scispace - formally typeset
Search or ask a question

Showing papers on "NS5B published in 2006"


Journal ArticleDOI
TL;DR: Hepatitis C virus (HCV) polymerase activity is essential for HCV replication and the corresponding 5′-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity.

228 citations


Journal ArticleDOI
TL;DR: It is observed that the presence of viral structural proteins does not influence the anti-HCV activity of CsA, and this provides an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents.
Abstract: Recently, a production system for infectious particles of hepatitis C virus (HCV) utilizing the genotype 2a JFH1 strain has been developed. This strain has a high capacity for replication in the cells. Cyclosporine (CsA) has a suppressive effect on HCV replication. In this report, we characterize the anti-HCV effect of CsA. We observe that the presence of viral structural proteins does not influence the anti-HCV activity of CsA. Among HCV strains, the replication of genotype 1b replicons was strongly suppressed by treatment with CsA. In contrast, JFH1 replication was less sensitive to CsA and its analog, NIM811. Replication of JFH1 did not require the cellular replication cofactor, cyclophilin B (CyPB). CyPB stimulated the RNA binding activity of NS5B in the genotype 1b replicon but not the genotype 2a JFH1 strain. These findings provide an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents.

161 citations


Journal ArticleDOI
TL;DR: The identification of this naturally occurring recombinant virus strengthens the concept that recombination may play a role in HCV epidemiology and evolution and the location of the recombination breakpoint may be relevant for constructing infectious chimeric viruses.
Abstract: Hepatitis C viruses (HCVs) display a high level of sequence diversity and are currently classified into six genotypes and an increasing number of subtypes. Most likely, this heterogeneity is caused by genetic drift; evidence for recombination is scarce. To study the molecular heterogeneity of HCV in Vietnam, we analyzed 58 HCV RNA-positive sera from Vietnamese blood donors by sequence analysis of the CORE and NS5B regions. Phylogenetic analyses revealed the presence of genotype 1 (38%), genotype 2 (10.3%), and genotype 6 viruses (51.7%). All samples showed concordant results except for two (D3 and D54). Sample D54 was a mixed infection of genotype 2i and 6h viruses. Whole-genome analysis and bootscan analysis of sample D3, on the other hand, revealed a recombinant virus with genotype 2i and genotype 6p sequences at the 5' and 3' ends, respectively. The crossover point was located between nucleotide positions 3405 to 3464 (numbering according to prototype strain HCV-H, M67463) at the NS2/NS3 junction. The identification of this naturally occurring recombinant virus strengthens the concept that recombination may play a role in HCV epidemiology and evolution. Furthermore, the location of the recombination breakpoint may be relevant for constructing infectious chimeric viruses.

156 citations


Journal ArticleDOI
TL;DR: Among the new compounds, compound 10n (JTK-109) exhibited favorable pharmacokinetic profiles, high selectivity for NS5B, and good safety profiles, suggesting the potential for a clinical candidate in the treatment of hepatitis C.
Abstract: Following the discovery of a new series of benzimidazole derivatives bearing a diarylmethyl group as inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase (HCV NS5B RdRp),1,2 we extended the structure-activity relationship (SAR) study to analogues bearing a substituted biphenyl group and succeeded in a significant advancement of activity. Starting from compound 1, optimization of the A, B, and C rings afforded potent inhibitors with low nanomolar potency against genotype 1b NS5B. The compounds, which have a substituent with a carbonyl function at the 4-position of the B-ring, efficiently blocked subgenomic viral RNA replication in the replicon cell assay at low submicromolar concentrations. Among the new compounds, compound 10n (JTK-109) exhibited favorable pharmacokinetic profiles, high selectivity for NS5B, and good safety profiles, suggesting the potential for a clinical candidate in the treatment of hepatitis C.

