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1.
Sci Signal ; 15(717): eabj4743, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-35041461

RESUMO

Most patients with hepatocellular carcinoma (HCC) are diagnosed at a late stage and have few therapeutic options and a poor prognosis. This is due to the lack of clearly defined underlying mechanisms or a dominant oncogene that can be targeted pharmacologically, unlike in other cancer types. Here, we report the identification of a previously uncharacterized oncogenic signaling pathway in HCC that is mediated by the tyrosine kinase Yes. Using genetic and pharmacological interventions in cellular and mouse models of HCC, we showed that Yes activity was necessary for HCC cell proliferation. Transgenic expression of activated Yes in mouse hepatocytes was sufficient to induce liver tumorigenesis. Yes phosphorylated the transcriptional coactivators YAP and TAZ (YAP/TAZ), promoting their nuclear accumulation and transcriptional activity in HCC cells and liver tumors. We also showed that YAP/TAZ were effectors of the Yes-dependent oncogenic transformation of hepatocytes. Src family kinase activation correlated with the tyrosine phosphorylation and nuclear localization of YAP in human HCC and was associated with increased tumor burden in mice. Specifically, high Yes activity predicted shorter overall survival in patients with HCC. Thus, our findings identify Yes as a potential therapeutic target in HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas de Sinalização YAP
2.
Sci Rep ; 10(1): 4040, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132633

RESUMO

Flaviviridae infections represent a major global health burden. By deciphering mechanistic aspects of hepatitis C virus (HCV)-host interactions, one could discover common strategy for inhibiting the replication of related flaviviruses. By elucidating the HCV interactome, we identified the 17-beta-hydroxysteroid dehydrogenase type 12 (HSD17B12) as a human hub of the very-long-chain fatty acid (VLCFA) synthesis pathway and core interactor. Here we show that HSD17B12 knockdown (KD) impairs HCV replication and reduces virion production. Mechanistically, depletion of HSD17B12 induces alterations in VLCFA-containing lipid species and a drastic reduction of lipid droplets (LDs) that play a critical role in virus assembly. Oleic acid supplementation rescues viral RNA replication and production of infectious particles in HSD17B12 depleted cells, supporting a specific role of VLCFA in HCV life cycle. Furthermore, the small-molecule HSD17B12 inhibitor, INH-12, significantly reduces replication and infectious particle production of HCV as well as dengue virus and Zika virus revealing a conserved requirement across Flaviviridae virus family. Overall, the data provide a strong rationale for the advanced evaluation of HSD17B12 inhibition as a promising broad-spectrum antiviral strategy for the treatment of Flaviviridae infections.


Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Hepacivirus/fisiologia , Hepatite C/enzimologia , Ácido Oleico/farmacologia , Replicação Viral/efeitos dos fármacos , 17-Hidroxiesteroide Desidrogenases/genética , Animais , Chlorocebus aethiops , Células HeLa , Células Hep G2 , Hepatite C/genética , Humanos , Células Vero , Replicação Viral/genética
3.
Cell Rep ; 17(2): 425-435, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27705791

RESUMO

Functional genomic analysis of gene expression in mice allowed us to identify a quantitative trait locus (QTL) linked in trans to the expression of 190 gene transcripts and in cis to the expression of only two genes, one of which was Ypel5. Most of the trans-expression QTL genes were interferon-stimulated genes (ISGs), and their expression in mouse macrophage cell lines was stimulated in an IFNB1-dependent manner by Ypel5 silencing. In human HEK293T cells, YPEL5 silencing enhanced the induction of IFNB1 by pattern recognition receptors and phosphorylation of TBK1/IKBKE kinases, whereas co-immunoprecipitation experiments revealed that YPEL5 interacted physically with IKBKE. We thus found that the Ypel5 gene (contained in a locus linked to a network of ISGs in mice) is a negative regulator of IFNB1 production and innate immune responses that interacts functionally and physically with TBK1/IKBKE kinases.


