1. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #1 Enrichment and cross-validation studies of the eHiTS high throughput screening software package. Darryl Reid, Zsolt Zsoldos, Aniko Simon, and A. Peter Johnson SimBioSys Inc., © 2004 Contents: ● Introduction: eHiTS overview, exhaustive search, scoring function ● Validation: Can eHiTS reproduce crystal structures? ● Cross-validation: Finding a suitable representative receptor ● Enrichment Study: Virtual High-throughput screening, finding the diamonds in the rough http://www.simbiosys.ca/

2. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #2 Introduction  Brief overview of eHiTS  Validation study with DHFR complexes  Prove docking ability / accuracy  Cross-validation study  Show receptor site compatibility  Enrichment study  Show applicability for virtual high-throughput screening

3. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #3 eHiTS - Overview eHiTS features an exhaustive systematic flexible docking algorithm

4. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #4 eHiTS - Search  Ligand is divided into  rigid fragments and  connecting flexible chains  All rigid fragments are docked independently  Graph matching  Flexible chain fitting  Local energy minimisation

5. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #5 eHiTS - Scoring  Empirical-based scoring  Many components; Hydrogen bonding, Hydrophobicity, Electrostatic potential, Van der Waals contact energy,Metal ion interactions, etc.  All parameters are configurable  Chemical properties mapped to Connolly surface  Flag compatibility matrix score for receptor-ligand contacts

6. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #6 Experiment Objectives  Show ability to reproduce crystal structures  Show that eHiTS can select active ligands of human DHFR from a drug database  Illustrate the ease of use of eHiTS  No pdb preparation, no ligand preparation  Show eHiTS can be used in HTS applications to discover active ligands

7. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #7 Dihydrofolate Reductase (DHFR)  Plays an essential role in the building of DNA  “juggles” two molecules in this reaction  Folate (purple) and NADPH (green)  The first enzyme targeted for cancer chemotherapy Oct. 2002 PDB Molecule of the Month: http://www.rcsb.org/pdb/molecules/pdb34_3.html

8. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #8 DHFR – Binding site  The drug methotrexate is designed to mimic folate, blocking the enzyme's action  Note the interaction between folate and NADPH, this is essential for the enzyme's function

9. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #9 “Actives” Selection  Searched for DHFR complexes in the PDB  Obtained 88 complexes, all sources  Upon quick visual inspection, eliminated 17 complexes  Contained no ligand in the binding site  Contained multiple ligands in the binding site  Selected 71 DHFR complexes for study  Including 19 human complexes

10. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #10 19 Human DHFR complex

11. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #11 Validation  Each DHFR ligand was removed from the protein and docked back into its binding site  EHiTS was allowed to do this split automatically  Results were then judged by evaluating the RMSD between the crystal structure binding position and the computed docking pose  Standard (default) parameters for eHiTS were used in all the runs

12. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #12 Validation – All Sources ❑ 71 PDBs ❑ 29 Unique Ligands

13. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #13 Validation - Human  19 PDBs  12 Unique Ligands

14. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #14 Results – The Good 1dyi Complex – x-ray ligand in white Top-Rank, -139.6 0.85 RMS Closest, -105.77 0.76 RMS

15. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #15 Results – The Bad 1ly4 Complex – x-ray ligand in white Top-Rank, --55.11 2.23 RMS Closest, -50.32 0.89 RMS

16. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #16 Results – The Ugly Top-Rank, -7.87 4.90 RMS Closest, 43.22 4.38 RMS 1rc4 Complex – x-ray ligand in white

17. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #17 Validation - Summary  EHiTS was able to reproduce accurately (RMS < 2.0) the crystal structure position of DHFR ligands 85% of the time  67% of the time, eHiTS' highest ranking (best scoring) pose had a RMS < 2.0  This number improves for Human DHFR ligands, 74%  This shows that eHiTS is able to predict docking poses for DHFR ligands

18. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #18 Cross-validation  Each ligand is docked against each receptor resulting in a matrix of dockings  A receptor that docks many ligands well is a good candidate for enrichment studies  Tests were ran using standard (default) parameters, with no preprocessing of the pdb data (eHiTS did all processing automatically)

19. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #19 Cross-Validation Color map of cross-validation matrix of 71 DHFR complexes. Green = negative score (good), Red = Positive score (bad)., Grey = no dock Proteins are listed to the right, ligands listed across the top. Ligands Proteins

20. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #20 Cross-Validation Human  Looking horizontally across matrix, can judge how well the receptor site will accept different ligands  As a representative sample (looking at both human and all sources, we chose 1DLS for our enrichment study  1DLS docks almost every ligand, gives average scores for ligands Color map of cross-validation matrix of 19 Human DHFR complexes. Green = negative score (good), Red = Positive score (bad), Grey = no dock Proteins are listed to the right, ligands listed across the top. Proteins Ligands

21. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #21 Enrichment Study  The object of virtual screening is to select a set of ligands “enriched” with actives, relative to the entire database  1DLS used as receptor site  Two groups of ligands were chosen for enrichment tests  21000 random ligands from MDDR database of “drug-like” ligands  16000 MDDR ligands of comparable size to DHFR ligands (actives) found in PDB, 40-60 atoms in size  Enrichment factor is the ratio between the % actives in sample portion and % actives in entire database

22. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #22 Enrichment – All Sources 21000 Random MDDR Ligands ❑ Total # ligands: 21239 ❑ # Ligands docked: 12133 ❑ # actives: 71 ❑ # actives docked: 67

23. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #23 Enrichment – Human Ligands 21000 Random MDDR Ligands ❑ Total # ligands: 21191 ❑ # Ligands docked: 12085 ❑ # actives: 19 ❑ # actives docked: 19

24. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #24 Enrichment – All Sources Selected 16000 MDDR Ligands, 40-60 atoms ❑ Total # ligands: 16636 ❑ # Ligands docked: 641 ❑ # actives: 71 ❑ # actives docked: 67

25. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #25 Enrichment – Human ligands Selected 16000 MDDR Ligands, 40-60 atoms ❑ Total # ligands: 16588 ❑ # Ligands docked: 593 ❑ # actives: 19 ❑ # actives docked: 19

26. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #26 Conclusions  EHiTS can accurately reproduce crystal structure poses  Cross-validation studies showed that 1DLS is a representative structure for DHFR family, especially for Human ligands  eHiTS gives very good enrichment results on our given dataset. Especially considering:  Our “actives” are hypothetical (some may not be active to 1DLR)  Our “decoys” could have activity towards 1DLR

27. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #27 Conclusions  EHiTS proved suitable for virtual high throughput screening  Docking times averaged ~5mins / ligand for standard parameter and 30 sec / ligand for Enrichment studies using “fast” parameter sets  21000 ligands were screened in under 12 hours on 160 cpu cluster  Good enrichment factors shows effectiveness of screening

28. SimBioSys Inc.© 2004 http://www.simbiosys.ca/ Slide #28 Acknowledgments  Zsolt Zoldos, SimBioSys Inc. CEO  Aniko Simon, Bashir Sadjad, Beihong Wu, Constantin Tanurkov, James Law, Sing Yoong Khew, Irina Szabo, Zsolt Szabo, David Fung.  Dr. Peter Johnson, Leeds University http://www.simbiosys.ca