RESUMEN
Rho-associated coiled-coil kinase (ROCK) is a major downstream effector of the small GTPase RhoA. The ROCK family, consisting of ROCK1 and ROCK2, plays a central role in the organization of the actin cytoskeleton, and is involved in a wide range of fundamental cellular functions such as contraction, adhesion, migration, proliferation, and apoptosis. Since the discovery of effective inhibitors such as fasudil and Y27632, the biological roles of ROCK have been extensively explored in numerous diseases, including cancer. Accumulating evidence supports the concept that ROCK plays important roles in tumor development and progression through regulating many key cellular functions associated with malignancy, including tumorigenicity, tumor growth, metastasis, angiogenesis, tumor cell apoptosis/survival and chemoresistance as well. This review focuses on the new advances of the most recent 5 years from the studies on the roles of ROCK in cancer development and progression; the discussion is mainly focused on the potential value of ROCK inhibitors in cancer therapy.
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Neoplasias/metabolismo , Quinasas Asociadas a rho/metabolismo , Apoptosis , Adhesión Celular , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Resistencia a Antineoplásicos , Humanos , MicroARNs/metabolismo , Mutación , Metástasis de la Neoplasia , Neoplasias/genética , Células Madre Neoplásicas/citología , Neovascularización Patológica , Polimorfismo Genético , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de Señal , Quinasas Asociadas a rho/genéticaRESUMEN
We have recently reported that ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has superior anti-apoptotic and pro-survival effects than antioxidants against doxorubicin, a chemotherapeutic drug. Although oxidative stress is the most widely accepted mechanism, our studies suggest that ROCK1-dependent actin cytoskeleton remodeling plays a more important role in mediating doxorubicin cytotoxicity on MEFs. To further explore the contributions of ROCK1-dependent actin cytoskeleton remodeling in response to stress, this study investigates the mechanistic differences between the cytotoxic effects of doxorubicin versus hydrogen peroxide (H2O2), with a focus on cytoskeleton alterations, apoptosis and necrosis induction. We found that both types of stress induce caspase activation but with different temporal patterns and magnitudes in MEFs: H2O2 induces the maximal levels (2 to 4-fold) of activation of caspases 3, 8, and 9 within 4 h, while doxorubicin induces much higher maximal levels (15 to 25-fold) of caspases activation at later time points (16-24 h). In addition, necrosis induced by H2O2 reaches maximal levels within 4 h while doxorubicin-induced necrosis largely occurs at 16-24 h secondary to apoptosis. Moreover, both types of stress induce actin cytoskeleton remodeling but with different characteristics: H2O2 induces disruption of stress fibers associated with cytosolic translocation of phosphorylated myosin light chain (p-MLC) from stress fibers, while doxorubicin induces cortical F-actin formation associated with cortical translocation of p-MLC from central stress fibers. Furthermore, N-acetylcysteine (an antioxidant) is a potent suppressor for H2O2-induced cytotoxic effects including caspase activation, necrosis, and cell detachment, but shows a much reduced inhibition on doxorubicin-induced changes. On the other hand, ROCK1 deficiency is a more potent suppressor for the cytotoxic effects induced by doxorubicin than by H2O2. These results support the notion that doxorubicin induces caspase activation, necrosis, and actin cytoskeleton alterations largely through ROCK1-dependent and oxidative stress-independent pathways.