Assuntos
Proteínas de Ciclo Celular/genética , Quinase I-kappa B/genética , Interferon beta-1a/genética , Interferons/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Células HEK293 , Humanos , Macrófagos/metabolismo , Camundongos , Ligação Proteica , Locos de Características Quantitativas/genética , Elementos Reguladores de Transcrição/genética
4.
PLoS Biol ; 12(3): e1001809, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24643257

RESUMO

The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing components can also specifically impact alternative splicing.


Assuntos
Processamento Alternativo , Proteínas de Drosophila/genética , Drosophila/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas ras/metabolismo , Animais , Linhagem Celular , Análise por Conglomerados , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Epistasia Genética , Regulação da Expressão Gênica , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/fisiologia , Interferência de RNA
5.
J Virol ; 87(21): 11704-20, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23986595

RESUMO

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.


Assuntos
Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Proteína 1 de Ligação a Y-Box/metabolismo , Linhagem Celular , Inativação Gênica , Hepatócitos/química , Humanos , Substâncias Macromoleculares , Espectrometria de Massas , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica , Multimerização Proteica , Proteínas não Estruturais Virais/genética
6.
PLoS Pathog ; 9(6): e1003416, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23785285

RESUMO

To identify new regulators of antiviral innate immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-ß (IFNB1) promoter following Sendai virus (SeV) infection. We now report a novel link between WNT signaling pathway and the modulation of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-dependent innate immune responses. Here we show that secretion of WNT2B and WNT9B and stabilization of ß-catenin (CTNNB1) upon virus infection negatively regulate expression of representative inducible genes IFNB1, IFIT1 and TNF in a CTNNB1-dependent effector mechanism. The antiviral response is drastically reduced by glycogen synthase kinase 3 (GSK3) inhibitors but restored in CTNNB1 knockdown cells. The findings confirm a novel regulation of antiviral innate immunity by a canonical-like WNT/CTNNB1 signaling pathway. The study identifies novel avenues for broad-spectrum antiviral targets and preventing immune-mediated diseases upon viral infection.


Assuntos
Glicoproteínas/imunologia , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Infecções por Respirovirus/imunologia , Vírus Sendai/imunologia , Proteínas Wnt/imunologia , Via de Sinalização Wnt/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/imunologia , RNA Helicases DEAD-box/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Estudo de Associação Genômica Ampla , Glicoproteínas/metabolismo , Humanos , Interferon beta/imunologia , Interferon beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Interferência de RNA , Proteínas de Ligação a RNA , Receptores Imunológicos , Infecções por Respirovirus/metabolismo , Infecções por Respirovirus/patologia , Vírus Sendai/metabolismo , Proteínas Wnt/metabolismo
7.
J Hepatol ; 56(1): 70-7, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21835140

RESUMO

BACKGROUND & AIMS: Innate sensing of viral infection activates a global defense response including type I interferon (IFN) and IFN-stimulated genes (ISGs) expression. We previously reported that HCV NS3/4A protease, an essential protein in viral polyprotein processing, can abrogate antiviral signaling pathways and effectors' response when ectopically expressed in human hepatocytes by cleaving antiviral adaptor CARDIF. However, whether HCV mediates evasion of innate immunity in patients with chronic infection remains unclear. METHODS: In this study, paired liver biopsies and corresponding purified hepatocytes of chronic hepatitis C patients and controls were subjected to transcriptional analysis of selected innate immune genes and to CARDIF protein detection. RESULTS: We report that an antiviral response is largely supported by infected hepatocytes as demonstrated by upregulation of the representative antiviral genes ISG15, ISG56, and OASL as well as chemokines genes CXCL9, CXCL10, and CXCL11 measured in both HCV-derived liver biopsies and hepatocytes; that the mRNA levels of these indicator ISGs correlate inversely with HCV RNA level; and more importantly that expression of the early responsive IRF3-dependent genes type I IFNß, type III IL28A/IL29, and chemokine CCL5 are severely compromised and associated to a global decrease of CARDIF adaptor in infected hepatocytes. CONCLUSIONS: Altogether the data argue for a strong viral strategy that counteracts the host's early antiviral response of hepatocytes from chronic patients without impairing ISGs induced via classical IFN pathway.