147 citations


01 Jan 2006
TL;DR: The HCV Genome and Life Cycle, Recombinant Vesicular Stomatitis Virus (VSV) and Other Strategies in HCV Vaccine Designs and Immunotherapy, and Animal Models for HCV Study are described.
Abstract: Introduction: Hepatitis C 1. HCV Genome and Life Cycle 2. HCV 5' and 3'UTR: When Translation Meets Replication 3. Assemble and Interact: Pleiotropic Functions of the HCV Core Protein 4. HCV Glycoproteins: Assembly of a Functional E1-E2 Heterodimer 5. HCV NS2/3 Protease 6. HCV NS3-4A Serine Protease 7. HCV Helicase: Structure, Function, and Inhibition 8. HCV NS4B: from Obscurity to Central Stage 9. HCV NS5A: A Multifunctional Regulator of Cellular Pathways and Virus Replication 10. Biochemical Activities of the HCV NS5B RNA-Dependent RNA Polymerase 11. HCV Replicon Systems 12. Animal Models for HCV Study 13. HCV Regulation of Host Defense 14. Regulation of Adaptive Immunity by HCV 15. Recombinant Vesicular Stomatitis Virus (VSV) and Other Strategies in HCV Vaccine Designs and Immunotherapy 16. Development of an Infectious HCV Cell Culture System

139 citations


Journal ArticleDOI
01 Aug 2006-Virology
TL;DR: The characterization of the inhibitory effect of 2'-C-Methyl-Cytidine shows that it is a potent inhibitor of both genotype 1b and 1a HCV replicon replication, both of laboratory-optimized as well as of NS5B clinical isolates-chimera replicons.

130 citations


Journal ArticleDOI
TL;DR: Novel non-nucleoside inhibitors of the HCV RNA polymerase (NS5b) with sub-micromolar biochemical potency have been identified which are selective for the inhibition of HCV NS5b over other polymerases.
Abstract: Novel non-nucleoside inhibitors of the HCV RNA polymerase (NS5b) with sub-micromolar biochemical potency have been identified which are selective for the inhibition of HCV NS5b over other polymerases. The structures of the complexes formed between several of these inhibitors and HCV NS5b were determined by X-ray crystallography, and the inhibitors were found to bind in an allosteric binding site separate from the active site. Structure-activity relationships and structural studies have identified the mechanism of action for compounds in this series, several of which possess drug-like properties, as unique, reversible, covalent inhibitors of HCV NS5b.

126 citations


Journal ArticleDOI
TL;DR: A series of 1-cycloalkyl-2-phenyl-1H-benzimidazole-5-carboxylic acid derivatives was synthesized and evaluated for inhibitory activity against HCV NS5B RNA-dependent RNA polymerase (RdRp) and led to identify the 2-[(4-diarylmethoxy)phenyl]-benzIMidazoles as potent inhibitors.

121 citations


Journal ArticleDOI
TL;DR: A new series of hepatitis C virus NS5B RNA polymerase inhibitors containing a conformationally constrained tetracyclic scaffold and displaying a very small shift in cellular potency when the replicon assay was performed in the presence of human serum albumin.
Abstract: We report a new series of hepatitis C virus NS5B RNA polymerase inhibitors containing a conformationally constrained tetracyclic scaffold. SAR studies led to the identification of 6,7-dihydro-5H-benzo[5,6][1,4]diazepino[7,1-a]indoles (19 and 20) bearing a basic pendent group with high biochemical and cellular potencies. These compounds displayed a very small shift in cellular potency when the replicon assay was performed in the presence of human serum albumin.

112 citations


Journal ArticleDOI
TL;DR: The immunosuppressive drug MPA is as potent as CsA as an inhibitor of HCV replication and was shown to have a distinct anti-HCV mechanism of action, independent of cell proliferation and guanosine depletion.

111 citations


Journal ArticleDOI
TL;DR: Lentiviral delivery of multiple shRNA, inhibiting HCV in a specific, IFN-independent, manner is demonstrated, suggesting the targeting of multiple viral and host cell elements simultaneously by RNAi could increase the potency of antiviral gene therapies.

