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

Abstract

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.

Selective detection of azelnidipine in pharmaceuticals via carbon dot mediated spectrofluorimetric method: A green approach

“…AZEL is a third-generation and long-acting dihydropyridine calcium channel antagonist used for the treatment of hypertension [1]. So far, numerous methodologies have been developed for the estimation of AZEL based on different analytical techniques such as UV-visible spectrophotometry [3][4][5][6][7][8], high-performance liquid chromatography (HPLC)/reverse phase (RP)-HPLC [9][10][11][12][13], ultraperformance/pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) [14], ultra-fast liquid chromatography (UFLC) [15], LC-ESI-MS/MS [16], HPLC-MS/MS [17], and high-performance thin layer chromatography (HPTLC) [18] for the estimation of AZEL alone, as well as in combination with other drugs in pharmaceutical dosage forms [19]. However, these methods have their limitations requiring more expensive instruments, a large quantity of samples, the use of costly organic solvents, complex mathematic dealing, and robust sample handling, and they are time consuming.…”