Inhibit Hepatitis C Virus Replication Research Articles

Proprotein convertase subtilisin/kexin type 9 inhibits hepatitis C virus replication through interacting with NS5A

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease actively involved in regulating lipid homeostasis. Although PCSK9 has been shown to inhibit hepatitis C virus (HCV) entry and replication, the underlying mechanisms have not been thoroughly characterized. Moreover, whether PCSK9 regulates HCV translation and assembly/secretion has not been determined. We therefore further studied the effects of PCSK9 on the HCV life cycle. We showed that PCSK9 did not affect HCV translation or assembly/secretion. Overexpression of PCSK9 inhibited HCV replication in HCV genomic replicon cells in a dose-dependent manner and after cell culture-derived HCV (HCVcc) infection. Knocking down PCSK9 increased HCV replication. The gain-of-function (D374Y) or loss-of-function (Δaa. 31-52) PCSK9 mutants for low-density lipoprotein receptor (LDLR) degradation had no effect on HCV replication, suggesting that HCV replication inhibition by PCSK9 was not due to LDLR degradation. The uncleaved ProPCSK9, but not cleaved PCSK9, down-regulated HCV replication, suggesting that the auto-cleavage of PCSK9 affected HCV replication. We also found that PCSK9 interacted with NS5A through NS5A aa. 95-215, and this region played an important role in NS5A dimerization, NS5A-RNA binding and was essential for HCV replication. More importantly, NS5A dimerization and NS5A-RNA binding were suppressed by PCSK9 upon interaction. These results suggested that PCSK9 inhibited HCV replication through interaction with NS5A. Our study should help optimize anti-HCV treatment regimen in patients with abnormal lipid profiles.

Discovery and evolution of aloperine derivatives as a new family of HCV inhibitors with novel mechanism

Aloperine (1), a Chinese natural product with a unique endocyclic scaffold, was first identified to be a potent hepatitis C virus (HCV) inhibitor in our laboratory. Thirty-four new aloperine derivatives were designed, synthesized and evaluated for their anti-HCV activities taking 1 as the lead. Among them, compound 7f exhibited the potential potency with EC50 values in a micromolar range against both wild-type and direct-acting antiviral agents (DAAs)-resistant variants, and synergistically inhibited HCV replication with approved DAAs. Furthermore, it also owned a good oral pharmacokinetic and safety profile, suggesting a highly druglike nature. The primary mechanism showed that 7f might target host components, distinctly different from the DAAs currently used in clinic. Therefore, we consider aloperine derivatives to be a novel class of anti-HCV agents, and compound 7f has been selected as a promising antiviral candidate for further investigation.

Intrahepatic Vγ9Vδ2 T-cells from HCV-infected patients show an exhausted phenotype but can inhibit HCV replication

Hepatitis C virus (HCV) persistence results from inefficiencies of both innate and adaptive immune responses to eradicate the infection. A functional impairment of circulating Vγ9Vδ2 T-cells was described but few data are available on Vγ9Vδ2 T-cells in the liver that, however, represents the battlefield in the HCV/host interaction.Aim of this work was to compare circulating and intrahepatic Vγ9Vδ2 T-cells in chronic HCV-infected patients (HCVpos) and in HCV-negative (HCVneg) subjects. Phenotypic and functional analysis was performed by flow cytometry. Anti-HCV activity was analyzed by using an in vitro autologous liver culture system.Independently from HCV infection, the liver was enriched of Vγ9Vδ2 T-cells expressing an effector/activated phenotype. In contrast, an enrichment of PD-1 expressing Vγ9Vδ2 T-cells was observed both in the peripheral blood and in the liver of HCVpos patients, probably due to a persistent antigenic stimulation. Moreover, a lower frequency of IFN-γ producing Vγ9Vδ2 T-cells was observed in the liver of HCVpos patients, suggesting a functional impairment in the cytokine production in HCVpos liver. Despite this hypo-responsiveness, intrahepatic Vγ9Vδ2 T-cells are able to exert an anti-HCV activity after specific stimulation.Altogether, our data show that HCV infection induced a dysregulation of intrahepatic Vγ9Vδ2 T cells that maintain their anti-HCV activity after specific stimulation. A study aimed to evaluate the mechanisms of the antiviral activity may be useful to identify new pathways able to improve Vγ9Vδ2 T-cells intrahepatic function during HCV infection.

