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Objectives: Many positive-stranded RNA viruses, including HCV, drastically remodel intracellular membranes to generate specialized environments for RNA replication. Phosphatidylinositol 4-kinase III (PI4KIII)α plays an essential role in the formation of HCV replication complexes and has therefore been explored as a potential drug target. Here, we characterized the anti-HCV activity of the PI4KIII inhibitors enviroxime and BF738735 and elucidated their mechanism of action., Methods: Antiviral assays were performed using HCV subgenomic replicons and infectious HCV. Enviroxime- and BF738735-resistant HCV replicons were generated by long-term culture with increasing compound concentrations. Intracellular localization of phosphatidylinositol 4-phosphate (PI4P) lipids was analysed by confocal microscopy., Results: HCV subgenomic replicons resistant to either enviroxime or BF738735 proved cross-resistant and carried mutations in the NS3, NS4B and NS5A genes. Knockdown of PI4KIIIβ by small interfering RNA (siRNA) did not affect the replication of the HCV subgenomic replicon in this study. Furthermore, the compounds did not affect PI4P lipid levels at the replication complexes nor the phosphorylation status of NS5A, activities attributed to PI4KIIIα. Interestingly, the broad-spectrum phosphoinositide 3-kinase (PI3K) inhibitor LY294002 proved to be 10-fold less effective against the resistant replicons. In addition, enviroxime and BF738735 inhibited several PI3Ks in enzymatic assays., Conclusions: Contrary to assumptions, our data indicate that PI4KIIIα and PI4KIIIβ are not the main targets for the anti-HCV activity of enviroxime and BF738735. Instead, we demonstrated that both molecules impede HCV replication at least partially by an inhibitory effect on PI3Ks. Moreover, HCV is able to bypass PI3K inhibition by acquiring mutations in its genome.

HCV is a major risk factor for hepatocellular carcinoma (HCC). HCC development in chronically infected HCV patients has until now been attributed to persistent inflammation and interference of viral proteins with host cell signaling. Since activation of the epidermal growth factor receptor (EGFR) presents a crucial step in HCV entry, we aimed at investigating whether EGFR signaling may contribute to the pathogenesis of HCV-related HCC. By applying microarray analysis, we generated a gene expression signature for secreted proteins in HCV-infected hepatoma cells. This gene signature was enriched for inflammatory and angiogenic processes; both crucially involved in HCC development. RT-qPCR analysis, conducted on the entire list of upregulated genes, confirmed induction of 11 genes (AREG, IL8, CCL20, CSF1, GDF15, IGFBP1, VNN3, THBS1 and PAI-1) in a virus titer- and replication-dependent manner. EGFR activation in hepatoma cells largely mimicked the gene signature seen in the infectious HCV model. Further, the EGFR-ERK pathway, but not Akt signaling, was responsible for this gene expression profile. Finally, microarray analysis conducted on clinical data from the GEO database, revealed that our validated gene expression profile is significantly represented in livers of patients with HCV-related liver pathogenesis (cirrhosis and HCC) compared to healthy livers. Taken together, our data indicate that persistent activation of EGFR-ERK signaling in chronically infected HCV patients may induce a specific pro-inflammatory and pro-angiogenic signature that presents a new mechanism by which HCV can promote liver cancer pathogenesis. A better understanding of the key factors in HCV-related oncogenesis, may efficiently direct HCC drug development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Combination therapy of directly acting antivirals (DAA's) for the treatment of chronic HCV infections has proven to be a highly effective strategy to cure chronic infections with this virus. Here we studied, using HCV as an example, how to best design in vitro studies that explore the combined antiviral efficiency of combinations of three or more DAA's. To that end we used a HCV NS3 protease inhibitor, a NS5A targeting compound and two non-nucleoside NS5B polymerase inhibitors (each one targeting a different drug binding site). We demonstrate, employing HCV subgenomic replicon containing Huh 9-13 hepatoma cells, that quadruple therapy with these 4 different DAA's each at 1x their EC 75 , results in a highly efficient inhibition of viral replication. This is further reflected in the rapid clearance of the HCV replicon from the host cell. By contrast, neither equipotent combinations that consist of either molecules alone at 4x EC 75 nor triple combinations at 1.33x the EC 75 resulted in clearance. In contrast to the quadruple combo, drug-resistant variants emerged under mono-treatment