RESUMEN
Influenza A virus (IAV) infections are still a major global threat for humans, especially for the risk groups of young children and the elderly. Annual epidemics and sporadically occurring pandemics highlight the necessity of effective antivirals that can limit viral replication. The currently licensed antiviral drugs target viral factors and are prone to provoke viral resistance. In infected host cells IAV induces various cellular signaling cascades. The Raf/MEK/ERK signaling cascade is indispensable for IAV replication because it triggers the nuclear export of newly assembled viral ribonucleoproteins (vRNPs). Inhibition of this cascade limits viral replication. Thus, next to their potential in anti-tumor therapy, inhibitors targeting the Raf/MEK/ERK signaling cascade came into focus as potential antiviral drugs. The first licensed MEK inhibitor Trametinib (GSK-1120212) is used for treatment of malignant melanoma, being highly selective and having a promising side effect profile. Since Trametinib may be qualified for a repurposing approach that would significantly shorten development time for an anti-flu use, we evaluated its antiviral potency and mode of action. In this study, we describe that Trametinib efficiently blocks replication of different IAV subtypes in vitro and in vivo. The broad antiviral activity against various IAV strains was due to its ability to interfere with export of progeny vRNPs from the nucleus. The compound also limited hyper-expression of several cytokines. Thus, we show for the first time that a clinically approved MEK inhibitor acts as a potent anti-influenza agent.
Asunto(s)
Antivirales/farmacología , Citocinas/antagonistas & inhibidores , Factores Inmunológicos/farmacología , Virus de la Influenza A/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Línea Celular , Reposicionamiento de Medicamentos , Humanos , Virus de la Influenza A/crecimiento & desarrolloRESUMEN
Cell division, a prerequisite for cell proliferation, is a process in which each daughter cell inherits one complete set of chromosomes. The mitotic spindle is a dedicated apparatus for the alignment and segregation of chromosomes. Extracellular signal-regulated kinase (ERK) 1/2 plays crucial roles in cell cycle progression, particularly during M-phase. Although, association with the mitotic spindle has been reported, the precise roles played by ERK in the dynamics of the mitotic spindle and in M-phase progression remain to be elucidated. In this study, we used MEK inhibitors U0126 and GSK1120212 to dissect the roles of ERK in M-phase progression and chromosome alignment. Fluorescence microscopy revealed that ERK is localized to the spindle microtubules in a manner independent of Src kinase, which is one of the kinases upstream of ERK at mitotic entry. ERK inhibition induces an increase in the number of prophase cells and a decrease in the number of anaphase cells. Time-lapse imaging revealed that ERK inhibition perturbs chromosome alignment, thereby preventing cells from entering anaphase. These results suggest that ERK plays a role in M-phase progression by regulating chromosome alignment and demonstrate one of the mechanisms by which the aberration of ERK signaling may produce cancer cells.
Asunto(s)
Cromosomas Humanos/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Huso Acromático/genética , Anafase/efectos de los fármacos , Animales , Butadienos/farmacología , Línea Celular , Humanos , Nitrilos/farmacología , Profase/efectos de los fármacos , Piridonas/farmacología , Pirimidinonas/farmacología , Huso Acromático/metabolismo , Porcinos , Imagen de Lapso de TiempoRESUMEN
PURPOSE: Over 90% of pancreatic adenocarcinoma PC express oncogenic mutant KRAS that constitutively activates the Raf-MEK-MAPK pathway conferring resistance to both radiation and chemotherapy. MEK inhibitors have shown promising anti-tumor responses in recent preclinical and clinical studies, and are currently being tested in combination with radiation in clinical trials. Here, we have evaluated the radiosensitizing potential of a novel MEK1/2 inhibitor GSK1120212 (GSK212,or trametinib) and evaluated whether MEK1/2 inhibition alters DNA repair mechanisms in multiple PC cell lines. METHODS: Radiosensitization and DNA double-strand break (DSB) repair were evaluated by clonogenic assays, comet assay, nuclear foci formation (γH2AX, DNA-PK, 53BP1, BRCA1, and RAD51), and by functional GFP-reporter assays for homologous recombination (HR) and non-homologous end-joining (NHEJ). Expression and activation of DNA repair proteins were measured by immunoblotting. RESULTS: GSK212 blocked ERK1/2 activity and radiosensitized multiple KRAS mutant PC cell lines. Prolonged pre-treatment with GSK212 for 24-48 hours was required to observe significant radiosensitization. GSK212 treatment resulted in delayed resolution of DNA damage by comet assays and persistent γH2AX nuclear foci. GSK212 treatment also resulted in altered BRCA1, RAD51, DNA-PK, and 53BP1 nuclear foci appearance and resolution after radiation. Using functional reporters, GSK212 caused repression of both HR and NHEJ repair activity. Moreover, GSK212 suppressed the expression and activation of a number of DSB repair pathway intermediates including BRCA1, DNA-PK, RAD51, RRM2, and Chk-1. CONCLUSION: GSK212 confers radiosensitization to KRAS-driven PC cells by suppressing major DNA-DSB repair pathways. These data provide support for the combination of MEK1/2 inhibition and radiation in the treatment of PC.
