some pyrazinamide analogues/prodrugs derived from N-phenyl/Nbenzylpyrazine-2-carboxamides (1,2) [2]. The promising pyrazine candidates for further antimycobacterial evaluation were discovered. Results give good view onto structure-activity relationships of these analogues and promise even better activity of new compounds prepared after some structure optimization experiments.
This study was supported by the Ministry of Education of the Czech Republic (MSM0021620822) and by the Ministry of Health of the Czech Republic (IGA NS 10367-3). REFERENCES
[1].The Stop TB Partnership. World Health Organization. Available from: http://www.stoptb.org/assets/documents/global/plan/SSP_DRUGWG.pdf [2].Articles: Authors surname, name initial, journal title (italicized), volume number (bold), pages, printing year. [2].Dolezal, M.; Zitko, J.; Osicka, Z.; Kunes, J.; Buchta, V.; Vejsova, M.; Dohnal, J.; Jampilek, J.; Kralova, K. Molecules, 15, 856781, 2010.

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Salicylanilide esters active against MDR tuberculosis

Vinov J a., Krtk M. a, Stola kov J. b, a Charles University in Prague, Faculty of Pharmacy in Hradec Krlov, Department of Inorganic and Organic Chemistry, Heyrovskho 1203, 500 05 Hradec Krlov, Czech Republic vinsova@faf.cuni.cz a Laboratory for Mycobacterial Diagnostics and TB, Institute of Public Health in Ostrava, 702 00 Ostrava, Czech Republic

Mycobacterium tuberculosis remains one of the most significant human pathogens since its discovery in 1882. WHO estimated that about one-third of the worlds population is currently infected with the bacillus in its latent form, 9.4 million of new cases develop each year and 1.7 million of deaths from TB were determined in 2009 [1]. Increasing number of multidrugresistant tuberculosis (MDR-TB), extensively drug-resistant (XDR-TB) and most recently emergence of totally resistant (TDR) tuberculosis is alarming [2]. Recently, we have found promising antimycobacterial activity of salicylanilide esters with N-protected amino acids [3,4] and salicylanilide carbamates [5] active against several atypical strains of Mycobacteria and MDR-TB strains. Thus, we have designed and synthesised a new group of salicylanilide esters, with activity against MDR strains in MIC 0.125-0.5 mol/L.
O R1 O O R3 N H R2

All the prepared compounds were tested in vitro for their antimycobacterial activity against M. tuberculosis (331/88) and some nontuberculous strains, M. avium (330/88) and M. kansasii (235/80 and 6509/96) and five resistant strains. The details about synthesis and activity of the series of the salicylanilide derivatives will be presented.
This work was financially supported by the Research project MSM 0021620822 and IGA NS 10367-3. REFERENCES

[1]. Global tuberculosis control: a short update to the 2010 report. http://whqlibdoc.who.int/publications/2010/9789241564069_eng.pdf [2]. Vinov J., Krtk M. Development of New MDR-Tuberculosis Drugs. Nova Science Publishers. New York, 2010, ISBN 978-1-61668-233-0 [3]. Krtk M., Vinov J. et al. Eur. J. Med. Chem. 45, 6106-6113, 2010. [4]. Imramovsk A., Vinov J. et al. Bioorg. Med. Chem. 17, 3572-3579, 2009. [5]. Frriz J. M., Vvrov K. et al. Bioorg. Med. Chem. 18, 1054-1061, 2010.

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Synthesis and Biological Evaluation of 4 Substituted 7, 10 Dihydro 6H [1, 3] diazepino [1, 2 e] purines
A. Thalassitisa, D. J. Hadjipavlou-Litinab, K. E. Litinas,a,aLaboratory of Organic Chemistry, Chemistry Department, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece, klitinas@chem.auth.gr, bDepartment of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece, hadjipav@pharm.auth.gr.

Nucleosides, modified nucleosides [1] and cyclonucleosides [2] represent classes of compounds that possess very interesting biological activities, especially antiviral and anticancer. We have reported recently the synthesis [3] of fused dihydropyrido[e]purines via ring closing metathesis reactions and the study [4] of the reactions of 9-allylpurines with mesityl nitriloxide followed by the evaluation of their products as lipid peroxidation and thrombin inhibitors. In continuation to these studies we like to present here the coupling of 6-morpholinyl derivative [3] 1a with allylamine (2) in the presence of catalytic amount of CuI, or under MW irradiation, to the derivative 3a and the RCM reaction of the later in the presence of the 2nd generation Grubbs catalyst (4) to the title 4-morpholinyl-dihydrodiazepine compound 5a. Analogous reactions sequence from 6-piperidinyl and 6-pyrrolidinyl derivatives 1b,c resulted to the 4-substituted dihydrodiazepines 5b,c respectively. These compounds have been tested presenting significant promising results as lipoxygenase, lipid peroxidation and thrombin inhibitors.

Acknowledgements: This work was supported by the Program HRAKLEITOS II (A. Thalassitis). REFERENCES [1]. Herdewijn, P. Modified Nucleosides in Biochemistry, Biotechnology and Medicine; Wiley- VCH: Weinheim, Germany, 2008. [2]. Mieczkowski, A.; Roy, V.; Agrofoglio, L. A. Chem. Rev. 110, 1828 (2010). [3]. Litinas, K. E.; Thalassitis, A. Tetrahedron Lett. 51, 6451 (2010). [4]. Thalassitis, A.; Hadjipavlou-Litina, D. J.; Litinas, K. E.; Miltiadou, P. Bioorg. Med. Chem. Lett., 19, 6433 (2009).

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New adamantane derivatives with sigma affinity and antiproliferative activity.

Stefanos Riganas,a,b Ioannis Papanastasiou,a George B. Foscolos,*a Andrew Tsotinis,a Vassilios N. Kourafalos,b Andreas Eleutheriades,b Vassilios I. Moutsos,b Humaira Khan,b Prassa Margarita,b Stavroula Georgakopoulou,b Angeliki Zaniou,b Stavroula Pondiki b and Alexandre Vamvakides b, a Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Athens, Panepistimioupoli-Zografou, 157 71 Athens, Greece, Fax: (+30) 210-7274747, b Anavex Life Sciences, 27 Marathonos Avenue, 153 51 Pallini, Athens, Greece. Presenting authors e-mail address: papanastasiou@pharm.uoa.gr.

Sigma ( ) receptors have been recently involved in anticancer activity [1, 2]. In this work, the synthesis of 4-(1-adamantyl)-4,4diarylbutylamines 1, 5-(1-adamantyl)-5,5-diarylpentylamines 2 and 6(1-adamantyl)-6,6-diarylhexylamines 3 is described and the 1, 2receptors and sodium channels binding affinity of compounds 1 are investigated. The in vitro activity of compounds 1, 2 and 3 against main cancer cell lines is significant. One of the most active analogs, 1a, had an interesting in vivo anticancer profile against the ovarian cancer cell line IGROV-1, which was associated with an anagelsic activity against the neuropathic pain induced by the main anticancer drugs [3].
REFERENCES [1]. Maurice, T.; Su, P.T. Pharmacol. Ther.,124, 195-206, 2009. [2]. Kashiwagi, H.; McDunn, E.J.; Simon Jr, O.P.; Goedegebuure, S.P.; Vangveravong, S.; Chang, K.; Hotchkiss, S.R.; Mach, H.R.; Hawkins, G.W. J. Transl. Med., 26(7), 24, 2009. [3]. de la Puente, B.; Nadal, X.; Portillo-Salido, E.; Snchez-Arroyos, R.; Ovalle, S.; Palacios, G.; Muro, A.; Romero, L.; Entrena, M.J.; Baeyens, M.J.; Lpez-Garca, A.J.; Maldonado, R.; Zamanillo, D.; Vela, M. J. Pain , 145, 294-303, 2009.