P.8.b.002 - Prevalence and clinical correlates of intermittent explosive disorder in psychiatric outpatients

Aloperine (1), a Chinese natural product with a unique endocyclic scaffold, was first identified to be a potent hepatitis C virus (HCV) inhibitor in our laboratory. Thirty-four new aloperine derivatives were designed, synthesized and evaluated for their anti-HCV activities taking 1 as the lead. Among them, compound 7f exhibited the potential potency with EC50 values in a micromolar range against both wild-type and direct-acting antiviral agents (DAAs)-resistant variants, and synergistically inhibited HCV replication with approved DAAs. Furthermore, it also owned a good oral pharmacokinetic and safety profile, suggesting a highly druglike nature. The primary mechanism showed that 7f might target host components, distinctly different from the DAAs currently used in clinic. Therefore, we consider aloperine derivatives to be a novel class of anti-HCV agents, and compound 7f has been selected as a promising antiviral candidate for further investigation.

MxA is a positive regulator of type I IFN signaling in HCV infection.

Type I interferons (IFNs) are a family of primordial cytokines that respond to various pathogen infections including Hepatitis C virus (HCV). Type I IFNs signal through Jak/STAT pathway leading to the production of a few hundred interferon stimulated genes (ISGs). The aim of this study was to explore the role of one of these ISGs, MxA in HCV infection and type I IFN production. Plasmid encoding MxA was cloned into PcDNA3.1-3×tag vector and MxA expression was confirmed both at mRNA (RT-PCR) and protein (Western blot, WB) levels. IFNα and IFNβ productions were quantified by RT-PCR from cell lysate and by ELISA kit from culture medium following MxA over-expression in Huh7.5.1 cells. The activation status of Jak/STAT signaling pathway was examined at three levels: p-STAT1 (WB), interferon sensitive response element (ISRE) activity (dual luciferase reporter gene assay), and levels of ISG expression (RT-qPCR). J6/JFH1 HCV culture system was used to study the role of MxA in HCV replication. Our findings indicated that MxA over-expression inhibited HCV replication and potentiated the IFNα-mediated anti-HCV activity; MxA stimulated the production of IFNα, IFNβ, and enhanced IFNα-induced activation of Jak-STAT signaling pathway. We concluded that MxA is a positive regulator of type I IFN signaling in HCV infection.

Celastrol inhibits hepatitis C virus replication by upregulating heme oxygenase-1 via the JNK MAPK/Nrf2 pathway in human hepatoma cells

background and purposeCelastrol, a quinone methide triterpene isolated from the root extracts of Tripterygium wilfordii, can greatly induce the gene expression activity of heme oxygenase-1 (HO-1) to achieve disease prevention and control. HO-1 induction was recently shown to result in anti-HCV activity by inducing type I interferon and inhibiting hepatitis C virus (HCV) NS3/4A protease activity. The aim of the present study is to evaluate the anti-HCV activity of celastrol and characterize its mechanism of inhibition. MethodsThe anti-HCV activity of celastrol was evaluated using the HCV subgenomic replicon and HCVcc infection systems. The anti-HCV mechanism of celastrol targeting HO-1 expression was clarified using specific inhibitors against several signaling pathways. The transcriptional regulation of celastrol on target gene expression was determined using promoter-based reporter activity assay. The synergistic effect of celastrol and a numbers of clinically used anti-HCV drugs was determined via a drug combination assay. ResultsCelastrol inhibited HCV replication in both the HCV subgenomic and HCVcc infection systems with EC50 values of 0.37 ± 0.022 and 0.43 ± 0.019 μM, respectively. Celastrol-induced heme oxygenase 1 (HO-1) expression promoted antiviral interferon responses and inhibition of NS3/4A protease activity, thereby blocking HCV replication. These antiviral effects were abrogated by treatment with the HO-1-specific inhibitor SnMP or silencing of HO-1 expression by transfection of shRNA, which indicates that HO-1 induction contributes to the anti-HCV activity of celastrol. JNK mitogen-activated protein kinase and nuclear factor erythroid 2-related factor 2 (Nrf2) were confirmed to be involved in the inductive effect of celastrol on HO-1 expression. Celastrol exhibited synergistic effects in combination with interferon-alpha, the NS5A inhibitor daclatasvir, and the NS5B inhibitor sofosbuvir. ConclusionCelastrol can serve as a potential supplement for blocking HCV replication. Targeting the JNK/Nrf2/HO-1 axis presents a promising strategy against HCV infection.

