RESUMEN
Increased stiffness in the extracellular matrix (ECM) contributes to tumor progression and metastasis. Therefore, stromal modulating therapies and accompanying biomarkers are being developed to target ECM stiffness. Magnetic resonance (MR) elastography can noninvasively and quantitatively map the viscoelastic properties of tumors in vivo and thus has clear clinical applications. Herein, we used MR elastography, coupled with computational histopathology, to interrogate the contribution of collagen to the tumor biomechanical phenotype and to evaluate its sensitivity to collagenase-induced stromal modulation. Elasticity (G d) and viscosity (G l) were significantly greater for orthotopic BT-474 (G d = 5.9 ± 0.2 kPa, G l = 4.7 ± 0.2 kPa, n = 7) and luc-MDA-MB-231-LM2-4 (G d = 7.9 ± 0.4 kPa, G l = 6.0 ± 0.2 kPa, n = 6) breast cancer xenografts, and luc-PANC1 (G d = 6.9 ± 0.3 kPa, G l = 6.2 ± 0.2 kPa, n = 7) pancreatic cancer xenografts, compared with tumors associated with the nervous system, including GTML/Trp53KI/KI medulloblastoma (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 7), orthotopic luc-D-212-MG (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 7), luc-RG2 (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 5), and luc-U-87-MG (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 8) glioblastoma xenografts, intracranially propagated luc-MDA-MB-231-LM2-4 (G d = 3.7 ± 0.2 kPa, G l = 2.2 ± 0.1 kPa, n = 7) breast cancer xenografts, and Th-MYCN neuroblastomas (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 5). Positive correlations between both elasticity (r = 0.72, P < 0.0001) and viscosity (r = 0.78, P < 0.0001) were determined with collagen fraction, but not with cellular or vascular density. Treatment with collagenase significantly reduced G d (P = 0.002) and G l (P = 0.0006) in orthotopic breast tumors. Texture analysis of extracted images of picrosirius red staining revealed significant negative correlations of entropy with G d (r = -0.69, P < 0.0001) and G l (r = -0.76, P < 0.0001), and positive correlations of fractal dimension with G d (r = 0.75, P < 0.0001) and G l (r = 0.78, P < 0.0001). MR elastography can thus provide sensitive imaging biomarkers of tumor collagen deposition and its therapeutic modulation. SIGNIFICANCE: MR elastography enables noninvasive detection of tumor stiffness and will aid in the development of ECM-targeting therapies.
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Neoplasias de la Mama/metabolismo , Colágeno/metabolismo , Animales , Línea Celular Tumoral , Elasticidad , Diagnóstico por Imagen de Elasticidad/métodos , Matriz Extracelular/metabolismo , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Ratones , FenotipoRESUMEN
Sunitinib and pazopanib are antiangiogenic tyrosine kinase inhibitors (TKI) used to treat metastatic renal cell carcinoma (RCC). However, the ability of these drugs to extend progression-free and overall survival in this patient population is limited by drug resistance. It is possible that treatment outcomes in RCC patients could be improved by rationally combining TKIs with other agents. Here, we address whether inhibition of the Ras-Raf-MEK-ERK1/2 pathway is a rational means to improve the response to TKIs in RCC. Using a xenograft model of RCC, we found that tumors that are resistant to sunitinib have a significantly increased angiogenic response compared with tumors that are sensitive to sunitinib in vivo. We also observed significantly increased levels of phosphorylated ERK1/2 in the vasculature of resistant tumors, when compared with sensitive tumors. These data suggested that the Ras-Raf-MEK-ERK1/2 pathway, an important driver of angiogenesis in endothelial cells, remains active in the vasculature of TKI-resistant tumors. Using an in vitro angiogenesis assay, we identified that the MEK inhibitor (MEKI) trametinib has potent antiangiogenic activity. We then show that, when trametinib is combined with a TKI in vivo, more effective suppression of tumor growth and tumor angiogenesis is achieved than when either drug is utilized alone. In conclusion, we provide preclinical evidence that combining a TKI, such as sunitinib or pazopanib, with a MEKI, such as trametinib, is a rational and efficacious treatment regimen for RCC.