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Inhibition of Monoamine Oxidase by C5 substituted Phthalimide Analogues

Clarina I. Manley-King, Jacobus J. Bergh, and Jacobus P. Petzer. Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, 2520, South Africa, email: kobus.bergh@nwu.ac.za.

Literature reports that isatin as well as C5- and C6-substituted isatin analogues are reversible inhibitors of human monoamine oxidase (MAO) A and B [1], [2], [3]. In general, C5- and C6-substitution of isatin leads to enhanced binding affinity to both MAO isozymes compared to isatin and in most instances result in selective binding to the MAO-B isoform. Crystallographic and modeling studies suggest that the isatin ring binds to the substrate cavities of MAO-A and -B and is stabilized by hydrogen bond interactions between the NH and the C2 carbonyl oxygen of the dioxoindolyl moiety and water molecules present in the substrate cavities of MAO-A and B [4]. Based on these observations and the close structural resemblances between isatin and its phthalimide isomer, a series of phthalimide analogues were synthesized and evaluated as MAO inhibitors.
R
O R NH O

C6H5O C6H5CH2O C6H5CH2CH2O C6H5(CH2)3O (E)-C6H5CH=CHCH2O 2-Naphthalenyl-O

4-BrC6H4CH2O 4-BrC6H4CH2CH2O 4-BrC6H4O Isatin 5-benzyloxyisatin 6-benzyloxyistin

Figure 1. Phthalimide analogues used in this study

While phthalimide and N-aryl-substituted phthalimides were found to be weak MAO inhibitors, phthalimide homologues containing C5 substituents were potent reversible inhibitors of recombinant human MAO-B with IC50 values ranging from 0.007 to 2.5 M and moderately potent reversible inhibitors of recombinant human MAO-A with IC50 values ranging from 0.22 to 9.0 M.
REFERENCES [1]. Hublek, F.; Binda, C.; Khalil, A.; Li, M.; Mattevi, A.; Castagnoli, N., Jr.; Edmondson, D. E. J. Biol. Chem., 280, 15761-15766, 2005. [2]. Van der Walt, E. M.; Milczek, E. M.; Malan, S. F.; Edmondson, D. E.; Castagnoli, N., Jr.; Bergh, J. J.; Petzer, J. P. Bioorg. Med. Chem. Lett., 19, 25092513, 2009. [3]. Manley-King, C. I.; Bergh, J. J.; Petzer, J. P. Bioorg. Med. Chem., 19, 261-274, 2011. [4] Binda, C.; Li, M.; Hublek, F.; Restelli, N.; Edmondson, D. E.; Mattevi, A. Proc. Natl. Acad. Sci. U. S. A., 100, 9750-9755, 2003.

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Determination of Residues in the Third Membrane Spanning Segment of CRF1, which are Accessible in the Ligand Binding Site Crevice of Receptor
K. Gkounteliasa, K. Spyridakia, P. Giannopoylosa, A. Akritidesa, M. Papadokostakia, T. Mavromoustakosb and G. Liapakisa, aDepartment of Pharmacology, Faculty of Medicine, University of Crete, Greece, liapakis@med.uoc.gr, bUniversity of Athens, Department of Chemistry, Laboratory of Organic Chemistry, Panepistimiopolis Zographou 15771, Greece.

The type 1 receptor (CRF1) for the corticotropin releasing factor (CRF) is a plasma-membrane receptor, which belongs to family B of G-protein coupled receptors (GPCRs) and consists of seven membrane-spanning segments (TMs). Previous studies have proposed that the third TM (TM3) as well as the other TMs of CRF1 most likely bind small nonpeptide ligands, such as antalarmin [1, 2]. Recently, we have shown that, similar to family A, GPCRs, the TMs of CRF1 form a wateraccessible binding crevice, which extends from the extracellular surface of the receptor into the plane of the membrane [3]. The surface of this crevice must be formed by residues that contact ligands, such as antalarmin, as well as, by other residues that may play a structural role and affect binding indirectly. In this study, we mapped the TM residues that form the surface of the binding-site crevice of CRF1, starting from the extracellular portion of the TM3. We achieved this by applying the cysteine-substituted accessibility method (SCAM) and using as background the Cys mutant of CRF1, which has near normal functional properties and it is relatively insensitive to the hydrophilic, positively charged sulfhydryl-specific reagent, methanethiosulfonate ethylammonium (MTSEA). We mutated eight TM3 residues of CRF1 to Cys, one at a time and heterologously expressed the mutants in HEK 293 cells. Four of these mutants, Thr192Cys, Ala193Cys, Tyr195Cys, and Asn196Cys reacted with the MTSEA, added extracellularly. We therefore suggest that the side chains of the residues at the reactive loci (Thr192, Ala193, Tyr195, and Asn196) are on the water-accessible surface of the binding-site crevice of CRF1. The pattern of accessibility is consistent with an alpha-helical conformation for this segment of CRF1.
[1] [2] [3] Liaw, C. W., Grigoriadis, D. E.; Lorang, M. T.; De Souza, E. B.; Maki, R. A. Mol Endocrinol, 11, 2048-53, 1997. Hoare, S. R.; Sullivan, S. K.; Ling, N.; Crowe, P. D.; Grigoriadis, D. E. Mol Pharmacol, 63, 751-65, 2003. Gkountelias, K.; Papadokostaki, M.; Javitch, J. A.; Liapakis, G. Mol Pharmacol, 78, 785-93, 2010.

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Synthesis, Competition Binding and Conformational Studies of Lnear and Cyclc Analogues of C Terminal Hexapeptide of Neurotensin
R. Exarchakoua, K. Spyridakid, , P. Zoumpoulakisb, C. Potamitisb, N. L. Assimomytisa, M. Venihakic, P. Cordopatisa, G. Liapakisd and V. Magafaa, aDepartment of Pharmacy, University of Patras, GR-26500 Patras, Hellas, bLaboratory of Molecular Analysis, Institute of Organic and Pharmaceutical Chemistry, NHRF, 48 Vas. Constantinou Ave, GR-11635 Athens, Hellas, cDepartment of Clinical Chemistry, dDepartment of Pharmacology, Faculty of Medicine, University of Crete, GR-71003 Heraklion, Crete, Hellas, liapakis@med.uoc.gr.

