in silico small molecule discovery

1. in silico small molecule discovery Sales Hit to lead Optimise lead Target gene Discover hit Clinical Target gene identified with a viable assay High throughput screen in silico

2. Case 1 – receptor structure known Novel in silicohits ~ 100 dock molecules into receptor Secondary assay ? hits IC50 < 10 µM Computer Database of Molecules 100,000 +

3. 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%

4. How does one get the receptor structure? • X-ray structure available already at RCSB databank • Set up a structure determination • Predict structure

5. X-ray crystallography pipeline Cloning Recombinant protein Expression Protein purification – mg quantities Crystallization Protein structure Electron density map X-ray diffraction pattern Protein crystals

6. Prediction protein structure by homology Matched fold Query sequence Match sequence against library of known folds

7. Phyre- www.sbg.bio.ic.ac.uk Phyre and predecessor 3DPSSM > 1,000 citations

8. Case 2: Ligand activity data available Structure-activity rules Observed activity Screen Novel in silico hits database

9. 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

10. D D C C B B B Logic-rules lead to new chemotypes 7Å C A Fragment C is bonded to fragment D Fragment B is bonded to fragment C Fragment A is 7Å from fragment B  INDDExcan learn complex rule from simpler facts 7Å A Fragment A is 7Å from fragment B which is bonded to fragment C which is bonded to fragment D

11. D C B Rules can be understood by chemists  Standard programs: Activity = 0.45 LogP + 0.56667 Lumo +1.65 V  ILPrule: In an active molecule: Fragment A is 7Å from fragment B which is bonded to fragment C which is bonded to fragment D 7Å A

12. Blind trial of hit discovery on GPCR-1 Data from literature 250 novel in silico hits Observed activity - From Literature INDDEx in silico at Equinox Chemistrt a Order Equinox outsourced wet chemistry and biology Test Cerep 30 Verified in vitro hits NEW CHEMOTYPES 157 Compounds

13. GPCR-1: training set Distribution of 686 training molecules collected from public domain Actives Inactives

14. GPCR Target 1 hits for optimatisation

15. GPCR-1: results of primary screening True hits False hits Number of in silico hits: 157 (10µM concentration) Number of actives: 76 Number of inactives: 81 Primary screen success rate = 48%

16. GPCR-1: new chemotypes Distribution of hits based on their diversity (Tanimoto coefficients) New chemotype

17. Equinox hit discovery on GPCR-2 - Data from BioPrint (Cerep) INDDEx 250 novel in silico hits Observed activity - From BioPrint in silico at Equinox Chemistrt a Order Equinox outsources wet chemistry and biology Test Cerep 28 Verified in vitro hits 94 Compounds

18. Confirmed hit rate of in silicopredictions on secondary screen c. 35%

19. Comparative hit rates

20. 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

21. In silico small molecule discovery • Michael Sternberg, Ata Amini, Paul Freemont & Michael Sternberg • Imperial CollgeLond • www.sbg.bio.ic.ac.uk & www.doc.ic.ac.uk/~shm • www.equinoxpharma.com