Asunto(s)
Adenocarcinoma/genética , Antineoplásicos/uso terapéutico , Neoplasias Pancreáticas/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridonas/uso terapéutico , Pirimidinonas/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/radioterapia , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Reparación del ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/radioterapia , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/genética , Tolerancia a Radiación/genéticaRESUMEN
PURPOSE: Radiotherapy (RT) is used frequently in patients with melanoma, but results are suboptimal because the disease is often radioresistant. This may be due to constitutive activation of MAPK pathway signalling through mutations involving RAS/RAF. Thus, we studied whether trametinib, a potent and selective allosteric inhibitor of MEK1/2 could improve the efficacy of RT. METHODS AND MATERIALS: Clonogenic survival assays were performed in human BRAF-mutant (A375), NRAS-mutant (D04, WM1631), KRAS-mutant (WM1791c) and wild-type (PMWK) melanoma cell lines. The effects of trametinib with and without radiation on protein levels of MEK effectors were measured by immunoblot analyses. Cell cycle effects, DNA damage repair, mitotic catastrophe and senescence were measured using flow cytometry, γH2Ax staining, nuclear fragmentation and ß-galactosidase staining, respectively. Additionally, athymic mice with D04 flank tumours were treated with fractionated RT after gavage with trametinib and monitored for tumour growth. RESULTS: All cell lines, except PMWK, exhibited enhanced cytotoxicity when RT was combined with trametinib compared to either agent alone. Sensitiser enhancement ratios were 1.70, 1.32, 1.10, and 1.70 for A375, D04, WM1361 and WM1791c, respectively. Trametinib efficiently blocked RT-induced phosphorylation of ERK at nanomolar concentrations. Increased radiosensitivity correlated with prolonged G1 arrest and reduction in the radioresistant S phase up to 48 h following RT. A larger population of senescence-activated ß-galactosidase-positive cells was seen in the trametinib pretreated group, and this correlated with activation of two of the major mediators of induced senescence, p53 and pRb. Mice receiving the combination treatment (trametinib 1mg/kg and RT over 3 days) showed a reduced mean tumour volume compared with mice receiving trametinib alone (p=0.016), or RT alone (p=0.047). No overt signs of drug toxicity were observed. CONCLUSION: Trametinib radiosensitised RAS-/RAF-mutated melanoma cells by inducing prolonged G1 arrest and premature senescence. In this pre-clinical study we demonstrate that combining trametinib and RT is well tolerated, and reduces tumour growth in vivo.
Asunto(s)
Envejecimiento/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Femenino , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Desnudos , Mutación , Proteínas Proto-Oncogénicas B-raf/efectos de los fármacos , Proteínas ras/efectos de los fármacosRESUMEN
Arctigenin, the main effective constituent of Arctium lappa L. fruit, has previously been proven to dramatically attenuate dextran sulfate sodium (DSS)-induced colitis in mice, a frequently used animal model of inflammatory bowel disease (IBD). As Th1 and Th17 cells play a crucial role in the pathogenesis of IBD, the present study addressed whether and how arctigenin exerted anti-colitis efficacy by interfering with the differentiation and activation of Th1/Th17 cells. In vitro, arctigenin was shown to markedly inhibit the differentiation of Th17 cells from naïve T cells, and moderately inhibit the differentiation of Th1 cells, which was accompanied by lowered phosphorylation of STAT3 and STAT4, respectively. In contrast, arctigenin was lack of marked effect on the differentiation of either Th2 or regulatory T cells. Furthermore, arctigenin was shown to suppress the mammalian target of rapamycin complex 1 (mTORC1) pathway in T cells as demonstrated by down-regulated phosphorylation of the downstream target genes p70S6K and RPS6, and it functioned independent of two well-known upstream kinases PI3K/AKT and ERK. Arctigenin was also able to inhibit the activity of mTORC1 by dissociating raptor from mTOR. Interestingly, the inhibitory effect of arctigenin on T cell differentiation disappeared under a status of mTORC1 overactivation via knockdown of tuberous sclerosis complex 2 (TSC2, a negative regulator of mTORC1) or pretreatment of leucine (an agonist of mTOR). In DSS-induced mice, the inhibition of Th1/Th17 responses and anti-colitis effect of arctigenin were abrogated by leucine treatment. In conclusion, arctigenin ameliorates colitis through down-regulating the differentiation of Th1 and Th17 cells via mTORC1 pathway.