Neurotensin (NT), a tridecapeptide (pGlu-Leu-Tyr-Glu-Asn-Lys-ProArg-Arg-Pro-Tyr-Ile-Leu) was first isolated from bovine hypothalamus. NT displays a wide spectrum of biological actions in the central nervous system as well as the periphery of different mammalian species [1]. At central level, NT plays the role of neurotransmitter and/or neuromodulator, whereas in the anterior pituitary it regulates hormone secretion. In addition NT plays an important role in the function of the digestive tract and the cardiovascular system of mammals as well as in the regulation of growth of normal and cancer cells. The plethora of NT actions is mediated through its binding to NT receptors (NTRs). The NTRs are plasma membrane proteins which belong to the family of Gprotein coupled receptors [2]. The C-terminal hexapeptide fragment of NT [NT(8-13)], which contains the necessary structural requirements to bind and elicit biological effects at NT receptors, is an obvious lead compound for development. However NT(8-13) is rapidly degraded by peptidases. Therefore, it is important to synthesize functional NT analogues with stabilized bonds against metabolic deactivation. Based on the above, we herein report the synthesis of new linear and cyclic analogues of NT with modifications in the basic structure of the Cterminal part of the molecule needed for function in order to improve the metabolic stability. The analogues contain D-Tyrosine(Ethyl) [DTyr(Et)] or D-1-naphtylalanine [1-Nal] in position 11, D-Arginine in position 8 or 9 and L-Lysine in position 8 or 9. They were synthesized by the Fmoc/But solid phase methodology [3] utilizing 2-chlorotrityl chloride resin and are now being tested for their functional properties in binding studies. Competition radioligand binding studies were
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performed in membrane homogenates from HT-29 cells endogenously expressing NTRs [4]. A series of NMR spectra including 1H, 2D TOCSY and 2D NOESY were recorded on a Varian 600MHz spectrometer for two cyclic analogues in order to elucidate their structure. The conformational properties of the two peptides, as well as similarities and differences between them are currently studied using NMR spectroscopy and Molecular Dynamics simulations.

REFERENCES [1]. Carraway, R. and Leeman, E.S. J.Biol. Chem. 248, 6854-6861 (1973). [2]. Vincent, J.P., Mazella, P., Kitabgi, P. Trends Pharmacol. Sci. 20, 302-309 (1999). [3]. Fields, B.G., Noble, L.R. Int. J. Pept. Prot Res. 35, 161-214 (1990). [4]. Morinville, A., Martin, S., Lavalle, M., Vincent, J.P, Beaudet, A. and Mazella, J. Int. J. Bioch. & Cell Biol. 36, 2153-2168 (2004).

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Design of Multi Targeted Antihyperlipidemic Agents

Angeliki Kourounakis*, Alexios Matralis, Maria Katselou and Anastasios Nikitakis

Over the past years, evidence has accumulated suggesting that free radicals, and the consequent lipid peroxidation they induce, could trigger most of the factors involved in atherosclerotic vascular injuries, such as cytotoxicity, inflammation, and formation of atheromatic plaques. Oxidative modification of LDL can lead to its uncontrolled uptake by macrophages. This event triggers a cascade of cellular processes that lead to the formation of fatty streaks and eventually atherosclerotic lesions in the arterial wall. As multiple mechanisms are involved in the development of atherosclerosis (hyperlipidemia, oxidative stress and inflammation), agents with at least two mechanisms of action may offer a therapeutic benefit compared to those only targeting a single mechanism. Thus, we applied a strategy that involves the design of a single chemical compound able to simultaneously modulate more than one targets. In this study, we present the design and synthesis of novel benzoxazine (1 and 3) and benzothiazine (2 and 4) derivatives, that inhibit in vitro hepatic microsomal lipid peroxidation (IC50 of the most potent is 9 ), LDL oxidation (complete inhibition at 10 ), as well as squalene synthase activity (IC50 values 4.9-16.5 ). Docking studies of these derivatives revealed a different mode of binding to the active site of SQS compared to reference compounds[1,2]. Further, these compounds show antidyslipidemic and antioxidant properties in vivo, decreasing total cholesterol, LDL, tri glyceride and MDA levels of hyperlipidemic rats by 26-74 %. Finally, by determining their in vivo concentration (up to 24 h) in target tissues (i.e. blood/liver) it is shown that both 1 and 2 reach their targets in the lower M range. The new compounds seem interesting multifunctional molecules aimed towards the development of a new pharmacophore for disease-modifying agents useful in the treatment of atherosclerosis. [1] Kourounakis, A.P. et al. Lipid-lowering (hetero) aromatic tetrahydro-1,4-oxazine derivatives with antioxidant and squalene synthase inhibitory activity. J. Med. Chem. 2008, 51, 5864-5865. [2] Kourounakis, A.P. et al. Design of more potent squalene synthase inhibitors with multiple activities. Bioorg. Med. Chem. 2010, 51, 7402-7412.

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Isoxazole Substituted Chromans Against Oxidative Stress Induced Neuronal Damage

Alexandra Tsatsaronia, Maria Koufakia, Xanthippi Alexia, Sofia Zervab, Michael N. Alexisb, Isoxazole Substituted Chromans Against Oxidative Stress-Induced Neuronal Damage. National Hellenic Research Foundation, Institute of Organic and Pharmaceutical Chemistry, 48 Vas. Constantinou Ave., 11635 Athens, Greece; .bNational Hellenic Research Foundation, Institute of Biological Research and Biotechnology, 48 Vas. Constantinou Ave., 11635 Athens, Greece.

The 1-benzopyran ring system constitutes the basic skeleton of a variety of natural compounds with interesting biological activities. In addition, several biologically active synthetic 3,4-dihydro-2H-1-benzopyrans (chromans) have been reported, suggesting that derivatives of this benzopyran motif may be capable of interacting with a variety of cellular targets. Our group has long been involved with the design and synthesis of cardioprotective and neuroprotective chroman derivatives [1-7]. In an effort to optimize our neuroprotective chromans, new 2- or 5-substituted chroman analogues bearing isoxazole moieties. were synthesized using the copper(I)-catalyzed cycloaddition reaction between in situ generated nitrile oxides and terminal acetylenes [8]. The activity of these compounds against oxidative stress-induced death (oxytosis) of neuronal HT22 cells was evaluated and interesting SARs for this group of analogues were derived. The majority of new chroman analogues exhibited high in vitro neuroprotective activity displaying EC50 values below 1 M and lacked cytotoxicity. In general, the position of the substituents on the isoxazole ring influences the activity of the regioisomers. The absence of hydrogen bond between the hydroxyl group at position 6 of the 5-substituted chroman derivatives and the isoxazole nitrogen in conjunction with the presence of electron donating substituents on the 3-aryl-isoxazoles or a second chroman moiety results in strong neuroprotective activity (EC50 ~ 0.3 M).
References [1] Koufaki M., Calogeropoulou T., Detsi A., Roditis A., Kourounakis A., Papazafiri P., Tsiakitzis K., Gaitanaki C., Beis I., Kourounakis P. J. Med. Chem. 2001, 44, 4300 [2] Koufaki M., Calogeropoulou T., Rekka E., Chryselis M., Papazafiri P., Gaitanaki C., Makriyannis A. Bioorg. Med. Chem. 2003, 11, 5209 [3] Koufaki M., Detsi A., Theodorou E., Kiziridi C., Calogeropoulou T., Vassilopoulos A., Kourounakis A., Rekka E., Kourounakis P., Gaitanaki C., Papazafiri P. Bioorg. Med. Chem. 2004, 12, 4835 [4] Koufaki M., Theodorou E., Galaris D., Nousis L., Katsanou E.S, Alexis M.N. J. Med. Chem. 2006, 49, 300 [5] Koufaki M., Kiziridi C., Papazafiri P., Vassilopoulos A., Varr A., Nagy Z., Farkas A., Makriyannis A. Bioorg. Med. Chem. 2006, 14, 6666 [6] Koufaki M., Kiziridi C., Alexi X., Alexis M.N., Bioorg. Med. Chem., 2009, 17, 6432 [7] Koufaki M., Theodorou E., Alexi X., Alexis M.N., Bioorg. Med. Chem., 2010, 18, 3898 [8] Hansen, T.V.; Wu, P.; Fokin, V.V. J. Org. Chem., 2005, 70, 7761

