Structure-based virtual screening –

Case studies
Prof. Dr. Wolfgang Sippl
Department of Pharmaceutical Chemistry

Virtual Screening

Virtual Screening decreases the number of

compounds to be experimentally tested

Filtering for compounds or libraries that are either

lead-like, or drug-like, or have the potential of oral
bioavailability

Compounds which are similar to a an already identified

active (lead) – Similarity-based Screening

Compounds which share a common pharmacophore -

Pharmacophore Sreening

Compound which fit to a protein binding pocket –

Target-based Screening, Docking

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 Virtual Screening

?
Biological Testing of a
small subset

Scoring function compound libraries

Binding mode

Filtering unwanted compounds

Docking

3D-Puzzle

Docking/Virtual Screening

3 Ligand Docking and Scoring

4

5 Binding strength?
Score

6 Score
7
X-Score
8 RMSD

0 5 10 15 20

Sampling Problem Scoring Problem

2
 Docking - Accuracy

200 Protein-Ligand Complexes

ParaDockS, University of Halle www.paradocks.org

Correlation scores – binding affinities

ParaDockS

poor correlation

3
 Virtual Screening

• Beside poor correlation between docking

scores and biol. data VS is able to provide
good enrichments in database screening

• Often combination of ligand- and protein-

based VS yields higher hit rates (lower
number of false positives)

Virtual Screening - Example

• Considered Database
• e.g. Chembridge Database ~400.000 compounds
• Reliable 3D Structures
• Generation of stereoisomers/protonation state
• Multi-conformations e.g. Omega (OpenEye)
• Pre-Docking Filters
• Pharmacokinetic properties (MW, TPSA, Lipinski, ..)
• Pharmacophore Screening (LigandScout)
• Multiconf databases necessary
• Docking (e.g. GOLD)
• Use of docking constraints
• Post-Docking Filters
• Post-docking filters
• Visual inspection of binding mode

4
 I. Target-basierte Suche nach
Hemmstoffen der Protein-Arg-
Methyltransferase (PRMT1)

R. Heinke, R. Meier, W. Sippl

Institut für Pharmazie
Martin-Luther-Universität Halle-Wittenberg
Martin-Luther-University Albert-Ludwigs-Universität
Halle-Wittenberg Freibug
A. Spannhoff, E. Huda, M. Jung
Institut für Pharmazeutische Wissenschaften
Albert-Ludwigs Universität Freiburg

Epigenetic Regulation

Epigenetic regulation

Chromatin DNA Methylation

Cytosin methylation represses
transcriptional activity

Histone modification
Histone tails
post-translational modifications
on terminal amino acid residues
Control of activation/silencing
Histone
gene expression

Chromosome

Nature 441, 2006

5
 Chromatine – DNA + Histone

Nucleosom X-ray structure

Acetylation Deacetylation

Methylation Demethylation
H3
…. ...
H2A H2B

H4

N-terminal residues in contact with DNA

Histone Methyltransferases

• Histone methylation modulates histone

interaction with DNA and chromatin associated
proteins
• Histone-Arginine-Methyltransferases (PRMT)
• PRMTs - SAM dependent Methyltransferases

NH2 NH2

N N
N N

COO N N COO
N
PRMTs
N O
O +
S
+ + S NH3
NH3
CH3
HO OH HO OH

SAM + Arg---Histone SAH + N-Methyl-Arg---Histone

6
 Available Information

• 3D structure of PRMT1 (incomplete, inactive)

• 3D structure of related homolog
• substrate known, cofactor known
• no SAR on ligands available

→ Search for drug-like, substrate-

competitive PRMT inhibitors
to further explore their therapeutic
potential

X-ray structures PRMT

X-ray structures of PRMTs:

• PRMT1 (rat):
Problem: solved at non-physiological pH
(buffer pH = 4.7, PRMTs inactive) - not all
residues resolved
• PRMT3 (rat)
Solved at pH = 7.1 (PRMTs active)
Problem: only moderate sequence identity to
PRMT1 Zhang X, Cheng X, Structure 11, 509, 2003

No drug-like PRMT inhibitors known so far

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 X-ray Structures PRMT1 - PRMT3

PRMT3 PRMT1

Co-crystallized S-Adenosylhomocystein

PRMT1 – Histone peptide complex

Histone
Peptide
Arg

SAM

8
 Virtual Screening Setup
• Considered database
• NCI Diversity Database ~2 000 compounds
• 3D structures
• Generation of stereoisomers/correction protonation state
• Omega (OpenEye Software)
• Pre-docking filters
• Reactive groups / at least one nitrogen atom
• Docking – GOLD
• Hydrogen-bond constraints (Glu144)
• Post-docking filters (GRID based pharmacophore)
• Visual inspection of binding mode
• Selection of 36 cpds. / experimental testing
• ~70 % actives in an in vitro Aspergillus nid. PRMT assay
• 7 potent inhibitors IC50 human PRMT1 1.5 – 76 µm
Spannhoff A, et al., J Med Chem 2007

