MET Tyrosine Kinase Inhibitor PF-04217903 is an orally bioavailabe, small-molecule tyrosine kinase inhibitor with potential antineoplastic activity.

c-Met:4.8 nM

尽管在舒尼替尼敏感的B16F1和Tib6肿瘤模型中不能抑制肿瘤生长,但与单独使用舒尼替尼或PF-04217903相比,PF-04217903和舒尼替尼联用显著抑制舒尼替尼耐受性EL4和LLC肿瘤模型的肿瘤生长,血管扩张,表明舒尼替尼耐药肿瘤中HGF/c-Met轴的功能作用.

PF-04217903明显抑制 LXFA 526L和LXFA 1647L克隆生长,IC50分别为16 nM和13 nM,与西妥昔单抗联用效果增强。PF-04217903有效抑制c-Met驱动的生物过程,如多种肿瘤细胞的生长,运动,侵袭和形态学变化。2 μM PF-04217903促进GTL-16 细胞死亡,其涉及磷酸化的4E-BP1,ERK/MAPK相关蛋白和 PI3K/AKT通路的下调。PF-04217903选择性比十字孢碱或PF-02341066高,PF-04217903作用于c-Met选择性比作用于其他一组208种激酶选择性高1000多倍,对c-Met致癌突变更敏感。除WT c-Met之外,PF-04217903显示出类似的抑制c-Met-H1094R,c-Met-R988C和c-Met-T1010I活性的效力,IC50为3.1 nM,6.4 nM和6.7 nM,但对c-Met-Y1230C没有抑制活性,IC50>10 μM。PF-04217903和舒尼替尼联用明显抑制内皮细胞增殖,但是不抑制肿瘤细胞B16F1,Tib6,EL4和LLC。

Cellular c-Met phosphorylation ELISA: A549 cells with endogenous human WT c-Met are seeded in 96-well plates in growth medium and cultured overnight. On the second day of the assay, the growth medium is replaced with serum-free medium (with 0.04% BSA). Serial dilutions of PF-04217903 are added to each well, and cells are incubated at 37 °C for 1 hour. Then 40 ng/mL HGF is added to the cells for 20 minutes. The cells are washed once with HBSS supplemented with 1 mM Na3VO4, and protein lysates are generated from cells using lysis buffer. Phosphorylation of c-Met is assessed by an ELISA method utilizing capture antibodies specific for c-Met and a detection antibody specific for phosphorylated tyrosine residues. Antibody-coated plates are incubated in the presence of protein lysates at 4 °C overnight and washed with 1% Tween 20 in PBS seven times. HRP-PY20 (horseradish peroxidase-conjugated anti-phosphotyrosine) is diluted 1:500 in blocking buffer and added to each plate for 30 minutes. Plates are then washed again, and TMB peroxidase substrate is added to initiate the HRP-dependent colorimetric reaction and the reaction stopped by addition of 0.09 N H2SO4. ELISA end points are the absorbance measured at 450 nm using a spectrophotometer. IC50 value is calculated by concentration-response curve fitting utilizing a Microsoft Excel-based four-parameter analytical met

Cells are treated with different concentrations PF-04217903 for 4 days. Cell proliferation is assessed by counting content of each well using a Coulter counter machine.(Only for Reference)