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Synthesis and Melatoninergic Action of New C4 and C6 Methoxylated N1 OMe N acyl tryptamines
Andrew Tsotinis*a, Rodanthi Kompogennitakia and David Sugdenb, Synthesis and Melatoninergic Action of New C4 and C6-Methoxylated N1-OMe N-acyl tryptamines.

of Athens, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Panepistimioupoli-Zografou, Athens 157 71, Greece; bDivision of Reproduction and Endocrinology, School of Biomedical & Health Sciences, Room 2.12N Hodgkin Building, King's College London, London Bridge, London SE1 1UL, U.K. Melatonin (N-acetyl-5-methoxytryptamine) is widely distributed in nature, from plants, bacteria and unicellular algae to humans [1]. Although melatonin is synthesised in numerous organs (pineal, retina, lymphocytes, gastrointestinal tract, etc.), circulating melatonin in mammals is mainly of pineal origin [1]. Melatonin has been shown to have a hypnotic action in animals and humans and there has been considerable recent interest in the therapeutic potential of melatonin analogues as hypnotics and as agents for restoring circadian rhythms disturbed by jet-lag, shiftwork and aging [2]. Melatonin exerts some of its effects by activating specific, high affinity, G-protein-coupled membrane receptors. Three distinct melatonin receptor subtypes have been cloned MT1, MT2, and Mel1c. MT1 and MT2 receptor mRNA has been identified in several mammalian tissues, but the Mel1c mRNA has not been found in mammals [3]. In our ongoing effort to probe the stereoelectronic requirements for optimal melatoninergic activity we have recently reported the synthesis of two N1-substituted indoles, compounds 1 and 2, which showed up to 5-fold potency of that of melatonin in the Xenopus laevis melanophore model. This enhancement in melatoninergic activity was ascribed to the presence of the second methoxyl at N1, which acts synergistically to the 5-methoxyl group as a result of the non-classical I, +R effect it exerts to the aromatic indole nucleus via the N1 heteroatom [4].

aUniversity

As an extension of this work we report herein the synthesis and melatoninergic activity of two new series of N1-OMe indoles (compounds 3 and 4). In these ligands, the pharmacophoric N1-OMe moiety, present in 1 and 2, has been retained, but the benzene methoxyl has been translocated to C4 and C6, respectively. 92
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Preliminary Xenopus laevis assay data suggest that the C6-methoxylated analogues 4 are antagonists. Conversely, two of their C4-OMe substituted congeners 3 (R=Me and Et) are partial agonists, whereas the butyramido analogue 3: R=n-Pr is a pure agonist (pEC50 = 7.061).
References [1] Reiter, R. J. Endocr. Rev. 1991, 12, 151. [2] Delagrange, P.; Guardiola-Lemaitre, B. Clin. Neuropharmacol. 1997, 20, 482. [3] Morgan, P. J.; Barratt, P.; Howell, H. E.; Helliwell, R. Neurochem. Int. 1994, 24, 101. [4] Tsotinis, A.; Eleutheriades, A.; Hough, K.; Sugden, D. Chem. Commun. 2003, 382.

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Synthesis and Melatoninergic Potency of New N [x (y Methoxy 6,11 dihydroindolo[1,2 b]isoquinolin 11 yl)alkyl]alkanamides
Andrew Tsotinis*1, Vasiliki Belekou1 and David Sugden2, 1University of Athens, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Panepistimioupoli-Zografou, Athens 157 71, Greece; 2Division of Reproduction and Endocrinology, School of Biomedical & Health Sciences, Room 2.12N Hodgkin Building, King's College London, London Bridge, London SE1 1UL, U.K.

Melatonin (N-acetyl-5-methoxytryptamine), a hormone synthesized and released from the pineal gland at night, has been shown to have a hypnotic action in animals and humans and there has been considerable recent interest in the therapeutic potential of melatonin analogues as hypnotics and as agents for restoring circadian rhythms disturbed by jet-lag, shiftwork and aging.1 The physiological actions of melatonin in regulating seasonal and circadian rhythms are thought to be mediated through a family of specific, high affinity, G-protein-coupled cell membrane receptors. Two receptor subtypes (MT1 and MT2) have been cloned in mammals2 which, when expressed in host cells, show the general pharmacological characteristics of native melatonin receptors. Notwithstanding the renewed interest in melatonin and its actions in recent years, the pharmacology of melatonin is in its infancy. In a series of studies we have sought to understand how melatonin binds to and activates its receptor and to use the knowledge gained to design subtype specific receptor agonists and antagonists, which will be useful tools for defining the full physiological and pathophysiological role of this hormone. In 2001 we published on the synthesis and biology of the tricyclic N-[2-(2-methoxy-6,7,8,9-tetrahydropyrido[1,2- ]indol-10yl)ethyl]alkanamides (I), which act as probes of the constraints at the hormones receptor site with regard to the lower N1-C2 region of the indole moiety of melatonin.3

I: R=CH3, C2H5, n-C3H7, c-C3H5, c-C4H7 II: n=1, 2; R=CH3, C2H5, n-C3H7

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As an extension of this work we synthesised the N-[x-(y-methoxy-6,11dihydroindolo[1,2-b]isoquinolin-11-yl)alkyl]alkanamides (II), in which the side chain of (I) was translocated from C3 (indole numbering) to C11 and a phenyl ring was annulated to the N1-C2 cyclohexane. The biological activity of the new analogues is currently underway; preliminary Xenopus laevis assay data suggest that the new molecules are antagonists.
References 1. Delagrange, P.; Guardiola-Lemaitre, B. Clin. Neuropharmacol. 1997, 20, 482-5102. 2. Reppert, S.M.; Godson, C.; Mahle, C.D.; Weaver, D.R.; Slaugenhaupt, S.A.; Gusella, J.F. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 8734-8738. 3. Tsotinis, A.; Panoussopoulou, M.; Sivavavthan, S.; Sugden, D. Il Farmaco, 2001, 56, 725-729.

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Bioconjugates to Specifically Render Inhibitors Water Soluble

T. Masini, S. Wieczorek, H. G. Brner, A. K. H. Hirsch

Water solubility is a recurring problem in chemical biology. In medicinal chemistry, an increase in affinity is often accompanied by a concomitant increase in hydrophobicity. The resulting inhibitors often suffer from low water solubility, precluding in vitro studies. A novel approach that renders inhibitors water-soluble is presented: a polymer peptide conjugate is designed, in which the polymer moiety affords water solubility, while the peptide sequence interacts specifically with the insoluble inhibitor,[1] leading to a soluble inhibitorbioconjugate complex.[2] This concept was applied to render small-molecule inhibitors of the kinase IspE,[3] an attractive antimalarial target, watersoluble. This strategy avoids compromising the inhibitor structure to afford water solubility, opening up a much wider part of chemical space.

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NM MR Spe ectroscop py and Dock d king stu udy Ag g(tpp)3(a asp) to so oybean l lipoxyge enase 1

of f

comp plex

E. V Vrontakia, S.K. Had djikakoub, S. Golic Grdadolni c,d, T. Ma G ik avromous stakosa

aCh hemistry

D Department Universi of Athe t, ity ens, Panepistimiopolis Zografo 15771, ou, bInorga Ath hens, Gree ece anic and A Analytical Chemistry Departm y, ment of Ch hemistry, Uni iversity of Ioannina 45110, Ioannina, Greece cLaboratory of Biom f a, y molecular Stru ucture, Na ational Institute of Chemistry, Hajdrih hova 19, SI-1001, Ljubljana, L Slov venia EN-F FIST Centr of Excell re lence, Dun najska 156, SI-1000 Ljubljana, Sl lovenia.