Assuntos
Hepatite C Crônica/imunologia , Imunidade Inata , Fígado/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Quimiocinas/genética , Feminino , Hepatite C Crônica/genética , Hepatite C Crônica/metabolismo , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Imunidade Inata/genética , Fatores Reguladores de Interferon/genética , Interferons/genética , Fígado/metabolismo , Fígado/virologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Regulação para Cima
8.
J Virol ; 85(21): 11022-37, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21849455

RESUMO

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.


Assuntos
Proteínas de Transporte/metabolismo , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Proteína 1 de Ligação a Y-Box/metabolismo , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Espectrometria de Massas , Microscopia de Fluorescência , Proteína 1 de Ligação a Y-Box/genética
9.
PLoS Pathog ; 6(6): e1000930, 2010 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-20532218

RESUMO

The innate immune response is essential to the host defense against viruses, through restriction of virus replication and coordination of the adaptive immune response. Induction of antiviral genes is a tightly regulated process initiated mainly through sensing of invading virus nucleic acids in the cytoplasm by RIG-I like helicases, RIG-I or Mda5, which transmit the signal through a common mitochondria-associated adaptor, MAVS. Although major breakthroughs have recently been made, much remains unknown about the mechanisms that translate virus recognition into antiviral genes expression. Beside the reputed detrimental role, reactive oxygen species (ROS) act as modulators of cellular signaling and gene regulation. NADPH oxidase (NOX) enzymes are a main source of deliberate cellular ROS production. Here, we found that NOX2 and ROS are required for the host cell to trigger an efficient RIG-I-mediated IRF-3 activation and downstream antiviral IFNbeta and IFIT1 gene expression. Additionally, we provide evidence that NOX2 is critical for the expression of the central mitochondria-associated adaptor MAVS. Taken together these data reveal a new facet to the regulation of the innate host defense against viruses through the identification of an unrecognized role of NOX2 and ROS.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Brônquios/imunologia , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica , Neoplasias Pulmonares/imunologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Western Blotting , Brônquios/citologia , Brônquios/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Luciferases/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/virologia , Glicoproteínas de Membrana/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Receptores Imunológicos , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/metabolismo , Infecções por Respirovirus/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus Sendai/fisiologia , Transdução de Sinais
10.
Viruses ; 2(8): 1752-1765, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21994705

RESUMO

Hepatitis C virus (HCV) infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease) that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection.

11.
BMC Cancer ; 8: 337, 2008 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-19014680

RESUMO

BACKGROUND: The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established. METHODS: Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells. RESULTS: We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect. CONCLUSION: MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.


Assuntos
Adenocarcinoma/secundário , Transformação Celular Neoplásica , Mucosa Intestinal/patologia , Neoplasias Intestinais/patologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Animais , Anoikis , Linhagem Celular Tumoral , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Humanos , Mucosa Intestinal/metabolismo , Neoplasias Intestinais/enzimologia , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/metabolismo , Interferência de RNA , Ratos
12.
J Virol ; 81(11): 5537-46, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17376921

