Substituted N-(4-(2-aminopyridin-4-yloxy)-3-fluoro-phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamides were identified as potent and selective Met kinase inhibitors. Substitution of the pyridine 3-position gave improved enzyme potency, while substitution of the pyridone 4-position led to improved aqueous solubility and kinase selectivity. Analogue 10 demonstrated complete tumor stasis in a Met-dependent GTL-16 human gastric carcinoma xenograft model following oral administration. Because of its excellent in vivo efficacy and favorable pharmacokinetic and preclinical safety profiles, 10 has been advanced into phase I clinical trials.
A chemical genetics approach identified a cellular target of several proapoptotic farnesyl transferase inhibitors (FTIs). Treatment with these FTIs caused p53-independent apoptosis in Caenorhabditis elegans, which was mimicked by knockdown of endosomal trafficking proteins, including Rab5, Rab7, the HOPS complex, and notably the enzyme Rab geranylgeranyl transferase (RabGGT). These FTIs were found to inhibit mammalian RabGGT with potencies that correlated with their proapoptotic activity. Knockdown of RabGGT induced apoptosis in mammalian cancer cell lines, and both RabGGT subunits were overexpressed in several tumor tissues. These findings validate RabGGT, and by extension endosomal function, as a therapeutically relevant target for modulation of apoptosis, and enhance our understanding of the mechanism of action of FTIs.
Several ras genes have been expressed at high levels in Escherichia coli and the resultant ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. We were able to detect a weak GTPase activity associated with the purified proteins. The normal cellular ras protein (p21N) exhibits -40 times higher GTPase activity than the "activated" proteins. Even though the turnover rate of the reaction is very low (0.02 mol of GTP hydrolyzed per mol of p21N protein per minute), the reaction appears to be catalytic; one molecule of p21N hydrolyzes more than one molecule of GTP. The GTPase and the GDP binding activities both have been recovered from a Mr 23,000 protein eluted following NaDodSO4/polyacrylamide gel electrophoresis, suggesting that these two activities are associated with the same protein. Mg2e ions and dithiotheitol are required for GTPase activity and the optimal pH is between 7 and 8. Guanidine HCl, which is required for solubilizing bacterially expressed ras protein, is strongly inhibitory to GTPase activity at concentrations higher than 0.5 M.Members of the ras gene family encode closely related proteins of "189 amino acid residues and Mr 21,000 termed p21 ras (1, 2). The ras genes were first identified as the oncogenic sequences of certain strains of acute transforming retroviruses (3). Their normal counterparts (cellular ras genes) are highly conserved in eukaryotic cells (4-10). Cellular ras genes acquire transforming properties by single point mutations within their coding sequences (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and the "activated" ras genes are detected in a significant fraction of human cancers and in experimentally obtained animal tumors (21)(22)(23). The observed conservation of cellular ras genes and analysis of their expression (24, 25) have led to speculations that p21 proteins are essential components of normal cells involved in cell division and differentiation (26). It has also been observed that elevated expression of the normal cellular p21 gene can induce transformation (27). More recently, Stacey and Kung (28) observed that both mutated and normal p21 proteins can induce transformation of NIH 3T3 cells, when introduced by microinjection, but that higher concentrations of the normal protein were required. These observations strongly suggest that high levels of cellular p21 protein, as well as low amounts of activated p21 protein have the same effect on NIH 3T3 cells and this might be due to altered or lack of regulation of cellular p21 activity consequent to its "activation" by mutation. However, various studies aimed at understanding the biochemical basis for the transforming activity of ras gene products gave no major differences in their subcellular localization, post-translational modification, and in vitro guanine nucleotide binding properties of normal and activated ras proteins (29,30).We have shown recently that bacterially produced p21 protein binds guanine nucleotides with high affinity using a simple nitro...
Continuing structure-activity studies were performed on the 2,3,4, 5-tetrahydro-1-(imidazol-4-ylalkyl)-1,4-benzodiazepine farnesyltransferase (FT) inhibitors. These studies demonstrated that a 3(R)-phenylmethyl group, a hydrophilic 7-cyano group, and a 4-sulfonyl group bearing a variety of substituents provide low-nanomolar FT inhibitors with cellular activity at concentrations below 100 nM. Maximal in vivo activity in the mutated K-Ras bearing HCT-116 human colon tumor model was achieved with analogues carrying hydrophobic side chains such as propyl, phenyl, or thienyl attached to the N-4 sulfonyl group. Several such compounds achieved curative efficacy when given orally in this model. On the basis of its excellent preclinical antitumor activity and promising pharmacokinetics, compound 20 (BMS-214662, (R)-7-cyano-2,3,4, 5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thie nyl sulfonyl)-1H-1,4-benzodiazepine) has been advanced into human clinical trials.
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