In a course o studies on metalotherape of s eutics, a n new mixed ligand silver (I) d s com mplex Ag g(tpp)3(asp) (Fig.1 was synthesize and c 1) ed characteri ized by elem mental an nalyses, sp pectroscopic techn niques and X-ray cr d rystallogr raphy at am mbient con nditions [1 We ha 1]. ave initia ated studi using STD (Sat ies turation 1H-NM Tra ansfer Diff ference) [2] MR experim ments to investigat if this complex te c is b binding to LOX us o sing differ rent exper rimental c conditions Both so s. onicated and not soni d icated sam mples show that aromatic ring of as wed spirin ado opts two con nformers t that depend on the experim e mental con nditions an their in nd ntensity rati ios are 2: and 0.5:1 corresp :1 pondingly. In additi ion, tpp w found to have was com mpletely d different c conformat tion than present as a single molecule or in a e e com mplex form This indicates t m. that not only aspir but tp adopt distinct o rin pp con nformation during their bind ns g ding proc cess. Proba ably, this is an evid dent that the cavity of the LOX induces a certain fit that lea the com e f f ads mplex to adjust a pro oper comp plementar conform ry mation.

Fig.1. M Molecular str ructure of complex Ag c g(tpp)3(asp)

REFEREN NCES [1]. M. P Poyraz, C.N Banti, N Kourkou N. N. umelis, V. Dokorou, M D M.J. Manos, M. Simic, S. Golic , , Grda adolnik, T. Mavromoustakos, A.D Giannouli I.I. Vergi D. is, inadis, K. C Charalabopo oulos, S.K. Had djikakou, DOI:10.1016/j.ica.2011.04 4.032 (accept manuscr ted ript) May B. Meyer J. Am. Che Soc., 123, 6108-6117, 2001. yer, r. em. ,

[2].

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Use of Dynamic Combinatorial Mass Spectrometry to Identify Potent Prolyl Hydroxylase Domain 2 Inhibitors
Marina Demetriades, Ivanhoe K.A. Leung, Rasheduzzaman Chowdhury, Esther Woon, Christopher J. Schofield, Organization/University: Oxford University.

Use of Dynamic Combinatorial Mass Spectrometry to Identify Potent Prolyl Hydroxylase Domain 2 Inhibitors Marina Demetriades, Ivanhoe K.A. Leung, Rasheduzzaman Chowdhury, Esther Woon, Christopher J. Schofield From the Chemistry Research Laboratory, Department of Chemistry,University of Oxford, Mansfield Road, Oxford OX1 3TA, United Kingdom marina.demetriades@chem.ox.ac.uk Dynamic combinatorial chemistry (DCC) involves the generation of a diverse compound library from a set of small amount of molecules that react reversibly.(1) The targets, e.g. enzymes, can act as templates shifting the equilibrium position towards the compounds that are the best binders. DCC has been applied to identify new ligands for proteins with the aim to assist in pharmaceutical design. Protein mass spectrometry (MS) is a fast and sensitive technique that requires relatively small amount of samples. These properties have attracted interest in the use of protein MS in DCC (DC-MS). HIF prolyl hydroxylases (PHDs) are Fe(II) and 2OG dependent oxygenases which catalyse hydroxylation on Pro402 and Pro564 of HIF- , promoting its degradation.(2) Under hypoxia condition these enzymes are less active, hence HIF- is stabilized and can form the HIF dimer to trigger responses to hypoxia, like erythropoiesis and angiogenesis. Thus, inhibition of PHDs, and more specifically PHD2, is an attractive target for the treatment of anemia, ischemic diseases and cancer.(3) Herein we report the development of a novel DC-MS approach to the discovery of potent inhibitors for this enzyme.
REFERENCES [1] Ramstrm O. and Lehn J.M., Nat. Rev. Drug Discov., 1, 2636, 2002. [2] Epstein A.C., Gleadle J. M., et al, Cell, 107, 4354, 2001. [3] Fraisl P., Aragons J. and Carmeliet P., Nat. Rev. Drug Discov., 8, 139-152, 2009.

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Neurohormone Melatonin: Solubility and Controlled Release from Cyclodextrin Vehicles

Marilena Vlachoua*, Marianna Papamichaela, Irene Fragkoulia and Rodanthi Kompogennitakib , aFaculty of Pharmacy, Department of Pharmaceutical Technology, National and Kapodistrian University of Athens, Panepistimioupoli-Zografou, 15771 Athens, Greece, bFaculty of Pharmacy, Department of Pharmaceutical Chemistry, National and Kapodistrian University of Athens, Panepistimioupoli-Zografou, 15771 Athens, Greece

Controlled drug delivery systems of the hormone melatonin for oral, transdermal and transmucosal route have been widely investigated to deliver melatonin in a physiological fashion.1 The very variable oral bioavailability of melatonin, possibly resulting from extensive first-pass metabolism and variable absorption,2 prompted us to investigate the solubilization and controlled release of the hormone from cyclodextrin vehicles. The controlled release studies were conducted in two dissolution media of pH 7.4 and 1.2 using a series of hydrophilic matrix tablets. Each tablet was comprised of HPMC K 15M, microcrystalline cellulose (Avicel PH 102), low viscosity sodium alginate, magnesium stearate and complexes of melatonin with each of the cyclodextrins: CD, -CD, -CD and the derivatives HP- -CD, HP- -CD, HP- -CD, methyl- -CD and sulfated- -CD in different stoichiometric melatonin:cyclodextrin ratios. Host-guest interactions in aqueous solution were studied by Nuclear Magnetic Resonance (NMR) Spectroscopy. The stability constants were calculated by monitoring the spectrophotometric properties of melatonin (UV-Vis) in the presence of increasing concentrations of the cyclodextrin used. The dissolution data obtained suggest that, in general, the complexes of melatonin exhibit faster dissolution than free melat nin in both dissolution media. This is due to the increased solubility of the drug:cyclodextrin inclusion complex.
REFERENCES [1] [2] Lee, B.-J.; Choi, H.-G.; Kim, C.-K.; Parrott, K.A.; Ayres, J.W.; Sack, R.L. Arch. Pharm. Res., 1997, 20, 560-565 and references cited therein. Lane, E.A.; Moss, H.B. J. Clin. Endocrinol. Metab., 1985, 61, 1214-1216.

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Structural Elucidation Conformational Properties and Molecular Docking Study at Bace 1 of Bioactive Molecule BV6
1University

C. Koukoulitsa1, G. Agelis2, A. Resvani2, J. Matsoukas2, T. Mavromoustakos1,3 of Athens, Department of Chemistry, Laboratory of Organic Chemistry, Athens 2Department of Chemistry, University of Patras, Patras 26500, Greece 3National Hellenic Research Foundation, Vas. Constantinou 48, 11635 Athens, Greece

A non peptide compound, BV6, has been synthesized as AT1 antagonist and its chemical structure shown in Fig.1, has been elucidated using a combination of 2D COSY and 2D NOESY experiments. Its conformational properties have been explored using a combination of conformational search methodologies and 2D NOESY experiments. BV6 has been found to mimic the action of losartan as the biological results showed. As a promising bioactive molecule was screened for several receptors in order to explore the possibility to serve as an inhibitor of other targets. The molecular docking results showed binding of BV6 at the human aspartic protease BACE-1. BACE-1 is the initial protease that processes amyloid precursor protein (APP) in the pathway, leading to A proteins. Thus, BACE-1 has emerged as a promising pharmaceutical target for combating Alzheimer disease. These results propose the testing of the molecule for potential beneficial properties against Alzheimer disease.