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Inhibidores de la Angiogénesis/farmacología , Carcinoma de Células Renales/patología , Neoplasias Renales/patología , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Animales , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Humanos , Indoles/farmacología , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Pirroles/farmacología , Sunitinib , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The purpose of this study is to evaluate if the differential exchange rates with bulk water between amine and amide protons can be exploited using chemical exchange saturation transfer magnetic resonance (CEST-MR) to monitor the release of glutamate induced by carboxypeptidase G2 (CPG2), an enzyme utilized in cancer gene therapy. PROCEDURES: Z spectra of solutions of the CPG2 substrate, 3,5-difluorobenzoyl-L-glutamate (amide), and glutamate (amine) were acquired at 11.7 T, 37 °C, across different pH (5-8). The ability of CEST-MR to monitor CPG2-mediated release of glutamate was assessed in extracts of CPG2-expressing cancer cells and purified solution of CPG2. RESULTS: The addition of CPG2 to a solution containing 3,5-difluorobenzoyl-L-glutamate led to a marked and progressively increasing CEST effect (+3 ppm), concomitant with the time-dependent release of glutamate induced by CPG2. CONCLUSION: CEST-MR allows the detection of CPG2 activity in vitro and supports the translation of CEST-MRI to assess CPG2-based gene therapy in vivo.
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Espectroscopía de Resonancia Magnética/métodos , Profármacos/metabolismo , gamma-Glutamil Hidrolasa/metabolismo , Benzoatos/metabolismo , Línea Celular Tumoral , Humanos , Mecloretamina/química , Mecloretamina/metabolismo , Profármacos/química , Procesamiento de Señales Asistido por Computador , SolucionesRESUMEN
The RAS-RAF-MEK-ERK pathway is hyperactivated in 30% of human cancers. BRAF is a serine-threonine kinase, belonging to this pathway that is mutated with high frequency in human melanoma and other cancers thus BRAF is an important therapeutic target in melanoma. We have designed inhibitors of BRAF based on 2,4,5-trisubstituted imidazoles with naphthyl and benzothiophene-4-substituents. Two compounds were discovered to be potent BRAF inhibitors: 1-(6-{2-[4-(2-dimethylamino-ethoxy)phenyl]-5-(pyridin-4-yl)-1H-imidazol-4-yl} benzo[b]thiophen-3-yl)-2,2,2-trifluoroethanol (1i) with BRAF IC(50)=190 nM and with cellular GI(50)=2100 nM, and 6-{2-[4-(2-dimethylamino-ethoxy)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-naphthalen-1-ol (1q) with IC(50)=9 nM and GI(50)=220 nM.
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Imidazoles/química , Naftoles/química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Tiofenos/química , Benzofuranos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Imidazoles/síntesis química , Imidazoles/farmacología , Melanoma/metabolismo , Melanoma/patología , Naftoles/síntesis química , Naftoles/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/farmacologíaRESUMEN
INTRODUCTION: After its identification as an oncogene in 2002, mutant BRAF has become the target of a number of drug discovery programmes, primarily aimed at the treatment of late stage or unresectable melanoma. Some of the drugs thus developed, such as vemurafenib and dabrafenib, show impressive responses in melanoma patients harbouring a BRAF mutation. AREAS COVERED: This review summarises the patent literature on BRAF from 2006 to 2012, focusing on the specific areas of inhibitors of mutant BRAF, drug combinations including BRAF inhibitors, diagnostic methods for use with mutant BRAF inhibitors & diagnosis and treatment of mutant BRAF cancers resistant to BRAF inhibitors. EXPERT OPINION: Whilst these first-generation BRAF inhibitors initially mediate excellent responses in late stage or unresectable melanoma patients bearing the V600 mutation, resistance usually occurs and patients eventually relapse. The patent literature for new BRAF inhibitors and therapies reflects the desire to develop second-generation drugs able to overcome this resistance and combination treatments that increase the efficiency of current mutant BRAF inhibitors.