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Citoesqueleto de Actina/metabolismo , Doxorrubicina/química , Estrés Oxidativo , Quinasas Asociadas a rho/metabolismo , Actinas/metabolismo , Animales , Antibióticos Antineoplásicos/química , Antioxidantes/metabolismo , Apoptosis , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Supervivencia Celular , Activación Enzimática , Fibroblastos/metabolismo , Peróxido de Hidrógeno/química , Ratones , Microscopía Fluorescente , Necrosis , Fosforilación , Fibras de Estrés/patologíaRESUMEN
PURPOSE: To identify and characterize novel, activating mutations in Notch receptors in breast cancer and to determine response to the gamma secretase inhibitor (GSI) PF-03084014. EXPERIMENTAL DESIGN: We used several computational approaches, including novel algorithms, to analyze next-generation sequencing data and related omic datasets from The Cancer Genome Atlas (TCGA) breast cancer cohort. Patient-derived xenograft (PDX) models were sequenced, and Notch-mutant models were treated with PF-03084014. Gene-expression and functional analyses were performed to study the mechanism of activation through mutation and inhibition by PF-03084014. RESULTS: We identified mutations within and upstream of the PEST domains of NOTCH1, NOTCH2, and NOTCH3 in the TCGA dataset. Mutations occurred via several genetic mechanisms and compromised the function of the PEST domain, a negative regulatory domain commonly mutated in other cancers. Focal amplifications of NOTCH2 and NOTCH3 were also observed, as were heterodimerization or extracellular domain mutations at lower incidence. Mutations and amplifications often activated the Notch pathway as evidenced by increased expression of canonical Notch target genes, and functional mutations were significantly enriched in the triple-negative breast cancer subtype (TNBC). PDX models were also identified that harbored PEST domain mutations, and these models were highly sensitive to PF-03084014. CONCLUSIONS: This work suggests that Notch-altered breast cancer constitutes a bona fide oncogenic driver segment with the most common alteration being PEST domain mutations present in multiple Notch receptors. Importantly, functional studies suggest that this newly identified class can be targeted with Notch inhibitors, including GSIs.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Receptor Notch1/genética , Receptor Notch2/genética , Receptores Notch/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Algoritmos , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Biología Computacional/métodos , Variaciones en el Número de Copia de ADN/genética , Femenino , Dosificación de Gen/genética , Humanos , Ratones , Ratones SCID , Estructura Terciaria de Proteína/genética , Receptor Notch3 , Análisis de Secuencia de ADN , Transducción de Señal/genética , Tetrahidronaftalenos/farmacología , Neoplasias de la Mama Triple Negativas/genética , Valina/análogos & derivados , Valina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes. We performed whole-genome sequencing in 100 tumor-normal pairs, along with DNA copy number, gene expression and methylation profiling, for integrative genomic analysis. We found subtype-specific genetic and epigenetic perturbations and unique mutational signatures. We identified previously known (TP53, ARID1A and CDH1) and new (MUC6, CTNNA2, GLI3, RNF43 and others) significantly mutated driver genes. Specifically, we found RHOA mutations in 14.3% of diffuse-type tumors but not in intestinal-type tumors (P < 0.001). The mutations clustered in recurrent hotspots affecting functional domains and caused defective RHOA signaling, promoting escape from anoikis in organoid cultures. The top perturbed pathways in gastric cancer included adherens junction and focal adhesion, in which RHOA and other mutated genes we identified participate as key players. These findings illustrate a multidimensional and comprehensive genomic landscape that highlights the molecular complexity of gastric cancer and provides a road map to facilitate genome-guided personalized therapy.
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Regulación Neoplásica de la Expresión Génica , Mutación , Neoplasias Gástricas/genética , Uniones Adherentes , Algoritmos , Animales , Metilación de ADN , Análisis Mutacional de ADN , Epigénesis Genética , Femenino , Dosificación de Gen , Perfilación de la Expresión Génica , Variación Genética , Genoma Humano , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Figitumumab (CP-751,871), a potent and fully human monoclonal anti-insulin-like growth factor 1 receptor (IGF1R) antibody, has been investigated in clinical trials of several solid tumors. To identify biomarkers of sensitivity and resistance to figitumumab, its in vitro antiproliferative activity was analyzed in a panel of 93 cancer cell lines by combining in vitro screens with extensive molecular profiling of genomic aberrations. Overall response was bimodal and the majority of cell lines were resistant to figitumumab. Nine of 15 sensitive cell lines were derived from colon cancers. Correlations between genomic characteristics of cancer cell lines with figitumumab antiproliferative