A Robust, Target-Driven, Cell-Based Assay for Checkpoint Kinase 1 Inhibitors

Author:

Ishii Tsuyoshi1,Sootome Hiroshi2,King Alastair J.3,Suda Mikiya4,Noro Nobuhiro1,Yamashita Keizo1,Noumi Takato1

Affiliation:

1. GlaxoSmithKline K.K., Tsukuba-shi Ibaraki, Japan

2. Bioscience Department GlaxoSmithKline K.K. 43 Wadai, Tsukuba-shi Ibaraki 300-4247, Japan,

3. GlaxoSmithKline, Upper Providence, Pennsylvania

4. GlaxoSmithKline K.K., Tsukuba-shi Ibaraki, Japan, Molecular Medicine Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan

Abstract

Checkpoint kinase 1 (Chk1), a serine/threonine kinase, plays an important role in DNA damage checkpoint control and is an attractive target for cancer treatment. To develop a Chk1-specific cell-based assay, stable clones were established in which Chk1 kinase domain fused at its N-terminus with p53 through 4 tandem repeats of Gly-Gly-Gly-Gly-Ser was expressed in an inducible manner. Chk1 kinase specificity of the phosphorylation of fused p53 was confirmed by the experiments with a kinase-inactive Chk1. Only in the presence of an inducer molecule was phosphorylation of p53 at Ser-15 in the stable clones induced. Furthermore, its assay performance proved acceptable for high-throughput screening applications, judging from the Z′ factor values (> 0.77). Finally, the cell-based assay thus established yielded structure-activity relationship data for a small set of test inhibitors of Chk1 within cells. Collectively, these results demonstrate that the established cell-based assay provides a novel and highly sensitive cellular platform for Chk1 inhibitor discovery. ( Journal of Biomolecular Screening 2007:809-817)

Publisher

Elsevier BV

Cited by 3 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

1. Stem cell contributions to neurological disease modeling and personalized medicine;Progress in Neuro-Psychopharmacology and Biological Psychiatry;2018-01

2. Cell-based assays for kinase drug discovery;Drug Discovery Today: Technologies;2010-03

3. A cell-based screening method for specifically detecting kinase activity;Analytical and Bioanalytical Chemistry;2007-11-06

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