Tivantinib

ARQ-197 has been shown to prevent HGF/c-met induced cellular responses in vitro. This compound possesses antitumor activity; inhibiting proliferation of A549, DBTRG and NCI-H441 cells with IC50 of 0.38, 0.45, 0.29 μM. Treatment with this agent results in a decrease in phosphorylation of the MAPK signaling cascade and prevention of invasion and migration. In addition, ectopic expression of c-Met in NCI-H661, a cell line having no endogenous expression of c-Met, causes it to acquire an invasive phenotype that is also suppressed by this chemical. Although the addition of increasing concentrations of this inhibitor does not significantly affect the K_m of ATP, exposure of c-Met to 0.5 μM of this substance decreased the V_max of c-Met by approximately 3-fold. The ability of this molecule to decrease the V_max without affecting the Km of ATP confirmed that it inhibits c-Met through a non–ATP-competitive mechanism and may therefore account for its high degree of kinase selectivity. It prevents human recombinant c-Met with a calculated inhibitory constant K_i of approximately 355 nM. Although the highest concentration of ATP used is 200 μM, the potency of this compound against c-Met is not reduced by using concentrations of ATP up to 1 mM. It blocks c-Met phosphorylation and downstream c-Met signaling pathways. This chemical suppresses constitutive and ligand-mediated c-Met autophosphorylation and, by extension, c-Met activity, in turn leading to the inhibition of downstream c-Met effectors. Its induction of caspase-dependent apoptosis is increased in c-Met–expressing human cancer cells including HT29, MKN-45, and MDA-MB-231 cells.^[1][2]

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 this compound (0.03-10 μM) for 24, 32, and 48 hours at 37 °C. After this compound 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 this compound 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 this compound 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.

All three xenograft models treated with Tivantinib display reductions in tumor growth: 66% in the HT29 model, 45% in the MKN-45 model, and 79% in the MDA-MB-231 model. In these xenograft studies, no significant body weight changes following oral administration of this compound at 200 mg/kg are observed. Pharmacodynamically, the phosphorylation of c-Met in human colon xenograft tumors (HT29) is strongly inhibited by this chemical, as assessed by a dramatic reduction of c-Met autophosphorylation 24 hours after a single oral dose of 200 mg/kg of this agent. This same dosage in mice exhibits that tumor xenografts are exposed to sustained plasma levels of the compound, consistent with the observed pharmacodynamic inhibition of c-Met phosphorylation and inhibition of proliferation of c-Met harboring cancer cell lines. Plasma levels of the agent 10 hours after dosing are determined to be 1.3 μM, more than 3-fold above the biochemical inhibitory constant of this substance for c-Met. Therefore, it is able to suppress its target in vivo in the xenografted human tumor tissue. In conclusion, this inhibitor blocks the growth of c-Met-dependent xenografted human tumors.^[1]