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. USA 1996, 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. A 1999, 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
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
closed
semi-open




Figure adapted from Hornak and Simmerling. Drug
Discov Today 2007, 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)

• Only 4 weak shifts
• No shifts in the traditional binding site
• 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
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. Proteins 2005,
58, 222-234
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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