title: p42/p44 MAPKs are intracellular targets of the CDK inhibitor purvalanol.
abstract: Chemical inhibitors of cyclin-dependent kinases (CDKs) have a great therapeutic potential against various proliferative and neurodegenerative disorders. Intensive screening of a combinatorial chemistry library of 2,6,9-trisubstituted purines has led to the identification of purvalanol, one of the most potent and selective CDK inhibitors to date. In preliminary studies, this compound demonstrates definite anti-mitotic properties, consistent with its nanomolar range efficiency towards purified CDK1 and CDK2. However, the actual intracellular targets of purvalanol remain to be identified, and a method for the determination of its in vivo selectivity was developed. In this technique, cell extracts were screened for purvalanol-interacting proteins by affinity chromatography on immobilized inhibitor. In addition to CDK1, p42/p44 MAPK were found to be two major purvalanol-interacting proteins in five different mammalian cell lines (CCL39, PC12, HBL100, MCF-7 and Jurkat cells), suggesting the generality of the purvalanol/p42/p44 MAPK interaction. The Chinese hamster lung fibroblast cell line CCL39 was used as a model to investigate the anti-proliferative properties of purvalanol. The compound inhibited cell growth with a GI(50) value of 2.5 microM and induced a G2/M block when added to exponentially growing cells. It did not appear to trigger massive activation of caspase. We next tested whether CDKs and p42/p44 MAPK were actually targeted by the compound in vivo. p42/p44 MAPK activity was visualized using an Elk-Gal4 luciferase reporter system and CDK1 activity was detected by the phosphonucleolin level. When cells were treated with purvalanol, p42/p44 MAPK and CDK1 activities were inhibited in a dose-dependent manner. Furthermore, purvalanol inhibited the nuclear accumulation of p42/p44 MAPK, an event dependent on the catalytic activity of these kinases. We conclude that the anti-proliferative properties of purvalanol are mediated by inhibition of both p42/p44 MAPK and CDKs. These observations highlight the potency of moderate selectivity compounds and encourage the search for new therapeutics which simultaneously target distinct but relevant pathways of cell proliferation.
authors.affiliation: Station Biologique de Roscoff, CNRS, B.P.74, 29682 Roscoff Cedex, Bretagne, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Knockaert
authors.forename: Philippe
authors.initials: P
authors.lastname: Lenormand
authors.forename: Nathanael
authors.initials: N
authors.lastname: Gray
authors.forename: Peter
authors.initials: P
authors.lastname: Schultz
authors.forename: Jacques
authors.initials: J
authors.lastname: Pouysségur
authors.forename: Laurent
authors.initials: L
authors.lastname: Meijer
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured / Q000187:drug effects; D006224:Cricetinae; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D008156:Luciferases / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D015394:Molecular Structure; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D016601:RNA-Binding Proteins / Q000378:metabolism; D014162:Transfection; D000097796:Nucleolin
chemical_list: D000225:Adenine; D018844:Cyclin-Dependent Kinases; D004791:Enzyme Inhibitors; D008156:Luciferases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin; C113584:purvalanol B