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Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Patentes como Asunto , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Antineoplásicos/química , Diseño de Fármacos , Resistencia a Antineoplásicos/genética , Humanos , Estructura Molecular , Terapia Molecular Dirigida , Mutación , Neoplasias/diagnóstico , Neoplasias/enzimología , Neoplasias/genética , Selección de Paciente , Medicina de Precisión , Valor Predictivo de las Pruebas , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
UNLABELLED: NRAS mutations are common in human melanoma. To produce a mouse model of NRAS-driven melanoma, we expressed oncogenic NRAS (NRAS(G12D)) in mouse melanocytes. When NRAS(G12D) was expressed in the melanocytes of developing embryos, it induced melanocyte proliferation and congenital melanocytic lesions reminiscent of human blue nevi but did not induce cutaneous melanoma. Unexpectedly, however, it did induce early-onset primary melanoma of the central nervous system (CNS). The tumors were rapidly proliferating and caused neurologic symptoms, rapid health deterioration, and death. NRAS is not a common driver oncogene of primary melanoma of the CNS in adults, but we report two cases of primary melanoma of the CNS in children, both of which carried oncogenic mutations in NRAS. We conclude that acquisition of somatic mutations in NRAS in CNS melanocytes is a predisposing risk factor for primary melanoma of the CNS in children, and we present a mouse model of this disease. SIGNIFICANCE: We show that the acquisition of NRAS mutations in melanocytes during embryogenesis is a risk factor for early-onset melanoma of the CNS. We have developed a powerful mouse model to study this rare but devastating childhood disease, and to develop therapeutic approaches for its treatment.
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Neoplasias del Sistema Nervioso Central/genética , Genes ras/genética , Melanocitos/metabolismo , Melanoma/genética , Animales , Niño , Preescolar , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Sunitinib is a potent and clinically approved tyrosine kinase inhibitor that can suppress tumour growth by inhibiting angiogenesis. However, conflicting data exist regarding the effects of this drug on the growth of metastases in preclinical models. Here we use 4T1 and RENCA tumour cells, which both form lung metastases in Balb/c mice, to re-address the effects of sunitinib on the progression of metastatic disease in mice. We show that treatment of mice with sunitinib prior to intravenous injection of tumour cells can promote the seeding and growth of 4T1 lung metastases, but not RENCA lung metastases, showing that this effect is cell line dependent. However, increased metastasis occurred only upon administration of a very high sunitinib dose, but not when lower, clinically relevant doses were used. Mechanistically, high dose sunitinib led to a pericyte depletion effect in the lung vasculature that correlated with increased seeding of metastasis. By administering sunitinib to mice after intravenous injection of tumour cells, we demonstrate that while sunitinib does not inhibit the growth of 4T1 lung tumour nodules, it does block the growth of RENCA lung tumour nodules. This contrasting response was correlated with increased myeloid cell recruitment and persistent vascularisation in 4T1 tumours, whereas RENCA tumours recruited less myeloid cells and were more profoundly devascularised upon sunitinib treatment. Finally, we show that progression of 4T1 tumours in sunitinib treated mice results in increased hypoxia and increased glucose metabolism in these tumours and that this is associated with a poor outcome. Taken together, these data suggest that the effects of sunitinib on tumour progression are dose-dependent and tumour model-dependent. These findings have relevance for understanding how anti-angiogenic agents may influence disease progression when used in the adjuvant or metastatic setting in cancer patients.
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Antineoplásicos/uso terapéutico , Indoles/uso terapéutico , Metástasis de la Neoplasia/tratamiento farmacológico , Pirroles/uso terapéutico , Animales , Ratones , Ratones Endogámicos BALB C , Tomografía de Emisión de Positrones , Sunitinib , Tomografía Computarizada por Rayos XRESUMEN
Over the last few years, BRAF has emerged as a validated target in melanoma. This review summarises recent advances in the development of BRAF inhibitors, focussing on agents that have been assessed clinically.
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Ensayos Clínicos como Asunto , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Humanos , Melanoma/metabolismo , Peso Molecular , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS: We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS: Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS: Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).
