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INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH

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IJPR included in UGC-Approved List of Journals - Ref. No. is SL. No. 4812 & J. No. 63703

Published by : Advanced Scientific Research
ISSN
0975-2366
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IJPR 9[3] July - September 2017 Special Issue

July - September 9[3] 2017

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Validation of Luminescent Assay for CDK2 Inhibitors.

Author: UPLOADED BY-ADMIN, MAULIK P. SUTHAR, RAKESH P. PATEL
Abstract: Background: Cyclin Dependent Kinases ensures the tight regulation of the cell cycle execution by mediating phosphorylation of cellular proteins. Mis-regulation of the CDK2 activity by cellular and external factors leads to many diseases like cancers. Objective: Present study is focused on development, optimization and validation of CDK2 assay which is suitable for identification potent and selective, ATP competitive inhibitors of CDK2. Method: Luminescent Signal was developed using beetle luciferin as a substrate for determination of residual ATP leftover after Histone H1 phosphorylation, mediated by CDK2 isoforms. Reactions were started by adding 100 µM ATP and 2.5mg/ml Histone H1, at room temperature for 20 minutes. 5µl of Kinase glo plus reagent was added and was allowed to stabilize for 1 min, and then luminescence was recorded. Results: Enzyme and substrate concentrations were optimized, which were 50 ng/well for CDK2/Cyclin A and 25 ng/well for CDK2/cyclin E. Histone H1 was used as a substrate. Z’ was determined, which were 0.817 for CDK2/cyclin A and 0.772 for CDK2/cyclin E assays. Conclusions: With Z’ factor higher than 0.7, both assays are suitable for screening of inhibitors. This can help to determine selectivity and potency of CDK2 inhibitors.
Keyword: CDK2, Luminescence, inhibitors
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