activity revealed that components of the IGF pathway, including IRS2 (insulin receptor substrate 2) and IGFBP5 (IGF-binding protein 5), played a pivotal role in determining the sensitivity of tumors to single-agent figitumumab. Tissue-specific differences among the top predictive genes highlight the need for tumor-specific patient selection strategies. For the first time, we report that alteration or expression of the MYB oncogene is associated with sensitivity to IGF1R inhibitors. MYB is dysregulated in hematologic and epithelial tumors, and IGF1R inhibition may represent a novel therapeutic opportunity. Although growth inhibitory activity with single-agent figitumumab was relatively rare, nine combinations comprising figitumumab plus chemotherapeutic agents or other targeted agents exhibited properties of synergy. Inhibitors of the ERBB family were frequently synergistic and potential biomarkers of drug synergy were identified. Several biomarkers of antiproliferative activity of figitumumab both alone and in combination with other therapies may inform the design of clinical trials evaluating IGF1R inhibitors.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Receptor IGF Tipo 1/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Análisis por Conglomerados , Variaciones en el Número de Copia de ADN , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Mutación , Sitios de Carácter Cuantitativo , Receptor IGF Tipo 1/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Progression of hepatocellular carcinoma (HCC) often leads to vascular invasion and intrahepatic metastasis, which correlate with recurrence after surgical treatment and poor prognosis. The molecular prognostic model that could be applied to the HCC patient population in general is needed for effectively predicting disease-free survival (DFS). METHODS: A cohort of 286 HCC patients from South Korea and a second cohort of 83 patients from Hong Kong, China, were used as training and validation sets, respectively. RNA extracted from both tumor and adjacent nontumor liver tissues was subjected to microarray gene expression profiling. DFS was the primary clinical end point. Gradient lasso algorithm was used to build prognostic signatures. RESULTS: High-quality gene expression profiles were obtained from 240 tumors and 193 adjacent nontumor liver tissues from the training set. Sets of 30 and 23 gene-based DFS signatures were developed from gene expression profiles of tumor and adjacent nontumor liver, respectively. DFS gene signature of tumor was significantly associated with DFS in an independent validation set of 83 tumors (P = 0.002). DFS gene signature of nontumor liver was not significantly associated with DFS in the validation set (P = 0.827). Multivariate analysis in the validation set showed that DFS gene signature of tumor was an independent predictor of shorter DFS (P = 0.018). CONCLUSIONS: We developed and validated survival gene signatures of tumor to successfully predict the length of DFS in HCC patients after surgical resection.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/mortalidad , Perfilación de la Expresión Génica , Neoplasias Hepáticas/mortalidad , Hígado/metabolismo , Anciano , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/cirugía , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/cirugía , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Tasa de Supervivencia , Estudios de Validación como AsuntoRESUMEN
UNLABELLED: Cancer is a genetic disease with frequent somatic DNA alterations. Studying recurrent copy number aberrations (CNAs) in human cancers would enable the elucidation of disease mechanisms and the prioritization of candidate oncogenic drivers with causal roles in oncogenesis. We have comprehensively and systematically characterized CNAs and the accompanying gene expression changes in tumors and matched nontumor liver tissues from 286 hepatocellular carcinoma (HCC) patients. Our analysis identified 29 recurrently amplified and 22 recurrently deleted regions with a high level of copy number changes. These regions harbor established oncogenes and tumor suppressors, including CCND1 (cyclin D1), MET (hepatocyte growth factor receptor), CDKN2A (cyclin-dependent kinase inhibitor 2A) and CDKN2B (cyclin-dependent kinase inhibitor 2B), as well as many other genes not previously reported to be involved in liver carcinogenesis. Pathway analysis of cis-acting genes in the amplification and deletion peaks implicates alterations of core cancer pathways, including cell-cycle, p53 signaling, phosphoinositide 3-kinase signaling, mitogen-activated protein kinase signaling, Wnt signaling, and transforming growth factor beta signaling, in a large proportion of HCC patients. We further credentialed two candidate driver genes (BCL9 and MTDH) from the recurrent focal amplification peaks and showed that they play a significant role in HCC growth and survival. CONCLUSION: We have demonstrated that characterizing the CNA landscape in HCC will facilitate the understanding of disease mechanisms and the identification of oncogenic drivers that may serve as potential therapeutic targets for the treatment of this devastating disease.
