Inhibit HCV Replication Research Articles

Open-Label Phase 1B Pilot Study to Assess the Antiviral Efficacy of Simvastatin Combined with Sertraline in Chronic Hepatitis C Patients

3-Hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors inhibit HCV replication in vitro. The combination of sertraline and simvastatin has synergistic antiviral activity in vitro, but there are no prior in vivo studies. Our aims were to prospectively assess the antiviral efficacy and safety of this drug combination in chronic hepatitis C (CHC) patients. A total of 15 CHC adults (including 1 control subject) that were treatment-naive or prior partial responders/relapsers to standard-of-care therapy were enrolled at four centres (2 in Singapore and 2 in the US). Patients received simvastatin 40 mg once daily and sertraline 50 mg once daily for 7 days, and then 80 mg once daily and 100 mg once daily, respectively, for another 21 days with a 14-day follow-up. Of the 15 CHC patients, 13 completed the study. Subjects were mostly Caucasian (8/15), mean age 49.1 ±9 years and the genotype distribution was 1=10, 2=2 and 3=3. No subject discontinued dosing due to adverse events. Mean HCV RNA change from baseline was from -0.005 to -0.236 log(10) IU/ml across study intervals. Three subjects had transient >1 log(10) HCV RNA declines. No subject achieved >2 log(10) HCV RNA decline. The combination of sertraline and simvastatin is well-tolerated over the short-term, but has no significant antiviral or anti-inflammatory response in CHC patients. This may reflect in vivo differences in synergy between statin and/or selective serotonin reuptake inhibitors and incomplete inhibition of membrane protein prenylation with statin therapy.

Resistance Analysis and Characterization of a Thiazole Analogue, BP008, as a Potent Hepatitis C Virus NS5A Inhibitor

Hepatitis C virus (HCV) is a global health problem, affecting approximately 3% of the world's population. The standard treatment for HCV infection is often poorly tolerated and ineffective. Therefore, the development of novel or more effective treatment strategies to treat chronic HCV infection is urgently needed. In this report, BP008, a potent small-molecule inhibitor of HCV replication, was developed from a class of compounds with thiazol core structures by means of utilizing a cell-based HCV replicon system. The compound reduced the reporter expression of the HCV1b replicon with a 50% effective concentration (EC(50)) and selective index value of 4.1 ± 0.7 nM and >12,195, respectively. Sequencing analyses of several individual clones derived from BP008-resistant RNAs purified from cells harboring HCV1b replicon revealed that amino acid substitutions mainly within the N-terminal region (domain I) of NS5A were associated with decreased inhibitor susceptibility. Q24L, P58S, and Y93H are the key substitutions for resistance selection; F149L and V153M play the compensatory role in the replication and drug resistance processes. Moreover, BP008 displayed synergistic effects with alpha interferon (IFN-α), NS3 protease inhibitor, and NS5B polymerase inhibitor, as well as good oral bioavailability in SD rats and favorable exposure in rat liver. In summary, our results pointed to an effective small-molecule inhibitor, BP008, that potentially targets HCV NS5A. BP008 can be considered a part of a more effective therapeutic strategy for HCV in the future.

Nucleotide Prodrugs for HCV Therapy

HCV infection is a significant worldwide health problem and is a major cause of hepatocellular carcinoma. The current standard of care, interferon and ribavirin, is only effective against a proportion of the patient population infected with HCV. To address the shortcomings of existing therapy, the development of direct acting antiviral agents is under investigation. The HCV RNA dependent RNA polymerase is an essential enzyme for viral replication and is therefore a logical target against which to develop novel anti-HCV agents. Nucleosides have been shown to be effective as antiviral agents for other viral diseases and therefore, have been investigated as inhibitors of HCV replication. The development of prodrugs of nucleoside 5'-monophosphates has been pursued to address limitations associated with poor nucleoside phosphorylation. This is required to produce the nucleoside 5'-triphosphate which is the anabolite that is the actual inhibitor of the polymerase enzyme. Prodrugs of nucleoside 5'-monophosphates have been developed that enable their delivery into cells and in vivo into the liver. The implementation of these prodrug strategies has ultimately led to the identification of several prodrugs of nucleoside 5'-monophosphates that are potent inhibitors of HCV replication in vitro. They have progressed into the clinic and the early data demonstrate greatly reduced viral load levels in HCV-infected patients. This review will survey the state of nucleotide prodrugs for the treatment of HCV.

Optimization of Pharmacokinetics through Manipulation of Physicochemical Properties in a Series of HCV Inhibitors.