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Carcinoma de Células Escamosas/genética , Genes ras , Indoles/uso terapéutico , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias Cutáneas/genética , Sulfonamidas/uso terapéutico , Anciano , Anciano de 80 o más Años , Animales , Carcinoma de Células Escamosas/tratamiento farmacológico , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Indoles/administración & dosificación , Masculino , Ratones , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Sulfonamidas/administración & dosificación , VemurafenibRESUMEN
The pseudomonad protein, carboxypeptidase G2 (CPG2), is a prodrug-activating enzyme utilized in the targeted chemotherapy strategies of antibody- and gene-directed enzyme prodrug therapy (ADEPT and GDEPT). We have developed a noninvasive imaging approach to monitor CPG2 activity in vivo that will facilitate the preclinical and clinical development of CPG2-based ADEPT and GDEPT strategies. Cleavage of the novel reporter probe, 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu), by CPG2, in human colon adenocarcinoma WiDr xenografts engineered to stably express CPG2, was monitored using (19)F MRSI. The high signal-to-noise ratio afforded by the two MR-equivalent (19)F nuclei of 3,5-DFBGlu, and the 1.4 ppm (19)F chemical shift difference on CPG2-mediated cleavage, enabled the dynamics and quantification of the apparent pharmacokinetics of 3,5-DFBGlu and its CPG2-mediated cleavage in the tumor to be evaluated. In addition, the apparent rate of increase of 3,5-difluorobenzoic acid concentration could also provide a biomarker of CPG2 activity levels in tumors of patients undergoing CPG2-based therapies, as well as a biomarker of treatment response. The addition of in vivo reporter probes, such as 3,5-DFBGlu, to the armamentarium of prodrugs cleaved by CPG2 affords new applications for CPG2 as a gene reporter of transgene expression.
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gamma-Glutamil Hidrolasa/metabolismo , Animales , Ácido Benzoico/química , Ácido Benzoico/metabolismo , Línea Celular Tumoral , Femenino , Flúor/metabolismo , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncogenic BRAF is a critical driver of proliferation and survival and is thus a validated therapeutic target in cancer. We have developed a potent inhibitor, termed 1t (CCT239065), of the mutant protein kinase, (V600E)BRAF. 1t inhibits signaling downstream of (V600E)BRAF in cancer cells, blocking DNA synthesis, and inhibiting proliferation. Importantly, we show that 1t is considerably more selective for mutated BRAF cancer cell lines compared with wild-type BRAF lines. The inhibitor is well tolerated in mice and exhibits excellent oral bioavailability (F = 71%). Suppression of (V600E)BRAF-mediated signaling in human tumor xenografts was observed following oral administration of a single dose of 1t. As expected, the growth rate in vivo of a wild-type BRAF human tumor xenograft model is unaffected by inhibitor 1t. In contrast, 1t elicits significant therapeutic responses in mutant BRAF-driven human melanoma xenografts.
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Melanoma/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas B-raf/genética , Administración Oral , Sustitución de Aminoácidos , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , División Celular , Línea Celular Tumoral , Supervivencia Celular , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Melanoma/patología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Hibridación de Ácido Nucleico , Fosforilación , Trasplante HeterólogoRESUMEN
Mutated BRAF serine/threonine kinase is implicated in several types of cancer, with particularly high frequency in melanoma and colorectal carcinoma. We recently reported on the development of BRAF inhibitors based on a tripartite A-B-C system featuring an imidazo[4,5]pyridin-2-one group hinge binder. Here we present the design, synthesis, and optimization of a new series of inhibitors with a different A-B-C system that has been modified by the introduction of a range of novel hinge binders (A ring). The optimization of the hinge binding moiety has enabled the development of compounds with low nanomolar potencies in both BRAF inhibition and cellular assays. These compounds display optimal pharmacokinetic properties that warrant further in vivo investigations.
