RESUMEN
A novel unsymmetrical structural class of orally bioavailable hepatitis C virus (HCV) nonstructural 5A protein (NS5A) inhibitors has been generated by improving both the solubility and membrane permeability of the lead compound found in our previous work. The representative compound 14, with a 5-hydroxymethylpyrazine group and a 3-t-butylpropargyl group on each side of the molecule, exhibited the best oral bioavailability in this study, inhibiting not only the HCV genotype 1a, 1b, 2a, and 3a replicons with EC50 values in the picomolar range, but also inhibited 1a Q30 mutants induced by launched symmetrical inhibitors with EC50 values in the low nanomolar range.
Asunto(s)
Antivirales/química , Hepacivirus/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Administración Oral , Antivirales/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , Genotipo , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/patología , Hepatitis C/virología , Humanos , Mutación , Pargilina/química , Pirazinas/química , Relación Estructura-Actividad , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
A novel unsymmetrical structural class of HCV NS5A inhibitors showing picomolar range antiviral activity has been identified. An unsymmetrical lead compound 2, generated from a substructure of a known symmetrical inhibitor 1, was optimized by extension of its substituents to interact with the hitherto unexplored site of the target protein. This approach afforded novel highly potent unsymmetrical inhibitor 20, which not only equally inhibited HCV genotypes1a, 1b, and 2a with EC50 values in the picomolar range, but also inhibited the 1a Q30K mutant induced by a launched symmetrical inhibitor daclatasvir with an EC50 in the low nanomolar range.
Asunto(s)
Antivirales/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Hepacivirus/química , Imidazoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/síntesis química , Línea Celular Tumoral , Humanos , Imidazoles/síntesis química , Pruebas de Sensibilidad Microbiana , Estructura MolecularRESUMEN
Some preceding studies have provided evidence that hypoxia-inducible factor (HIF)-prolyl hydroxylase (PH) inhibitors have therapeutic potential against tubular interstitial fibrosis (TIF). Recently, transformation of renal interstitial fibroblasts (RIFs) into α-smooth muscle actin-positive myofibroblasts with loss of their hypoxia-inducible erythropoietin (EPO) expression has been hypothesized as the central mechanism responsible for TIF with renal anemia (the RIF hypothesis). These reports have suggested that HIF-PH inhibitors may suppress TIF via suppressing transformation of RIFs. However, the direct effect of HIF-PH inhibitors on transformation of RIFs has not been demonstrated because there has been no appropriate assay system. Here, we established a novel in vitro model of the transformation of RIFs. This model expresses key phenotypic changes such as transformation of RIFs accompanied by loss of their hypoxia-inducible EPO expression, as proposed by the RIF hypothesis. Using this model, we demonstrated that JTZ-951, a newly developed HIF-PH inhibitor, stabilized HIF protein in RIFs, suppressed transformation of RIFs, and maintained their hypoxia-inducible EPO expression. JTZ-951 also suppressed the expression of FGF2, FGF7, and FGF18, which are upregulated during transformation of RIFs. Furthermore, expression of Fgf2, Fgf7, and Fgf18 was correlated with TIF in an animal model of TIF. We also demonstrated that not only FGF2, which is a well-known growth-promoting factor, but also FGF18 promoted proliferation of RIFs. These data suggest that JTZ-951 has therapeutic potential against TIF with renal anemia. Furthermore, FGF2, FGF7, and FGF18, which faithfully reflect the anti-TIF effects of JTZ-951, have potential as TIF biomarkers.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Riñón/efectos de los fármacos , Glicinas N-Sustituídas/farmacología , Inhibidores de Prolil-Hidroxilasa/farmacología , Piridinas/farmacología , Triazoles/farmacología , Regulación hacia Arriba/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/genética , Fibroblastos/metabolismo , Humanos , Riñón/metabolismoRESUMEN
Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs.IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.
Asunto(s)
Hepacivirus/genética , Hepatitis C/metabolismo , Hepatitis C/virología , Gotas Lipídicas/metabolismo , ARN Viral/genética , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Regulación de la Expresión Génica/fisiología , Células HEK293 , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Hígado/virología , Interferencia de ARN/fisiología , Transducción de Señal/genética , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación/genética , Células Vero , Replicación Viral/genéticaRESUMEN
The envelope proteins of the hepatitis C virus (HCV), E1 and E2, have been revealed to be essential for invasion of HCV. Thus, we were engaged in the search for the inhibitors against HCV invasion through the assay system using the model virus expressing recombinant HCV envelopes, E1 and E2. Now, we disclosed dimeric hydrolysable tannin oenothein B (1) from MeOH extract of Oenothera erythrosepala as an active principle for inhibition of HCV invasion and its potency was almost the same as that of monomeric hydrolysable tannin, tellimagrandin I (2). Furthermore, by use of stereoselectively prepared 1-ß- and 1-α-O-methyl tellimagrandin Is (4 and 5), the introduction of methyl moiety into 1-hydroxy group of 2 was clarified to result in slightly reduction of activity and ß-isomer was revealed to exhibit a little stronger activity than α-one.