Journal ArticleDOI
TL;DR: It is demonstrated that a subgenomic RNA replicon of genotype 2a HCV replicated efficiently in mouse embryonic fibroblasts (MEFs), as determined by cell colony formation efficiency and the detection of HCV proteins and both positive- and negative-strand RNAs.
Abstract: Hepatitis C virus (HCV) is a member of the Flaviviridae family, with a single-stranded and positive-sense RNA genome of approximately 9.6 kb in length (12, 51). The viral RNA genome contains a single open reading frame flanked by untranslated regions (UTR) at both the 5′ and 3′ ends. A large viral polyprotein precursor of about 3,010 amino acids is translated and is subsequently cleaved by both cellular and viral proteases into at least 10 different structural (C, E1, E2, and p7) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (42, 50). Replication of HCV RNA occurs in the cytoplasmic membrane-bound multiple-protein complex containing the HCV RNA and nonstructural proteins NS3 to NS5B (15, 42). Owing to the lack of a proofreading activity, the virally encoded RNA-dependent RNA polymerase (RdRp) catalyzes an error-prone RNA replication and thereby results in a mixture of HCV variants (quasispecies) in the infected individual (6). Based on genome sequence similarity, HCV is grouped into six major genotypes and numerous subtypes (55). HCV is a common cause of chronic viral hepatitis, affecting approximately 170 million people worldwide (9, 68). The vast majority (∼85%) of individuals exposed to HCV become chronically infected and carry a higher risk of developing cirrhosis and hepatocellular carcinoma (9, 13). The pegylated alpha interferon (PEG-IFN-α) in combination with ribavirin is currently the optimal therapy to treat HCV infection (13). However, the sustained virologic response (SVR) to IFN-based therapy varied significantly among patients infected with different HCV genotypes (19, 30, 44). In general, HCV genotypes 2 and 3 are sensitive to PEG-IFN-α and ribavirin combination therapy, with an SVR of more than 80%. By contrast, genotypes 1 and 4 are refractory to IFN-based therapy, with an SVR of only about 40 to 50%. The underlying mechanisms of chronic HCV infection, IFN-induced antiviral state against HCV replication, and IFN resistance have not been defined. Many studies have demonstrated that virus infection, replication, and double-stranded RNA (dsRNA) can activate cellular antiviral pathways and induce IFN production (53, 54). IFN stimulates a large number of cellular genes (IFN-stimulated genes [ISGs]) (14, 25), many of which possess direct or indirect antiviral activities, including the well-characterized protein kinase R (PKR), 2′,5′-oligoadenylate synthetase/RNase L, and Mx (53, 54, 67). PKR is implicated in controlling HCV replication and mediating the IFN-induced antiviral state against HCV replication (21, 52). Several independent studies have found that PKR was induced by HCV RNA replication or dsRNA in the HCV-infected chimpanzee liver and in the HCV replicon RNA-harboring Huh7 cells (3, 56, 74). The activated PKR phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α), by which the translation of HCV proteins was inhibited (65). Additionally, other ISGs such as ISG6-16, ISG15, and ISG56 (p56) were also activated by HCV RNA replication and induced by IFN in Huh7 cells in which HCV RNA was persistently replicated (58, 59, 65, 74). P56 binds the e subunit of human eIF3 (eIF3e) and the c subunit of mouse eIF3 (eIF3c), leading to the blockage of protein translation initiation (31, 62). Therefore, the activation of both PKR and p56 can lead to the inhibition of HCV replication by disrupting the translation initiation of HCV proteins (21, 65). On the contrary, findings derived from other independent studies suggest that HCV replication can be suppressed by antiviral pathways independently of PKR (18, 28, 29). However, the roles of PKR-dependent and -independent antiviral pathways in cellular defense against HCV replication are still not well understood. In an effort to better understand the role of PKR in the establishment of persistent HCV RNA replication and the mediation of IFN-induced antiviral action, we developed mouse cell culture systems of HCV RNA replication. The replication of a subgenomic genotype 2a (JFH1) HCV RNA in PKR knockout (PKR−/−) and wild-type (PKR+/+) mouse embryonic fibroblasts (MEFs) was determined by cell colony formation assay and the detection of high levels of HCV proteins and both positive- and negative-stranded RNAs. Findings derived from our studies provide a foundation for the development of transgenic mouse models of HCV replication as well as a powerful in vitro system to study the virus-host interactions in MEFs derived from diverse gene knockout mice. Additionally, the JFH1 RNA was found to replicate more efficiently in the PKR−/− MEFs than in wild-type (PKR+/+) MEFs but failed to induce cell colony formation in MEFs that constitutively overexpressed a human PKR. In PKR+/+ MEFs, the knockdown expression of PKR by specific small interfering RNAs (siRNAs) significantly enhanced the level of HCV RNA replication. Collectively, these findings demonstrate that PKR plays an important role in controlling HCV replication. Furthermore, IFN was able to inhibit HCV RNA replication in the PKR−/− MEF as efficiently as in the PKR+/+ MEF, suggesting that PKR-independent antiviral pathways are evolved for the control of HCV replication and the mediation of IFN-induced antiviral response against HCV infection. Moreover, our studies set a stage to determine the roles of cellular genes in the establishment of a chronic HCV infection and the mediation of IFN-induced intracellular innate antiviral response.