Asunto(s)
Antiinflamatorios/uso terapéutico , Colitis/tratamiento farmacológico , Furanos/uso terapéutico , Lignanos/uso terapéutico , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Células TH1/efectos de los fármacos , Células Th17/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Colitis/inducido químicamente , Colitis/patología , Sulfato de Dextran , Humanos , Células Jurkat , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Endogámicos C57BL , Transducción de Señal , Células TH1/patología , Células Th17/patologíaRESUMEN
INTRODUCTION: Aberrant MAPK pathway signaling is a hallmark of melanoma. Mitogen/extracellular signal-regulated kinase (MEK) 1/2 are integral components of MAPK signaling. Several MEK inhibitors have demonstrated activity as single agents and in combination with other therapies. Trametinib was the first MEK inhibitor approved for use in treatment of advanced BRAF(V600) mutant melanoma as a single agent and in combination with BRAF inhibitor, dabrafenib. AREAS COVERED: In this article, we discuss the underlying biology of MEK inhibition and its rationale in melanoma treatment with special emphasis on the clinical development of trametinib, from initial Phase I studies to randomized Phase II and III studies, both as monotherapy and in combination with other therapeutics. Furthermore, we briefly comment on trametinib for NRAS mutant and other non-BRAF mutant subsets of melanoma. EXPERT OPINION: Trametinib is a novel oral MEK inhibitor with clinical activity in BRAF(V600) mutant metastatic melanoma alone and in combination with dabrafenib. Trametinib is currently being explored in other genetic subsets as well, particularly those with NRAS mutations or atypical BRAF alterations. Furthermore, to maximize efficacy and overcome acquired resistance, studies evaluating the combination of trametinib with other targeted agents and immunotherapy are underway.
Asunto(s)
Antineoplásicos/uso terapéutico , Melanoma/tratamiento farmacológico , Melanoma/enzimología , Piridonas/uso terapéutico , Pirimidinonas/uso terapéutico , Animales , Ensayos Clínicos como Asunto/métodos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Inmunoterapia/métodos , Inmunoterapia/tendencias , Melanoma/diagnóstico , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Resultado del TratamientoRESUMEN
Multiplexed isobaric tag based quantitative proteomics and phosphoproteomics strategies can comprehensively analyze drug treatments effects on biological systems. Given the role of mitogen-activated protein/extracellular signal-regulated kinase (MEK) signaling in cancer and mitogen-activated protein kinase (MAPK)-dependent diseases, we sought to determine if this pathway could be inhibited safely by examining the downstream molecular consequences. We used a series of tandem mass tag 10-plex experiments to analyze the effect of two MEK inhibitors (GSK1120212 and PD0325901) on three tissues (kidney, liver, and pancreas) from nine mice. We quantified â¼ 6000 proteins in each tissue, but significant protein-level alterations were minimal with inhibitor treatment. Of particular interest was kidney tissue, as edema is an adverse effect of these inhibitors. From kidney tissue, we enriched phosphopeptides using titanium dioxide (TiO2 ) and quantified 10 562 phosphorylation events. Further analysis by phosphotyrosine peptide immunoprecipitation quantified an additional 592 phosphorylation events. Phosphorylation motif analysis revealed that the inhibitors decreased phosphorylation levels of proline-x-serine-proline (PxSP) and serine-proline (SP) sites, consistent with extracellular-signal-regulated kinase (ERK) inhibition. The MEK inhibitors had the greatest decrease on the phosphorylation of two proteins, Barttin and Slc12a3, which have roles in ion transport and fluid balance. Further studies will provide insight into the effect of these MEK inhibitors with respect to edema and other adverse events in mouse models and human patients.