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Antineoplásicos/síntesis química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Pirazinas/síntesis química , Piridinas/síntesis química , Administración Oral , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Bencenosulfonatos/química , Disponibilidad Biológica , Cristalografía por Rayos X , Femenino , Humanos , Enlace de Hidrógeno , Imidazoles/química , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Trasplante de Neoplasias , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Unión Proteica , Proteínas Proto-Oncogénicas B-raf/química , Pirazinas/farmacocinética , Pirazinas/farmacología , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacología , Sorafenib , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine-specific protein kinase that is mutated with high frequency in cutaneous melanoma, and many other cancers. Inhibition of mutant BRAF is an attractive therapeutic approach for the treatment of melanoma. A triarylimidazole BRAF inhibitor bearing a phenylpyrazole group (dimethyl-[2-(4-{5-[4-(1H-pyrazol-3-yl)-phenyl]-4-pyridin-4-yl-1H-imidazol-2-yl}-phenoxy)-ethyl]-amine, 1a) was identified as an active BRAF inhibitor. Based on this starting point, we synthesized a series of analogues leading to the discovery of 6-{2-[4-(4-methyl-piperazin-1-yl)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-2,4-dihydro-indeno[1,2-c]pyrazole (1j), with nanomolar activity in three assays: inhibition of purified mutant BRAF activity in vitro; inhibition of oncogenic BRAF-driven extracellular regulated kinase (ERK) activation in BRAF mutant melanoma cell lines; and inhibition of proliferation in these cells.
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Furanos/química , Imidazoles/química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Pirazoles/química , Animales , Sitios de Unión , Simulación por Computador , Femenino , Humanos , Ratones , Mutación , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pirazoles/síntesis química , Pirazoles/farmacocinética , Relación Estructura-ActividadRESUMEN
We describe the design, synthesis, and optimization of a series of new inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF), a kinase whose mutant form (V600E) is implicated in several types of cancer, with a particularly high frequency in melanoma. Our previously described inhibitors with a tripartite A-B-C system (where A is a hinge binding pyrido[4,5-b]imidazolone system, B is an aryl spacer group, and C is a heteroaromatic group) were potent against purified (V600E)BRAF in vitro but were less potent in accompanying cellular assays. Substitution of different aromatic heterocycles for the phenyl based C-ring is evaluated herein as a potential means of improving the cellular potencies of these inhibitors. Substituted pyrazoles, particularly 3-tert-butyl-1-aryl-1H-pyrazoles, increase the cellular potencies without detrimental effects on the potency on isolated (V600E)BRAF. Thus, compounds have been synthesized that inhibit, with low nanomolar concentrations, (V600E)BRAF, its downstream signaling in cells [as measured by the reduction of the phosphorylation of extracellular regulated kinase (ERK)], and the proliferation of mutant BRAF-dependent cells. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo.
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Diseño de Fármacos , Proteínas Oncogénicas v-raf/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Virus del Sarcoma Murino/enzimología , Homología de Secuencia , Animales , Línea Celular Tumoral , Femenino , Humanos , Concentración 50 Inhibidora , Ratones , Modelos Moleculares , Conformación Molecular , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Relación Estructura-ActividadRESUMEN
We describe a mechanism of tumorigenesis mediated by kinase-dead BRAF in the presence of oncogenic RAS. We show that drugs that selectively inhibit BRAF drive RAS-dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling. This does not occur when oncogenic BRAF is inhibited, demonstrating that BRAF inhibition per se does not drive pathway activation; it only occurs when BRAF is inhibited in the presence of oncogenic RAS. Kinase-dead BRAF mimics the effects of the BRAF-selective drugs and kinase-dead Braf and oncogenic Ras cooperate to induce melanoma in mice. Our data reveal another paradigm of BRAF-mediated signaling that promotes tumor progression. They highlight the importance of understanding pathway signaling in clinical practice and of genotyping tumors prior to administering BRAF-selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects.
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Antineoplásicos/efectos adversos , Melanoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas ras/metabolismo , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
We recently reported on the development of a novel series of BRAF inhibitors based on a tripartite A-B-C system characterized by a para-substituted central aromatic core connected to an imidazo[4,5]pyridin-2-one scaffold and a substituted urea linker. Here, we present a new series of BRAF inhibitors in which the central phenyl ring connects to the hinge binder and substrate pocket of BRAF with a meta-substitution pattern. The optimization of this new scaffold led to the development of low-nanomolar inhibitors that permits the use of a wider range of linkers and terminal C rings while enhancing the selectivity for the BRAF enzyme in comparison to the para series.
