Tivantinib (ARQ 197) is an orally bioavailable small molecule inhibitor of c-Met with potential antineoplastic activity.

c-Met:0.355 μM(Ki)

用ARQ-197处理的所有三种异种移植物模型显示肿瘤生长减少：HT29模型中66％,MKN-45模型中45％,MDA-MB-231模型中79％.口服给药200 mg/kg ARQ-197处理HT29,MKN-45和MDA-MB-231三种移植瘤模型,没有显著的体重变化.药效学方面,ARQ-197强效抑制人类结肠移植瘤HT29中c-Met的磷酸化,单独口服200 mg/kg ARQ-19724小时后,c-Met自磷酸化强烈下降.总之,ARQ-197抑制ARQ-197抑制c-Met依赖性异种移植人类肿瘤的生长.

ARQ-197具有抗肿瘤活性,抑制A549,DBTRG和NCI-H441细胞增殖,IC50分别为0.38,0.45,0.29 μM。用ARQ-197处理,导致MAPK信号级联反应的磷酸化减少及预防侵入和迁移。此外,在没有内源性c-Met表达的细胞系NCI-H661中c-Met的异位表达导致其获得侵袭性表型,也被ARQ-197抑制。ARQ-197抑制人类重组c-Met,具有恒定的Ki值,为355 nM。ARQ-197抑制c-Met磷酸化,且阻断下游c-Met信号通路。尽管增加浓度的ARQ-197不会显著影响ATP的Km,但c-Met暴露于0.5 μMARQ-197会使c-Met的Vmax降低约3倍。ARQ-197降低Vmax而不影响ATP的Km值说明ARQ-197抑制c-Met是非ATP竞争抑制,也说明ARQ-197具有高度激酶选择性。ARQ-197抑制组成型和配体介导的c-Met自磷酸化,并且进而抑制c-Met活性,继而导致抑制下游c-Met效应物。在表达c-Met的人类癌细胞（包括HT29,MKN-45和MDA-MB-231细胞）中ARQ-197诱导胱天蛋白酶依赖性细胞凋亡增加。

c-Met SDS-PAGE in vitro kinase assay: Recombinant c-Met protein (100 ng) is preincubated with increasing concentrations of ARQ-197 for 30 minutes at room temperature. Following preincubation, 100 μM of poly-Glu-Tyr substrate and various concentrations of ATP containing 5 μCi of [γ-32P]ATP are added to the reaction mixture. The reaction is incubated for 5 minutes at room temperature and then stopped by the addition of 5 μL of SDS-polyacrylamide gel, reducing sample buffer. The samples are then loaded onto a 7.5% acrylamide gel and SDS-PAGE is performed. The phosphorylated poly-Glu-Tyr substrates are ultimately visualized by autoradiography. c-Met activity is quantified by densitometry.

HT29, MKN-45, and MDA-MB-231 cells are seeded in black 96-well plates at 5 × 103 cells per well overnight in a medium with 10% FBS. The next day, cells are treated with increasing concentrations of ARQ-197 (0.03-10 μM) for 24, 32, and 48 hours at 37 °C. After ARQ-197 treatment, the drug-containing medium is removed and cells are incubated for at least 10 minutes in a labeling solution (10 mM HEPES, 140 mM NaCl, and 6 mM CaCl2) containing 2 μg/mL Hoescht 33342 (blue channel), 500-times diluted Annexin V-FITC (green channel), and 1 μg/mL propidium iodide (red channel). High-content image acquisition and analysis are carried out. The program is set to take four images per well. The exposure time is set at 16.7 ms/10% gain, 500 ms/35% gain, and 300 ms/30% gain for the 4,6-diamidino-2-phenylindole, FITC, and rhodamine channels, respectively. Images are processed and the numbers of positive cells for each channel and each condition are determined. In addition, HT29 cells are treated with increasing concentrations of ARQ-197 for 32 hours in the absence or the presence of 25, 50, and 100 μM ZvAD-FMK (irreversible general caspase inhibitor), and the same procedures are undertaken. All experiments are done in triplicate. To determine whether the apoptotic effect is due to c-Met inhibition, the effect of ARQ-197 when glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and c-Met are knocked down using siRNA is investigated. HT29, MKN-45, and MDA-MB-231 cells are transfected with a nontargeted control siRNA, a gapgh-targeted control siRNA, or a met-targeted siRNA. After 3 days, c-Met, GAPDH, and β-actin expression levels are determined using specific antibodies. To determine if the effect is caspase dependent, HT29, MKN-45, and MDA-MB-231 cells are transfected with a met-targeted siRNA for 2 days and incubated in the absence or the presence of increasing concentrations of ZvAD-FMK for 1 additional day.A nontargeted siRNA and a gapgh-targeted siRNA (siRNA GAPDH) are also transfected in parallel, as controls. Cells are then stained with Annexin V-FITC and propidium iodide, and the percentage of apoptotic cells is determined.(Only for Reference)