Chemical genomic profiling to identify intracellular targets of protein kinase inhibitors.

摘要

Protein phosphorylation catalyzed by protein kinases is a major mechanism by which cells transmit signals within cells. Over the past decade, significant efforts have been devoted to identifying potent and highly selective inhibitors of kinases associated with disease. Specific kinase inhibitors would also serve as powerful chemical tools to probe cellular signaling in a temporal and spatially controlled manner.;Due to the large size of the protein kinase superfamily and the fact that most kinase inhibitors bind in the highly conserved ATP-binding pocket, such inhibitors have proven to be difficult to identify. Thus, the inhibitors used as chemical tools to probe the often poorly understood roles of kinases in signaling pathways are paradoxically themselves of incompletely characterized specificity, and are widely acknowledged to inhibit multiple kinases within the cell ("multiplex" inhibition). One important question that must be answered is whether kinase inhibitors exert their cellular effects because of, or in spite of, this lack of specificity.;Here, we have used and developed chemical genetic tools to address these critical scientific questions. We describe the chemical genetic analysis of protein kinases as a general route towards kinase inhibition biomarker discovery and as a tool for discovery of novel functions of protein kinases. We have also adapted these chemical genetic tools in combination with whole-genome microarray profiling into a screen that can be used to assess the precise cellular targets of protein kinase inhibitors. We show that the cellular responses elicited in S. cerevisiae by the cyclin-dependent kinase inhibitor GW400426 are the result of supra-additive effects of simultaneous inhibition of two CDKs, Cdk1 and Pho85. We also show that the CDK inhibitor GW297361 acts as a specific inhibitor of Pho85 despite a much greater in vitro potency against Cdk1 due to a difference in the intrinsic response thresholds of the signaling pathways controlled by these two kinases. Finally, we describe a novel chemical genetic strategy to enable the rapid sensitization of protein kinases to irreversible inhibition using an electrophilic small molecule inhibitor.