Journal ArticleDOI
TL;DR: The determination of the molecular mechanism by which the S282T mutation confers resistance to 2′-modified nucleotide analogues suggests not only that “2′-conformer” analogues target distinct steps in RNA synthesis but also that these analogues have interesting potential in combination therapies.
Abstract: The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is an important target for antiviral therapies. NS5B is able to initiate viral RNA synthesis de novo and then switch to a fast and processive RNA elongation synthesis mode. The nucleotide analogue 2'-C-methyl CTP (2'-C-Me-CTP) is the active metabolite of NM283, a drug currently in clinical phase II trials. The resistance mutation S282T can be selected in HCV replicon studies. Likewise, 2'-O-Me nucleotides are active both against the purified polymerase and in replicon studies. We have determined the molecular mechanism by which the S282T mutation confers resistance to 2'-modified nucleotide analogues. 2'-C-Me-CTP is no longer incorporated during the initiation step of RNA synthesis and is discriminated 21-fold during RNA elongation by the NS5B S282T mutant. Strikingly, 2'-O-methyl CTP sensitivity does not change during initiation, but the analogue is no longer incorporated during elongation. This mutually exclusive resistance mechanism suggests not only that "2'-conformer" analogues target distinct steps in RNA synthesis but also that these analogues have interesting potential in combination therapies. In addition, the presence of the S282T mutation induces a general cost in terms of polymerase efficiency that may translate to decreased viral fitness: natural nucleotides become 5- to 20-fold less efficiently incorporated into RNA by the NS5B S282T mutant. As in the case for human immunodeficiency virus, our results might provide a mechanistic basis for the rational combination of drugs for low-fitness viruses.

Journal ArticleDOI
TL;DR: The details of NS5B polymerase/inhibitor binding interactions coupled with the observed induced conformational changes provide new insights into the design of novel NNIs of HCV.

Journal ArticleDOI
TL;DR: It is reported that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells.

Journal ArticleDOI
TL;DR: The data suggest that the HCV polyprotein delivered with adjuvants induces broad B- and T-cell responses and could be a vaccine candidate against HCV.
Abstract: Although approximately 3 % of the world's population is infected with Hepatitis C virus (HCV), there is no prophylactic vaccine available. This study reports the design, cloning and purification of a single polyprotein comprising the HCV core protein and non-structural proteins NS3, NS4a, NS4b, NS5a and NS5b. The immunogenicity of this polyprotein, which was formulated in alum, oil-in-water emulsion MF59 or poly(dl-lactide co-glycolide) in the presence or absence of CpG adjuvant, was then determined in a murine model for induction of B- and T-cell responses. The addition of adjuvants or a delivery system to the HCV polyprotein enhanced serum antibody and T-cell proliferative responses, as well as IFN-gamma responses, by CD4+ T cells. The antibody responses were mainly against the NS3 and NS5 components of the polyprotein and relatively poor responses were elicited against NS4 and the core components. IFN-gamma responses, however, were induced against all of the individual components of the polyprotein. These data suggest that the HCV polyprotein delivered with adjuvants induces broad B- and T-cell responses and could be a vaccine candidate against HCV.