and in most triple combo's. These data thus demonstrate that quadruple combinations at total suboptimal concentrations [i.e. concentrations at which neither mono- nor triple therapy is sufficiently potent] result rapidly in a pronounced antiviral efficacy. Altogether, this work provides an example as to how to design studies to explore the antiviral efficacy of combinations of more than two compounds., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A ligand-based approach was applied to screen in silico a library of commercially available compounds, with the aim to find novel inhibitors of the HCV replication starting from the study of the viral NS3 helicase. Six structures were selected for evaluation in the HCV subgenomic replicon assay and one hit was found to inhibit the HCV replicon replication in the low micromolar range. A small series of new pyrrolone compounds was designed and synthesised, and novel structures were identified with improved antiviral activity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A structure-based virtual screening of commercial compounds was carried out on the HCV NS3 helicase structure, with the aim to identify novel inhibitors of HCV replication. Among a selection of 13 commercial structures, one compound was found to inhibit the subgenomic HCV replicon in the low micromolar range. Different series of new piperazine-based analogues were designed and synthesised, and among them, several novel structures exhibited antiviral activity in the HCV replicon assay. Some of the new compounds were also found to inhibit HCV NS3 helicase function in vitro, and one directly bound NS3 with a dissociation constant of 570 ± 270 nM., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wild-type and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A series of new conjugated compounds with a -SCH₂- linkage were synthesized by chemical methods from imidazole and coumarin derivatives. The experimental results indicate that of the twenty newly synthesized imidazole-coumarin conjugates, three of them exhibited appealing EC50 values (5.1-8.4 μM) and selective indices >20 against hepatitis C virus. Their potency and selectivity were increased substantially by modification of their structure with two factors: imidazole nucleus with a hydrogen atom at the N(1) position and coumarin nucleus with a substituent, such as Cl, F, Br, Me, and OMe. These guidelines provide valuable information for further development of conjugated compounds as anti-viral agents.

Several novel compounds have been identified that inhibit the replication of hepatitis C virus in a replicon assay with EC50 values as low as 0.6 μM. Lead compounds were modified to investigate the possible role that zinc binding may play in inhibitor efficacy. In addition, the structure-activity relationship was explored to increase inhibitor efficacy and possibly identify favorable interactions within the currently unknown inhibitor binding pocket. The rationale for inhibitor design and biological results are presented herein., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Certain food additives and drugs used for other indications have been shown to inhibit in vitro replication of HCV and have been proposed as cheap options for the treatment of HCV infections in resource-poor countries. We here report that the in vitro anti-HCV (genotypes 1a, 1b, 2a and 4b) activity of nitazoxanide, silymarin, silibinin and the green tea extract EGCG is very weak when compared to directly acting antivirals. HCV-infected patients in resource-poor countries should receive the best possible treatment (if possible via expanded access programs); it is therefore advisable not to plan clinical studies with drugs/compounds with weak anti-HCV activity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Despite the great progress made in the last 10 years, alternative strategies might help improving definitive treatment options against hepatitis C virus infection., Methods: With the aim of identifying novel inhibitors of the hepatitis C virus-1b replication targeting the viral NS3 helicase, the structures of previously reported symmetrical inhibitors of this enzyme were rationally modified, and according to docking-based studies, four novel scaffolds were selected for synthesis and evaluation in the hepatitis C virus-1b subgenomic replicon assay., Results: Among the newly designed compounds, one new structural family was found to inhibit the hepatitis C virus-1b replication in the micromolar range. This scaffold was chosen for further exploration and different novel analogues were synthesised and evaluated., Conclusions: Different new inhibitors of the hepatitis C virus genotype 1b replication were identified. Some of the new compounds show mild inhibition of the NS3 helicase enzyme.

