Thomas K. Harris

In many cancers, oncogenic transformation has been found to correlate with increased activation of a number of signal transduction pathways mediated internally by members of the serine-threonine protein kinase family; and large efforts are being directed towards design of potent and selective inhibitors of well established protein kinase drug targets. Since the overwhelming majority of protein kinase inhibitors bind in or near the ATP binding pocket shared by the catalytic domain of all kinases, very few serine-threonine protein kinase inhibitors have been clinically approved due to their broad specificity and overall high toxicity. Therefore, we are pursuing the hypothesis that serine-threonine protein kinase inhibitor selectivity may be better achieved by designing compounds that target interfacial clefts and crevices formed between contiguous regulatory and catalytic kinase domains, thereby 'allosterically' stabilizing inactive or autoinhibited conformations of multi-domain protein kinases. The idea of improving selectivity by targeting interfacial clefts is motivated by inspection of primary structure alignments of a number of well established serine-threonine protein kinase drug targets. While the catalytic kinase domains share a high degree of structural homology, the most distinguishing feature among these kinases is the distribution of various types of regulatory domains, many of which are contiguous with the catalytic kinase domain. We are employing two complementary approaches that seek to establish both the functional role and the structural basis by which regulatory domains affect kinase activity. The foregoing approach is determination of the kinetic mechanisms of target protein kinases, which focus on establishing the degree of activation or inhibition that a regulatory domain exerts on one or more specific elementary reaction steps such as substrate binding or chemical phosphorylation. For example in the PDK1, PKB/Akt, and S6K1 protein kinase drug targets, we have demonstrated (i) how contiguous regulatory domains stabilize inactive conformations of their respective catalytic kinase domain and (ii) how allosteric effectors such as second messenger binding or phosphorylation can lead to kinase activation. Subsequent elucidation of the structures entailing inactive conformations will facilitate design of the hypothesized highly selective 'allosteric' inhibitors.