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Virtual Screening

Virtual Screening. Identify and prepare a protein structure Systematically dock a database of structures Score the best orientations Put best hits through the real screen. Lead Optimization. Dock leads into active site (or use x-ray coords) Introduce synthetically feasible modifications

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Virtual Screening

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  1. Virtual Screening • Identify and prepare a protein structure • Systematically dock a database of structures • Score the best orientations • Put best hits through the real screen Lead Optimization • Dock leads into active site (or use x-ray coords) • Introduce synthetically feasible modifications • Dock the modified structure • Score the best orientation • Propose synthesis

  2. Caveats • How good are the scores? • How good is the Structure? • False Positives/Negatives. • Ligand Flexibility. • Protein Flexibility. The system needs to be validated before committing to an expensive, exhaustive search.

  3. Factors Influencing Docking Scores • Orientation of Residues • Protonation states (ligand and Protein) • Tautomeric forms • Protein flexibility • Involvement of water • Internal energy of the ligand • Desolvation penalties • Crystal packing of initial x-ray structure

  4. PTP-1B as a Test System • Target for Type II Diabetes • Deregulates Insulin Receptor • Several Crystal Structures Available • In-House Crystallography

  5. Orientation of Hard to Resolve Residues • Some residues have multiple orientations that fit density • Glutamine is classic example • Hydrogen bonding pattern seriously effected • Need to setup and run both orientations

  6. Orientation of Hard to Resolve Residues

  7. Unexpected Protonation State of Certain Residues • Aspartic acid is deprotonated by default • ASP 181 is a known general acid in PTP-1B • X-Ray data shows Asp181 involved in H- • bonds with other acidic resdiues • Must treat ASP181 as protonated or the ligand. • Ligand is harder in screening runs

  8. ASP 181 as an H-Bond Donor 2.73 3.26

  9. Effect of Protonation of Asp181 Asp181 Charged Asp181 Neutral X-Ray Structure

  10. Flexibility of Unexpected Residues Most residues are treated as rigid Key H-bond residues can be flexible Residues known to move can be flexible Unexpected movements can alter a docking result Isoleucine 220 in PTP-1B

  11. Flexibility of Unexpected Residues Starting Protein FLO Orientation X-Ray Structure

  12. Tautomerization of the Ligand(or Protein) • Tautomers provide different H-Bonding patterns • Structure often drawn in one tautomer in Corporate database • Rigid protein side chains can freeze out the wrong tautomer and enolates

  13. Involvement of Water • Explicit waters can usually be handled • Their presence can stabilize some orientations • They can prevent other orientations • Becomes a combinatorial problem

  14. Evaluation of Water with Grid Leau = 0 versus Leau = 1 Leau = 0: Does a reasonable job identifying key water in active site But misses carbonyl group region Leau = 1: Identifies the key water in active site but also identifies the water bridged carbonyl group region

  15. Internal Strain of the Ligand • Internal energy in docking score • Some torsions are given too much weight • Good solutions are rejected • Bent biphenyls are a problem • Serious effect on PTP-1B virtual screen • Exaggerated electrostatics can give false positives • Compensating effects give good scores • for the wrong reason

  16. Internal Energy of WAY Compound

  17. Docking of WAY Compound with Flo Flo Orientation X-Ray Structure

  18. Effect of Solvation on Binding ScoreHIV-1 Protease/L-700,417

  19. Scoring L-700,417 in the Binding Site ASP29 (solvent exposed) • x-ray structure available (2.10Å) • Low B-factors for key residues • Large data set exists (L-689,502 analogs) • Provides good test case • IC50= 0.67 nM (MM predicts 3 orders of magnitude more potent) Eric Manas 3/6/2002

  20. Observed PIC50 vs. DGEl+Hphob, outliers removed L-700,417 L-700,417 Eric Manas 3/6/2002

  21. Crystal Packing Interactions

  22. BPPM Crystal Structure Puius, Y. A., Zhao, Y., Sullivan, M., Lawrence, D. S., Almo S. C., and Zhang, Z. Y. (1997) Proc. Natl. Acad. Sci. U S A. 94, 13420-5

  23. Docking BPPM into PTP1B Protein Dimer Single Protein X-Ray Structure

  24. Packing in Staph MurB Crystal

  25. Overlap of Symmetry Related Protein with MurB Substrate

  26. Conclusions • Don’t treat docking software as a black box • Be prepared to carry out multiple runs • Validate runs with x-ray data - if possible • Develop software to handle tautomers • protonation states, and the presence of water Acknowledgements X-Ray - Weixin Xu, Rajiv Chopra Dock Scripts - Juan Alvarez, Burt Thomas Solvation Free Energies – Eric Manas

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