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Journal of Biomolecular Screening
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A Robust, Target-Driven, Cell-Based Assay for Checkpoint Kinase 1 Inhibitors

Tsuyoshi Ishii

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

Hiroshi Sootome

Bioscience Department GlaxoSmithKline K.K. 43 Wadai, Tsukuba-shi Ibaraki 300-4247, Japan, hiroshi_sootome{at}merck.com

Alastair J. King

GlaxoSmithKline, Upper Providence, Pennsylvania

Mikiya Suda

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

Nobuhiro Noro

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

Keizo Yamashita

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

Takato Noumi

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

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)

Key Words: checkpoint kinase 1 • GeneSwitch system • target specific • cell-based assay

This version was published on September 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 6, 809-817 (2007)
DOI: 10.1177/1087057107303323
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