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用于筛选细胞周期蛋白依赖性激酶2抑制剂的发光测定法的优化。

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

作者信息

Suthar M P, Patel M M

机构信息

Department of Biotechnology, Shree S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Kherva-382 711, India.

出版信息

Indian J Pharm Sci. 2010 May;72(3):290-4. doi: 10.4103/0250-474X.70472.

DOI:10.4103/0250-474X.70472
PMID:21188035
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3003159/
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.

摘要

细胞周期蛋白依赖性激酶是癌症化疗中研究最为广泛的靶点，因为肿瘤细胞存在错误的检查点，即使基因组受损也能增殖。细胞周期蛋白依赖性激酶通过介导细胞蛋白的磷酸化来确保细胞周期执行的严格调控。细胞内和外部因素导致的细胞周期蛋白依赖性激酶2活性失调会引发许多疾病，如癌症。有多种方法可用于筛选针对激酶的化合物文库，如放射性标记、荧光和发光法。然而，生物发光法具有一些优势，如背景低且不受荧光化合物干扰。本研究聚焦于细胞周期蛋白依赖性激酶2检测方法的开发、优化和验证，该方法适用于鉴定细胞周期蛋白依赖性激酶2的强效、选择性、ATP竞争性和非竞争性抑制剂。本研究的目的是利用基于生物发光的生化反应优化细胞周期蛋白依赖性激酶2/细胞周期蛋白A和细胞周期蛋白依赖性激酶2/细胞周期蛋白E的检测方法。细胞周期蛋白依赖性激酶2的两种复合物，即细胞周期蛋白依赖性激酶2/细胞周期蛋白A和细胞周期蛋白依赖性激酶2/细胞周期蛋白E，对ATP具有不同的亲和力。因此，分别对细胞周期蛋白依赖性激酶2的两种异构体类似物进行了优化。发现细胞周期蛋白依赖性激酶2/细胞周期蛋白A和细胞周期蛋白依赖性激酶2/细胞周期蛋白E的最佳浓度分别为250 ng/孔和200 ng/孔。两种细胞周期蛋白依赖性激酶2类似物的最佳底物（组蛋白H1）浓度均为2.5 mg/ml。两种细胞周期蛋白依赖性激酶2/细胞周期蛋白复合物的最佳反应时间均为20分钟。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/dadf4a33a872/IJPhS-72-290-g028.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/9cd3cc73df5e/IJPhS-72-290-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/fc6c7cd73156/IJPhS-72-290-g012.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/74773a8e3fbf/IJPhS-72-290-g016.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/c4a54c7ff5eb/IJPhS-72-290-g019.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/ff829ccbb478/IJPhS-72-290-g022.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/5c15c2ab2d5e/IJPhS-72-290-g025.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/dadf4a33a872/IJPhS-72-290-g028.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/9cd3cc73df5e/IJPhS-72-290-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/fc6c7cd73156/IJPhS-72-290-g012.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/74773a8e3fbf/IJPhS-72-290-g016.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/c4a54c7ff5eb/IJPhS-72-290-g019.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/ff829ccbb478/IJPhS-72-290-g022.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/5c15c2ab2d5e/IJPhS-72-290-g025.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6117/3003159/dadf4a33a872/IJPhS-72-290-g028.jpg

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