RESUMO
The clinical benefit of MAPK pathway inhibition in melanoma patients carrying BRAF mutations is temporal. After the initial response to treatment, the majority of tumors will develop resistance and patients will relapse. Here we demonstrate that the endothelin-endothelin receptor B (ETBR) signaling pathway confers resistance to MAPK pathway inhibitors in BRAF mutated melanoma. MAPK blockade, in addition to being anti-proliferative, induces a phenotypic change which is characterized by increased expression of melanocyte-specific genes including ETBR. In the presence of MAPK inhibitors, activation of ETBR by endothelin enables the sustained proliferation of melanoma cells. In mouse models of melanoma, including patient-derived xenograft models, concurrent inhibition of the MAPK pathway and ETBR signaling resulted in a more effective anti-tumor response compared to MAPK pathway inhibition alone. The combination treatment significantly reduced tumor growth and prolonged survival compared to therapies with MAPK pathway inhibitors alone. The phosphoproteomic analysis revealed that ETBR signaling did not induce resistance towards MAPK pathway inhibitors by restoring MAPK activity, but instead via multiple alternative signaling pathways downstream of the small G proteins GNAq/11. Together these data indicate that a combination of MAPK pathway inhibitors with ETBR antagonists could have a synergistically beneficial effect in melanoma patients with hyperactivated MAPK signaling pathways.
Assuntos
Melanoma/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/genética , Receptor de Endotelina B/genética , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Antagonistas do Receptor de Endotelina B/farmacologia , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação/genética , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Endothelin receptors (ETRs) are often overexpressed in ovarian tumors, which can be resistant to conventional therapies. Thus, we investigated whether blockage of the ETR pathways using the dual ETR antagonist macitentan combined with taxol or cisplatinum can produce therapy for orthotopically growing multidrug-resistant (MDR) human ovarian carcinoma. In several studies, nude mice were injected in the peritoneal cavity with HeyA8-MDR human ovarian cancer cells. Ten days later, mice were randomized to receive vehicle (saline), macitentan (oral, daily), taxol (intraperitoneal, weekly), cisplatinum (intraperitoneal, weekly), macitentan plus taxol, or macitentan plus cisplatinum. Moribund mice were killed, and tumors were collected, weighed, and prepared for immunohistochemical analysis. The HeyA8-MDR tumors did not respond to taxol, cisplatinum, or macitentan administered as single agents. In contrast, combination therapy with macitentan and taxol or macitentan and cisplatinum significantly decreased the tumor incidence and weight and significantly increased the survival of mice and their general condition. Multiple immunohistochemical analyses revealed that treatment with macitentan and macitentan plus taxol or cisplatinum inhibited the phosphorylation of ETRs, decreased the levels of pVEGFR2, pAkt, and pMAPK in tumor cells after 2 weeks of treatment and induced a first wave of apoptosis in tumor-associated endothelial cells followed by apoptosis in surrounding tumor cells. Our study shows that ovarian cancer cells, which express the endothelin axis and are multidrug resistant, are exquisitely sensitive to treatment with a dual ET antagonist and can be resensitized to both taxol and cisplatinum. This combined therapy led to a significant reduction in tumor weight.
RESUMO
The discovery of a new series of piperidine-based renin inhibitors is described herein. SAR optimization upon the P3 renin sub-pocket is described, leading to the discovery of 9 and 41, two bioavailable renin inhibitors orally active at low doses in a transgenic rat model of hypertension.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Piperidinas/síntese química , Piperidinas/farmacologia , Renina/antagonistas & inibidores , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Desenho de Fármacos , Modelos Moleculares , Piperidinas/química , Conformação Proteica , Ratos , Relação Estrutura-Atividade , Difração de Raios XRESUMO
The optimization of the 4-position of recently described new 3,4-disubstituted piperidine-based renin inhibitors is reported herein. The synthesis and characterization of compounds leading to the discovery of 11 (ACT-178882, MK-1597), a renin inhibitor with a suitable profile for development is described.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Piperidinas/síntese química , Piperidinas/farmacologia , Renina/antagonistas & inibidores , Angiotensinogênio/genética , Animais , Animais Geneticamente Modificados , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/química , Humanos , Indicadores e Reagentes , Modelos Moleculares , Piperidinas/química , Ratos , Renina/genética , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
New classes of de novo designed renin inhibitors are reported. Some of these compounds display excellent in vitro and in vivo activities toward human renin in a TGR model. The synthesis of these new types of mono- and bicyclic scaffolds are reported, and properties of selected compounds discussed.
Assuntos
Compostos Bicíclicos com Pontes/classificação , Compostos Bicíclicos com Pontes/farmacologia , Inibidores Enzimáticos/classificação , Inibidores Enzimáticos/farmacologia , Renina/antagonistas & inibidores , Compostos Bicíclicos com Pontes/química , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Starting from known piperidine renin inhibitors, a new series of 3,9-diazabicyclo[3.3.1]nonene derivatives was rationally designed and prepared. Optimization of the positions 3, 6, and 7 of the diazabicyclonene template led to potent renin inhibitors. The substituents attached at the positions 6 and 7 were essential for the binding affinity of these compounds for renin. The introduction of a substituent attached at the position 3 did not modify the binding affinity but allowed the modulation of the ADME properties. Our efforts led to the discovery of compound (+)-26g that inhibits renin with an IC(50) of 0.20 nM in buffer and 19 nM in plasma. The pharmacokinetics properties of this and other similar compounds are discussed. Compound (+)-26g is well absorbed in rats and efficacious at 10 mg/kg in vivo.
Assuntos
Compostos Azabicíclicos/síntese química , Compostos Azabicíclicos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Renina/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Modelos Moleculares , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
Plasma renin activity (PRA) is a well-established biomarker for assessing the efficacy of various antihypertensive agents such as direct renin inhibitors, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors (ACEIs). PRA measurements are obtained through the detection and quantification of angiotensin I (Ang I) produced by the action of renin on its natural substrate angiotensinogen. The most accepted and reproducible method for PRA measurement uses an antibody capture Ang I methodology that employs specific antibodies that recognize and protect Ang I against angiotensinase activities contained in plasma. The amount of Ang I is then quantified by either radioimmunoassay (RIA) or enzyme immunoassay (EIA). In the current report, we describe the optimization of a novel homogeneous immunoassay based on the AlphaScreen technology for the detection and quantification of antibody-captured Ang I using AlphaLISA acceptor beads in buffer and in the plasma of various species (human, rat, and mouse). Ex vivo measurements of renin activity were performed using 10 microl or less of a reaction mixture, and concentrations as low as 1 nM Ang I were quantified. Titration curves obtained for the quantification of Ang I in buffer and plasma gave similar EC(50) values of 5.6 and 14.4 nM, respectively. Both matrices generated an equivalent dynamic range that varies from approximately 1 to 50 nM. Renin inhibitors have been successfully titrated and IC(50) values obtained correlated well with those obtained using EIA methodology (r(2)=0.80). This assay is sensitive, robust, fast, and less tedious than measurements performed using nonhomogeneous EIA. The AlphaLISA methodology is homogeneous, does not require wash steps prior to the addition of reagents, and does not generate radioactive waste.