Journal ArticleDOI
TL;DR: The results of these studies have confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb domain, independent of GTP concentrations and is likely mediated by an allosteric blockade introduced by the inhibitor during the transition to RNA elongation after the formation of an initiation complex.
Abstract: A new pyranoindole class of small-molecule inhibitors was studied to understand viral resistance and elucidate the mechanism of inhibition in hepatitis C virus (HCV) replication. HCV replicon variants less susceptible to inhibition by the pyranoindoles were selected in Huh-7 hepatoma cells. Variant replicons contained clusters of mutations in the NS5B polymerase gene corresponding to the drug-binding pocket on the surface of the thumb domain identified by X-ray crystallography. An additional cluster of mutations present in part of a unique β-hairpin loop was also identified. The mutations were characterized by using recombinant replicon variants engineered with the corresponding amino acid substitutions. A single mutation (L419M or M423V), located at the pyranoindole-binding site, resulted in an 8- to 10-fold more resistant replicon, while a combination mutant (T19P, M71V, A338V, M423V, A442T) showed a 17-fold increase in drug resistance. The results of a competition experiment with purified NS5B enzyme with GTP showed that the inhibitory activity of the pyranoindole inhibitor was not affected by GTP at concentrations up to 250 μM. Following de novo initiation, the presence of a pyranoindole inhibitor resulted in the accumulation of a five-nucleotide oligomer, with a concomitant decrease in higher-molecular-weight products. The results of these studies have confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb domain. This inhibition is independent of GTP concentrations and is likely mediated by an allosteric blockade introduced by the inhibitor during the transition to RNA elongation after the formation of an initiation complex.

Journal ArticleDOI
TL;DR: This study is the first description of an inhibitor specific to the HCV subtype 3a polymerase, which demonstrated some inhibitory effect on norovirus and φ6 polymerase activity.
Abstract: Research into antiviral agents directed at hepatitis C virus (HCV) proteins is commonly based and tested on a single genotype, namely, genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than genotype 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach used in this study that allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNA-dependent RNA polymerase (RdRp) (nonstructural protein 5B [NS5B]) of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 as the partitioning system. Two aptamers, r10/43 and r10/47, were chosen for further studies on the basis of their abilities to bind the HCV RdRp and inhibit polymerase activity. The affinities (equilibrium dissociation constants) of these aptamers for the HCV subtype 3a polymerase were estimated to be 1.3 +/- 0.3 nM (r10/43) and 23.5 +/- 6.7 nM (r10/47). The inhibition constants of r10/43 and r10/47 were estimated to be 1.4 +/- 2.4 nM and 6.0 +/- 2.3 nM, respectively. Inhibition of HCV 3a polymerase was specific for r10/47, while r10/43 also demonstrated some inhibitory effect on norovirus and phi6 polymerase activity. Neither r10/43 nor r10/47 was able to inhibit the RdRp activity of HCV genotype 1a and 1b polymerases. This study is the first description of an inhibitor specific to the HCV subtype 3a polymerase.

Journal ArticleDOI
TL;DR: Through high throughput screening, substituted proline sulfonamide 6 was identified as HCV NS5b RNA-dependent RNA polymerase inhibitor and optimization of various regions of the lead molecule resulted in compounds that displayed good potency and selectivity.
Abstract: Through high throughput screening, substituted proline sulfonamide 6 was identified as HCV NS5b RNA-dependent RNA polymerase inhibitor. Optimization of various regions of the lead molecule resulted in compounds that displayed good potency and selectivity. The crystal structure of 6 and NS5b polymerase complex confirmed the binding near the active site region. The optimization approach and SAR are discussed in detail.

Journal ArticleDOI
15 Mar 2006-Virology
TL;DR: It is found that HCV NS5B, an RNA-dependent RNA polymerase, delayed cell cycle progression through the S phase in PH5CH8 immortalized human hepatocyte cells, and induced IFN-beta through the TLR3 signaling pathway in immortalizedhuman hepatocytes even without replicating viral genomes.

Journal ArticleDOI
Uwe Koch1, Frank Narjes
TL;DR: A review of the recent developments in the development of small molecule inhibitors of NS5B RdRp, based on the success of allosteric inhibitors in the treatment of HIV infection, have been developed into compounds which show activity in the subgenomic cell-culture assay of HCV replication.
Abstract: The human and monetary costs of chronic hepatitis C and the complications arising from this disease emphasize the urgency to find a treatment for Hepatitis C Virus (HCV) infected patients. The current standard of treatment for patients chronically infected with HCV is combination therapy with pegylated interferon plus ribavirin. Recently, viral enzymes have become the target of efforts to develop small molecule inhibitors interfering with the essential steps in the life cycle of the virus. Amongst these enzymes the HCV-encoded NS5B RNA-dependent RNA polymerase (NS5B RdRp) is essential for viral replication and has been recognized as a prime target for therapeutic intervention. Several distinct classes of inhibitors of NS5B RdRp have been disclosed in the literature, including active site inhibitors such as nucleosides and pyrophosphate mimetics, as well as non-nucleoside inhibitors. The latter, based on the success of allosteric inhibitors in the treatment of HIV infection, have been developed into compounds which show activity in the subgenomic cell-culture assay of HCV replication. This review provides an account of the recent developments in this field.

