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

2. Freire. Proc. Natl. Acad. Sci. USA 1996, 96, 10118-10122. Binding Sites have Dual Characteristics 4 Blue regions are rigid and red regions are flexible 4 Arrows mark the binding sites 4 Freire and coworkers have analyzed many protein structures and found that binding sites are a mix of rigid regions and flexible regions

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

4. MPS Pharmacophore Models 4 Generate MPS (MD, NMR, crystal structures) 4 Map each binding site with probe molecules 4 Combine the MPS binding sites 4 Identify regions of consensus 4 Translate them into pharmacophore models First HIV-1 Protease Study: Meagher and Carlson. J. Am. Chem. Soc. 2004, 126, 13276-13281. Carlson et al. J. Med. Chem. 2000, 43, 2100-2114. Original HIV-1 Integrase Studies: Carlson et al. J. Phys. Chem. A 1999, 103, 10213-10219. 4 Sites are centered at the average position of probes 4 Radii based on the RMSD of probes 4 Excluded volumes are centered at the average position of key/catalytic residues (radius = 1.5 Å)

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

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

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. closed semi-open new site Figure adapted from Hornak and Simmerling. Drug Discov Today 2007, 12, 132-138.

9. Pharmacophore Model of “Eye” Region 4 3 Aromatic: Green 4 2 Hydrophobic: Cyan 4 1 Hydrogen-Bond Donating: Red 4 1 Hydrogen-Bond Accepting: Blue 7 pharmacophore elements

10. Ligand Behavior in LD Simulations 4 Multiple disassociations then returns back to “Eye” site Run 1 Run 2 4 Dissociates into the central active site 4 Crosses the binding site 4 And finally binds into the “Eye” site of opposite side monomer!

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

12. Experimental Results (FRET-Based Assay) 4 Compound 1 was auto-fluorescent 4 A derivative (also identified in the virtual screen) was tested Compound 2 Compound 2 inhibits HIV-1p IC 50 = 18 μM (no optimization whatsoever) (no optimization whatsoever) Collaboration with Jason E. Gestwicki, UMich Life Sciences Institute Log [Inhibitor] in μM 0.1 1 10 100 R 2 = 0.9967 compound 2 ● Pepstatin A □

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

14. An unusual crystal structure shows an inorganic ligand with some contacts to the eye 1ZTZ (Cobalt metallacarborane ligands) 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 top 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? 4Dimerization Inhibitor? 4“Elbow” Inhibitor? 4Traditional Active Site Inhibitor? 4Current Protease Inhibitors 4MW range: 505 – 720 Da 4Our Inhibitor (MW 323) 4New chemical class 4Few Rotatable Bonds 4Optimization could potentially lead to a drug with better pharmacokinetic properties Saquinavir Darunavir

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. Proteins 2005, 58, 222-234

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

21. GLIDE Flexible Docking

22. Pushing the Flexible Core of MDM2

23. Summary of MPS Method 4 Unbound HIVp provides a pharmacophore for bound structures – despite large conformational changes upon binding 4 Discovered a potential new site for inhibiting HIVp 4 New scaffolds for MDM2

24. Acknowledgements 4 Dr. Kristin L. Meagher 4 Dr. Kelly L. Damm 4 Dr. Anna L. Bowman  CCG (MOE)  William L. Jorgensen (BOSS) $ NIH $ Beckman Young Investigator Program Testing Compounds Jerome Quintero Jerome Quintero Man-Un “Peter” Ung Man-Un “Peter” Ung Prof. Jason E. Gestwicki Prof. Jason E. Gestwicki Prof. Reiko Ishima Prof. Reiko Ishima Dr. Zaneta Nikolovska- Coleska Dr. Zaneta Nikolovska- Coleska Prof. Shaomeng Wang Prof. Shaomeng Wang