RESUMO

The role of peripheral dendritic cells (DCs) in hepatitis C virus (HCV) infection is unclear. To determine if persistent infection exerts an inhibitory pressure on HCV-specific innate responses, we analyzed DC function in blood through quantification of cell-associated HCV RNA levels in conjunction with multiparametric flow cytometry analysis of pathogen recognition receptor-induced cytokine expression. Independently of the serum viral load, fluorescence-activated cell sorter-purified total DCs had a wide range of cell-associated HCV genomic RNA copy numbers (mean log(10), 5.0 per 10(6) cells; range, 4.3 to 5.8). Here we report that for viremic patients with high viral loads in their total DCs, the myeloid DC (MDC) subset displayed impaired expression of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) but normal IL-6 or chemokine CCL3 expression in response to poly(I:C) and lipopolysaccharide (LPS). IL-6-expressing cells from this subgroup of viremic patients demonstrated a significant increase (sixfold more) in TNF-alpha(-) IL-12(-) cell frequency compared to healthy donors (mean, 38.8% versus 6.5%; P < 0.0001), indicating a functional defect in a subpopulation of cytokine-producing MDCs ( approximately 6% of MDCs). Attenuation of poly(I:C) and LPS innate sensing was HCV RNA density dependent and did not correlate with viremia or deficits in circulating MDC frequencies in HCV-infected patients. Monocytes from these patients were functionally intact, responding normally on a per-cell basis following stimulation, independent of cell-associated HCV RNA levels. Taken together, these data indicate that detection of HCV genomic RNA in DCs and loss of function in the danger signal responsiveness of a small proportion of DCs in vivo are interrelated rather than independent phenomena.


Assuntos
Células Dendríticas/metabolismo , Hepacivirus/fisiologia , Hepatite C Crônica/sangue , Lipopolissacarídeos/metabolismo , Células Mieloides/metabolismo , Poli I-C/metabolismo , Adulto , Idoso , Células Cultivadas , Células Dendríticas/patologia , Células Dendríticas/virologia , Feminino , Hepacivirus/genética , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Células Mieloides/patologia , RNA Viral/sangue
13.
J Med Chem ; 47(10): 2511-22, 2004 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-15115394

RESUMO

The structure-activity relationship at the C-terminal position of peptide-based inhibitors of the hepatitis C virus NS3 protease is presented. The observation that the N-terminal cleavage product (DDIVPC-OH) of a substrate derived from the NS5A/5B cleavage site was a competitive inhibitor of the NS3 protease was previously described. The chemically unstable cysteine residue found at the P1 position of these peptide-based inhibitors could be replaced with a norvaline residue, at the expense of a substantial drop in the enzymatic activity. The fact that an aminocyclopropane carboxylic acid (ACCA) residue at the P1 position of a tetrapeptide such as 1 led to a significant gain in the inhibitory enzymatic activity, as compared to the corresponding norvaline derivative 2, prompted a systematic study of substituent effects on the three-membered ring. We report herein that the incorporation of a vinyl group with the proper configuration onto this small cycle produced inhibitors of the protease with much improved in vitro potency. The vinyl-ACCA is the first reported carboxylic acid containing a P1 residue that produced NS3 protease inhibitors that are significantly more active than inhibitors containing a cysteine at the same position.


Assuntos
Inibidores Enzimáticos/química , Hepacivirus/química , Oligopeptídeos/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Aminoácidos Cíclicos/síntese química , Aminoácidos Cíclicos/química , Ciclopropanos/síntese química , Ciclopropanos/química , Inibidores Enzimáticos/síntese química , Modelos Moleculares , Conformação Molecular , Oligopeptídeos/síntese química , Estereoisomerismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química
14.
J Virol ; 78(1): 491-501, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14671129

RESUMO

Hepatitis C virus (HCV) infects liver cells and its replication in other cells is incompletely defined. Human hepatoma Huh-7 cells harboring subgenomic HCV replicons were used in somatic cell fusion experiments with human embryonic kidney 293 cells as a means of examining the permissiveness of 293 cells for HCV subgenomic RNA replication. 293 cells were generally not permissive for replication of Huh-7 cell-adapted replicons. However, upon coculturing of the two cell lines, we selected rare replicon-containing cells, termed 293Rep cells, that resembled parental 293 cells. Direct metabolic labeling of cells with (33)P in the presence of actinomycin D and Northern blotting to detect the negative strand of the replicon demonstrated functional RNA replicons in 293Rep cells. Furthermore, Western blots revealed that 293Rep cells expressed the HCV nonstructural proteins as well as markers of the naïve 293 cells but not Huh-7 cells. Propidium iodide staining and fluorescence-activated cell sorting analysis of 293Rep cells revealed that clone 293Rep17 closely resembled naïve 293 cells. Transfection of total RNA from 293Rep17 into naïve 293 cells produced replicon-containing 293 cell lines with characteristics distinct from those of Huh-7-derived replicon cell lines. Relative to Huh-7 replicons, the 293 cell replicons were less sensitive to inhibition by alpha interferon and substantially more sensitive to inhibition by poly(I)-poly(C) double-stranded RNA. This study established HCV subgenomic replicons in nonhepatic 293 cells and demonstrated their utility in expanding the study of cellular HCV RNA replication.