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Imidazoles/química , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Piridonas/química , Piridonas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Imidazoles/síntesis química , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Moleculares , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridonas/síntesis química , Relación Estructura-ActividadRESUMEN
We recently demonstrated that expression of (V600E)Braf in mature mouse melanocytes induces melanoma. Here, we show that expression of (V600E)Braf using the tyrosinase promoter leads to an unexpected embryonic lethality, with the animals dying before, at, or shortly after birth. The mice suffer from a range of developmental defects in the skin, the brain, the eyes and the heart, tissues that are normally colonized by melanocytes. We show that the (V600E)Braf expressing cells are potential melanocytic precursors that are fully transformed, suggesting that (V600E)Braf stimulates proliferation and blocks differentiation of these cells. Our data suggests that the presence of these cells in the organs that are normally occupied by melanocytes leads to severe developmental disruption, resulting in catastrophic defects and leading to death of the individual.
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Transformación Celular Neoplásica/genética , Anomalías Congénitas/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genes Letales/genética , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Diferenciación Celular/genética , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Anomalías Congénitas/metabolismo , Desarrollo Embrionario/genética , Técnicas de Sustitución del Gen , Integrasas/genética , Melanocitos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Ratones , Ratones Transgénicos , Monofenol Monooxigenasa/genética , Regiones Promotoras Genéticas/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Células Madre/metabolismoRESUMEN
Carboxypeptidase G2 (CPG2) is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene-directed enzyme prodrug therapy (GDEPT). Employing dynamic nuclear polarization (DNP) and natural abundance (13)C magnetic resonance spectroscopy (MRS), we observed the CPG2-mediated conversion of a novel hyperpolarized reporter probe 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu) to 3,5-difluorobenzoic acid (3,5-DFBA) and L-glutamic acid (L-Glu) in vitro. Isotopic labeling of the relevant nuclei with (13)C in 3,5-DFBGlu or related substrates will yield a further factor of 100 increase in the signal-to-noise. We discuss the feasibility of translating these experiments to generate metabolic images of CPG2 activity in vivo.
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Algoritmos , Espectroscopía de Resonancia Magnética/métodos , gamma-Glutamil Hidrolasa/análisis , gamma-Glutamil Hidrolasa/química , Isótopos de Carbono/análisis , Isótopos de Carbono/química , Activación EnzimáticaRESUMEN
BRAF, a serine/threonine specific protein kinase that is part of the MAPK pathway and acts as a downstream effector of RAS, is a potential therapeutic target in melanoma. We have developed a series of small-molecule BRAF inhibitors based on a 1H-imidazo[4,5-b]pyridine-2(3H)-one scaffold (ring A) as the hinge binding moiety and a number of substituted phenyl rings C that interact with the allosteric binding site. The introduction of various groups on the central phenyl ring B combined with appropriate A- and C-ring modifications afford very potent compounds that inhibit (V600E)BRAF kinase activity in vitro and oncogenic BRAF signaling in melanoma cells. Substitution on the central phenyl ring of a 3-fluoro, a naphthyl, or a 3-thiomethyl group improves activity to yield compounds with an IC(50) of 1 nM for purified (V600E)BRAF and nanomolar activity in cells.
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Antineoplásicos/química , Fenoles/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Piridinas/química , Sitio Alostérico , Antineoplásicos/farmacología , Concentración 50 Inhibidora , Melanoma/tratamiento farmacológico , Mutación Missense , Fenoles/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Piridinas/farmacología , Relación Estructura-ActividadRESUMEN
BRAF is a serine/threonine kinase that is mutated in a range of cancers, including 50-70% of melanomas, and has been validated as a therapeutic target. We have designed and synthesized mutant BRAF inhibitors containing pyridoimidazolone as a new hinge-binding scaffold. Compounds have been obtained which have low nanomolar potency for mutant BRAF (12 nM for compound 5i) and low micromolar cellular potency against a mutant BRAF melanoma cell line, WM266.4. The series benefits from very low metabolism, and pharmacokinetics (PK) that can be modulated by methylation of the NH groups of the imidazolone, resulting in compounds with fewer H-donors and a better PK profile. These compounds have great potential in the treatment of mutant BRAF melanomas.