Cell growth and differentiation is a highly controlled process which, when lost, can lead to aberrant cell function, potentially resulting in disease. Multiple cellular signals can stimulate growth, differentiation, and apoptosis. A key mechanism for regulating these processes involves the cell cycle. Progression through the eukaryotic cell cycle is controlled by the cyclin dependent kinase (CDK) family. Uncontrolled regulation of the cell cycle pathway is thought to be a source of human cancers and inhibition of unregulated CDK activity by small molecule inhibitors should be beneficial in the treatment of cancer. Most CDK2 research efforts have focused on screening the active active CDK2/Cyclin A complex for direct kinase inhibition. With the intent of finding new modes of inhibition we describe a new strategy of screening inactive CDK2 in the absence of Cyclin A in order to find direct binders which may modulate protein-protein interactions (i.e. cyclin binding), inhibit phosphorylation of CDK2, or identify non-ATP competitive inhibitors. By utilizing the AS/MS-based ALIS technology, a high-throughput screening campaign using basal CDK2 succeeded in identifying an initial hit series which only binds to CDK2 in the absent of Cyclin A. Optimization of the initial hit utilized several affinity-based assays, including a Fluorescence Polarization (FP) Assay; Isothermal Titration Calorimetry (ITC) and Temperature-dependent Circular Dichroism (TdCD). The formation of a complex of the inhibitor with the CDK2 basal form has been confirmed with a co-crystal structure. The inhibition of binding between CDK2 and Cyclin A has also been demonstrated. This process of screening and binding profiling may serve as an example for future kinase programs and offer alternatives to traditional approaches.

Novel approaches to CDK2 modulation: Hit identification, characterization, and optimization

240th National Meeting (2010)

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