MECHANISM BASED INHIBITION OF CYCLIN DEPENDENT KINASES

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DESCRIPTION (verbatim from the applicant's abstract): Cyclin-dependent kinases (CDKs) play an important role in the control and regulation of cell cycle events. Inhibitors specific for CDKs could potentially be used as pharmacological probes for the elucidation of signal transduction pathways and as therapeutic agents in the control and management of cell cycle-related diseases. However, most current chemical inhibitors of CDKs act as a competitive inhibitor of the common substrate ATP and often lack the desired specificity and potency. This prompted us to develop specific CDK inhibitors with a novel mechanism of action and a generally applicable approach to the design of protein serine/threonine kinase inhibitors. Mechanism-based inhibitors designed using a specific substrate sequence, as a template should offer the best probability of retaining specificity of the template without compromising the necessary inhibitory potency. To test this hypothesis, several potential mechanism-based inhibitors are designed by incorporation of dipeptide Gly-Ser replacements into a sequence known to be a good substrate of CDKs. The use of modified peptide groups surrounding the targeted serine will be explored in converting the phosphorylated peptide analog to a more highly reactive species, which could react with the targeted kinase to produce a covalently inactivated enzyme (specific aim 1). Replacements are designed to cover a wide range of activation in order to discover the fundamental chemistry necessary for making "suicide" inhibitors of CDKs. These inhibitors will be tested for their type and nature of inhibition against a recombinant human cyclin B/CDC2 kinase (specific aim 2). The specificity of inhibitors will be evaluated and compared with the substrate specificity of the original template towards cyclin B/CDC2, relative to cAMP-dependent protein kinase and protein kinase C (specific aim 3). Future efforts will focus on identifying the active site residue(s) modified and on improving the inhibitors' permeability across cellular membranes and their stability towards proteolysis. This approach should be generally applicable to the quick development of mechanism-based inhibitors specific for any protein serine/threonine kinase with a known specific substrate sequence.