title: A selective cellular screening assay for B-Raf and c-Raf kinases.
abstract: The Ras/Raf signaling pathway has been recognized as an important process in cancer biology. Recently, activating mutations in the BRAF gene were reported to be present in approximately 66% of malignant melanomas as well as other malignancies such as colon cancer. Here, the authors report the development of a B-Raf-specific cellular assay to profile cell-active B-Raf inhibitors. Expression of the active B-Raf mutant (V600E) and the kinase-inactive form of its substrate, MEK1, was regulated by mifepristone, and the catalytic activity of B-Raf was monitored by following MEK1 phosphorylation. Target specificity was ensured because the phosphorylation of MEK1 was significantly inhibited when kinase-inactive B-Raf was used in place of the active kinase. A cellular c-Raf assay was similarly established to monitor the selectivity between B-Raf and c-Raf. Z' factor values were consistently above 0.50 with either kinase, indicating that assay performance was sufficiently robust for use as cellular profiling assays. The authors used this system to demonstrate that the selectivity profile of compounds targeted against B-Raf and c-Raf kinases could be quantitatively determined. This platform provides a quantitative cellular readout for a spectrum of specific inhibitors of B-Raf and c-Raf kinases that is particularly suitable for use in drug discovery.
authors.affiliation: GlaxoSmithKline K.K., Ibaraki, Japan. tsu_ishii01@yahoo.co.jp
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Ish
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sootome
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Yagi
authors.forename: Keizo
authors.initials: K
authors.lastname: Yamashita
authors.forename: Takato
authors.initials: T
authors.lastname: Noumi
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Noro
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Ishii
mesh_terms: D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005240:Feasibility Studies; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors* / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D047428:Protein Kinase Inhibitors; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human