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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. Binding Sites have Dual Characteristics. Blue regions are rigid and red regions are flexible

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Ensemble approaches yield new scaffolds and new binding sites l.jpg

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

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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.

Plasticity is evident using multiple protein structures mps l.jpg
Plasticity is Evident Using Multiple Protein Structures (MPS)



Collection of Conformational States

Carlson and McCammon. Mol. Pharmacol. 2000, 57, 213-218.

Mps pharmacophore models l.jpg
MPS Pharmacophore Models (MPS)

  • 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.

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Val 32 (MPS)

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

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More Flexibility = Better Performance! (MPS)








  •  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.

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Newer Directions (MPS)

Fragment-based discovery of inhibitors of HIV-1 protease with a possible new mode of inhibition

Damm et al. Biopolymers, ASAP.

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new site (MPS)



Figure adapted from Hornak and Simmerling. Drug Discov Today2007, 12, 132-138.

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Pharmacophore Model of “Eye” Region (MPS)

7 pharmacophore elements

  • 3 Aromatic: Green

  • 2 Hydrophobic: Cyan

  • 1 Hydrogen-Bond Donating: Red

  • 1 Hydrogen-Bond Accepting: Blue

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Ligand Behavior in LD Simulations (MPS)

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!

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MD Simulations – Alternate Closed Form? (MPS)

side view


Closed (1PRO)

top view

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Experimental Results (FRET-Based Assay) (MPS)

  • 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

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1H-15N HSQC Spectra from Reiko Ishima (Pitt) (MPS)




Q58 sc

  • Only 4 weak shifts

  • No shifts in the traditional binding site

  • Support, but not proof

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top (MPS)

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


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Furthermore… (MPS)

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

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What if Inhibitor Binds Elsewhere? (MPS)

  • 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



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Newer Directions (MPS)

Inhibitors of p53-MDM2

P53 mdm2 complex structure l.jpg
p53/MDM2 Complex Structure (MPS)

Kussie et al, Science, 1996, 274, 948-953.

P53 mdm2 complex structure19 l.jpg
p53/MDM2 Complex Structure (MPS)

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

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4 of 23 Tested Compounds were Inhibitors (MPS)

  • 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

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Summary of MPS Method (MPS)

  • 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

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Acknowledgements (MPS)

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