Characterization and structure-activity relationship study of iminodipyridinopyrimidines as novel hepatitis C virus inhibitor

Upon high-throughput screening of synthetic small molecule libraries with the infectious hepatitis C virus (HCV) cell culture system, we identified an iminodipyridinopyrimidine (IDPP) scaffold. IDPP did not inhibit HCV replication, but exhibited very potent inhibitory activity on early and late steps of HCV life cycle. Applying an intensive structure-activity relationship (SAR) study, a promising IDPP Lead compound (12c) with excellent potency (EC50 = 10 nM), high safety margin (SI > 2000), and an acceptable stability in human and rat liver microsomes (t1/2 >60 min) was identified. Overall, our results suggest that the IDPP scaffold could be used for the development of novel HCV interventions.

Searching for synergy: Identifying optimal antiviral combination therapy using Hepatitis C virus (HCV) agents in a replicon system

Direct acting antiviral agents (DAAs) are potent inhibitors of Hepatitis C virus (HCV) that have revolutionized the treatment landscape for this important viral disease. There are currently four classes of DAAs that inhibit HCV replication via distinct mechanisms of action: nonstructural protein (NS) 3/4a protease inhibitors, NS5A inhibitors, NS5B nucleoside polymerase inhibitors, and NS5B non-nucleoside polymerase inhibitors. Combination therapy with two or more DAAs has great potential to further enhance antiviral potency. The purpose of this study was to identify optimal combinations of DAAs against genotype 1 HCV replicons that maximized the inhibition of replicon replication. All possible two-drug combinations were evaluated against genotype 1a and 1b HCV replicons using a 96-well plate luciferase-based assay in triplicate. The Greco Universal Response Surface Area mathematical model was fit to the luciferase data to identify drug-drug interactions (i.e.: synergy, additivity, and antagonism) for antiviral effect against both genotypes. This information was used to rank-order combinations of DAAs based on their ability to inhibit replicon replication against genotype 1a and 1b HCV. These preclinical findings can provide information as to which antiviral regimens should move on in the development process.

Hepatitis C virus down-regulates SERPINE1/PAI-1 expression to facilitate its replication.