PRMT1 in vitro TRF Assay

Spannhoff A, et al., Bioorg Med Chem Lettt 2007

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 Substrate-competititve PRMT1 Inhibitors

O O

O N O NH2
S N N HN
H H
O O NH
H 2N
(1) (2)
H2N
1.7 µM IC50 hPRMT1 56.6 µM

Substrate competitive inhibitors

18
1/v (x 10 6)

16
No competition with
control 14 the cosubstrate SAM
NSC 35605
12

10

0
-0,01 -0,005 0 0,005 0,01

1/S

NH2
Lineweaver-Burk Diagramme: HN
hPRMT1 Stilbamidine NH
H2N
Native Histon H4 Substrate
Stilbamidine

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 Pharmacophore-based VS

Chembridge Database

• Considered database
• Chembridge database ~330 000 compounds
• 3D structures/Multiconformer database
• Generation of stereoisomers/correction protonation state
• Omega (OpenEye Software)
• Multiconf. database (50 Mio conformations)
• Pre-docking filters
• at least two nitrogen atom
• MW 200-400 (lead like compounds), TPSA < 150 Å2
• Pharmacophore-based VS
• Structure-based VS – LigandScout
• Export in MOE (manual adjustion of features)
• Docking of ~2000 compounds
• Selection of nine molecules among the top ranked 200

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 Pharmacophore-based VS

VS Hits
N
NH2 O N O N
H2N H2N N S H2N N S Cl
N N N
H
N N
NH2 NH2 NH2

6000016 7112201 7155176

O O O O O O
S S S
N N N
H H
H2N H2N H2N

7736382 6689772 7280948

O O O H
S N
N
H N
H2N N
H2N N
N
O H H2N
O

7789734 5784982 5756663

All nine are active in the in vitro PRMT1 assay (10-30 µM)

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 Biological Charterization

Hypomethylation (HepG2 Cells) Selectivity H4R3

100 100
Methylation level (%)

Methylation level (%)

75 75

50
50

25
25
0
0
25
50

25
50

25
50

25
50
0

0
15

15

15

15
1 2 3 9 µM

225
50

25
50
0

0
15

15
c(µM) H4R3 H3K4 (SET7/9)
Estrogen receptor inhibition

150
MCF-7-2a cells
125

100

75
PRMT1 Coactivator ER
ptor
50
tion (%)
25

0
5 50 100 150 5 50 100 150 5 50 100 150 5 50 100 150
-25
1 2 3 9
-50

c(µM )
Spannhoff A, et al., J Med Chem. 2007

High-Throughput-Screening

Experimental HT Screening
• Screening of a chemical library of 9 000 diverse
molecules (Chembridge Diversity) yielded 9 active
inhibitors – mainly SAM analogs (Hit rate ~0.1 %)
• Only one active „non-SAM derivative“ was
identified – all others inhibited also Histone-Lysine-
Methyltransferases

OH OH

O
Na
+ + AMI-1
- N SO
- Na
O3S N 3
H H

Bedford et al. J Biol Chem 279, 23892, 2004

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II. Homology modelling of the histamine
H3 receptor and virtual screening of
novel antagonists

B. Schlegel, C. Laggner, R. Meier, T.

Langer, D. Schnell, R. Seifert, H.
Martin-Luther-University
Halle-Wittenberg
Stark, H.-D. Höltje, and W. Sippl

hH3 Receptor

• Belongs to the GPCR superfamily

• Mainly expressed in the CNS
• Autoreceptor / Heteroreceptor
• Control of other neurotransmitter
systems
• It is suggested that the H3 receptor represents a novel
drug target for diseases including obesity, cognitive
disorders and insomnia

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 Which Design Strategy?