Asunto(s)
Benzamidas/farmacología , Difenilamina/análogos & derivados , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Fosfopéptidos/análisis , Inhibidores de Proteínas Quinasas/farmacología , Proteoma/metabolismo , Piridonas/farmacología , Pirimidinonas/farmacología , Secuencia de Aminoácidos , Animales , Difenilamina/farmacología , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Fosfopéptidos/metabolismo , Fosforilación/efectos de los fármacos , Proteoma/química , Proteómica , Espectrometría de Masas en TándemRESUMEN
High levels of cholecystokinin (CCK) can stimulate pancreatic adaptive growth in which mature acinar cells divide, leading to enhanced pancreatic mass with parallel increases in protein, DNA, RNA, and digestive enzyme content. Prolonged release of CCK can be induced by feeding trypsin inhibitor (TI) to disrupt normal feedback control. This leads to exocrine growth in a CCK-dependent manner. The extracellular signal-related kinase (ERK) pathway regulates many proliferative processes in various tissues and disease models. The aim of this study was to evaluate the role of ERK signaling in pancreatic adaptive growth using the MEK inhibitors PD-0325901 and trametinib (GSK-1120212). It was determined that PD-0325901 given two times daily by gavage or mixed into powdered chow was an effective and specific inhibitor of ERK signaling in vivo. TI-containing chow led to a robust increase in pancreatic mass, protein, DNA, and RNA content. This pancreatic adaptive growth was blocked in mice fed chow containing the MEK inhibitors. PD-0325901 blocked TI-induced ERK-regulated early response genes, cell-cycle proteins, and mitogenesis by acinar cells. It was determined that ERK signaling is necessary for the initiation of pancreatic adaptive growth but not necessary to maintain it. PD-0325901 blocked adaptive growth when given before cell-cycle initiation but not after mitogenesis had been established. Furthermore, GSK-1120212, a chemically distinct inhibitor of the ERK pathway that is now approved for clinical use, inhibited growth similar to PD-0325901. These data demonstrate that the ERK pathway is required for CCK-stimulated pancreatic adaptive growth.
Asunto(s)
Proliferación Celular , Colecistoquinina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Páncreas/enzimología , Animales , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Replicación del ADN , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones Endogámicos ICR , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Páncreas/efectos de los fármacos , Páncreas/crecimiento & desarrollo , Inhibidores de Proteínas Quinasas/farmacología , ARN/biosíntesis , Factores de Tiempo , Inhibidores de Tripsina/farmacologíaRESUMEN
The purpose of this study is to review the development of BRAF inhibitors, with emphasis on the trials conducted with dabrafenib (GSK2118436) and the evolving role of dabrafenib in treatment for melanoma patients. Fifty percent of cutaneous melanomas have mutations in BRAF, resulting in elevated activity of the mitogen-activated protein kinase signaling pathway. Dabrafenib inhibits the mutant BRAF (BRAF(mut)) protein in melanomas with BRAF(V600E) and BRAF(V600K) genotypes. BRAF(V600E) metastatic melanoma patients who receive dabrafenib treatment exhibit high clinical response rates and compared with dacarbazine chemotherapy, progression-free survival. Efficacy has also been demonstrated in BRAF(V600K) patients and in those with brain metastases. Dabrafenib has a generally mild and manageable toxicity profile. Cutaneous squamous cell carcinomas and pyrexia are the most significant adverse effects. Dabrafenib appears similar to vemurafenib with regard to efficacy but it is associated with less toxicity. It is expected that new combinations of targeted drugs, such as the combination of dabrafenib and trametinib (GSK1120212, a MEK inhibitor), will provide higher response rates and more durable clinical benefit than dabrafenib monotherapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Imidazoles/uso terapéutico , Melanoma/tratamiento farmacológico , Oximas/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Ensayos Clínicos como Asunto , Supervivencia sin Enfermedad , Diseño de Fármacos , Humanos , Imidazoles/efectos adversos , Imidazoles/farmacología , Indoles/efectos adversos , Indoles/farmacología , Indoles/uso terapéutico , Melanoma/patología , Metástasis de la Neoplasia , Oximas/efectos adversos , Oximas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias Cutáneas/patología , Sulfonamidas/efectos adversos , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , VemurafenibRESUMEN
Inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) represents a promising strategy for the discovery of a new generation of anticancer chemotherapeutics. Our synthetic efforts, beginning from the lead compound 2, were directed at improving antiproliferative activity against cancer cells as well as various drug properties. These efforts led to the discovery of N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide dimethylsulfoxide solvate (GSK1120212, JTP-74057 DMSO solvate; 1), a selective and highly potent MEK inhibitor with improved drug properties. We further confirmed that the antiproliferative activity correlates with cellular MEK inhibition and observed significant antitumor activity with daily oral dosing of 1 in a tumor xenograft model. These qualities led to the selection of 1 for clinical development.