Identification of host factors involved in viral replication is critical for understanding the molecular mechanism of viral replication and pathogenesis. Genes differentially expressed in HuH-7 cells with or without a hepatitis C virus (HCV) sub-genomic replicon were screened by microarray analysis. SERPINE1/PAI-1 was found to be down-regulated after HCV infection in this analysis. Down-regulation of SERPINE1/PAI-1 expression at the transcriptional level was verified by the real-time reverse transcriptase (RT)-PCR assay. Reduced SERPINE1/PAI-1 protein secretion was detected in the supernatant of HCV replicon cells and in sera from HCV-infected patients. SERPINE1 gene expression was down-regulated by HCV NS3/4A and NS5A proteins through the transforming growth factor-β (TGF-β) signalling pathway at the transcriptional level. Down-regulated genes in HCV replicon cells could be the factors supressing HCV replication. Indeed, over-expressed PAI-1 inhibited HCV replication but the mechanism is unknown. It has been demonstrated that HCV induces the expression of TGF-β, and TGF-β enhances HCV replication by a not-yet-defined mechanism. SERPINE1/PAI-1 is also known to be potently induced by TGF-β at the transcriptional level through both Smad-dependent and Smad-independent pathways. The exogenously expressed SERPINE1/PAI-1 suppressed the expression of the endogenous SERPINE1 gene at the transcriptional level through the TGF-β signalling but not the Smad pathway. Thus, SERPINE1/PAI-1 could suppress HCV replication possibly by negatively regulating TGF-β signalling. A model is proposed for the interplay betweenthe TGF-β signalling pathway, HCV and SERPINE1/PAI-1 to keep the homeostasis of the cells.

Tylophorine Analogs Allosterically Regulates Heat Shock Cognate Protein 70 And Inhibits Hepatitis C Virus Replication

Tylophorine analogs have been shown to exhibit diverse activities against cancer, inflammation, arthritis, and lupus in vivo. In this study, we demonstrated that two tylophorine analogs, DCB-3503 and rac-cryptopleurine, exhibit potent inhibitory activity against hepatitis C virus (HCV) replication in genotype 1b Con 1 isolate. The inhibition of HCV replication is at least partially mediated through cellular heat shock cognate protein 70 (Hsc70). Hsc70 associates with the HCV replication complex by primarily binding to the poly U/UC motifs in HCV RNA. The interaction of DCB-3503 and rac-cryptopleurine with Hsc70 promotes the ATP hydrolysis activity of Hsc70 in the presence of the 3′ poly U/UC motif of HCV RNA. Regulating the ATPase activity of Hsc70 may be one of the mechanisms by which tylophorine analogs inhibit HCV replication. This study demonstrates the novel anti-HCV activity of tylophorine analogs. Our results also highlight the importance of Hsc70 in HCV replication.

Metformin activates type I interferon signaling against HCV via activation of adenosine monophosphate-activated protein kinase.

Activation of the type I interferon (IFN) signaling pathway is essential for the eradication of hepatitis C virus (HCV). Metformin can activate adenosine monophosphate-activated protein kinase (AMPK) to reduce insulin resistance. Cross talks between AMPK and IFN signaling remain unclear. To understand the influence of metformin on the type I IFN signaling pathway and HCV infection, the full-length HCV replicon OR6 cells and the infectious HCV clones JFH1 were used to assess the anti-HCV effect of the insulin sensitizers, metformin and pioglitazone. Immunofluorescence staining and the immunoblotting of HCV viral protein demonstrated that metformin, but not pioglitazone, inhibited HCV replication in OR-6 and JFH-1-infected Huh 7.5.1 cells. Immunoblotting data showed that metformin activated the phosphorylation of STAT-1 and STAT-2 in OR-6 and JFH-1 infected Huh 7.5.1 cells. Metformin enhanced the phosphorylation of AMPK, and the metformin-activated IFN signaling was down-regulated by AMPK inhibitor. After treatment of AMPK inhibitor, the level of HCV core protein decreased by metformin can be rescued. In conclusion, metformin activates type I interferon signaling and inhibits the replication of HCV via activation of AMPK.

Treatment with PTEN-Long protein inhibits hepatitis C virus replication

Hepatitis C virus (HCV) infection is a confirmed risk factor for hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) possesses tumor suppression function that is frequently defective in HCC tumors. PTEN-Long, a translation isoform of PTEN, functions in a cell non-autonomous manner. In this study, we demonstrated that intracellular overexpression of PTEN-Long inhibits HCV replication. More importantly, we showed that treatment with extracellular PTEN-Long protein inhibits HCV replication in a dose-dependent manner. Furthermore, we showed that PTEN-Long interacts with HCV core protein and this interaction is required for HCV replication inhibition by PTEN-Long. In summary, we demonstrated, for the first time, that PTEN-Long protein, an isoform of the canonical PTEN and in the form of extracellular protein treatment, inhibits HCV replication. Our study offers an opportunity for developing additional anti-HCV agents.

