title: A robust, target-driven, cell-based assay for checkpoint kinase 1 inhibitors.
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.
authors.affiliation: GlaxoSmithKline K.K., Tsukuba-shi Ibaraki, Japan. hiroshi_sootome@merck.com
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Ish
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sootome
authors.forename: Alastair J
authors.initials: AJ
authors.lastname: King
authors.forename: Mikiya
authors.initials: M
authors.lastname: Suda
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Noro
authors.forename: Keizo
authors.initials: K
authors.lastname: Yamashita
authors.forename: Takato
authors.initials: T
authors.lastname: Noumi
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Ishii
mesh_terms: D002460:Cell Line; D000071877:Checkpoint Kinase 1; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005240:Feasibility Studies; D005786:Gene Expression Regulation; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D016158:Genes, p53; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D008954:Models, Biological; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1