Asunto(s)
Carcinoma Hepatocelular/genética , Moléculas de Adhesión Celular/genética , Variaciones en el Número de Copia de ADN/genética , Estudio de Asociación del Genoma Completo , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Carcinogénesis/genética , Carcinoma Hepatocelular/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/patología , Masculino , Proteínas de la Membrana , Persona de Mediana Edad , Oncogenes/genética , Proteínas de Unión al ARN , Factores de TranscripciónRESUMEN
PF-03814735 is a novel, reversible inhibitor of Aurora kinases A and B that finished a phase I clinical trial for the treatment of advanced solid tumors. To find predictive biomarkers of drug sensitivity, we screened a diverse panel of 87 cancer cell lines for growth inhibition upon PF-03814735 treatment. Small cell lung cancer (SCLC) and, to a lesser extent, colon cancer lines were very sensitive to PF-03814735. The status of the Myc gene family and retinoblastoma pathway members significantly correlated with the efficacy of PF-03814735. Whereas RB1 inactivation, intact CDKN2A/p16, and normal CCND1/Cyclin D1 status are hallmarks of SCLC, activation or amplification of any of the three Myc genes (MYC, MYCL1, and MYCN) clearly differentiated cell line sensitivity within the SCLC panel. By contrast, we found that expression of Aurora A and B were weak predictors of response. We observed a decrease in histone H3 phosphorylation and polyploidization of sensitive lines, consistent with the phenotype of Aurora B inhibition. In vivo experiments with two SCLC xenograft models confirmed the sensitivity of Myc gene-driven models to PF-03814735 and a possible schedule dependence of MYC/c-Myc-driven tumors. Altogether our results suggest that SCLC and other malignancies driven by the Myc family genes may be suitable indications for treatment by Aurora B kinase inhibitors.
Asunto(s)
Biomarcadores de Tumor/genética , Compuestos Heterocíclicos con 3 Anillos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Animales , Antineoplásicos/farmacología , Aurora Quinasa A , Aurora Quinasa B , Aurora Quinasas , Biomarcadores de Tumor/metabolismo , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Genómica/métodos , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gastric cancer is a heterogeneous disease with multiple environmental etiologies and alternative pathways of carcinogenesis. Beyond mutations in TP53, alterations in other genes or pathways account for only small subsets of the disease. We performed exome sequencing of 22 gastric cancer samples and identified previously unreported mutated genes and pathway alterations; in particular, we found genes involved in chromatin modification to be commonly mutated. A downstream validation study confirmed frequent inactivating mutations or protein deficiency of ARID1A, which encodes a member of the SWI-SNF chromatin remodeling family, in 83% of gastric cancers with microsatellite instability (MSI), 73% of those with Epstein-Barr virus (EBV) infection and 11% of those that were not infected with EBV and microsatellite stable (MSS). The mutation spectrum for ARID1A differs between molecular subtypes of gastric cancer, and mutation prevalence is negatively associated with mutations in TP53. Clinically, ARID1A alterations were associated with better prognosis in a stage-independent manner. These results reveal the genomic landscape, and highlight the importance of chromatin remodeling, in the molecular taxonomy of gastric cancer.
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Exoma , Mutación , Proteínas Nucleares/genética , Neoplasias Gástricas/genética , Factores de Transcripción/genética , Adulto , Anciano , Anciano de 80 o más Años , Proteínas de Ciclo Celular/genética , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN , Femenino , Genes Relacionados con las Neoplasias , Estudios de Asociación Genética , Humanos , Uniones Intercelulares , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Análisis de Secuencia de ADN , Transducción de Señal , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/mortalidad , Adulto JovenRESUMEN
The current standard of care for hepatitis C virus (HCV) infection is a combination of PEGylated interferon and ribavirin, which offer limited efficacy and significant side effects. Novel HCV-specific inhibitors, including those directed at the viral polymerase, have become the focus of HCV drug-discovery efforts in the past decade. In addition to the active site targeted by traditional nucleoside inhibitors, at least four different allosteric-binding sites have been reported for the HCV polymerase, which offer ample opportunities for small-molecule inhibitors. In this review, we summarize the recent progress in the discovery of non-nucleoside HCV polymerase inhibitors with a focus on novel chemical matters, their clinical efficacy, safety and potential for combination therapy.
