Abstract
Cyclin dependent kinases (CDKs) are key regulators of the cell cycle progression and therefore constitute excellent targets for the design of anticancer agents. Most of the inhibitors identified to date inhibit kinase activity by interfering with the ATP-binding site of CDKs. We recently proposed that the protein/protein interface and conformational changes required in the molecular mechanism of CDK2-cyclin A activation were potential targets for the design of specific inhibitors of cell cycle progression. To this aim, we have designed and characterized a small peptide, termed C4, derived from amino acids 285-306 in the alpha5 helix of cyclin A. We demonstrate that this peptide does not interfere with complex formation but forms stable complexes with CDK2-cyclin A. The C4 peptide significantly inhibits kinase activity of complexes harboring CDK2 in a competitive fashion with respect to substrates but does not behave as an ATP antagonist. Moreover, when coupled with the protein transduction domain of Tat, the C4 peptide blocks the proliferation of tumor cell lines, thereby constituting a potent lead for the development of specific CDK-cyclin inhibitors.
Highlights
In higher eukaryotes, cell cycle progression is coordinated by several closely related Ser/Thr protein kinases resulting from the association of a catalytic cyclin-dependent kinase (CDK)1 subunit with a regulatory cyclin subunit [1]
We recently proposed that the protein/protein interface and conformational changes required in the molecular mechanism of CDK2-cyclin A activation were potential targets for the design of specific inhibitors of cell cycle progression
Based on these structural and biochemical characterizations, we propose a novel approach to inhibiting the kinase activity of CDK-cyclin complexes by targeting proteinprotein interfaces or conformational changes involved in their activation
Summary
Chemicals—N,N-Dimethylformamide, piperidine, trifluoroacetic acid, and CH3CN (high performance liquid chromatography grade) were purchased from SDS (Peypin, France). Where Vmax is the maximum velocity, [S] is the concentration of the varied substrate (ATP or histone H1), and Ki is the dissociation constant for the CDK2-cyclin A-peptide complex. GST complexes were pulled down with glutathione-Sepharose beads and washed and eluted with buffer A containing 20 mM glutathione Both the flow-through and the elution were analyzed by SDS-PAGE and Western blotting using antibodies against CDK2 (affinity-purified PSTAIRE polyclonal antibody) and cyclin A (H-432; Santa Cruz Biotechnology, Santa Cruz, CA). Peptide samples were prepared at different concentrations in the range of 50 to 1000 nM in 50 mM Tris HCl (pH 8.0) buffer containing 150 mM NaCl, 2 mM EDTA, and 50 mM KCl and were injected (30 l) over the sensor surface at a flow rate of 10 l1⁄7sϪ1. The modeling package Discover/Insight II (MSI Inc.; San Diego, CA) and DeLano Scientific LLC PyMol software version 0.93 (DeLano Scientific, San Carlos CA) were used to analyze the structure of CDK2-cyclin A
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