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Discovery of a Potential Allosteric Ligand Binding Site in CDK2

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Stephane Betzi†, Riazul Alam†, Mathew Martin†, Donna J. Lubbers†, Huijong Han†, Sudhakar R. Jakkaraj‡, Gunda I. Georg‡, and Ernst Schönbrunn†*

^† Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, Florida 33612, United States

^‡ Department of Medicinal Chemistry, University of Minnesota, 717 Delaware Street SE, Minneapolis, Minnesota 55414, United States

ACS Chem. Biol., 2011, 6 (5), pp 492–501

DOI: 10.1021/cb100410m

Publication Date (Web): February 3, 2011

*E-mail: ernst.schonbrunn@moffitt.org.

Section:

Enzymes

Abstract

Cyclin-dependent kinases (CDKs) are key regulatory enzymes in cell cycle progression and transcription. Aberrant activity of CDKs has been implicated in a number of medical conditions, and numerous small molecule CDK inhibitors have been reported as potential drug leads. However, these inhibitors exclusively bind to the ATP site, which is largely conserved among protein kinases, and clinical trials have not resulted in viable drug candidates, attributed in part to the lack of target selectivity. CDKs are unique among protein kinases, as their functionality strictly depends on association with their partner proteins, the cyclins. In an effort to identify potential target sites for disruption of the CDK−cyclin interaction, we probed the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS) with human CDK2 and cyclin A using fluorescence spectroscopy and protein crystallography. ANS interacts with free CDK2 in a saturation-dependent manner with an apparent K_d of 37 μM, and cyclin A displaced ANS from CDK2 with an EC₅₀ value of 0.6 μM. Co-crystal structures with ANS alone and in ternary complex with ATP site-directed inhibitors revealed two ANS molecules bound adjacent to one another, away from the ATP site, in a large pocket that extends from the DFG region above the C-helix. Binding of ANS is accompanied by substantial structural changes in CDK2, resulting in a C-helix conformation that is incompatible for cyclin A association. These findings indicate the potential of the ANS binding pocket as a new target site for allosteric inhibitors disrupting the interaction of CDKs and cyclins.

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This article has been cited by 1 ACS Journal articles (1 most recent appear below).

A Novel Mechanism by Which Small Molecule Inhibitors Induce the DFG Flip in Aurora A
Mathew P. Martin, Jin-Yi Zhu, Harshani R. Lawrence, Roberta Pireddu, Yunting Luo, Riazul Alam, Sevil Ozcan, Said M. Sebti, Nicholas J. Lawrence, and Ernst Schönbrunn
ACS Chemical Biology2012 Article ASAP
- A Novel Mechanism by Which Small Molecule Inhibitors Induce the DFG Flip in Aurora A
  Mathew P. Martin, Jin-Yi Zhu, Harshani R. Lawrence, Roberta Pireddu, Yunting Luo, Riazul Alam, Sevil Ozcan, Said M. Sebti, Nicholas J. Lawrence, and Ernst Schönbrunn
  ACS Chemical Biology2012 Article ASAP
  Most protein kinases share a DFG (Asp-Phe-Gly) motif in the ATP site that can assume two distinct conformations, the active DFG-in and the inactive DFG-out states. Small molecule inhibitors able to induce the DFG-out state have received considerable ...
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