• No 3D structure of hH3R known

• Only 3D structure of bovine rhodospin
• Many hH3 receptor antagonists known
• 418 ligand in house data set available
(affinity between 5 nM and 10 µM)
• Clear SAR available

→ Search for novel chemotypes of

hH3 receptor antagonists

MD / Membrane Model

Generation of a homology model of hH3R based on

Rhodopsin
Insertion into a membrane model

7637 water molecules

128 DPPC molecules

MD simulation, GROMACS
Schlegel et al, J Mol Mod 2005

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 Generation of Binding Pockets

• „inverse“ docking

Epot

Selection of top-ranked poses

12000

10000

8000
156
N 6000

4000

2000

0
-50 -40 -30 -20 -10 0 10 20 30 40 50 60 70 80 Goldscore
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4

Docking in 43740
generated binding
pockets Scoring

Receptor-Ligand Complexes

Docking of other active hH3TM 3

antagonists

N
O

Cl N O N
H

FUB181 UCL2190

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 Enrichment

• GOLD docking:
ƒ 654 randomly selected ligands (WDI)
ƒ 418 H3-receptor ligands (antagonists)
1 00

100 9 0

Goldscore
80
8 0

1
7 0
% H3-ligands

60 Goldscore *
6 0

5 0

NN( (heavyatoms)
Schweratome)
40 4 0

3 0

20 2 0

1 0

0 0

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 00

0 20 40 60 80 100
% WDI-ligands

Scoring Distribution

Maybridge/WDI
basic cpds.
(13.000)
versus
hH3 ligands

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 Pharmacophore (Catalyst)

FUB836

Virtual Screening Setup

• Considered databases
• Maybridge Database ~55 000 compounds
• 3D structures
• Generation of stereoisomers/correction protonation state
• Omega (OpenEye Software)
• Pharmacophore - CATALYST
• based on 3 active hH3R ligands
• testing using 418 actives data set
• Docking GOLD (~1700 cpds.)
• Hydrogen-bond constraint (D3.32)
• Visual inspection of binding mode
• Selection of 7 cpds. for experimental testing
(among the 100 top-ranked docking solutions)
Schlegel, B. et al. JCAM 2007

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 Virtual Screening Hits

- all active between 79 nM – 6 µM

- 2 potent antagonists (nM) showing novel chemotype

Experimental Testing

Similarity / hH3 ligand data set

Compound Ki (nM) GoldScore Tanimoto c. Tanimoto c.

(Graph-3-point (MACCS
pharmacophore) keys)
HTS-07217 2459 (1.510 - 4.004) 72.89 0.45 0.65

PD-00043 1024 (599 - 1749) 79.21 0.55 0.73

RJC-03033 383 (249 – 589) 81.10 0.59 0.83

BTB-12683 3655 (2266 – 5896) 65.97 0.51 0.73

CD-04850 6258 (3775 - 10370) 60.13 0.47 0.60

CD-06177 2958 (1940 - 4510) 82.21 0.56 0.63

BTB-08079 79 (47 - 131) 87.89 0.45 0.57

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 BTB-08079

Conclusions

• Virtual screening based on homology

models is able to provide true actives
• Combination of pharmacophore-based
screening, docking and medicinal-
chemistry guided selection yielded high hit
rates
• Chosen procedures guided the selection of
novel PRMT1, hH3R leads with minimal
experimental effort

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 Virtual Screening

Protein Target Protein family VS Hits Actively Activity Hit

tested range rate
Histone modifyng
PRMT1 ~100 51 0.5-100 µM 50%
enzyme
Histone modifying
Sirtuin Sirt2 10 7 0.8-100 µM 70%
enzyme
Histone modifying
HDAC6 5 2 5-50 µM 40%
enzyme
PCAF Histone modifying
6 4 0.4-50 µM 66%
(irreversible) enzyme
Histone modifying
LSD1 23 7 10-100 µM 28%
enzyme
hCAR (model) Nuclear receptor 54 6 100-150% 11%
hCAR (X-ray) Nuclear receptor 30 17 100-800% 56%
USP7 Deubiquitinase enzyme 49 14 0.5 – 50 µM 30%
ADP-
MTX Toxin 40 2 15-30 µM 5%
Ribosyltransferase
Histamine H3
GPCR 7 7 0.07-5 µM 100%
receptor

Some (personal) suggestions

• Don‘t use virtual screening as a „black-

box“
• Consider virtual screening as
„hypothesis-generator“ (rather than for
prediction of biol. activity!)
• Combination of different approaches
often reduces the number of „false
positives“ (however sometimes you end
up with no hits!)

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 Acknowledgements

• Dr. Björn Windshügel

• Rene Meier
• Ralf Heinke
Martin-Luther-Universität
Halle-Wittenberg
• Sonja Schlimme

• Prof. M. Jung
• Astrid Spannhoff • Prof. H.-D. Höltje
• Prof. Dr. R. Schüle • Dr. Birgit Schlegel
Albert-Ludwigs-Universität Heinrich-Heine-Universität
Freiburg Düsseldorf

Leopold- • Prof. R. Seifert

Franzens • Prof. Thierry Langer Universität Regensburg
• David Schnell
Universität • Dr. Christian Laggner
Innsbruck
• Dr. Gerhard Wolber Johann-Wolfgang-Goethe • Prof. H. Stark
Universität Frankfurt

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