Targeting CDC25B-CDK2/Cyclin A Activity Using Chemical Biology Approaches

Abstract

The cell cycle is a fundamental process of cell biology, and its progression is highly regulated. A critical mode of regulation for proper advancement of the cell cycle is the activation of the CDKs by the CDC25 family of dual specificity phosphatases. The CDC25 proteins are often overexpressed or misregulated in cancer, resulting in dysregulated cell growth, genomic instability and evasion of apoptosis. The oncogenic role of the CDC25 proteins has inspired over two decades of drug discovery efforts to inhibit their enzymatic activity. Despite these efforts, no therapeutic agents targeting family of CDC25 phosphatases emerged. In order to identify new classes of CDC25B inhibitors, new approaches to target CDC25 are needed. We have employed a novel approach to inhibit the CDC25 family member CDC25B by targeting its interaction with its native substrate, the CDK2/Cyclin A complex. We used two different methods, fragment-based drug discovery and “gray-box” high-throughput screening, to identify inhibitors of the CDC25B-CDK2/Cyclin A protein-protein interaction. Using NMR- based fragment based screening, we identified a small molecule ligand of the CDC25B catalytic domain. We solved the co-crystal structure with this ligand bound to CDC25B, and used this structure to develop more potent analogs. We have shown that fragment-derived compounds can disrupt the CDC25B-CDK2/Cyclin A interaction and inhibit CDC25B catalytic activity. To our knowledge, our inhibitor-bound crystal structure of CDC25B is the first crystal structure with CDC25B bound to a small molecule ligand. We have also developed several protein-protein interaction assays to quantify the interaction between CDC25B and CDK2/Cyclin A. We employed these assays in three high- throughput screens to identify several classes of CDC25B-CDK2/Cyclin A protein-protein interaction inhibitors. The inhibitors we identified do not target CDC25B, but disrupt the protein- protein interaction by targeting CDK2/Cyclin A. Importantly, we have developed a high quality screening assay for the identification of CDC25B-CDK2/Cyclin A interaction inhibitors. This assay will be useful for future drug discovery efforts targeting the CDC25B-CDK2/Cyclin A interaction. In summary, we have developed two new approaches to inhibit CDC25B. These results pave the way towards the development of new chemical probes and potential therapeutic agents targeting CDC25B.PhDMolecular and Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113311/1/lundgeo_1.pd