| Assay Method Information | |
| | Evaluation of Enzyme Inhibitory Activity for PDGFR-α, PDGFR-β, c-KIT Kinase |
| Description: | In order to evaluate the inhibitory activity of the compounds synthesized according to the above examples on PDGFR-α(wt), PDGFR-β(wt), c-KIT(wt), and c-KIT(D816V) kinases, the following method was performed. The compounds of examples were reacted with purified human PDGFR-α(wt) (Signalchem #P12-18G) enzyme, human PDGFR-β(wt) (Signalchem #P13-11G) enzyme, human c-KIT(wt) (Signalchem #K06-11BG) enzyme, human c-KIT(D816V) (Signalchem #C06-12LG) enzyme, and the enzyme inhibitory abilities were evaluated by the following method.The reaction buffer was used as a composition of 40 mM Tris-HCl pH7.4, 20 mM MgCl2, 0.5 mg/ml BSA, 50 μM DTT, and the reactions of all the tests were carried out on the reaction buffer. The compound was diluted in 12 steps using a serial dilution method from a 10 mM DMSO stock, and the enzyme activity was measured at a final compound concentration of 10˜0.00005645 μM. During the test, the enzyme activity was confirmed using in vitro ADP-Glo™ kinase assay (Promega) after reacting with the proper concentration of human PDGFR-α(wt) enzyme, human PDGFR-β(wt), human c-KIT(wt) enzyme, and human c-KIT(D816V) enzyme with purified ATP and enzyme substrate at 25° C. for 1˜2 hours. At a ratio of 2:2:1, an enzyme-active reaction solution, an ADP-Glo reaction solution, and an enzyme-active detection solution were reacted to measure the inhibition of the enzyme's activity with luminoscence. |
| Affinity data for this assay | |
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