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

Research output: Contribution to Journal/MagazineJournal articlepeer-review

  • Marie Knockaert
  • N. Gray
  • E. Damiens
  • Y.-T. Chang
  • P. Grellier
  • Karen M. Grant
  • David Fergusson
  • Jeremy C. Mottram
  • M. Soete
  • J.-F. Dubremetz
  • K. Le Roch
  • C. Doerig
  • P. G. Schultz
  • Laurent Meijer
<mark>Journal publication date</mark>1/06/2000
<mark>Journal</mark>Chemistry and Biology
Issue number6
Number of pages12
Pages (from-to)411-422
Publication StatusPublished
<mark>Original language</mark>English


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.