A novel series of HCV replication inhibitors based on a pyrido[3,2-d]pyrimidine core were optimized for pharmacokinetics (PK) in rats. Several associations between physicochemical properties and PK were identified and exploited to guide the design of compounds. In addition, a simple new metric that may aid in the prediction of bioavailability for compounds with higher polar surface area is described (3*HBD-cLogP).

PP-137 Suppression of HCV replication in hepatocytes through a selective induction of IRF7

PP-137 Suppression of HCV replication in hepatocytes through a selective induction of IRF7

Effect of Tacrolimus on Survival in Hepatitis C–Infected Patients After Liver Transplantation

The observation that cyclosporine inhibits HCV replication in vitro has led some programs to use cyclosporine as the calcineurin inhibitor (CNI) of choice after orthotopic liver transplantation (OLT). Previous studies comparing outcomes with different CNIs used small HCV cohorts or had short-term follow-up. We examined patient survival and fibrosis progression in all HCV-infected adult primary OLT recipients from 1995 to 2004 at the Annette C. and Harold C. Simmons Transplant Institute (n = 516). Patients were categorized by their CNI on day 7 post-OLT, and they were excluded if they died before day 14. Patient and donor age, sex, race, and prevalence of cytomegalovirus infection post-OLT were similar in the tacrolimus and cyclosporine patients. As expected, acute cellular rejection and steroid-resistant rejection were less common in tacrolimus-treated patients. Although no difference in 1-year survival was seen, tacrolimus patients (n = 268) had superior 5-year survival compared to cyclosporine patients (n = 248) (75% vs. 67%; P = 0.02). Fibrosis progression was no different between the groups. In our retrospective analysis of 516 post-OLT patients, tacrolimus improved long-term survival compared to cyclosporine in HCV-infected patients, although it did not impact HCV fibrosis progression.

Antiviral drugs against hepatitis C virus

Hepatitis C virus (HCV) infection is a major worldwide problem causes acute and chronic HCV infection. Current treatment of HCV includes pegylated interferon-α (PEG IFN- α) plus ribavirin (RBV) which has significant side effects depending upon the type of genotype. Currently, there is a need to develop antiviral agents, both from synthetic chemistry and Herbal sources. In the last decade, various novel HCV replication, helicase and entry inhibitors have been synthesized and some of which have been entered in different phases of clinical trials. Successful results have been acquired by executing combinational therapy of compounds with standard regime in different HCV replicons. Even though, diverse groups of compounds have been described as antiviral targets against HCV via Specifically Targeted Antiviral Therapy for hepatitis C (STAT-C) approach (in which compounds are designed to directly block HCV or host proteins concerned in HCV replication), still there is a need to improve the properties of existing antiviral compounds. In this review, we sum up potent antiviral compounds against entry, unwinding and replication of HCV and discussed their activity in combination with standard therapy. Conclusively, further innovative research on chemical compounds will lead to consistent standard therapy with fewer side effects.

SiRNAs: Potential therapeutic agents against Hepatitis C Virus

Hepatitis C virus is a major cause of chronic liver diseases which can lead to permanent liver damage, hepatocellular carcinoma and death. The presently available treatment with interferon plus ribavirin, has limited benefits due to adverse side effects such as anemia, depression and "flu-like" symptoms. Needless to mention, the effectiveness of interferon therapy is predominantly, if not exclusively, limited to virus type 3a and 3b whereas in Europe and North America the majority of viral type is 1a and 2a. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option. Several reports have indicated the efficiency and specificity of synthetic and vector based siRNAs inhibiting HCV replication. In the present review, we focused that combination of siRNAs against virus and host genes will be a better option to treat HCV

Nitazoxanide is an Indirect Inhibitor of HCV Replication Through Modulation of Cellular Kinase CKI Alpha to Enhance HCV NS5A Hyperphosphorylation

Nitazoxanide is an Indirect Inhibitor of HCV Replication Through Modulation of Cellular Kinase CKI Alpha to Enhance HCV NS5A Hyperphosphorylation

