In vitro activity: Pamufetinib/TAS-115 is a novel and potent inhibitor of VEGFR and hepatocyte growth factor receptor (MET)-targeted kinase with an improved safety profile and with IC50s of 30 and 32 nM for rVEGFR2 and rMET, respectively. VEGF receptor (VEGFR) signaling plays a key role in tumor angiogenesis. Although some VEGFR signal-targeted drugs have been approved for clinical use, their utility is limited by associated toxicities or resistance to such therapy. To overcome these limitations, TAS-115 was developed to inhibit the kinase activity of both VEGFR2 and MET and their signal-dependent cell growth as strongly as other known VEGFR or MET inhibitors. The kinase selectivity of TAS-115 was more specific than that of sunitinib and TAS-115 produced relatively weak inhibition of growth (GI50> 10 μmol/L) in VEGFR signal- or MET signal-independent cells. Furthermore, TAS-115 induced less damage in various normal cells than did other VEGFR inhibitors. These data suggest that TAS-115 is extremely selective and specific, at least in vitro. In in vivo studies, TAS-115 completely suppressed the progression of MET-inactivated tumor by blocking angiogenesis without toxicity when given every day for 6 weeks, even at a serum-saturating dose of TAS-115. The marked selectivity of TAS-115 for kinases and targeted cells was associated with improved tolerability and contributed to the ability to sustain treatment without dose reduction or a washout period. Furthermore, TAS-115 induced marked tumor shrinkage and prolonged survival in MET-amplified human cancer-bearing mice. These data suggest that TAS-115 is a unique VEGFR/MET-targeted inhibitor with improved antitumor efficacy and decreased toxicity. In additoin, it has been reported that the triple inhibition of EGFR, HGF/Met, and VEGF/VEGF receptor 2, by either a triplet of clinical drugs or TAS-115 combined with erlotinib, may be useful for controlling progression of EGFR-mutant lung cancer by reversing EGFR-TKI resistance and for inhibiting angiogenesis.

Kinase Assay: Enzyme inhibition studies were performed using a mobility shift assay. Briefly, 0.3 μg/mL of recombinant MET (rMET, N-terminal glutathione S-transferase (GST) Tag; Carna Biosciences) and 1.5 μmol/L of FL-Peptide 2 (Caliper Life Sciences) or 2 μg/mL of recombinant VEGFR2 (rVEGFR2, amino acid 790-end, N-terminal 6His Tagged; Upstate) and 1.5 μmol/L of FL-Peptide 22 (Caliper Life Sciences) were added to a 25 μL mixture containing 1/2 the Michaelis constant (Km) level of ATP, 100 mmol/L of HEPES (pH 7.2), 0.003% (w/v) Brij35, 0.04% (v/v) Tween 20, 10 mmol/L of MgCl2, 1 mmol/L of dithiothreitol, a Complete Mini EDTA-free Protease Inhibitor Cocktail Tablet (Roche Diagnostics, K.K.), and a PhosSTOP Phosphatase Inhibitor Cocktail Tablet (Roche Diagnostics, K.K.), with the addition of 0.05% (w/v) CHAPSO only in the case of rVEGFR2. The reaction mixture was incubated for 90 minutes at 28°C and was stopped by the addition of 15 mmol/L of EDTA. Phosphorylated peptide was calculated using a LabChip EZ Reader, Version 2.1.82.0 (UCC Version: 1.96, CCD Version: 102). On the basis of the amount of phosphorylated peptide formed in the control well and the drug-treated well, the 50% inhibitory concentration (IC50) was calculated using a logistic regression analysis. A total of 192 kinase panel assays was performed using the ProfilerPro Kit 1-8 (Caliper Life Sciences) and was analyzed using a mobility shift assay.

Cell Assay: MKN45 cells were plated in 6-well plates. On the following day, TAS-115 was added to the wells at the desired final concentrations, and the cells were incubated for 30 minutes. After washing, the cells were lysed using Cell Extraction Buffer (Invitrogen Corporation). Cell lysate was analyzed using immunoblotting with the antibodies mentioned in the reagents and antibodies section. To investigate cellular VEGFR2 phosphorylation, HUVECs were plated in collagen-coated dishes (BD Biosciences, Inc.). On the following day, serum starvation was performed during overnight incubation. Then, TAS-115 and VEGF were added at the desired final concentrations. After incubation for 5 minutes, cell lysate was prepared with Mammalian Protein Extraction Reagent (M-PER; Pierce Thermo Scientific) and VEGFR2 phosphorylation was detected using immunoblotting. Blotting images were analyzed using an LAS1000 plus with Multi Gauge, Ver. 3.0 (Fuji Photo Film). The relative signal value from the image was calculated using the signal intensity of GAPDH.