Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors

Background: Chemical inhibitors of cyclin-dependent kinases (CDKs) have gre at therapeutic potential against various proliferative and neurodegenerativ e disorders. Olomoucine, a 2,6,9-trisubstituted purine, has been optimized for activity against CDK1/cyclin B by combinatorial and medicinal chemistry efforts to yield the purvalanol inhibitors. Although many studies support the action of purvalanols against CDKs, the actual intracellular targets of 2,6,9-trisubstituted purines remain unverified. Results: To address this issue, purvalanol B (95) and an NG-methylated, CDK -inactive derivative (95M) were immobilized on an agarose matrix. Extracts from a diverse collection of cell types and organisms were screened for pro teins binding purvalanol B. In addition to validating CDKs as intracellular targets, a variety of unexpected protein kinases were recovered from the 9 5 matrix. Casein kinase 1 (CK1) was identified as a principal 95 matrix bin ding protein in Plasmodium falciparum, Leishmania mexicana, Toxoplasma gond ii and Trypanosoma cruzi. Purvalanol compounds also inhibit the proliferati on of these parasites, suggesting that CK1 is a valuable target for further screening with 2,6,9-trisubstituted purine libraries. Conclusions: That a simple batchwise affinity chromatography approach using two purine derivatives facilitated isolation of a small set of highly puri fied kinases suggests that this could be a general method for identifying i ntracellular targets relevant to a particular Glass of ligands. This method allows a close correlation to be established between the pattern of protei ns bound to a small family of related compounds and the pattern of cellular responses to these compounds.