A Robust, Target-Driven, Cell-Based Assay for Checkpoint Kinase 1 Inhibitors
Tsuyoshi Ishii,
Hiroshi Sootome*,
Alastair J. King,
Mikiya Suda,
Nobuhiro Noro,
Keizo Yamashita,
Takato Noumi
GlaxoSmithKline K.K
* To whom correspondence should be addressed. E-mail: hiroshi_sootome{at}merck.com.
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Abstract |
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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 XXXX:xx-xx)