Fluoroquinolones Inhibit HCV by Targeting Its Helicase

HCV has infected >170 million individuals worldwide. Effective therapy against HCV is still lacking and there is a need to develop potent drugs against the virus. In the present study, we have employed two culture models to test the activity of fluoroquinolone drugs against HCV: a subgenomic replicon that is able to replicate independently in the cell line Huh-8 and the Huh-7 cell culture model that employs cells transfected with synthetic HCV RNA to produce the infectious HCV particles. Fluoroquinolones have also been shown to have inhibitory activity against certain viruses, possibly by targeting the viral helicase. To tease out the mechanism of the antiviral activity of fluoroquinolones, their effect on HCV NS3 helicase protein was also tested. Huh-7 cells producing the HCV virion as well as Huh-8 cells were grown in the presence or absence of 12 different fluoroquinolones. Afterwards, Huh-7 and Huh-8 cells were lysed and viral RNA was extracted. The extracted RNA was reverse transcribed and quantified by real-time quantitative PCR. Fluoroquinolones were also tested on purified NS3 protein in a molecular-beacon-based in vitro helicase assay. To varying degrees, all of the tested fluoroquinolones effectively inhibited HCV replication in both Huh-7 and Huh-8 culture models. The inhibition of HCV NS3 helicase activity was also observed with all 12 of the fluoroquinolones. Fluoroquinolones inhibit HCV replication possibly by targeting the HCV NS3 helicase. These drugs hold promise for the treatment of HCV infection.

Inhibition of hepatitis C virus replication by semi-synthetic derivatives of glycopeptide antibiotics

ObjectivesSome semi-synthetic derivatives of glycopeptide antibiotics have been shown to exert in vitro antiviral activity against HIV and coronaviruses. Here we report and characterize the in vitro anti-hepatitis C virus (HCV) activity of several semi-synthetic derivatives of teicoplanin aglycone.MethodsAnti-HCV activity was analysed in: (i) three different subgenomic HCV replicon systems using a luciferase or quantitative RT–PCR (qRT–PCR) assay; and (ii) an infectious HCV cell culture system by means of qRT–PCR and immunofluorescence assays.ResultsSeveral teicoplanin aglycone derivatives elicited selective anti-HCV activity in replicons as well as infectious cell culture systems, with LCTA-949 being the most potent derivative. LCTA-949 proved, in contrast to several directly acting antivirals for HCV, efficient in clearing cells of their replicons. When LCTA-949 was combined with HCV protease or polymerase inhibitors an overall additive effect was observed. Likewise, LCTA-949 was equipotent against wild-type replicons as well as against replicons resistant to polymerase and protease inhibitors. Following up to 4 months of selective pressure, no drug-resistant replicons were selected. When combined with the HCV NS3 protease inhibitor VX-950, LCTA-949 prevented the development of VX-950-resistant variants.ConclusionsSemi-synthetic derivatives of teicoplanin aglycone constitute a novel class of HCV replication inhibitors that are not cross-resistant with various HCV protease and polymerase inhibitors and in particular are potent in clearing hepatoma cells of their replicons. This class of molecules also provides a good tool to obtain novel insights into the replication cycle of HCV and into cellular factors/processes that are crucial for viral replication.

Inhibitors of HCV NS5A: From Iminothiazolidinones to Symmetrical Stilbenes.

The iminothiazolidinone BMS-858 (2) was identified as a specific inhibitor of HCV replication in a genotype 1b replicon assay via a high-throughput screening campaign. A more potent analogue, BMS-824 (18), was used in resistance mapping studies, which revealed that inhibitory activity was related to disrupting the function of the HCV nonstructural protein 5A. Despite the development of coherent and interpretable SAR, it was subsequently discovered that in DMSO 18 underwent an oxidation and structural rearrangement to afford the thiohydantoin 47, a compound with reduced HCV inhibitory activity. However, HPLC bioassay fractionation studies performed after incubation of 18 in assay media led to the identification of fractions containing a dimeric species 48 that exhibited potent antiviral activity. Excision of the key elements hypothesized to be responsible for antiviral activity based on SAR observations reduced 48 to a simplified, symmetrical, pharmacophore realized most effectively with the stilbene 55, a compound that demonstrated potent inhibition of HCV in a genotype 1b replicon with an EC50 = 86 pM.