Tegobuvir (GS-9190) is a non-nucleoside inhibitor of HCV RNA replication with proven antiviral activity in HCV-infected patients. The in vitro antiviral activity of Tegobuvir, when combined with one or two other direct acting antivirals (DAA) was assessed. When Tegobuvir was combined with either interferon α-2b, ribavirin, the protease inhibitor (PI) VX-950, the nucleoside polymerase inhibitor (NI) 2'-C-methylcytidine or various non-nucleoside polymerase inhibitors, an overall additive antiviral activity was observed. Adding Tegobuvir (at concentrations of 6, 30 or 150nM) to replicon-containing cells in the presence of suboptimal concentrations of the PI or of the various polymerase inhibitors either markedly delayed or completely prevented resistance development against these latter compounds. Tegobuvir (15nM), when combined with the PI, was able to cure replicon-containing cells from their replicon after a single passage, whereas either compound alone (at 2-fold higher concentration) was not. The triple combination of Tegobuvir (10nM), the PI and the NI resulted in clearance of replicon RNA after only two passages. In contrast, the inhibitors when used alone at 3-fold higher concentrations were not able to cure the cells from the replicon, after as long as 6 passages. Combinations containing low concentrations of Tegobuvir are thus highly effective in curing cells from HCV replicon and in delaying or preventing the development of resistance against other DAA., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A number of statins, the cholesterol-lowering drugs, inhibit the in vitro replication of hepatitis C virus (HCV). In HCV-infected patients, addition of statins to the earlier standard of care therapy (pegIFN-α and ribavirin) resulted in increased sustained virological response rates. The mechanism by which statins inhibit HCV replication has not yet been elucidated. In an attempt to gain insight in the underlying mechanism, hepatoma cells carrying an HCV replicon were passaged in the presence of increasing concentrations of fluvastatin. Fluvastatin-resistant replicon containing cells could be generated and proved ∼8-fold less susceptible to fluvastatin than wild-type cultures. The growth efficiency of the resistant replicon containing cells was comparable to that of wild-type replicon cells. The fluvastatin-resistant phenotype was not conferred by mutations in the viral genome but is caused by cellular changes. The resistant cell line had a markedly increased HMG-CoA reductase expression upon statin treatment. Furthermore, the expression of the efflux transporter P-gp was increased in fluvastatin-resistant replicon cells (determined by qRT-PCR and flow cytometry). This increased expression resulted also in an increased functional transport activity as measured by the P-gp mediated efflux of calcein AM. In conclusion, we demonstrate that statin resistance in HCV replicon containing hepatoma cells is conferred by changes in the cellular environment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Linear and branched compounds that contain two, three or five units of galloyl (3,4,5-trihydroxybenzoyl) or its isomer 2,3,4-trihydroxybenzoyl, as well as other mono- or dihydroxybenzoyl moieties have been synthesized. These molecules have been evaluated for their in vitro inhibitory effects against a wide panel of viruses showing preferential activity against HIV and HCV. Our structure-activity relationship studies demonstrated that the 2,3,4-trihydroxybenzoyl moiety provides better antiviral activities than the galloyl (3,4,5-trihydroxybenzoyl) moiety that is present in natural green tea catechins. This observation can be of interest for the further rational exploration of compounds with anti-HCV/HIV properties. The most notable finding with respect to HIV is that the tripodal compounds 43 and 45, with three 2,3,4-trihydroxybenzoyl moieties, showed higher activities than linear compounds with only one or two. With respect to HCV, the linear compounds, 52 and 41, containing a 12 polymethylene chain and two 2,3 di- or 2,3,4 tri-hydroxybenzoyl groups respectively at the ends of the molecule showed good antiviral efficiency. Furthermore, the anti-HCV activity of both compounds was observed at