Optimization of luminescent assay for screening of cyclin-dependent kinase 2 inhibitors.

Cyclin-dependent kinases are most extensively studied targets for cancer chemotherapy since the tumor cells exhibit false checkpoints and can proliferate even if the genome is compromised. Cyclin-dependent kinases ensure the tight regulation of the cell cycle execution by mediating phosphorylation of cellular proteins. Deregulation of the cyclin-dependent kinase 2 activity by cellular and external factors leads to many diseases like cancers. Different methods like radiolabeled, fluorescence and luminescence are available for screening of library of compounds against kinases. However, bioluminescent methods offer several advantages like low background and no effect of fluorescent compound interference. Present study is focused on development, optimization and validation of cyclin-dependent kinase 2 assay which is suitable for identification potent and selective, ATP competitive and non-competitive inhibitors of cyclin-dependent kinase 2. The aim of present investigation was to optimize the assay for cyclin-dependent kinase 2/cylin A and cyclin-dependent kinase 2/cyclin E with use of bioluminescence based biochemical reaction. Both cyclin-dependent kinase 2 which are cyclin-dependent kinase 2/cyclin A and cyclin-dependent kinase 2/cyclin E complexes, have different affinity for ATP. Therefore, both isoform analogs of cyclin-dependent kinase 2 were optimized separately. Optimum cyclin-dependent kinase 2/cyclin A and cyclin-dependent kinase 2/cyclin E concentration were found to be 250 ng/well and 200 ng/well, respectively. Optimum substrate (histone H1) concentration was found to be 2.5 mg/ml for both cyclin-dependent kinase 2 analogs. Optimum reaction time was found to be 20 min for both cyclin-dependent kinase 2/cyclin complexes.