In vitro activity: Savolitinib has exquisite kinase selectivity and excellent potency. It displays a high selectivity across a gastric cell line panel, potently inhibiting cell growth only in those lines with dysregulated cMET (EC50 values 0.6 nM/L-12.5 nM/L). Savolitinib has high membrane permeability without efflux transport across Caco-2 cell monolayer and exhibits negligible P-gp inhibition (IC50> 17 μM). Savolitinib shows no significant reversible or mechanism-based CYP inhibition in human liver microsomes, and no induction of CYP1A2 and CYP3A4 in human hepatocytes.

Kinase Assay: Savolitinib has IC50 values of 5 nM and 3 nM for c-Met and p-Met respectively. It exhibits high selectivity for c-Met over 274 other kinases.

Cell Assay: After incubation overnight, NCI-H441 cells are then treated with serially diluted test compounds at 37 ℃ for 1 h. Then the medium is removed, and cells are lysed in 100 μL/well lysis buffer (1% NP-40, 20 mM Tris/pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM activated sodium orthovanadate, 10 mg/mL Aprotinin, 10 mg/mL Leupeptin). The plates containing cell lysate are kept at -80℃ overnight. The next day, the plates are thawed on ice, mixed gently. 25 μL/well of lysates are added into the assay plates pre-coated with anti-p-Met antibody to detect p-c-Met signal. p-c-Met level is determined at 450 nm and 570 nm.