Cinnamic acid derivatives inhibit hepatitis C virus replication via the induction of oxidative stress

Several cinnamic acid derivatives have been reported to exhibit antiviral activity. In this study, we prepared 17 synthetic cinnamic acid derivatives and screened them to identify an effective antiviral compound against hepatitis C virus (HCV). Compound 6, one of two hit compounds, suppressed the viral replications of genotypes 1b, 2a, 3a, and 4a with EC50 values of 1.5–8.1 μM and SI values of 16.2–94.2. The effect of compound 6 on the phosphorylation of Tyr705 in signal transducer and activator of transcription 3 (STAT3) was investigated because a cinnamic acid derivative AG490 was reported to suppress HCV replication and the activity of Janus kinase (JAK) 2. Compound 6 potently suppressed HCV replication, but it did not inhibit the JAK1/2-dependent phosphorylation of STAT3 Tyr705 at the same concentration. Furthermore, a pan-JAK inhibitor tofacitinib potently impaired phosphorylation of STAT3 Tyr 705, but it did not inhibit HCV replication in the replicon cells and HCV-infected cells at the same concentration, supporting the notion that the phosphorylated state of STAT3 Tyr705 is not necessarily correlated with HCV replication. The production of reactive oxygen species (ROS) was induced by treatment with compound 6, whereas N-acetyl-cysteine restored HCV replication and impaired ROS production in the replicon cells treated with compound 6. These data suggest that compound 6 inhibits HCV replication via the induction of oxidative stress.

Emergence of drug resistance-associated variants and changes in serum lipid profiles in sofosbuvir plus ledipasvir-treated chronic hepatitis C patients

Combination of sofosbuvir plus ledipasvir therapy has been expected to enhance sustained virological response (SVR) rates in hepatitis C virus (HCV) genotype 1 chronic infected patients. We analyzed the emergence of drug resistance-associated variants (RAVs) in treatment failure and changes in lipid profiles in sofosbuvir/ledipasvir-treated patients. A total of 176 patients with chronic HCV genotype 1 infection without decompensated liver cirrhosis were treated with sofosbuvir/ledipasvir for 12 weeks. NS5A and NS5B RAVs were determined by either Invader assay or direct sequencing. Serum lipid-related markers were measured at the start of treatment and at week 4 in patients who received sofosbuvir/ledipasvir and ombitasvir/paritaprevir/ritonavir therapies. SVR was achieved in 94.9% (167 out of 176) of patients. SVR12 rate was 97.1% for patietns with low frequncy (<25%) of baseline NS5A RAVs, but 82.8% for patients with high frequency (>75%) of NS5A RAVs. In multivariate regression analysis, higher albumin (odds ratio [OR] = 0.020 for presence; P = 0.007), and NS5A-L31/Y93 RAVs with a population frequency <75% (OR = 29.860 for presence; P = 0.023) were identified as significant independent predictors for SVR12. NS5A-Y93H substitutions were detected in all nine treatment failures at HCV relapse, and three out of six patients with NS5A inhibitor-naïve patients achieved additional NS5A RAVs. Serum low-density lipoprotein cholesterol and apolipoprotein B levels were significantly elevated at week 4 in sofosbuvir/ledipasvir-treated patients. These elevations were greater than in ombitasvir/paritaprevir/ritonavir-treated patients. In conclusion, NS5A multi-RAVs are likely to develop in patients who fail to respond to sofosbuvir/ledipasvir therapy. Inhibition of HCV replication with sofosbuvir might affect lipid metabolism.

Hepatitis C virus in vitro replication is efficiently inhibited by acridone Fac4.

