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

Knockaert, Marie and Gray, N. and Damiens, E. and Chang, Y.-T. and Grellier, P. and Grant, Karen M. and Fergusson, David and Mottram, Jeremy C. and Soete, M. and Dubremetz, J.-F. and Le Roch, K. and Doerig, C. and Schultz, P. G. and Meijer, Laurent (2000) Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors. Chemistry and Biology, 7 (6). pp. 411-422. ISSN 1074-5521

Abstract

Background: Chemical inhibitors of cyclin-dependent kinases (CDKs) have great therapeutic potential against various proliferative and neurodegenerative 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 N6-methylated, CDK-inactive derivative (95M) were immobilized on an agarose matrix. Extracts from a diverse collection of cell types and organisms were screened for proteins binding purvalanol B. In addition to validating CDKs as intracellular targets, a variety of unexpected protein kinases were recovered from the 95 matrix. Casein kinase 1 (CK1) was identified as a principal 95 matrix binding protein in Plasmodium falciparum, Leishmania mexicana, Toxoplasma gondii and Trypanosoma cruzi. Purvalanol compounds also inhibit the proliferation 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 purified kinases suggests that this could be a general method for identifying intracellular targets relevant to a particular class of ligands. This method allows a close correlation to be established between the pattern of proteins bound to a small family of related compounds and the pattern of cellular responses to these compounds.