Asunto(s)
Hepacivirus/patogenicidad , Hepatitis C/tratamiento farmacológico , Taninos Hidrolizables/química , Oenothera/química , HumanosRESUMEN
BACKGROUND: Standard treatment of chronic hepatitis C with pegylated interferon and ribavirin is associated with suboptimal virological response rates and substantial side effects. This study describes the in vitro and in vivo development of JTK-652, a novel pyrrolopyridazin-derived HCV infection inhibitor. METHODS: JTK-652 was evaluated in multiple cell lines using an in vitro HCV infection model consisting of HCV pseudotype vesicular stomatitis virus bearing HCV E1/E2 envelope proteins. Safety, tolerability, pharmacokinetics and efficacy of JTK-652 were tested in a randomized double-blind and placebo-controlled study in healthy male volunteers (n=36) and chronic hepatitis C patients. A total of 10 HCV genotype-1-infected patients (treatment-naive [n=2] and treatment-experienced [n=8]) with HCV RNA>1x10(5) IU/ml received an oral dose of 100 mg JTK-652 three times daily or placebo (8:2 ratio) for 4 weeks. RESULTS: JTK-652 showed potent inhibitory activity against HCV genotype 1a and 1b pseudotype viruses bearing HCV E1/E2 envelope proteins in HepG2 cells and in human primary hepatocytes. No significant clinical laboratory, vital sign, ECG or physical examination abnormalities were observed during the Phase I trial. JTK-652 was found to be well tolerated. No significant changes in HCV RNA levels compared with baseline were observed at the end of treatment. CONCLUSIONS: Although results from the preclinical studies indicated that JTK-652 has well-established antiviral properties and a Phase I clinical trial has showed that JTK-652 was safe and well tolerated at a 100 mg three times daily dose level, plasma HCV RNA levels in chronically HCV-infected patients did not decrease during 28 days of dosing at a 100 mg three times daily dose level.
Asunto(s)
Antivirales/efectos adversos , Antivirales/uso terapéutico , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/tratamiento farmacológico , Piridazinas/efectos adversos , Piridazinas/uso terapéutico , Administración Oral , Adulto , Antivirales/administración & dosificación , Antivirales/farmacocinética , Línea Celular Tumoral , Células Cultivadas , Método Doble Ciego , Femenino , Hepacivirus/patogenicidad , Hepatitis C Crónica/virología , Hepatocitos/virología , Humanos , Masculino , Persona de Mediana Edad , Piridazinas/química , Piridazinas/farmacocinética , ARN Viral/sangre , Resultado del TratamientoRESUMEN
By use of the model virus, expressing the HCV envelope proteins E1 and E2, bioassay guided separation of the MeOH extract from Rosa rugosa Thunb. disclosed tellimagrandin I (1) together with eugeniin (2) and casuarictin (3) as the potent HCV invasion inhibitors. Furthermore, structure-activity relationship analysis of some relative tannins including the synthesized analogs elucidated the partial structures crucial for potent activity of 1.
Asunto(s)
Antivirales/química , Ácido Gálico/análogos & derivados , Glucósidos/química , Rosaceae/química , Proteínas del Envoltorio Viral/metabolismo , Integración Viral/efectos de los fármacos , Antivirales/aislamiento & purificación , Antivirales/farmacología , Compuestos de Bifenilo/química , Compuestos de Bifenilo/aislamiento & purificación , Compuestos de Bifenilo/farmacología , Ácido Gálico/química , Ácido Gálico/aislamiento & purificación , Ácido Gálico/farmacología , Glucósidos/aislamiento & purificación , Glucósidos/farmacología , Humanos , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/antagonistas & inhibidoresRESUMEN
Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala(191) by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe(177) by mass spectrometry. Mutations introduced into two residues (Ile(176) and Phe(177)) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Hepacivirus/metabolismo , Membranas Intracelulares/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas del Núcleo Viral/metabolismo , Sustitución de Aminoácidos , Línea Celular , Células Clonales , Técnica del Anticuerpo Fluorescente Directa , Vectores Genéticos , Hepacivirus/genética , Humanos , Riñón/citología , Mutación , Plásmidos , Transfección , Valina/metabolismo , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/inmunologíaRESUMEN
Although in vitro replication of the hepatitis C virus (HCV) JFH1 clone of genotype 2a (HCVcc) has been developed, a robust cell culture system for the 1a and 1b genotypes, which are the most prevalent viruses in the world and resistant to interferon therapy, has not yet been established. As a surrogate virus system, pseudotype viruses transiently bearing HCV envelope proteins based on the vesicular stomatitis virus (VSV) and retrovirus have been developed. Here, we have developed a replication-competent recombinant VSV with a genome encoding unmodified HCV E1 and E2 proteins in place of the VSV envelope protein (HCVrv) in human cell lines. HCVrv and a pseudotype VSV bearing the unmodified HCV envelope proteins (HCVpv) generated in 293T or Huh7 cells exhibited high infectivity in Huh7 cells. Generation of infectious HCVrv was limited in some cell lines examined. Furthermore, HCVrv but not HCVpv was able to propagate and form foci in Huh7 cells. The infection of Huh7 cells with HCVpv and HCVrv was neutralized by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. The infectivity of HCVrv was inhibited by an endoplasmic reticulum alpha-glucosidase inhibitor, N-(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin. Focus formation of HCVrv in Huh7 cells was impaired by Nn-DNJ treatment. These results indicate that the HCVrv developed in this study can be used to study HCV envelope proteins with respect to not only the biological functions in the entry process but also their maturation step.
