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Optimization of luminescent assay for screening of cyclin-dependent kinase 2 inhibitors
1Department of Biotechnology, Shree S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Kherva-382 711, India.
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.
Insights
This study optimized a bioluminescent assay for cyclin-dependent kinase 2 (CDK2) activity, crucial for cancer drug discovery. The validated assay enables efficient screening of potential CDK2 inhibitors for cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Cyclin-dependent kinases (CDKs) regulate the cell cycle and are key targets in cancer chemotherapy.
- Deregulation of cyclin-dependent kinase 2 (CDK2) activity is implicated in various cancers.
- Bioluminescent assays offer advantages over other screening methods due to low background and minimal compound interference.
Purpose of the Study:
- To develop, optimize, and validate a bioluminescent assay for cyclin-dependent kinase 2 (CDK2).
- To enable the identification of potent and selective ATP-competitive and non-competitive CDK2 inhibitors.
- To optimize assays for CDK2/cyclin A and CDK2/cyclin E complexes separately due to differing ATP affinities.
Main Methods:
- Utilized a bioluminescence-based biochemical reaction for assay development.
- Optimized concentrations of CDK2/cyclin A (250 ng/well) and CDK2/cyclin E (200 ng/well).
- Determined optimal substrate (histone H1) concentration (2.5 mg/ml) and reaction time (20 min) for both complexes.
Main Results:
- Successfully developed and optimized a bioluminescent assay for CDK2 activity.
- Established optimal conditions for CDK2/cyclin A and CDK2/cyclin E assays.
- Validated assay parameters for reliable screening of kinase inhibitors.
Conclusions:
- The optimized bioluminescent assay is suitable for identifying potent and selective CDK2 inhibitors.
- This assay provides a robust platform for drug discovery targeting CDK2 in cancer.
- The validated method facilitates efficient screening of compound libraries against CDK2.
