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

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.

• Authors: 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
• Journal: Chemistry & biology (Chem Biol) Vol. 7 Issue 6 Pg. 411-22 (Jun 2000) ISSN: 1074-5521 [Print] United States
• PMID: 10873834 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Enzyme Inhibitors
• Cyclin-Dependent Kinases

Topics

• Amino Acid Sequence
• Animals
• Chromatography, Affinity (methods)
• Cyclin-Dependent Kinases (antagonists & inhibitors)
• Enzyme Inhibitors (pharmacology)
• Eukaryota (enzymology)
• Molecular Sequence Data
• Oocytes (drug effects, enzymology)
• Rats
• Starfish (cytology)
• Substrate Specificity
• Swine
• Xenopus laevis