In vitro activity: SKLB1002 shows strikingly lower cytotoxicity on normal human cells L-02. SKLB1002 significantly inhibits HUVEC proliferation, migration, invasion, and tube formation, by inhibiting VEGF-induced phosphorylation of VEGFR2 kinase and the downstream protein kinases including ERK, FAK, and Src.

Kinase Assay: Kinase inhibition is measured by the use of radiometric assays conducted by Kinase Profiler service. Briefly, in the presence or absence of SKLB1002, VGFR2 (5–10 mU) is incubated in 25-μL reaction solution containing 8 mmol/L 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.0, 0.2 mmol/L EDTA, 0.33 mg/mL myelin basic protein, 10 mmol/L Mg acetate, and γ-[33P]ATP. After incubation for 40 minutes at room temperature, the reaction is stopped and 10 μL of the reaction solution is then spotted onto a P30 filtermat and washed 3 times for 5 minutes in 75 mmol/L phosphoric acid and once in methanol prior to scintillation counting.

Cell Assay: Cell proliferation is measured using MTT assay. Various cells including HUVECs, L-02, B16-F10, HepG2, and SW620 are treated with indicated concentrations of SKLB1002 for 24 hours. Vandetanib and sunitinib serve as positive controls. Each assay is replicated 3 times.

Clin Cancer Res. 2011 Jul 1;17(13):4439-50; Clin Exp Med. 2014 May;14(2):203-13.