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ACS Chem Biol. 2015 Sep 18;10(9):2116-25. doi: 10.1021/acschembio.5b00398. Epub 2015 Jul 20.

Type II Inhibitors Targeting CDK2.

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Structural Genomics Consortium, University of Oxford , Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, United Kingdom.
Department of Statistics, University of Oxford , 1 South Parks Road, Oxford, OX1 3TG, United Kingdom.
Novartis Institutes of Biomedical Research, Basel, Switzerland, Novartis Pharma AG , Postfach, CH-4002 Basel, Switzerland.
Target Discovery Institute, University of Oxford , NDM Research Building, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom.
Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University , Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.


Kinases can switch between active and inactive conformations of the ATP/Mg(2+) binding motif DFG, which has been explored for the development of type I or type II inhibitors. However, factors modulating DFG conformations remain poorly understood. We chose CDK2 as a model system to study the DFG in-out transition on a target that was thought to have an inaccessible DFG-out conformation. We used site-directed mutagenesis of key residues identified in structural comparisons in conjunction with biochemical and biophysical characterization of the generated mutants. As a result, we identified key residues that facilitate the DFG-out movement, facilitating binding of type II inhibitors. However, surprisingly, we also found that wild type CDK2 is able to bind type II inhibitors. Using protein crystallography structural analysis of the CDK2 complex with an aminopyrimidine-phenyl urea inhibitor (K03861) revealed a canonical type II binding mode and the first available type II inhibitor CDK2 cocrystal structure. We found that the identified type II inhibitors compete with binding of activating cyclins. In addition, analysis of the binding kinetics of the identified inhibitors revealed slow off-rates. The study highlights the importance of residues that may be distant to the ATP binding pocket in modulating the energetics of the DFG-out transition and hence inhibitor binding. The presented data also provide the foundation for a new class of slow off-rate cyclin-competitive CDK2 inhibitors targeting the inactive DFG-out state of this important kinase target.

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