Fig. 1 Chemical structure of BV6

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Do ocking, M Molecula Dynam ar mics and NMR S d Spectrosc copy Stu udy of two n new Cy ytotoxic C2_Sub bstituted Pyrro d olo [2,3 f] 3 Qu uinolines s
Var rvarigou N Nikolettaa, Megariotis Gregoryc, Tsotinis Andrewb, Papadopu ulos c,, M Manthos Mavromoustakos Thomasa. aChe emistry De epartment, University of Ath hens, Panep pistimiopo olis-Zograf fou, 15771; bFaculty o Pharma ; of acy, Univer rsity of A Athens, Pa anepistimioupolis-Zo ografou, 15 5771 Athe ens, Greece cInstitut of e; te Org ganic and Pharma d aceutical Chemistry Nation y, nal Hellen nic Resea arch Fou undation, A Athens, Gr reece;

DN cross-l NA linking ag gents are known for their significan antitum e nt mor acti ivity, whic has bee attribut to thei ability to form irre ch en ted ir o eparable base b pair adducts at precise defined genomic locations Most of the repor ely d c s. f rted g nvolve three or four aromatic chromophores in th r heir cross-linking probes in eleton and are of suf fficient siz to recog ze gnize two or three b base pairs [1]. ske An extension of this lim n mited sequ uence reco ognition, w which is at ttained by the y e cles, has be found to drastic een d cally enha ance use of larger aromatic heterocyc cyto otoxicity due to th formati he ion of mo rigid i ore irreversibl interstr le rand bon [2]. In the course of our program di nds irected tow wards the developm ment of new DNA A-complex xing agen nts, we ha ave previo ously rep ported on the nthesis of the follow wing C2-su ubstituted pyrrolo[2 2,3-f]quino olines 1 an 2 nd syn (Sch heme 1) [3 The pr 3]. romising b biological properties of the co s ompounds led s us to apply a combina ation of 2 NMR (NOESY) data and Monte Carlo 2D ( nformation analys in orde to explore the co nal sis er onformatio onal space of e con these molecu ules and correlate th obtaine results with thos of with the he ed se ntrolled re elease in s simulated aqueous gastric an intestin media In nd nal a. con add dition, mo olecular d dynamics simulation were c ns conducted in aque d eous env vironment in order to study their inte eractions w with lipid bilayers and doc cking stud with D dies DNA.
1. X= =(CH2)3NH H(CH2)4NH H(CH2)3 N2-{ {[3-(4-{3-[(1H H-pyrrolo[2 2,3-f]quinoli ine-2-carbon nylamino)] propy ylamino}but tylamino)pr ropyl]}-1H-p pyrrolo[2,3-f f]quinoline-2 2carbo oxamide 2. X= =(CH2)4NH H(CH2)3 N2-{ {3-[{4-[(1H-p pyrrolo[2,3-f -f]quinoline-2carbo onylamino)]butylamino} }propyl]}-1H H-pyrrolo[2, ,3-f]quinolin ne-2carbo oxamide

Ref ferences 1) Mattes, W.B.; Hartley, J.A.; Kohn, K.W Mathes W.; son, D.W. Carcinogen nesis 1988, 9, 2 2065-2072. 2) Subhas Bose, D.; Thompson A.S.; Ching, J.; H n, C Hartley, J.A.; Berard dini, M.D.; Jen nkins, T.C Neidle, S Hurley, L.H.; Thu C.; S.; , urston, D.E J. Am. Ch E. hem. Soc. 1992 114, 4939 2, 9-4941. 3) Tsotinis, A.; Vlac , chou, M.; Gerasim mopoulou, M.; Eikos sipentaki, A.; Ioannidi C.; Klou is, uvidaki, A. Afroudakis P.A.; M .; Moreau D.; Roussakis C. ; s, Lett. Dru Des. Discov. 2007, 4 87-91. ug 4, 94101

Thermal and Dynamic Properties of Drug AT1 Antangonist Olmesartan in Lipid Bilayers using variousphysical chemical Methodologies
Dimitrios Ntountaniotis,1,2 Gregor Mali,3 Simona Golic Grdadolnik,4,5 Halabalaki Maria,6 Alexios-Leandros Skaltsounis,6 Constantinos Potamitis,2 Eleni Siapi,2 Petros Hatzigeorgiou7, Michael Rappolt8, Thomas Mavromoustakos9 1Department of Chemistry, University of Patras, Patras 26500, Greece, 2National Hellenic Research Foundation, Institute of Organic and Pharmaceutical Chemistry, 48 Vas. Constantinou, 11635, Athens, Greece 3laboratory For Inorganic Chemistry And Technology, National Institute Of Chemistry, Hajdrihova 19, Si-1001 Ljubljana, Slovenia 4Laboratory of Biomolecular Structure, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia 5EN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia 6Division of Pharmacognosy and Natural Products Chemistry, School of Pharmacy, University of Athens, Zografou, Athens 15711, Greece 7University of Athens, Department of Chemistry, Laboratory of Physical Chemistry, Zographou 15771, Athens, Greece 8 Institute of Biophysics and Nanosystems Research (IBN), Austrian Academy of Sciences, 8042 Graz, Austria 9University of Athens, Department of Chemistry, Laboratory of Organic Chemistry, Zographou 15771, Athens, Greece.

Angiotensin II receptor blockers (ARBs) have been designed to inhibit the binding of angiotensin II (AII) to the AT1 receptors. Olmesartan medoxomil belongs to the antihypertensive class of ARBs. This drug is an ester prodrug of the active metabolite (olmesartan), which is deesterified in the gastrointestinal tract. In this study, solid state NMR (ssNMR), Raman spectroscopy, x-ray diffraction as well as Differential Scanning calorimetry (DSC) were applied to investigate the thermal and dynamic properties of olmesartan in L -dipalmitoylphosphatidylcholine (DPPC) bilayers in the absence and presence of cholesterol. Cholesterol is a major component of the cell plasma membrane and its role is essential to establish proper membrane permeability and fluidity. The aim of this research work is to study olmesartan/lipid interactions in a temperature range from 20oC up to 50oC covering all mesomorphic phases. The results are compared with those obtained with other AT1 antagonists in order to investigate the role of lipid bilayers in the drug action.
OH O N

