Golvatinib (E-7050) is an orally bioavailable dual kinase inhibitor of c-Met (hepatocyte growth factor receptor) and VEGFR-2 (vascular endothelial growth factor receptor-2) tyrosine kinases with potential antineoplastic activity. c-Met/VEGFR kinase inhibitor E7050 binds to and inhibits the activities of both c-Met and VEGFR-2, which may inhibit tumor cell growth and survival of tumor cells that overexpress these receptor tyrosine kinases. c-Met and VEGFR-2 are upregulated in a variety of tumor cell types and play important roles in tumor cell growth, migration and angiogenesis.

VEGFR2:16 nM, c-Met:14 nM

体外研究表明，Golvatinib 强效抑制 c-Met 和 VEGFR-2 的磷酸化作用。Golvatinib 同时也强效抑制了经 HGF 或 VEGF 刺激的 c-met 扩增肿瘤细胞和内皮细胞的生长。[1] Golvatinib 通过在体外阻断 Met/Gab1/PI3K/Akt 途径，克服了由外源和/或内源 HGF 引发的，对所有可逆的、不可逆的及突变选择性的 EGFR-TKIs 耐药性 EGFR 突变型肺癌细胞系的抵抗。Golvatinib 还能阻止因持续暴露于 HGF 而诱导的 gefitinib 抵抗性 HCC827 细胞的出现。[2]

在体内研究中，使用Golvatinib展示了对肿瘤中c-Met与VEGFR-2磷酸化的抑制，以及异种移植模型中肿瘤生长和肿瘤血管生成的显著抑制。对含有c-met扩增的某些肿瘤细胞系用高剂量Golvatinib（50-200 mg/kg）处理，可诱导肿瘤退缩和消失。在腹膜播散模型中，Golvatinib对腹膜肿瘤显示出抗肿瘤效果，并显著延长了受治疗小鼠的寿命。[1] 另一项异种移植模型研究中，由HGF转染的Ma-1（Ma-1/HGF）细胞产生的肿瘤比载体对照肿瘤更易于血管生成，并表现出对ZD1839的抗性。Golvatinib单独应用可抑制血管生成并延缓Ma-1/HGF肿瘤的生长。Golvatinib与ZD1839联合使用可显著抑制肿瘤生长。[3]

Western blot analysis: The phosphorylation status of c-Met and VEGFR-2 is detected by Western blot analysis. For c-Met, MKN45 cells are incubated with a serial dilution of E7050 in complete medium at 37 °C for 2 h. For VEGFR-2, HUVEC are starved with human endothelial serum free medium containing 0.5% FBS for 24 h. Subsequently HUVEC are incubated with a serial dilution of E7050 for 1 h and then incubated with 20 ng/mL of human VEGF for 5 min. Cells are lysed by lysis buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA [pH 8.0], 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride 1 mM sodium orthovanadate, 10 μg/mL aprotinin, 50 μg/mL leupeptin, and 1 μg/mL pepstatin A). The resected tumor samples are homogenized with lysis buffer containing 25 mM β-glycerophosphate and 0.5% (v/v) phosphatase inhibitor cocktail 2 at 4 °C. Cellular debris is removed by centrifugation at 17 860 g for 20 min at 4 °C. Aliquots of the supernatants containing 5-20 μg of protein are subjected to SDS-PAGE under reducing conditions. The proteins are then transferred onto PVDF membranes, blocked with TBS containing 0.05% Tween-20 and either 5% skim milk or 5% BSA. The membranes are probed with the following antibodies: anti-c-Met polyclonal antibody (C-28) and anti-VEGFR-2 polyclonal antibody (C-20); mouse anti-phosphotyrosine clone 4 g10; and anti-VEGFR-2 polyclonal antibody, anti-phospho-VEGFR-2 (Tyr996) polyclonal antibody, and anti-phospho-c-Met (Tyr1234/1235) polyclonal antibody. Detection is performed using a Super Signal enhanced chemiluminescence kit. Immunoreactive bands are visualized by chemiluminescence with an Image Master-VDS-CL detection system. The intensity of each band is measured by using an image analyzer.

Cells (1–3 × 103 cells/100 μL/well) are seeded on 96-well culture plates with various concentrations of E7050 and cultured for 3 days. Then, 10 μL of WST-8 reagent is added to each well, and absorbance is measured at 450 nm compared with a reference measurement at 660 nm using a MTP-500 microplate reader. HUVEC (2 × 103 cells/well) are cultured for 3 days in medium containing HGF (30 ng/mL), VEGF (20 ng/mL), or basic fibroblast growth factor (bFGF) (20 ng/mL) together with serially diluted E7050.(Only for Reference)