The antiviral protein viperin inhibits HCV replication via interaction with NS5A

The antiviral protein viperin inhibits HCV replication via interaction with NS5A

Hepatitis C virus complete life cycle screen for identification of small molecules with pro- or antiviral activity

Infection with the hepatitis C virus represents a global public health threat given that an estimated 170 million individuals are chronically infected and thus at risk for cirrhosis and hepatocellular carcinoma. A number of direct antiviral molecules are in clinical development. However, side effects, drug resistance and viral genotype-specific differences in efficacy may limit these novel therapeutics. Therefore, a combination of well tolerated drugs with distinct mechanisms of action targeting different steps of the viral replication cycle will likely improve viral response rates and therapy success. To identify small molecules that interfere with different steps of the HCV replication cycle, we developed a novel dual reporter gene assay of the complete HCV life cycle and adapted it to 384-well high-throughput format. The system is based on a highly permissive Huh-7 cell line stably expressing a secreted luciferase. Using these cells and an efficient HCV luciferase reporter virus, perturbations of each step of the viral replication cycle as well as cell viability can be easily and quantitatively determined. The system was validated with a selected set of known HCV entry, replication and assembly inhibitors and then utilized to screen a library of small molecules derived from myxobacteria. Using this approach we identified a number of molecules that specifically inhibit HCV cell entry, or primarily virus assembly and release. Moreover, we also identified molecules that increase viral propagation. These compounds may be useful leads for development of novel HCV inhibitors and could be instrumental for the identification of as yet unknown host-derived viral resistance and dependency factors.

IL28B inhibits hepatitis C virus replication through the JAK–STAT pathway

The combination of pegylated interferon (IFN) α and ribavirin (RBV) is the standard therapy for patients with chronic HCV infection. However, it produces a sustained virologic response (SVR) in only half of the treated individuals and is associated with significant side effects. Recently, several single-nucleotide polymorphisms (SNPs) near the IL28B locus, also known as IFNλ3, were identified to be strong predictors of SVR in patients receiving PEG-IFN and RBV. We sought to determine whether IL28B was capable of inhibiting HCV replication and to determine the pathway by which IL28B exhibits anti-HCV activity. Using the full-length HCV replicon OR6 and the infectious HCV clones JFH1 and Jc1, we assessed the anti-HCV effect of IL28B on HCV and characterized the key steps of the JAK-STAT pathway by real time PCR, luciferase assay, and Western blot. Finally, we evaluated the anti-HCV effect of IL28B in the presence of JAK-STAT pathway inhibitors such as blocking antibodies, a pharmacological inhibitor, and siRNAs. We found that IL28B inhibits HCV replication in a dose- and time-dependent manner. Like IFNα, IL28B induces the phosphorylation of STAT1 and STAT2, ISRE-driven transcription, and expression of known ISGs. The anti-HCV effects of IL28A, IL28B, and IL29 were abrogated by an IL10R2 blocking antibody, a pharmacological inhibitor of JAK1/TYK2, and by siRNA against IL28R1, STAT1, STAT2, and IRF9. Our data demonstrate that IL28A, IL28B, and IL29 signal through the JAK-STAT pathway to inhibit HCV. These data suggest possible applications of new approaches in HCV treatment.

Comparative analysis of the lambda-interferons IL-28A and IL-29 regarding their transcriptome and their antiviral properties against hepatitis C virus.

BackgroundSpecific differences in signaling and antiviral properties between the different Lambda-interferons, a novel group of interferons composed of IL-28A, IL-28B and IL-29, are currently unknown. This is the first study comparatively investigating the transcriptome and the antiviral properties of the Lambda-interferons IL-28A and IL-29.Methodology/Principal FindingsExpression studies were performed by microarray analysis, quantitative PCR (qPCR), reporter gene assays and immunoluminometric assays. Signaling was analyzed by Western blot. HCV replication was measured in Huh-7 cells expressing subgenomic HCV replicon. All hepatic cell lines investigated as well as primary hepatocytes expressed both IFN-λ receptor subunits IL-10R2 and IFN-λR1. Both, IL-28A and IL-29 activated STAT1 signaling. As revealed by microarray analysis, similar genes were induced by both cytokines in Huh-7 cells (IL-28A: 117 genes; IL-29: 111 genes), many of them playing a role in antiviral immunity. However, only IL-28A was able to significantly down-regulate gene expression (n = 272 down-regulated genes). Both cytokines significantly decreased HCV replication in Huh-7 cells. In comparison to liver biopsies of patients with non-viral liver disease, liver biopsies of patients with HCV showed significantly increased mRNA expression of IL-28A and IL-29. Moreover, IL-28A serum protein levels were elevated in HCV patients. In a murine model of viral hepatitis, IL-28 expression was significantly increased.Conclusions/SignificanceIL-28A and IL-29 are up-regulated in HCV patients and are similarly effective in inducing antiviral genes and inhibiting HCV replication. In contrast to IL-29, IL-28A is a potent gene repressor. Both IFN-λs may have therapeutic potential in the treatment of chronic HCV.

