| Assay Method Information | |
| | Kinase Enzyme Assay |
| Description: | The compounds of the invention were screened in vitro for their ability to inhibit c-Met kinase activity. The IC50 values for the inhibition of c-Met kinase were determined as described in the literature with some modifications (Wang, X. et al, Mol. Cancer. Ther. 2003, 2(11):1085-1092; Calic, M. et al., Croatica Chemical ACTA. 2005, 78(3):367-374). Briefly, histidine-tagged c-Met catalytic domain fusion protein (Invitrogen, #PV3143) was used for the assay. IC50 measurements were based on the degree of phosphorylation of poly Glu-Tyr (Sigma-Aldrich, #P0275) that was coated (0.01 mg/per well) on 96-well microplates (R&D systems, #DY990). The reaction was carried out in a 50 uL solution containing 50 mM HEPES (pH 7.5), 10 mM MnCl2, 10 mM MgCl2, 0.5 mM DTT, 100 uM Na3VO4, 5 uM ATP (Cell Signaling Technology, #9804) and serial dilutions of the test compound. The reaction lasted for 25 minutes at 30° C. After the reaction was completed, the contents of the plates were.discarded. |
| Affinity data for this assay | |
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