concentrations well below the cytotoxicity threshold. A representative member of these compounds, 41, showed that the anti-HCV activity was largely independent of the genetic make-up of the HCV subgenomic replicon and cell lines used., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Objective: Although direct-acting antiviral agents (DAAs) have markedly improved the outcome of treatment in chronic HCV infection, there continues to be an unmet medical need for improved therapies in difficult-to-treat patients as well as liver graft infection. Viral entry is a promising target for antiviral therapy., Design: Aiming to explore the role of entry inhibitors for future clinical development, we investigated the antiviral efficacy and toxicity of entry inhibitors in combination with DAAs or other host-targeting agents (HTAs). Screening a large series of combinations of entry inhibitors with DAAs or other HTAs, we uncovered novel combinations of antivirals for prevention and treatment of HCV infection., Results: Combinations of DAAs or HTAs and entry inhibitors including CD81-, scavenger receptor class B type I (SR-BI)- or claudin-1 (CLDN1)-specific antibodies or small-molecule inhibitors erlotinib and dasatinib were characterised by a marked and synergistic inhibition of HCV infection over a broad range of concentrations with undetectable toxicity in experimental designs for prevention and treatment both in cell culture models and in human liver-chimeric uPA/SCID mice., Conclusions: Our results provide a rationale for the development of antiviral strategies combining entry inhibitors with DAAs or HTAs by taking advantage of synergy. The uncovered combinations provide perspectives for efficient strategies to prevent liver graft infection and novel interferon-free regimens., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

We report here the synthesis and mechanism of inhibition of pyrazolecarboxamide derivatives as a new class of HCV inhibitors. Compounds 6, 7, 8 and 16 inhibited the subgenomic HCV replicon 1b genotype at EC50 values between 5 and 8 μM and displayed an even higher potency against the infectious Jc1 HCV 2a genotype. Compound 6 exhibited an EC50 of 6.7 μM and selectivity index of 23 against HCV 1b, and reduced the RNA copies of the infectious Jc1 chimeric 2a clone by 82% at 7 μM. Evaluation of the mode of anti-HCV activity of 6 revealed that it suppressed HCV-induced COX-2 mRNA and protein expression, displaying an IC50 of 3.2 μM in COX-2 promoter-linked luciferase reporter assay. Conversely, the anti-HCV activity of 6 was abrogated upon over-expression of COX-2. These findings suggest that 6 as a representative of these pyrazolecarboxamides function as anti-HCV agents via targeting COX-2 at both the transcription and translation levels., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Statins are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors that are widely used for the treatment of hypercholesterolemia. Besides their cholesterol-lowering effect, statins have been reported to have antiviral activity against a variety of viruses, including hepatitis C virus (HCV). Several statins inhibit the in vitro replication of subgenomic HCV replicons and also suppress in vitro RNA replication of infectious HCV. The precise mechanism of the anti-HCV activity of statins has not yet been defined. Recent studies suggest that the antiviral effect may result from the inhibition of geranylgeranylation of cellular proteins, rather than the inhibition of cholesterol synthesis. Despite the antiviral effect observed in vitro, statin monotherapy seems to be insufficient for the treatment of chronic HCV infection. However, several prospective and retrospective studies have demonstrated that the addition of statins to IFN-α and ribavirin therapy increases SVR, RVR, and EVR rates without the occurrence of additional adverse events. These clinical data, together with the excellent safety profile and low cost, suggest that statins may play a role in HCV therapy until more potent and safe direct-acting antivirals become available. This article forms part of a symposium in Antiviral Research on "Hepatitis C: next steps toward global eradication.", (Copyright © 2014. Published by Elsevier B.V.)