Hepatitis C virus (HCV) affects about 170 million people worldwide. The current treatment has a high cost and variable response rates according to the virus genotype. Acridones, a group of compounds extracted from natural sources, showed potential antiviral actions against HCV. Thus, this study aimed to evaluate the effect of a panel of 14 synthetic acridones on the HCV life cycle. The compounds were screened using an Huh7.5 cell line stably harbouring the HCV genotype 2a subgenomic replicon SGR-Feo-JFH-1. Cells were incubated in the presence or absence of compounds for 72 h and cell viability and replication levels were assessed by MTT and luciferase assays, respectively. At a concentration of 5 µM the acridone Fac4 exhibited a >90 % inhibition of HCV replication with no effect on cell viability. The effects of Fac4 on virus replication, entry and release steps were evaluated in Huh7.5 cells infected with the JFH-1 isolate of HCV (HCVcc). Fac4 inhibited JFH-1 replication to approximately 70 %, while no effect was observed on virus entry. The antiviral activity of Fac4 was also observed on viral release, with almost 80 % of inhibition. No inhibitory effect was observed against genotype 3 replication. Fac4 was able to intercalate into dsRNA, however did not inhibit NS5B polymerase activity or translation driven by the HCV IRES. Although its mode of action is partly understood, Fac4 presents significant inhibition of HCV replication and can therefore be considered as a candidate for the development of a future anti-HCV treatment.

The role of PTEN - HCV core interaction in hepatitis C virus replication

Hepatitis C virus (HCV) infection leads to severe liver diseases including hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumour suppressor, is frequently mutated or deleted in HCC tumors. PTEN has previously been demonstrated to inhibit HCV secretion. In this study, we determined the effects of PTEN on the other steps in HCV life cycle, including entry, translation, and replication. We showed that PTEN inhibits HCV entry through its lipid phosphatase activity. PTEN has no effect on HCV RNA translation. PTEN decreases HCV replication and the protein phosphatase activity of PTEN is essential for this function. PTEN interacts with the HCV core protein and requires R50 in domain I of HCV core and PTEN residues 1–185 for this interaction. This interaction is required for PTEN-mediated inhibition of HCV replication. This gives rise to a reduction in PTEN levels and intracellular lipid abundance, which may in turn regulate HCV replication. HCV core domain I protein increases the lipid phosphatase activity of PTEN in an in vitro assay, suggesting that HCV infection can also regulate PTEN. Taken together, our results demonstrated an important regulatory role of PTEN in the HCV life cycle.

Establishment and Application of a High Throughput Screening System Targeting the Interaction between HCV Internal Ribosome Entry Site and Human Eukaryotic Translation Initiation Factor 3.

Viruses are intracellular obligate parasites and the host cellular machinery is usually recruited for their replication. Human eukaryotic translation initiation factor 3 (eIF3) could be directly recruited by the hepatitis C virus (HCV) internal ribosome entry site (IRES) to promote the translation of viral proteins. In this study, we establish a fluorescence polarization (FP) based high throughput screening (HTS) system targeting the interaction between HCV IRES and eIF3. By screening a total of 894 compounds with this HTS system, two compounds (Mucl39526 and NP39) are found to disturb the interaction between HCV IRES and eIF3. And these two compounds are further demonstrated to inhibit the HCV IRES-dependent translation in vitro. Thus, this HTS system is functional to screen the potential HCV replication inhibitors targeting human eIF3, which is helpful to overcome the problem of viral resistance. Surprisingly, one compound HP-3, a kind of oxytocin antagonist, is discovered to significantly enhance the interaction between HCV IRES and eIF3 by this HTS system. HP-3 is demonstrated to directly interact with HCV IRES and promote the HCV IRES-dependent translation both in vitro and in vivo, which strongly suggests that HP-3 has potentials to promote HCV replication. Therefore, this HTS system is also useful to screen the potential HCV replication enhancers, which is meaningful for understanding the viral replication and screening novel antiviral drugs. To our knowledge, this is the first HTS system targeting the interaction between eIF3 and HCV IRES, which could be applied to screen both potential HCV replication inhibitors and enhancers.

