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: Marie Knockaert, Philippe Lenormand, Nathanael Gray, Peter Schultz, Jacques Pouysségur, Laurent Meijer
• Journal: Oncogene (Oncogene) Vol. 21 Issue 42 Pg. 6413-24 (Sep 19 2002) ISSN: 0950-9232 [Print] England
• PMID: 12226745 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Enzyme Inhibitors
• Phosphoproteins
• RNA-Binding Proteins
• nucleolin
• purvalanol B
• Luciferases
• Cyclin-Dependent Kinases
• Mitogen-Activated Protein Kinase 1
• Mitogen-Activated Protein Kinase 3
• Mitogen-Activated Protein Kinases
• Adenine

Topics

• Adenine (analogs & derivatives, chemistry, pharmacology)
• Animals
• Blotting, Western
• Cell Cycle (drug effects)
• Cell Division (drug effects)
• Cell Survival (drug effects)
• Cells, Cultured (drug effects)
• Cricetinae
• Cyclin-Dependent Kinases (antagonists & inhibitors)
• Enzyme Inhibitors (pharmacology)
• Fluorescent Antibody Technique, Indirect
• Luciferases (metabolism)
• Mitogen-Activated Protein Kinase 1 (metabolism)
• Mitogen-Activated Protein Kinase 3
• Mitogen-Activated Protein Kinases (metabolism)
• Molecular Structure
• Phosphoproteins (metabolism)
• Phosphorylation
• RNA-Binding Proteins (metabolism)
• Transfection