in silico small molecule discovery

SalesTargetgene

Discoverhit

Hit tolead

Optimiselead

Clinical

Target gene identified with a viable assay

High throughput screen

in silico

Case 1 – receptor structure known

Novel in silico hits~ 100

ComputerDatabase ofMolecules100,000 +

dock moleculesinto receptor

Secondary assay? hits

IC50 < 10 µM

How successful is this method?

• From Shoichet’s group on target – protein tyrosine phosphate 1B

• None of the in silico hits found by HTS• But unpredictable - other systems yielding < 1%

Method Compounds tested

Hits with IC50 < 10µ M Hit rate

High throughput screening (HTS)

400,000 6 0.001%

In silico docking 365 from docking

18 5%

How does one get the receptor structure?

• X-ray structure available already at RCSB databank

• Set up a structure determination

• Predict structure

Cloning Recombinant protein Expression

Protein purification –

mg quantities

Protein crystalsElectron density map

X-ray diffraction patternProtein structure

Crystallization

X-ray crystallography pipeline

Prediction protein structure by homology

Query sequence

Matchedfold

Match sequence against library of known folds

Phyre- www.sbg.bio.ic.ac.uk

Phyre and predecessor 3DPSSM > 1,000 citations

Case 2: Ligand activity data available

Novel in silico hits

database

Observed activity Structure-activity rules

Screen

INDDExTM –A logic-based method

• Muggleton & Sternberg developed a logic-based strategy

• Method now incorporated into INDDEx within an Imperial spin-out Equinox Pharma

• INDDEx designed to exploit availability of active and inactive data on a at least c. 5 but ideally more ligands

Logic-rules lead to new chemotypes

C D7Å

AB B C

Fragment B is bonded to fragment C

Fragment C is bonded tofragment D

AB C D

7Å

INDDEx can learn complex rule from simpler facts Fragment A is 7Å from fragment B which is bondedto fragment C which is bonded to fragment D

Fragment A is 7Å from fragment B

Rules can be understood by chemists

Standard programs:

Activity = 0.45 LogP + 0.56667 Lumo +1.65 V

AB C D

7Å

ILP rule:In an active molecule:Fragment A is 7Å from fragment B which is bondedto fragment C which is bonded to fragment D

Chemistrt a

Blind trial of hit discovery on GPCR-1 Data from literature

250 novel in silico hits

Order

Observed activity- From Literature

157 Compounds30 Verified in vitro hitsNEW CHEMOTYPES Test

Cerep

in silico at Equinox

Equinox outsourced wet chemistry and biology

INDDEx

GPCR-1: training set

Distribution of 686 training molecules collected from public domain

ActivesInactives

GPCR Target 1 hits for optimatisation

4.7M molecules in Zinc database

400,000 drug like molecules

500 in silico hits

250 hits & new chemotypes

157 tested for inhibition

76 actives

39 for IC50

30 confirmed

30 chemotypes

30

GPCR-1: results of primary screening

Number of in silico hits: 157 (10µM concentration)Number of actives: 76 Number of inactives: 81Primary screen success rate = 48%

10 919 22

16

81

010

2030

4050

6070

8090

Num

ber o

f hits

>70% 60%-70% 50%-60% 40%-50% 30%-40% <30%

Percent of specific binding

CB1 results - primary screening

True hitsFalse hits

GPCR-1: new chemotypes

Distribution of hits based on their diversity (Tanimoto coefficients)

8

14

8

0

2

4

6

8

10

12

14

Num

ber o

f hits

<0.60 0.60-0.70 0.70-0.75

Tanimoto coefficient

CB1 results - new chemotype

New chemotype

Chemistrt a

INDDEx

Equinox hit discovery on GPCR-2 - Data from BioPrint (Cerep)

250 novel in silico hits

Order

Observed activity- From BioPrint

94 Compounds28 Verified in vitro hitsTest

Cerep

in silico at Equinox

Equinox outsources wet chemistry and biology

Confirmed hit rate of in silico predictions on secondary screen c. 35%

Target 1 Target 2In silco hits 157 94Primary screen hits(>30% binding at 10µM)

76 42

No. compounds tested for IC50 39 28IC50 results (<12µM) 30 28Estimated secondary hits if all primary hits tested

40 42

Estimated hit rate = estimated secondary hitsIn silico hits

38/157 = 24 %

42 /94= 45 %

Comparative hit rates

Company / approach Target Hit Rate Technology

INDDEx GPCR 1 & 2 + unknown target

35 % Ligand-based

Structure-based Multiple targets Average< 2% Docking into 3D

structure

High throughput Multiple targets Average 0.001% Experimental

screening

Concluding remarks

• If protein structure available can initiative an in silico screening approach to find hits.– Success rate generally <.2%– X-ray structure determination requires mgs of material– Prediction of structure if sequence identity > 50%

• If structure- activity data available then in silico methods can yield far better hit rates c. 35%

• in silco methods complement high throughput and can find different hits

In silico small molecule discovery

• Michael Sternberg, Ata Amini, Paul Freemont & Michael Sternberg

• Imperial Collge Lond– www.sbg.bio.ic.ac.uk & www.doc.ic.ac.uk/~shm– www.equinoxpharma.com