In Vitro Antiviral Activity and Resistance Profile of the Next-Generation Hepatitis C Virus NS5A Inhibitor Pibrentasvir.

ABSTRACTPibrentasvir (ABT-530) is a novel and pan-genotypic hepatitis C virus (HCV) NS5A inhibitor with 50% effective concentration (EC50) values ranging from 1.4 to 5.0 pM against HCV replicons containing NS5A from genotypes 1 to 6. Pibrentasvir demonstrated similar activity against a panel of chimeric replicons containing HCV NS5A of genotypes 1 to 6 from clinical samples. Resistance selection studies were conducted using HCV replicon cells with NS5A from genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a, or 6a at a concentration of pibrentasvir that was 10- or 100-fold over its EC50 for the respective replicon. With pibrentasvir at 10-fold over the respective EC50, only a small number of colonies (0.00015 to 0.0065% of input cells) with resistance-associated amino acid substitutions were selected in replicons containing genotype 1a, 2a, or 3a NS5A, and no viable colonies were selected in replicons containing NS5A from other genotypes. With pibrentasvir at 100-fold over the respective EC50, very few colonies (0.0002% of input cells) were selected by pibrentasvir in genotype 1a replicon cells while no colonies were selected in other replicons. Pibrentasvir is active against common resistance-conferring substitutions in HCV genotypes 1 to 6 that were identified for other NS5A inhibitors, including those at key amino acid positions 28, 30, 31, or 93. The combination of pibrentasvir with HCV inhibitors of other classes produced synergistic inhibition of HCV replication. In summary, pibrentasvir is a next-generation HCV NS5A inhibitor with potent and pan-genotypic activity, and it maintains activity against common amino acid substitutions of HCV genotypes 1 to 6 that are known to confer resistance to currently approved NS5A inhibitors.

Synthesis and evaluation of novel HCV replication inhibitors.

Direct acting antiviral agents to cure hepatitis C virus (HCV) infection has emerged as the gold standard therapy. Along with protease inhibitors, nucleoside polymerase inhibitors and non-nucleoside polymerase inhibitors, the inhibition of NS5a has proved to be an effective way to treat HCV patients. Here we report on novel HCV NS5a inhibitors which were synthesized and evaluated in the HCV replicon assay. A series of inhibitors were formed by a cycloaddition reaction in parallel to establish new leads and explore the effects of unsymmetrical cap substitution. This led to the identification of several triazoles with picomolar potency in vitro against hepatitis C virus.

Interferon alpha treatment stimulates interferon gamma expression in type I NKT cells and enhances their antiviral effect against hepatitis C virus.

Interferon (IFN) inhibits hepatitis C virus (HCV) replication through up-regulation of intrahepatic IFN-stimulated gene expression but also through activation of host immune cells. In the present study, we analyzed the immune cell-mediated antiviral effects of IFN-α using HCV-infected mice. Urokinase-type plasminogen activator (uPA)-severe combined immunodeficiency (SCID) mice with transplanted human hepatocytes were infected with genotype 1b HCV and injected with human peripheral blood mononuclear cells (PBMCs). IFN-α treatment following human PBMC transplantation resulted in a significant reduction in serum HCV RNA titers and a higher human CD45-positive mononuclear cell chimerism compared to mice without human PBMC transplantation. In mice with human PBMCs treated with IFN-α, serum concentrations of IFN-γ increased, and natural killer T (NKT) cells, especially type I NKT cells, produced IFN-γ. Mice in which IFN-γ signaling was blocked using antibody or in which transplanted PBMCs were depleted for type I NKT cells showed similar levels of anti-HCV effect compared with mice treated only with IFN-α. These results show that IFN-α stimulates IFN-γ expression in type 1 NKT cells and enhances the inhibition of HCV replication. We propose that type 1 NKT cells might represent a new therapeutic target for chronic hepatitis C patients.