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A Selective Cellular Screening Assay for B-Raf and c-Raf Kinases

GlaxoSmithKline K.K., Ibaraki, Japan

Hiroshi Sootome

GlaxoSmithKline K.K., Ibaraki, Japan

Yukiko Yagi

GlaxoSmithKline K.K., Ibaraki, Japan

Keizo Yamashita

GlaxoSmithKline K.K., Ibaraki, Japan

Takato Noumi

GlaxoSmithKline K.K., Ibaraki, Japan

Nobuhiro Noro

GlaxoSmithKline K.K., Ibaraki, Japan

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 ~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. (Journal of Biomolecular Screening 2007:818-827)

Key Words: B-Raf • c-Raf • Raf-1 • cell-based assay • GeneSwitch^TM

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This version was published on September 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 6, 818-827 (2007)
DOI: 10.1177/1087057107302308


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This Article Abstract Free Full Text (Free PDF) Free Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Ishii, T. Articles by Noro, N. Search for Related Content PubMed PubMed Citation Articles by Ishii, T. Articles by Noro, N. Social Bookmarking                         What's this?