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Antivirales/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Hepacivirus/enzimología , Hepatitis C/tratamiento farmacológico , Proteínas no Estructurales Virales/antagonistas & inhibidores , Sitio Alostérico , Animales , Antivirales/química , Sitios de Unión , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Hepatitis C/enzimología , Humanos , Hígado/metabolismo , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Proteínas no Estructurales Virales/químicaRESUMEN
PF-00868554 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, which exerts its inhibitory effect by binding to the thumb base domain of the protein. It is a potent and selective inhibitor, with a mean 50% inhibitory concentration of 0.019 microM against genotype 1 polymerases and a mean 50% effective concentration (EC(50)) of 0.075 microM against the genotype 1b-Con1 replicon. To determine the in vitro antiviral activity of PF-00868554 against various HCV strains, a panel of chimeric replicons was generated, in which polymerase sequences derived from genotype 1a and 1b clinical isolates were cloned into the 1b-Con1 subgenomic reporter replicon. Our results indicate that PF-00868554 has potent in vitro antiviral activity against a majority (95.8%) of genotype 1a and 1b replicons, with an overall mean EC(50) of 0.059 microM. PF-00868554 showed no cytotoxic effect in several human cell lines, up to the highest concentration evaluated (320 microM). Furthermore, the antiviral activity of PF-00868554 was retained in the presence of human serum proteins. An in vitro resistance study of PF-00868554 identified M423T as the predominant resistance mutation, resulting in a 761-fold reduction in susceptibility to PF-00868554 but no change in susceptibility to alpha interferon and a polymerase inhibitor that binds to a different region. PF-00868554 also showed good pharmacokinetic properties in preclinical animal species. Our results demonstrate that PF-00868554 has potent and broad-spectrum antiviral activity against genotype 1 HCV strains, supporting its use as an oral antiviral agent in HCV-infected patients.
Asunto(s)
Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Animales , Antivirales/farmacocinética , Línea Celular , Perros , Farmacorresistencia Viral , Genotipo , Hepacivirus/enzimología , Humanos , Macaca fascicularis , Masculino , Fenotipo , Unión Proteica , Ratas , Ratas Sprague-Dawley , Replicón/efectos de los fármacosRESUMEN
The HCV RNA-dependent RNA polymerase has emerged as one of the key targets for novel anti-HCV therapy development. Herein, we report the optimization of the dihydropyrone series inhibitors to improve compound aqueous solubility and reduce CYP2D6 inhibition, which led to the discovery of compound 24 (PF-00868554). Compound 24 is a potent and selective HCV polymerase inhibitor with a favorable pharmacokinetic profile and has recently entered a phase II clinical evaluation in patients with genotype 1 HCV.
Asunto(s)
Antivirales/síntesis química , Hepacivirus/enzimología , Pironas/síntesis química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Triazoles/síntesis química , Administración Oral , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Cristalografía por Rayos X , Inhibidores del Citocromo P-450 CYP2D6 , Perros , Macaca fascicularis , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Pironas/farmacocinética , Pironas/farmacología , Ratas , Ratas Sprague-Dawley , Solubilidad , Estereoisomerismo , Relación Estructura-Actividad , Triazoles/farmacocinética , Triazoles/farmacologíaRESUMEN
To address the need for broad-spectrum antiviral activity characterization of hepatitis C virus (HCV) polymerase inhibitors, we created a panel of intergenotypic chimeric replicons containing nonstructural (NS) protein NS5B sequences from genotype 2b (GT2b), GT3a, GT4a, GT5a, and GT6a HCV isolates. Viral RNA extracted from non-GT1 HCV patient plasma was subjected to reverse transcription. The NS5B region was amplified by nested PCR and introduced into the corresponding region of the GT1b (Con-1) subgenomic reporter replicon by Splicing by Overlap Extension (SOEing) PCR. Stable cell lines were generated with replication-competent chimeras for in vitro antiviral activity determination of HCV nonnucleoside polymerase inhibitors (NNIs) that target different regions of the protein. Compounds that bind to the NNI2 (thiophene carboxylic acid) or NNI3 (benzothiadiazine) allosteric sites showed 8- to >1,280-fold reductions in antiviral activity against non-GT1 NS5B chimeric replicons compared to that against the GT1b subgenomic replicon. Smaller reductions in susceptibility, ranging from 0.2- to 33-fold, were observed for the inhibitor binding to the NNI1 (benzimidazole) site. The inhibitor binding to the NNI4 (benzofuran) site showed broad-spectrum antiviral activity against all chimeric replicons evaluated in this study. In conclusion, evaluation of HCV NNIs against intergenotypic chimeric replicons showed differences in activity spectrum for inhibitors that target different regions of the enzyme, some of which could be associated with specific residues that differ between GT1 and non-GT1 polymerases. Our study demonstrates the utility of chimeric replicons for broad-spectrum activity determination of HCV inhibitors.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antivirales/química , Línea Celular , Quimera/genética , Variación Genética , Genotipo , Hepacivirus/clasificación , Hepacivirus/enzimología , Hepatitis C/virología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Replicón , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/químicaRESUMEN
A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 A away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 muM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.
