Purpose: The mesenchymal-epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process that is physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with a variety of human malignancies including cancers of the lung, kidney, stomach, liver, and brain. In this study, we investigated the properties of two novel compounds developed to selectively inhibit the c-Met receptor in antitumor therapeutic interventions.Experimental Design: The pharmacologic properties, c-Met inhibitory activity, and antitumor effects of EMD 1214063 and EMD 1204831 were investigated in vitro and in vivo, using human cancer cell lines and mouse xenograft models.Results: EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase activity. Their inhibitory activity was potent [inhibitory 50% concentration (IC 50 ), 3 nmol/L and 9 nmol/L, respectively] and highly selective, when compared with their effect on a panel of 242 human kinases. Both EMD 1214063 and EMD 1204831 inhibited c-Met phosphorylation and downstream signaling in a dose-dependent fashion, but differed in the duration of their inhibitory activity. In murine xenograft models, both compounds induced regression of human tumors, regardless of whether c-Met activation was HGF dependent or independent. Both drugs were well tolerated and induced no substantial weight loss after more than 3 weeks of treatment.Conclusions: Our results indicate selective c-Met inhibition by EMD 1214063 and EMD 1204831 and strongly support clinical testing of these compounds in the context of molecularly targeted anticancer strategies.

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<p>PDF file - 53K, Characteristics of the antibodies used in the study. The specificity, isotype, clone number, and commercial source of the antibodies used throughout the study are indicated. All antibodies were used according to the manufacturer's instructions.</p>

<p>PDF file - 240K, Dose-dependent inhibition of c-Met auto-phosphorylation in vivo. Panel A: Mice bearing established tumors, derived from s.c. injection of Hs746T gastric cancer cells, received a single dose of 3 mg/kg , 10 mg/kg , 30 mg/kg , and 100 mg/kg EMD 1214063. The pharmacokinetic profile of EMD 1214063 was defined by measuring the compound levels in plasma and tumor tissue. The lower limits of detection for EMD 1214063 were 0.007 �M (4 ng/mL) in the plasma and 0.037 �M (20 ng/mL) in tumor samples. The levels of c-Met auto-phosphorylation (Y1234/Y1235) were assessed by Western blot analysis and used as a pharmacodynamic marker of activity. Each data point represents the mean � SD of the values obtained from four mice per experimental group. A data point was not shown if any of the values within the experimental group was below the detection limit. Panel B: A similar experimental approach to that already described for EMD 1214063 was used to define the pharmacokinetic profile and pharmacodynamic effects of EMD 1204831. In brief, mice bearing established Hs746T subcutaneous tumors received a single dose of 3 mg/kg, 10 mg/kg, 30 mg/kg, or 100 mg/kg EMD 1204831. In the pharmacokinetic studies, the lower limits of detection for EMD 1204831 were 0.014 �M (8 ng/mL) in the plasma and tumor samples. The pharmacodynamic effects were determined by Western blot analyses of c-Met auto-phosphorylation (Y1234/Y1235). Each data point represents the mean � SD of the values obtained from four mice per experimental group. A data point was not shown if any of the values within the experimental group was below the detection limit.</p>

<p>PDF file - 45K, ATP competition studies with EMD 1214063. EMD 1214063 inhibited c-Met phosphorylation in an ATP-competitive fashion. In brief, binding competition of EMD 1214063 and ATP to c-Met was analyzed by a kinase reaction followed by Western blotting. ATP in the indicated concentrations was mixed with 1 nM EMD 1214063 and added to 100 ng recombinant human c-Met kinase (Carna Biosciences 08-151). The reaction mixtures were incubated at room temperature for 30 min. The reaction was stopped by addition of 4x LDS Sample Buffer (Invitrogen NP-0007) containing 25 mM DTT and heating at 80{degree sign}C for 20 min. The reaction mixtures were analyzed by Western blotting using 1 ng c-Met kinase per lane in gel electrophoresis. After blotting on nitrocellulose, detection was performed with an anti-phosphotyrosine antibody (clone PY99, Santa Cruz sc-7020) and an anti-GST-tag antibody (clone B-14, Santa Cruz sc-138) as a loading control. Chemiluminescence read out was measured with a VersDoc analyzer (BioRad) and band densities were quantified with the inherent software (ImageLab).</p>