cMet is known to play a significant role in tumor development and progression. Identification of inhibitors of cMet signaling may be of therapeutic value in the treatment of cancer. As part of an effort to characterize possible inhibitors of hepatocyte growth factor (HGF) and cMet interactions, a kinase receptor activation (KIRA) bioassay was developed to detect cMet phosphorylation. After stimulation with wild type HGF or mutant HGF in the presence and absence of inhibitors, the cells were lysed, and phosphorylated cMet was detected immunochemically; here we describe the development and optimization of the assay. A cMet-expressing cell line was selected that yielded suitable signal-to-noise (S/N) ratios, and the time course of cMet phosphorylation was characterized. Finally the detection portion of the assay was converted from an ELISA to an electrochemiluminescent technology, which simplified the assay, increased assay throughput and further improved the S/N ratio. The flexibility of the assay allowed for the analysis of modulators of cMet signaling as diverse as small molecule kinase inhibitors, HGF mutants, and anti-HGF or anti-cMet antibodies.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]