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Farmacorresistencia Viral , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Pironas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Sitio Alostérico , Sitios de Unión , Línea Celular Tumoral , Farmacorresistencia Viral/genética , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/toxicidad , Hepacivirus/genética , Hepacivirus/crecimiento & desarrollo , Humanos , Modelos Moleculares , Mutación , Pironas/química , Pironas/metabolismo , Pironas/toxicidad , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Replicón , Replicación ViralRESUMEN
The discovery and optimization of a novel class of carbon-linked dihydropyrones as allosteric HCV NS5B polymerase inhibitors are presented. Replacement of the sulfur linker atom with carbon reduced compound acidity and greatly increased cell permeation. Further structure-activity relationship (SAR) studies led to the identification of compounds, exemplified by 23 and 24, with significantly improved antiviral activities in the cell-based replicon assay and favorable pharmacokinetic profiles.
Asunto(s)
Antivirales/síntesis química , Hepacivirus/enzimología , Pironas/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Administración Oral , Regulación Alostérica , Animales , Antivirales/química , Antivirales/farmacología , Disponibilidad Biológica , Células CACO-2 , Línea Celular Tumoral , Semivida , Humanos , Permeabilidad , Pironas/química , Pironas/farmacología , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Proteínas no Estructurales Virales/genéticaRESUMEN
A novel class of non-nucleoside HCV NS5B polymerase inhibitors has been identified from screening. A co-crystal structure revealed an allosteric binding site in the protein that required a unique conformational change to accommodate inhibitor binding. Herein we report the structure-activity relationships (SARs) of this novel class of dihydropyrone-containing compounds that show potent inhibitory activities against the HCV RNA polymerase in biochemical assays.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Hidrógeno/química , Pironas/química , Pironas/farmacología , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Pironas/síntesis química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , Relación Estructura-Actividad , Proteínas no Estructurales Virales/químicaRESUMEN
Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has previously been shown to bind mouse hepatitis virus (MHV) RNA at the 3' end of both plus and minus strands and modulate MHV RNA synthesis. However, a mouse erythroleukemia cell line, CB3, does not express hnRNP A1 but still supports MHV replication, suggesting that alternative proteins can replace hnRNP A1 in cellular functions and viral infection. In this study, we set out to identify these proteins. UV cross-linking experiments revealed that several CB3 cellular proteins similar in size to hnRNP A1 interacted with the MHV RNA. These proteins were purified by RNA affinity column with biotinylated negative-strand MHV leader RNA and identified by mass spectrometry to be hnRNP A2/B1, hnRNP A/B, and hnRNP A3, all of which belong to the type A/B hnRNPs. All of these proteins contain amino acid sequences with strong similarity to the RNA-binding domains of hnRNP A1. Some of these hnRNPs have previously been shown to replace hnRNP A1 in regulating RNA splicing. These proteins displayed MHV RNA-binding affinity and specificity similar to those of hnRNP A1. hnRNP A2/B1, which is predominantly localized to the nucleus and shuttles between the nucleus and the cytoplasm, was shown to relocalize to the cytoplasm in MHV-infected CB3 cells. Furthermore, overexpression of hnRNP A/B in cells enhanced MHV RNA synthesis. Our findings demonstrate that the functions of hnRNP A1 in MHV RNA synthesis can be replaced by other closely related hnRNPs, further supporting the roles of cellular proteins in MHV RNA synthesis.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/fisiología , Virus de la Hepatitis Murina/genética , ARN Viral/biosíntesis , Secuencia de Aminoácidos , Animales , Ribonucleoproteína Nuclear Heterogénea A1 , Ratones , Datos de Secuencia Molecular , Proteínas de Unión al ARN/análisis , Células Tumorales CultivadasRESUMEN
