title: 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.affiliation: Station Biologique de Roscoff, CNRS, Roscoff cedex, 29682, France.
authors.forename: M
authors.initials: M
authors.lastname: Knockaert
authors.forename: N
authors.initials: N
authors.lastname: Gray
authors.forename: E
authors.initials: E
authors.lastname: Damiens
authors.forename: Y T
authors.initials: YT
authors.lastname: Chang
authors.forename: P
authors.initials: P
authors.lastname: Grellier
authors.forename: K
authors.initials: K
authors.lastname: Grant
authors.forename: D
authors.initials: D
authors.lastname: Fergusson
authors.forename: J
authors.initials: J
authors.lastname: Mottram
authors.forename: M
authors.initials: M
authors.lastname: Soete
authors.forename: J F
authors.initials: JF
authors.lastname: Dubremetz
authors.forename: K
authors.initials: K
authors.lastname: Le Roch
authors.forename: C
authors.initials: C
authors.lastname: Doerig
authors.forename: P
authors.initials: P
authors.lastname: Schultz
authors.forename: L
authors.initials: L
authors.lastname: Meijer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D056890:Eukaryota / Q000201:enzymology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000201:enzymology; D051381:Rats; D013215:Starfish / Q000166:cytology; D013379:Substrate Specificity; D013552:Swine; D014982:Xenopus laevis
chemical_list: D004791:Enzyme Inhibitors; D018844:Cyclin-Dependent Kinases