RESUMO
Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of a structurally diverse series of carbon-linked quinoline triazolopyridinones, which demonstrates nanomolar inhibition of c-Met kinase activity. This novel series of inhibitors exhibits favorable pharmacokinetics as well as potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.
Assuntos
Antineoplásicos/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piridonas/síntese química , Quinolinas/síntese química , Triazóis/síntese química , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Descoberta de Drogas , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridonas/farmacologia , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Triazóis/farmacologiaRESUMO
The MET receptor tyrosine kinase is involved in cell growth, survival, and invasion. Clinical studies with small molecule MET inhibitors have shown the role of biomarkers in identifying patients most likely to benefit from MET-targeted therapy. AMG 337 is an oral, small molecule, ATP-competitive, highly selective inhibitor of the MET receptor. Herein, we describe AMG 337 preclinical activity and mechanism of action in MET-dependent tumor models. These studies suggest MET is the only therapeutic target for AMG 337. In an unbiased tumor cell line proliferation screen (260 cell lines), a closely related analogue of AMG 337, Compound 5, exhibited activity in 2 of 260 cell lines; both were MET-amplified. Additional studies examining the effects of AMG 337 on the proliferation of a limited panel of cell lines with varying MET copy numbers revealed that high-level focal MET amplification (>12 copies) was required to confer MET oncogene addiction and AMG 337 sensitivity. One MET-amplified cell line, H1573 (>12 copies), was AMG 337 insensitive, possibly because of a downstream G12A KRAS mutation. Mechanism-of-action studies in sensitive MET-amplified cell lines demonstrated that AMG 337 inhibited MET and adaptor protein Gab-1 phosphorylation, subsequently blocking the downstream PI3K and MAPK pathways. AMG 337 exhibited potency in pharmacodynamic assays evaluating MET signaling in tumor xenograft models; >90% inhibition of Gab-1 phosphorylation was observed at 0.75 mg/kg. These findings describe the preclinical activity and mechanism of action of AMG 337 in MET-dependent tumor models and indicate its potential as a novel therapeutic for the treatment of MET-dependent tumors. Mol Cancer Ther; 15(7); 1568-79. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Amplificação de Genes , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Necrose , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piridonas/síntese química , Piridonas/farmacologia , Triazóis/síntese química , Triazóis/farmacologia , Animais , Antineoplásicos/farmacocinética , Cristalografia por Raios X , Desenho de Fármacos , Descoberta de Drogas , Humanos , Camundongos , Modelos Moleculares , Piridonas/farmacocinética , Relação Estrutura-Atividade , Triazóis/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Src family kinases (SFKs) Src and Yes are believed to play critical roles in tumor growth, angiogenesis, invasion, and dissemination. Using a panel of highly selective and structurally diverse Src inhibitors, we found that phosphorylation of signal transducer and activator of transcription 3 [STAT3 (Y705)] and focal adhesion kinase [FAK (Y861)] was SFK dependent in cultured human colon, breast, lung, and ovarian tumor cells. These findings were reproduced in vivo in target modulation studies using tumors derived from fibroblasts overexpressing activated Src. Additionally, treatment of mice with multiple Src inhibitors resulted in inhibition of phosphorylation of FAK (Y861) and of a putative Src autophosphorylation epitope (Y419) in HT-29 human colon tumor xenografts. Next we pharmacologically examined the requirement for SFKs in asynchronous proliferation of human tumor cells. At concentrations sufficient to selectively inhibit Src, structurally diverse Src inhibitors inhibited growth of cultured human colon, breast, and lung cells on plastic under low serum conditions. In addition, these compounds inhibited anchorage-independent growth of HT-29 human colon tumor cells in soft agar. The role of SFK activity in vascular endothelial growth factor signaling was also evaluated. Inhibition of SFK signaling using structurally distinct Src inhibitors resulted in complete inhibition of vascular endothelial growth factor-dependent vascular permeability in vivo. These data demonstrate that STAT3 (Y705) and FAK (Y861) phosphoepitopes are SFK-dependent in tumor cells and reveal a requirement for SFK function in tumor cell proliferation and vascular permeability.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias Experimentais/patologia , Proteínas Tirosina Quinases/metabolismo , Transativadores/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases da Família src/fisiologia , Animais , Apoptose , Adesão Celular/efeitos dos fármacos , Divisão Celular , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/metabolismo , Fosforilação , Fator de Transcrição STAT3 , Transdução de Sinais , Células Tumorais Cultivadas , Tirosina/metabolismo , Quinases da Família src/antagonistas & inibidoresRESUMO
The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, we report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM), selective inhibitors of c-Met with desirable pharmacokinetic properties that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.
Assuntos
Descoberta de Drogas , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Quinolinas/farmacologia , Triazóis/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Quinolinas/química , Quinolinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual , Triazóis/química , Triazóis/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Raf inhibitors are under clinical investigation, specifically in patients with tumor types harboring frequent activating mutations in B-Raf. Here, we show that cell lines and tumors harboring mutant B-Raf were sensitive to a novel series of Raf inhibitors (e.g., (V600E)B-Raf A375, IC(50) on cells = 2 nmol/L; ED(50) on tumor xenografts = 1.3 mg/kg). However, in cells and tumors with wild-type B-Raf, exposure to Raf inhibitors resulted in a dose-dependent and sustained activation of mitogen-activated protein kinase signaling. In some of these cell lines, Raf inhibition led to entry into the cell cycle, enhanced proliferation, and significantly stimulated tumor growth in vivo. Inhibition with structurally distinct Raf inhibitors or isoform-specific small interfering RNA knockdown of Raf showed that these effects were mediated directly through Raf. Either A-Raf or C-Raf mediated the Raf inhibitor-induced mitogen-activated protein kinase pathway activation in an inhibitor-specific manner. These paradoxical effects of Raf inhibition were seen in malignant and normal cells in vitro and in vivo. Hyperplasia of normal epithelial cells in the esophagus and the stomach was evident in mice with all efficacious Raf inhibitors (n = 8) tested. An implication of these results is that Raf inhibitors may induce unexpected normal cell and tumor tissue proliferation in patients.