Assuntos
Genoma Viral , Hepacivirus/genética , Rim/virologia , Replicon , Replicação Viral/fisiologia , Fusão Celular , Linhagem Celular , Hepacivirus/fisiologia , Humanos , Rim/citologia , Rim/embriologia , RNA Viral/biossíntese , Células Tumorais Cultivadas
15.
Nature ; 426(6963): 186-9, 2003 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-14578911

RESUMO

Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 1, and they are poorly tolerated, highlighting the unmet medical need for new therapeutics. The HCV-encoded NS3 protease is essential for viral replication and has long been considered an attractive target for therapeutic intervention in HCV-infected patients. Here we identify a class of specific and potent NS3 protease inhibitors and report the evaluation of BILN 2061, a small molecule inhibitor biologically available through oral ingestion and the first of its class in human trials. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Our results further illustrate the potential of the viral-enzyme-targeted drug discovery approach for the development of new HCV therapeutics.


Assuntos
Antivirais/uso terapêutico , Carbamatos/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Compostos Macrocíclicos , Quinolinas , Inibidores de Serina Proteinase/uso terapêutico , Tiazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Antivirais/administração & dosagem , Antivirais/farmacocinética , Antivirais/farmacologia , Carbamatos/administração & dosagem , Carbamatos/química , Carbamatos/farmacocinética , Método Duplo-Cego , Hepacivirus/enzimologia , Hepacivirus/genética , Hepatite C/virologia , Humanos , Masculino , Poliproteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Inibidores de Serina Proteinase/administração & dosagem , Inibidores de Serina Proteinase/farmacocinética , Inibidores de Serina Proteinase/farmacologia , Tiazóis/administração & dosagem , Tiazóis/química , Tiazóis/farmacocinética , Carga Viral , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo
17.
J Biol Chem ; 278(22): 20374-80, 2003 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-12646587

RESUMO

The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-alpha (IFN-alpha), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-alpha showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-alpha also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection.


Assuntos
Hepacivirus/efeitos dos fármacos , RNA Viral/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Antivirais/uso terapêutico , Hepacivirus/enzimologia , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Humanos , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico , RNA Viral/biossíntese , Inibidores de Serina Proteinase/uso terapêutico
18.
Biochem Biophys Res Commun ; 295(3): 682-8, 2002 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-12099694

RESUMO

A variety of 3'-untranslated regions (UTRs) were cloned from infectious hepatitis C virus human samples and examined in NS5B polymerase de novo initiation reactions. We isolated and characterized four distinct 3'-UTRs that harbor the conserved terminal 98 nucleotides, but have poly(U/UC) tracts of 25, 93, 98, and 101 nucleotides, respectively. Reconstitution of de novo initiation by the mature NS5B with the different 3'-UTR RNA substrates revealed distinctively sized products that are consistent with internal initiation at specific sites within the polypyrimidine tract. These sites were further mapped by demonstrating that nucleotide substitutions of the cytidylate stretches within the poly(U/UC) template eliminate specific products of de novo synthesis and by showing that these products could be radiolabeled by [gamma-32P]GTP. We also examine analogs that can substitute for GTP in this reaction.


Assuntos
Regiões 3' não Traduzidas , Hepacivirus/genética , RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Proteínas não Estruturais Virais/genética , Sequência de Bases , Clonagem Molecular , Relação Dose-Resposta a Droga , Guanosina Trifosfato/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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