A chemical switch for inhibitor-sensitive alleles of any protein kinase

Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry(1). The lack of these reagents has complicated efforts to assign specific signallin g roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not in hibit wildtype kinases(2). From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibito r-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of th e wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mito tic cell-cycle arrest that is distinct from the G1 arrest typically observe d in temperature-sensitive cdc28 mutants(3). The mutation that confers inhi bitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alle les of protein kinases, allowing for rapid functional characterization of m embers of this important gene family.