Asunto(s)
Hepacivirus/fisiología , Modelos Biológicos , Virus de la Estomatitis Vesicular Indiana/genética , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Animales , Antígenos CD/metabolismo , Línea Celular , Inhibidores Enzimáticos/farmacología , Inhibidores de Glicósido Hidrolasas , Hepacivirus/efectos de los fármacos , Anticuerpos contra la Hepatitis C/farmacología , Humanos , Macrólidos/farmacología , Tetraspanina 28 , Proteínas del Envoltorio Viral/genética , Internalización del Virus/efectos de los fármacos , Replicación ViralRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). To develop a better animal model for the investigation of HTLV-1 infection, we established a transgenic (Tg) rat carrying the human CRM1 (hCRM1) gene, which encodes a viral RNA transporter that is a species-specific restriction factor. At first we found that CRM1 expression is elaborately regulated through a pathway involving protein kinase C during lymphocyte activation, initially by posttranscriptional and subsequently by transcriptional mechanisms. This fact led us to use an hCRM1-containing bacterial artificial chromosome clone, which would harbor the entire regulatory and coding regions of the CRM1 gene. The Tg rats expressed hCRM1 protein in a manner similar to expression of intrinsic rat CRM1 in various organs. HTLV-1-infected T-cell lines derived from these Tg rats produced 100- to 10,000-fold more HTLV-1 than did T cells from wild-type rats, and the absolute levels of HTLV-1 were similar to those produced by human T cells. We also observed enhancement of the dissemination of HTLV-1 to the thymus in the Tg rats after intraperitoneal inoculation, although the proviral loads were low in both wild-type and Tg rats. These results support the essential role of hCRM1 in proper HTLV-1 replication and suggest the importance of this Tg rat as an animal model for HTLV-1.
Asunto(s)
Animales Modificados Genéticamente , Regulación Neoplásica de la Expresión Génica/fisiología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Carioferinas/genética , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/virología , Receptores Citoplasmáticos y Nucleares/genética , Subgrupos de Linfocitos T/virología , Replicación Viral/fisiología , Animales , Línea Celular Transformada , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Carioferinas/biosíntesis , Ratas , Receptores Citoplasmáticos y Nucleares/biosíntesis , Subgrupos de Linfocitos T/metabolismo , Proteína Exportina 1RESUMEN
Although processing of the hepatitis C virus (HCV) polyprotein and characterization of each of its viral proteins have been described in detail, analysis of the structure and assembly of HCV particles has been hampered by the lack of a robust cell culture system to support efficient replication of HCV. In this study, we generated HCV-like particles (HCV-LP) using a recombinant baculovirus encoding structural and a part of non-structural proteins in a human hepatoma cell line. The HCV-LP exhibited a buoyant density of 1.17 g/ml in CsCl equilibrium gradient and particles of 40 to 50 nm in diameter. Binding of the HCV-LP to human hepatoma cells was partially inhibited by the treatment with anti-hCD81 antibody, in contrast to the hCD81-independent binding of HCV-LP produced in insect cells. These results indicate that HCV-LP generated in different types of cells exhibit different cellular tropism for binding to target cells.
Asunto(s)
Baculoviridae/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Ingeniería de Proteínas/métodos , Transfección/métodos , Virión/genética , Virión/metabolismo , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Vectores Genéticos , Humanos , Proteínas Recombinantes/metabolismoRESUMEN
Dendritic cells (DC) are the most potent antigen-presenting cells that regulate immune responses. One of the mechanisms for hepatitis C virus (HCV) persistence is the ability of HCV to suppress DC function. Direct HCV infection to blood DC has been implicated for DC dysfunction. To clarify the susceptibility of each DC subset to HCV, we used pseudotype vesicular stomatitis virus (VSV) coated with chimeric HCV envelope glycoproteins (E1 and E2). We demonstrate that pseudotype VSV enters myeloid DC (MDC) but not plasmacytoid DC (PDC). The highest efficiency of pseudotype VSV entry to MDC was observed when MDC were cultured with GM-CSF. Such efficiency decreased when MDC are matured with the treatment of IL-4, CpG oligodeoxynucleotide, or CD40 ligand. Mannan inhibited pseudotype VSV entry to MDC, but Ca(2+) chelators failed to do so. These results show that pseudotype VSV possessing HCV-E1 and E2 enters immature MDC through the interaction with lectins in a Ca(2+)-independent manner.