Ensemble Approaches Yield New Scaffolds and New Binding Sites

1. Ensemble Approaches YieldNew Scaffolds andNew Binding Sites Heather A. Carlson Department of Medicinal Chemistry College of Pharmacy University of Michigan Ann Arbor, Michigan 48109-1065

2. Binding Sites have Dual Characteristics • Blue regions are rigid and red regions are flexible • Arrows mark the binding sites • Freire and coworkers have analyzed many protein structures and found that binding sites are a mix of rigid regions and flexible regions Freire. Proc. Natl. Acad. Sci. USA1996, 96, 10118-10122.

3. Plasticity is Evident Using Multiple Protein Structures (MPS) Ensemble Sub-Ensemble Collection of Conformational States Carlson and McCammon. Mol. Pharmacol. 2000, 57, 213-218.

4. MPS Pharmacophore Models • Generate MPS (MD, NMR, crystal structures) • Map each binding site with probe molecules • Combine the MPS binding sites • Identify regions of consensus • Translate them into pharmacophore models • Sites are centered at the average position of probes • Radii based on the RMSD of probes • Excluded volumes are centered at the average position of key/catalytic residues (radius = 1.5 Å) Original HIV-1 Integrase Studies: Carlson et al. J. Phys. Chem. A1999, 103, 10213-10219. Carlson et al. J. Med. Chem.2000, 43, 2100-2114. First HIV-1 Protease Study: Meagher and Carlson. J. Am. Chem. Soc. 2004, 126, 13276-13281.

5. Val 32 Ile 84 Pro 81′ Leu 76 Val 82′ Gly 27 Asp 30 Asp 29 Leu 23′ Arg 8′ Consensus Maps Out S1 and S2 Pockets

6. More Flexibility = Better Performance! 3s 2.66s 2.33s 2s 1.66s 1.33s 1s •  1 ns 6 of 6 • 1 ns 5 of 6  1 ns 4 of 6 sites •  2 ns 6 of 6 • 2 ns 5 of 6 •  2 ns 4 of 6 sites •  3 ns 6 of 6 • 3 ns 5 of 6  3 ns 4 of 6 sites 89 unique, diverse inhibitors Percent Active Compounds Percent Inactive Compounds 85 unique, highly diverse, medicinal compounds Meagher and Carlson. J. Am. Chem. Soc. 2004, 126, 13276-13281.

7. Newer Directions Fragment-based discovery of inhibitors of HIV-1 protease with a possible new mode of inhibition Damm et al. Biopolymers, ASAP.

8. new site semi-open closed Figure adapted from Hornak and Simmerling. Drug Discov Today2007, 12, 132-138.

9. Pharmacophore Model of “Eye” Region 7 pharmacophore elements • 3 Aromatic: Green • 2 Hydrophobic: Cyan • 1 Hydrogen-Bond Donating: Red • 1 Hydrogen-Bond Accepting: Blue

10. Ligand Behavior in LD Simulations Run 1 • Multiple disassociations then returns back to “Eye” site Run 2 • Dissociates into the central active site • Crosses the binding site • And finally binds into the “Eye” site of opposite side monomer!

11. MD Simulations – Alternate Closed Form? side view AVE MD Closed (1PRO) top view

12. Experimental Results (FRET-Based Assay) • Compound 1 was auto-fluorescent • A derivative (also identified in the virtual screen) was tested R2 = 0.9967 Compound 2 compound 2 ● Pepstatin A □ 0.1 1 10 100 Log [Inhibitor] in μM Compound 2 inhibits HIV-1p IC50 = 18 μM (no optimization whatsoever) Collaboration with Jason E. Gestwicki, UMich Life Sciences Institute

13. 1H-15N HSQC Spectra from Reiko Ishima (Pitt) G52 I54 G48 Q58 sc • Only 4 weak shifts • No shifts in the traditional binding site • Support, but not proof

14. top An unusual crystal structure shows an inorganic ligand with some contacts to the eye 1ZTZ (Cobalt metallacarboraneligands) Cígler et al. PNAS 2005, 102, 15394-15399. Note a substrate mimic is bound with two metalo compounds per dimer, and… the presence of the ligands warps the flap tips outward side

15. Furthermore… The presence of the ligands also creates unusual inter-locking contacts between multiple copies of the protease.

16. What if Inhibitor Binds Elsewhere? • Dimerization Inhibitor? • “Elbow” Inhibitor? • Traditional Active Site Inhibitor? • Current Protease Inhibitors • MW range: 505 – 720 Da • Our Inhibitor (MW 323) • New chemical class • Few Rotatable Bonds • Optimization could potentially lead to a drug with better pharmacokinetic properties Darunavir Saquinavir

17. Newer Directions Inhibitors of p53-MDM2

18. p53/MDM2 Complex Structure Kussie et al, Science, 1996, 274, 948-953.

19. p53/MDM2 Complex Structure 6-site MPS model based on snapshots from a 2-ns MD Bowman et al. J. Am. Chem. Soc.2007, 129, 12809-12814. Zhong and Carlson. Proteins2005, 58, 222-234

20. 4 of 23 Tested Compounds were Inhibitors • 17% hit rate • Each is a new scaffold Ki = 0.11 µM Ki = 0.29 µM Collaboration with Shaomeng Wang, UMich Medical School Ki = 9.9 µM Ki = 37 µM

21. GLIDE Flexible Docking

22. Pushing the Flexible Core of MDM2

23. Summary of MPS Method • Unbound HIVp provides a pharmacophore for bound structures – despite large conformational changes upon binding • Discovered a potential new site for inhibiting HIVp • New scaffolds for MDM2

24. Acknowledgements Testing Compounds Jerome Quintero Man-Un “Peter” Ung Prof. Jason E. Gestwicki Prof. Reiko Ishima Dr. Zaneta Nikolovska-Coleska Prof. Shaomeng Wang • Dr. Kristin L. Meagher • Dr. Kelly L. Damm • Dr. Anna L. Bowman • CCG (MOE) • William L. Jorgensen (BOSS) • NIH • Beckman Young Investigator Program