24 22

OH

3
O N O O O N

23
O

5 2 6 1 4 7 8 9 10
N

21
OH

13 12 11 14 15 16 19 18 17

N N N N H N

20
N N

a) Olm esartan m edoxom il

b) Olmesartan

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Using Protein Binding Site Comparison to Guide Drug Repositioning; From Human to Trypanosome Phosphodiesterase Inhibitors
Chimed Jansena, Kristina Orrlinga, Iwan de Escha, Rob Leursa, Chris de Graafa Center for Drug Research, Division of Medicinal Chemistry, VU Amsterdam, The Netherlands, email: chimed.jansen@vu.nl Drug repositioning has proven to be a valuable route to the discovery of novel treatments. Applying marketed drugs to new indications allows the costs of drug development to largely be side stepped. Clearly the binding sites of the known and novel targets must be highly similar in order to bind the same drug. This logic has encouraged the application of binding site comparison tools in the search for new opportunities for drug repositioning. An example of drug repositioning from the field of neglected diseases is that of eflornithine. Developed as a potential cancer drug in 1977 by Merrell Toraude & Co, eflornithine found application as a topical hair growth inhibitor. The same target enzyme, ornithine decarboxylase, plays a vital role in polyamine production in Trypanosoma brucei.1 This resulted in the eflornithine becoming one of the most successful treatments for African sleeping sickness to date. Using the same drug target, or looking at the same protein family, in a different species is a way to anticipate drug site similarity. The trypanosomal Phosphodiesterases (PDEs) B1 and B2, were recently validated as new drug targets for the control of Trypanosoma brucei by knock down experiments.2 This inspired the hope of repositioning drugs targeting human PDEs as trypanocidals. A binding site comparison was performed to identify the human PDE which showed greatest overlap with the trypanosome PDEs. In this study a database of 126 published phosphodiesterase (PDE) crystal structures was compared with a homology model of a trypanosome PDEB1. The study identified human PDE4B as the most likely candidate from which to attempt to reposition drugs for use against trypanosomal PDEB1. A new method of comparing protein binding sites was employed for this study. This involved the application of ROCs3 to the search for similarity in protein binding sites. ROCs was designed for the comparison of small molecules. This novel implementation of ROCs proved successful at identifying pocket similarity.
,aLeiden/Amsterdam

REFERENCES [1].Bacchi, C.; Nathan, H.; Hutner, S.; McCann, P.; Sjoerdsma, A., Science, 210, 332-334, 1980. [2].Oberholzer, M.; Marti, G.; Baresic, M.; Kunz, S.; Hemphill, A.; Seebeck, T., The FASEB Journal, 21, 720-731, 2007. [3].Grant, J. A.; Gallardo, M. A.; Pickup, B. T., Journal of Computational Chemistry, 17, 16531666, 1996.

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CURRICULUM VITAE

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Afantitis Antreas PhD, MBA: Currently he is the director of NovaMechanics Ltd. He has a strong scientific background in the field of chemoinformatics, bioinformatics and medicinal & materials chemistry. Recently he has developed and published a virtual activity evaluation procedure in order to develop potent therapeutic agents for the treatment of different diseases by understanding the interactions at the molecular level. The main field of the application of his research is drug discovery, property design and prediction. His scientific work has been published in 26 original research articles in international peer reviewed journals (h-index: 13). He is a reviewer in leading scientific journals and an evaluator of international competitive grants. Currently he is the coordinator and scientific officer of the following funded grants: Development of Robust Computational Models of Chemical Toxicity for Health and Environmental Risk Assessment 2009-2011, In Silico Modelling, Prediction, Synthesis and Biological Evaluation of Novel Rheumatoid Arthritis Inhibitors 2009-2011, "New Algorithms for Host Pathogen Systems Biology" (PATHOSYS) FP7 Health funded project (20102014 Cypriot Principal Investigator) and "Methods for Acceleration of Ageing and Corrosion tests" Eureka Project in collaboration with Saab, Scania, Ericsson etc (2011-2012 Cypriot Project Coordinator & Scientific Officer). Belekou Vasiliki. Ph D, Faculty of Pharmacy Department of pharmaceutical Chemistry (2009), Master, Faculty of Pharmacy Department of pharmaceutical Chemistry (2008), Degree Pharmacy Department (2005), National and Kapodistrian University of Athens. Attended the 17th European Symposium of Organic Chemistry, Crete (2011), Annual Meeting and Exposition, USA, (2010, 2009), XX th International Symposium on Medicinal Chemistry (EFMCISMC 2008) Vienna, Austria, 5 th International Symposium on Pharmaceutical Chemistry Istanbul, Turkey (2007). Boss Christoph received his Ph. D. in Organic Chemistry in 1996 (University of Bern, Switzerland). After working as a postdoctoral fellow at The Scripps Research Institute in LaJolla, he worked in process development for 1 year and then moved on to Actelion Pharmaceuticals in early 1999 to take over the position as Labhead in Medicinal Chemistry. He was involved in cardiovascular projects (endothelin antagonists and renin inhibitors) and in CNS projects with a focus on orexin antagonists. His favorite hobby project is trying to find novel antimalarials by educating students in a collaborative effort with the SwissTPH. Davis Andy read Chemistry at Imperial College of Science and Technology and studied the mechanisms of rearrangements of penicillins for a PhD in Physical Organic Chemistry with Professor MI Page at University of Huddersfield. He joined Fisons PLC Pharmaceuticals Division in 1988, which became part of the Swedish pharmaceutical company Astra AB in 1995. Astra merged with Zeneca PLC in 1999 to form Astra Zeneca PLC. His scientific interests the energetic of drug-receptor interactions, structure-based ligand design, the importance of physical properties to compound quality, QSAR methods and the co-operative application of physical organic and

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computational chemistry to drug discovery. He has recently moved to Sweden with AstraZeneca as senior principal Scientist in Chemistry with the Respiratory and Inflammation Innovative Medicines in Gothenburg. Davies Tom is an Associate Director at Astex Therapeutics, with a joint position in the structural biology and computational chemistry departments. Joining Astex in 2001 as a protein crystallographer, he has been responsible for leading fragment-based drug discovery projects against several therapeutic targets. He has extensive experience of structure-based design, with particular expertise in kinases, and interests in molecular recognition and energetics. Before Astex, he carried out postdoctoral research at the Laboratory of Molecular Biophysics, University of Oxford on cyclindependent kinase inhibitors. He holds a BSc in Chemistry and a DPhil in protein crystallography from the University of York, UK. Goutopoulos Andreas received his Pharmacy degree from the University of Thessaloniki, Greece, and his Ph.D. in Medicinal Chemistry at the University of Connecticut under the supervision of Professor Alexandros Makriyannis. Right after the completion of his doctoral work in the area of cannabinoids in 2000, he joined the US site of Serono as Medicinal Chemist. In 2007, after Serono was acquired by Merck KGaA, he continued working at EMD Serono, the US subsidiary of Merck Serono. He has worked on medicinal chemistry programs in a variety of target classes including kinases, GPCRs, nuclear receptors, hydrolases, and others, some of which resulted in compounds that entered clinical trials. His current title is Director of Medicinal Chemistry. Hadjipavlou-Litina Dimitra: B.S. in Pharmacy; PhD in Pharmaceutical Chemistry. Postdoc with C. Hansch, Pomona College, California. Professor of Pharmaceutical Chemistry, School of Pharmacy, University of Thessaloniki, Greece. Research interests: computer-aided drug design, synthesis, biological evaluation of new anti-inflammatory, cardiovascular /antioxidant agents, inhibitors of enzymes implicated in inflammation, agents and QSAR. Award by the American Chemical Society for the most assessed paper for 2005 (Chem. Rev). Editor and editorial board in several scientific Journals and author for more than 150 papers. Hubbard Rod has spent his entire academic career at the University of York. During the 1980s, he developed and applied methods in molecular graphics and modelling. Through the 1990's he helped to build and directed the York Structural Biology Laboratory, studying the structure and function of many classes of proteins, including many therapeutic targets. Since 2001, he has spent varying amounts of his time at the company now called Vernalis, adopting and adapting methods of structure based drug discovery. Jacobs T. Robert is Director of Chemistry at SCYNEXIS Inc. in Research Triangle Park, NC, with responsibility for Neglected Disease research projects including the human African trypanosomiasis effort sponsored by the Drugs for Neglected Diseases initiative (DNDi). Prior to joining SCYNEXIS in 2005, Bob was Associate Director of Medicinal Chemistry at AstraZeneca

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Pharmaceuticals, where he managed Lead Optimization Chemistry activities for projects targeting CNS diseases. Bob received his Ph.D. in Organic Chemistry from Cornell University, and was a postdoctoral associate with Prof. Scott Denmark at the University of Illinois, Urbana-Champaign.
Kleanthous Marina, director of the Molecular Biology and Thalassaemia Department Kleanthous Marina, at The Cyprus Institute of Neurology and Genetics has extensive experience in the diagnosis, prenatal diagnosis and Preimplantation Genetic Diagnosis (PGD) for haemoglobinopathies and other rare diseases. Her research interests involve the development of non-invasive prenatal diagnosis (NIPD) for thalassaemia, identification of new HbF inducing agents and the development of gene therapy for thalassaemia treatment.