The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly understood. In this study, we labeled de novo-synthesized viral RNA in situ with bromouridine triphosphate (BrUTP) in Huh7 cells expressing an HCV subgenomic replicon. By immunofluorescence staining using an anti-BrUTP antibody and confocal microscopy, we showed that the newly synthesized HCV RNA was localized to distinct speckle-like structures, which also contain all of the HCV nonstructural (NS) proteins. These speckles are distinct from lipid droplets and are separated from the endoplasmic reticulum (ER), where some HCV NS proteins also reside. Membrane flotation analysis demonstrated that almost all of the NS5A and part of the NS5B proteins and all of the viral RNA were present in membrane fractions which are resistant to treatment with 1% NP-40 at 4 degrees C. They were cofractionated with caveolin-2, a lipid-raft-associated intracellular membrane protein, in the presence or absence of the detergent. In contrast, the ER-resident proteins were detergent soluble. These properties suggest that the membranes on which HCV RNA replication occurs are lipid rafts recruited from the intracellular membranes. The protein synthesis inhibitors cycloheximide and puromycin did not inhibit viral RNA synthesis, indicating that HCV RNA replication does not require continuous protein synthesis. We suggest that HCV RNA synthesis occurs on a lipid raft membrane structure.
Asunto(s)
Hepacivirus/fisiología , ARN Viral/biosíntesis , Caveolina 2 , Caveolinas/metabolismo , Línea Celular , Detergentes , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Hepacivirus/genética , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virología , Lípidos de la Membrana/metabolismo , Octoxinol , Polietilenglicoles , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Viral/genética , Solubilidad , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Hepacivirus/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Ubiquitinas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Relacionadas con la Autofagia , Sitios de Unión , Línea Celular , Hepacivirus/genética , Humanos , Estructura Terciaria de Proteína , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Replicón , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , Ubiquitinas/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
Hepatitis delta virus (HDV) contains a viroid-like circular RNA that is presumed to replicate via a rolling circle replication mechanism mediated by cellular RNA polymerases. However, the exact mechanism of rolling circle replication for HDV RNA and viroids is not clear. Using our recently described cDNA-free transfection system (L. E. Modahl and M. M. Lai, J. Virol. 72:5449-5456, 1998), we have succeeded in detecting HDV RNA replication by metabolic labeling with [32P]orthophosphate in vivo and obtained direct evidence that HDV RNA replication generates high-molecular-weight multimeric species of HDV RNA, which are processed into monomeric and dimeric forms. Thus, these multimeric RNAs are the true intermediates of HDV RNA replication. We also found that HDV RNA synthesis is highly temperature sensitive, occurring most efficiently at 37 to 40 degrees C and becoming virtually undetectable at temperatures below 30 degrees C. Moreover, genomic HDV RNA synthesis was found to occur at a rate roughly 30-fold higher than that of antigenomic RNA synthesis. Finally, in lysolecithin-permeabilized cells, the synthesis of full-length antigenomic HDV RNA was completely resistant to high concentrations (100 microg/ml) of alpha-amanitin. In contrast, synthesis of genomic HDV RNA was totally inhibited by alpha-amanitin at concentrations as low as 2.5 microg/ml. Thus, these results suggest that genomic and antigenomic HDV RNA syntheses are performed by two different host cell enzymes. This observation, combined with our previous finding that hepatitis delta antigen mRNA synthesis is likely performed by RNA polymerase II, suggests that the different HDV RNA species are synthesized by different cellular transcriptional machineries.