Inhibition of core gene of HCV 3a genotype using synthetic and vector derived siRNAs

BackgroundHepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option.ResultsThe current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed.ConclusionsOur results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

Measuring antiviral activity of benzimidazole molecules that alter IRES RNA structure with an infectious hepatitis C virus chimera expressing Renilla luciferase

Major progress has been made in developing infectious HCV cell culture systems and these systems have been useful in identifying novel HCV antivirals. However, more rapid and sensitive assays using infectious cell based HCV systems would facilitate the development of additional antivirals, including small molecules directed at unique targets such as the HCV RNA internal ribosomal entry site (IRES). We have found that the V3 region (28 aa) of NS5A of HCV JFH1 can be deleted from the genome with only modest effects on the titer of infectious virus produced in cell culture. Moreover, the V3 region can be replaced with the Renilla reniformis luciferase (Rluc) gene resulting in an infectious virus that stably expresses an NS5A–Rluc fusion protein. Infected cells cultured in 96-well plates provided a robust luciferase signal that accurately reflected the production of infectious virus. This infectious HCV reporter system was used to test the activity of three benzimidazole compounds that bind the HCV RNA IRES. Compounds in this chemical class of small molecules bind and alter the IRES RNA structure at low to sub-micromolar concentrations and interfere with viral replication. The current study shows that these compounds inhibit HCV replication in an infectious HCV cell culture system, defines their IC 50 in this system, and provides a platform for the rapid testing of next generation inhibitors.

IL28B genotype is associated with differential expression of intrahepatic interferon-stimulated genes in patients with chronic hepatitis C

Genetic variation in the IL28B (interleukin 28B; interferon lambda 3) region has been associated with sustained virological response (SVR) rates in patients with chronic hepatitis C (CHC) who were treated with peginterferon-α and ribavirin. We hypothesized that IL28B polymorphism is associated with intrahepatic expression of interferon-stimulated genes (ISGs), known to influence treatment outcome. IL28B genotyping (rs12979860) and whole-genome RNA expression were performed using liver biopsies from 61 North American patients with CHC. After correction for multiple testing (false discovery rate < 0.10), 164 transcripts were found to be differentially expressed by IL28B-type. The interferon signaling pathway was the most enriched canonical pathway differentially expressed by IL28B-type (P < 10(-5)), with most genes showing higher expression in livers of individuals carrying the poor-response IL28B-type. In 25 patients for which treatment response data were available, IL28B-type was associated with SVR (P = 0.0054). ISG expression was also associated with SVR; however, this was not independent of IL28B-type. Analysis of miR-122 expression in liver biopsies showed reduced miR-122 levels associated with poorer treatment outcome, independently of IL28B-type. No association was observed between IL28B-type and levels of liver IL28B or IL28A messenger RNA expression. IL28B protein sequence variants associated with rs12979860 were therefore investigated in vitro: no differences in ISG induction or inhibition of HCV replication were observed in Huh7.5 cells. The good response IL28B variant was strongly associated with lower level ISG expression. The results suggest that IL28B genotype may explain the relationship between hepatic ISG expression and HCV treatment outcome, and this is independent of miR-122 expression. IL28B-type was not associated with intrahepatic IL28B messenger RNA expression in vivo. Further investigation of the precise molecular mechanism(s) by which IL28B genetic variation influences HCV outcomes is warranted.

Suppression of Hepatitis C Virus Genome Replication in Cells with RNA-Cleaving DNA Enzymes and Short-Hairpin RNA

A class of antisense oligodeoxyribozymes, known as the 10-23 DNA enzymes (DNAzyme), has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. Herein we have utilized a strategy to identify accessible cleavage sites for DNAzyme in the target RNA, the hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of randomized DNAzyme library. The screening procedure identified 18 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA cleavage in vitro. Using positively charged dendrimer nanoparticles, the target RNA-cleaving DNAzymes that are 31-mer oliogonucleotides are delivered into the human hepatoma cells harboring the HCV subgenomic replicon RNA. DNAzymes introduced into the cells efficiently inhibited HCV RNA replication by reducing the expression of HCV NS3. In addition, we designed short-hairpin RNA (shRNA) that targets the same cleavage site for the selected DNAzyme and confirmed that the shRNA also inhibited HCV NS3 gene expression in the HCV replicon cells. These selected DNAzyme and shRNA may be a viable therapeutic intervention to inhibit HCV replication in hepatic cells. We suggest that the method used in this study can be applicable for identification of available sites in any target RNA for antisense oligonucleotides and siRNAs.