Kompogennitaki Rodanthi, PhD in Medicinal Chemistry (2009) Department of Pharmaceutical Chemistry, University of Athens, MSc in Medicinal Chemistry (2008) Department of Pharmaceutical Chemistry, University of Athens, BS in Chemistry (2006),Department of ChemistryUniversity of Crete. Kontoyianni Maria is an Assistant Professor in Medicinal Chemistry, in the School of Pharmacy. She holds a Ph.D. in computational chemistry from the University of North Carolina, Chapel Hill, where she worked under the supervision of Professor Phil Bowen. After a post-doctoral fellowship with Professor Terry Lybrand at the University of Washington, she joined ZymoGenetics, where she focused on ligand-based design and homology modeling. She then moved to Johnson & Johnson and Procter & Gamble, assuming positions of greater responsibility focusing on various therapeutic targets in all stages of drug discovery. In her most recent post, she was the Head of Drug Discovery in a small biotechnology firm in the Science Park of Barcelona. She holds seven patents, is a Clinical Advisors consultant and an expert evaluator of the European Union large scale (multi-million) grant applications integrating academic and industrial partnerships. Her laboratory focuses on the classification of structural data pertaining to ligand-protein complexes, development of computational tools to better understand ligand recognition by macromolecular targets, and drug discovery approaches to specific disease areas. Langer Thierry began his career at Leopold-Franzens-University of Innsbruck in 1992 after completing a post doctoral fellowship with Prof. C.-G. Wermuth. He established the Computer Aided Molecular Design Group, and was appointed Professor of Pharmaceutical Chemistry in 1997. His scientific work, which has culminated in 160 articles, together with more than 200 lectures and poster presentations at scientific meetings, led to the founding of the spin-off company Inte:Ligand GmbH. Since 2008, he is CEO of Prestwick Chemical, Strasbourg-Illkirch, France. Makriyannis Alexandros, Ph.D., holds the George D. Behrakis Trustee Chair in Pharmaceutical Biotechnology at Northeastern University and is Professor of Pharmaceutical Sciences, Chemistry, and Chemical Biology and Director of Northeastern Universitys Center for Drug Discovery. Dr. Makriyannis has spent the past two decades inventing and applying chemical, biochemical and

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biophysical approaches to study the endocannabinoid signalling system and exploring its therapeutic potential. He obtained his Ph.D. degree at the University of Kansas and his post-doctoral training at the University of California, Berkley. He has published over 360 papers and has trained more than 250 students and fellows. Mavromoustakos Thomas, Born in Cyprus, graduate Chemist, University of Athens, Msc-Medicinal Chemistry, University of Connecticut, Ph.D, in 1990. B.Sc-Theology School of Athens University-2007. Research director of Laboratory of Molecular Analysis at the Institute of Organic and Pharmaceutical Chemistry (IOPC) of National Hellenic Research Foundation (1991-2007) where he is continuing exerting research activity as scientific collaborator. Associate Professor at the University of Athens since 2007. Mouchlis Varnavas: Earned his PhD in Computer-Aided Drug Design and Organic Synthetic Chemistry at the University of Athens (Greece), under the supervision of Professor George Kokotos. While completing his PhD, he had four articles published in international, peer-reviewed journals. Funding included grants from the Research Promotion Foundation of Cyprus in two bilateral agreements (Cyprus-Slovenia and Cyprus-Romania). He joined NovaMechanics Ltd and worked in the Computational Toxicology programme, focusing on estrogen receptors alpha and beta. His scientific background includes 3D-QSAR, molecular docking, molecular dynamics and organic synthetic chemistry. Murray James graduated from the Biology Department of the Leeds University in 1996 having studied the structure and function of Ribozymes. He then took a Post-doctoral position with Professor William Scott at University of California, Santa Cruz, focussing on the crystallisation of catalytically relevant conformations of ribozymes. In early 2001 he joined Vernalis to establish a crystallography group, initially focussed on bacterial ribosomal sub-units, before moving onto protein targets and an interest in other biophysical methods and their application in drug discovery. Senderowitz Hanoch completed his Ph.D. studies in computational organic chemistry with Professor Benzion Fuchs at Tel Aviv University in 1993. Between 1993 and 1997, He was a Post Doctorate Fulbright Fellow with Professor Clark Still in the Macro Model development team at Columbia University. In 1997, He joined the Israeli pharmaceutical industry first at Peptor Ltd. and later at EPIX Pharmaceutical. In 2009 He joined Bar Ilan University as an associate professor at the Department of Chemistry where He head a laboratory of molecular modeling, computer aided drug design and cheminformatics. Sparkes Andrew graduated in 1994 from Southampton and Zurich Universities having studied bioorganic Chemistry and with a PhD project titled the Biosynthesis of Archaemycin antibiotics supported by GSK. He then took a position with Professor Tom Simpson at Bristol University, focussing on elucidation of the biosynthetic pathways of various antibiotic

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producing bacteria. He joined Chemical design in 1996 as a support scientist; then Tripos in 1999 as Application scientist and in 2004 joined Schrodinger as Account Manager. Theodosiou Jason joined Aureus in mid 2006. Jason brings extensive experience in the Life Science Informatics business. He has held various senior management positions at MDL Information Systems, SciQuest, LION bioscience AG and Elsevier MDL. Jason holds a BSc/MSc and PhD in Organic Chemistry from the University of Aix-Marseilles and an Executive MBA from the ESCP Business School of Paris. Topiol Sid received his B.S. from CCNY and his Ph.D. from NYU. He trained as a theoretical quantum chemist. He post-docked at Northwestern with Mark Ratner and Arthur Frost, and at Carnegie-Mellon with John Pople (Noble laureate.) He first joined The Mount Sinai School of Medicine in the Department of Pharmacology and moved to pharma, starting at Berlex, then Novartis and then Lundbeck. He has worked on all aspects of Computer Aided Drug Design in interdisciplinary project environments. In 2010, he formed 3D-2Drug, a company which provides the means for the most efficient discovery activities through advanced scientific approaches. Webborn Peter graduated in Biochemistry from Cardiff University in 1982 and subsequently completed a PhD studying the metabolism of alkylsulphates in vivo and in isolated perfused organs. With over 20 years in drug discovery/development with AstraZeneca, He has a longstanding interest in using pharmacokinetic knowledge in drug design, driven by the recognition that its key to integrating, interpreting and extrapolating DMPK/Phys chem information. Over the last decade he has developed an interest in how to assess and analyse the impact of transporters on drug elimination.

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