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Intracellular Targets of Cyclin-Dependent Kinase Inhibitors: Identification by Affinity Chromatography Using Immobilised Inhibitors

Intracellular Targets of Cyclin-Dependent Kinase Inhibitors: Identification by Affinity Chromatography Using Immobilised Inhibitors

M Knockaert, N Gray, E Damiens, Y T Chang, P Grellier, K Grant, D Fergusson, J Mottram, M Soete, J F Dubremetz, K Le Roch, C Doerig, P Schultz, L Meijer

Chem Biol. 2000 Jun;7(6):411-22.

PMID: 10873834

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP212844547	Purvalanol B		212844-54-7	Price

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