Journal ArticleDOI
TL;DR: The plasmid pET-21d-2c-5BDelta55 effectively expressing a C-terminally truncated form of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was constructed and a 29-fold enhancement of the protein yield was accompanied by only a 2.5-fold increase of the corresponding mRNA level.

Journal ArticleDOI
TL;DR: In vivo immunoprecipitation showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication.
Abstract: We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

Journal ArticleDOI
TL;DR: It is demonstrated that TNF-α-induced NF-κB activation is inhibited by NS5B protein in HEK293 and hepatic cells, and it is found that NS5 B protein synergistically activated TNF -α-mediated JNK activity in HEk293 and hepatatic cells.
Abstract: Hepatitis C virus (HCV) NS5B protein is a membrane-associated phosphoprotein that possesses an RNA-dependent RNA polymerase activity. We recently reported that NS5A protein interacts with TRAF2 and modulates tumor necrosis factor alpha (TNF-α)-induced NF-κB and Jun N-terminal protein kinase (JNK). Since NS5A and NS5B are the essential components of the HCV replication complex, we examined whether NS5B could modulate TNF-α-induced NF-κB and JNK activation. In this study, we have demonstrated that TNF-α-induced NF-κB activation is inhibited by NS5B protein in HEK293 and hepatic cells. Furthermore, NS5B protein inhibited both TRAF2- and IKK-induced NF-κB activation. Using coimmunoprecipitation assays, we show that NS5B interacts with IKKα. Most importantly, NS5B protein in HCV subgenomic replicon cells interacted with endogenous IKKα, and then TNF-α-mediated IKKα kinase activation was significantly decreased by NS5B. Using in vitro kinase assay, we have further found that NS5B protein synergistically activated TNF-α-mediated JNK activity in HEK293 and hepatic cells. These data suggest that NS5B protein modulates TNF-α signaling pathways and may contribute to HCV pathogenesis.

Journal ArticleDOI
TL;DR: From random screening of the compound libraries, a hit compound with an IC50 of 27 microM against hepatitis C viral NS5B RNA-dependent RNA polymerase was identified and a series of its derivatives were synthesized.

Journal ArticleDOI
TL;DR: Isothiazole analogs were discovered as a novel class of active-site inhibitors of HCV NS5B polymerase and it is revealed that the inhibitor is covalently linked with Cys 366 of the 'primer-grip'.

Journal ArticleDOI
TL;DR: Thieno[3,2,b]pyrroles are a novel class of allosteric inhibitors of HCV NS5B RNA-dependent RNA polymerase which show potent affinity for the NS5b enzyme as discussed by the authors.

Journal ArticleDOI
TL;DR: A novel class of HCV NS5B RNA dependent RNA polymerase inhibitors containing 3,4-dihydro-1H-[1]-benzothieno[2,3-c]pyran and 3,8-disubstitution pattern is preferred for the aromatic region.

Journal ArticleDOI
Gary Lee1, Derek E. Piper1, Zhulun Wang1, John Anzola1, Jay P. Powers1, Nigel Walker1, Yang Li1 
TL;DR: Interestingly, these inhibitors appear to form a reversible covalent bond with the NS5B cysteine 366, a residue that is not only conserved among all HCV genotypes and a large family of viruses but also required for full NS 5B RdRp activity.

Patent
06 Sep 2006
TL;DR: In this article, the authors describe compounds and salts of Formulas I, II, III, and IV as well as compositions and methods of using the compounds for hepatitis C virus (HCV).
Abstract: The invention encompasses compounds and salts of Formulas I, II, III, and IV as well as compositions and methods of using the compounds. The compounds have activity against hepatitis C virus (HCV) and are useful in treating those infected with HCV.