We have previously reported that the 6-aminoquinolone chemotype is a privileged scaffold to obtain antibacterial and antiviral agents. Herein we describe the design, synthesis, and enzymatic and cellular characterization of new 6-aminoquinolone derivatives as potent inhibitors of NS5B polymerase, an attractive and viable therapeutic target to develop safe anti-HCV agents. The 6-amino-7-[4-(2-pyridinyl)-1-piperazinyl]quinolone derivative 8 proved to be the best compound of this series, exhibiting an IC50 value of 0.069 μM against NS5B polymerase and selective antiviral effect (EC50 = 3.03 μM) coupled with the absence of any cytostatic effect (CC50 > 163 μM; SI > 54) in Huh 9-13 cells carrying a HCV genotype 1b, as measured by MTS assay. These results indicate that the 6-aminoquinolone scaffold is worthy of further investigation in the context of NS5B-targeted HCV drug discovery programs.

We reported previously that Artemisinin (ART), a widely used anti-malarial drug, is an inhibitor of in vitro HCV subgenomic replicon replication. We here demonstrate that ART exerts its antiviral activity also in hepatoma cells infected with full length infectious HCV JFH-1. We identified a number of ART analogues that are up to 10-fold more potent and selective as in vitro inhibitors of HCV replication than ART. The iron donor Hemin only marginally potentiates the anti-HCV activity of ART in HCV-infected cultures. Carbon-centered radicals have been shown to be critical for the anti-malarial activity of ART. We demonstrate that carbon-centered radicals-trapping (the so-called TEMPO) compounds only marginally affect the anti-HCV activity of ART. This provides evidence that carbon-centered radicals are not the main effectors of the anti-HCV activity of the Artemisinin. ART and analogues may possibly exert their anti-HCV activity by the induction of reactive oxygen species (ROS). The combined anti-HCV activity of ART or its analogues with L-N-Acetylcysteine (L-NAC) [a molecule that inhibits ROS generation] was studied. L-NAC significantly reduced the in vitro anti-HCV activity of ART and derivatives. Taken together, the in vitro anti-HCV activity of ART and analogues can, at least in part, be explained by the induction of ROS; carbon-centered radicals may not be important in the anti-HCV effect of these molecules.

Using Ttou 84 as starting point, a novel class of biphenyl derivatives of imidazo[1,2-a]pyridine and imidazo[1,2-b]pyridazine was designed to optimize the inhibitory properties on the replication of the bovine viral diarrhoea virus (BVDV) and hepatitis C virus (HCV). Three sites of pharmacomodulation were chosen i.e. positions 2, 3 and 6 on the central heterocyclic core structure. From the 49 analogues tested, only compound 18j (3-(2'-hydroxybiphen-3-yl)-2-(2-methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-b]pyridazine) showed antiviral activity in the HCV replicon system reminiscent of selective inhibition (60-70% inhibition). Compound 4f (3-(biphen-3-yl)-2-(4-fluorophenyl)-6-phenylthioimidazo[1,2-a]pyridine) proved to be the most selective inhibitor of BVDV replication and showed no or only marginal cross-resistance with known inhibitors of pestivirus replication. The cross-resistance profile of 4f might indicate that 4f does not interact with the same binding site as BPIP, VP32947, AG110 or LZ37. From 42 analogues tested against both viruses, QSAR studies were discussed in regard to BVDV antiviral activity., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The NS5B RNA-dependent RNA polymerase is an attractive target for the development of novel and selective inhibitors of hepatitis C virus replication. To identify novel structural hits as anti-HCV agents, we performed structure-based virtual screening of our in-house library followed by rational drug design, organic synthesis, and biological testing. These studies led to the identification of pyrazolobenzothiazine scaffold as a suitable template for obtaining novel anti-HCV agents targeting the NS5B polymerase. The best compound of this series was the meta-fluoro-N-1-phenyl pyrazolobenzothiazine derivative 4a, which exhibited an EC50 = 3.6 μM, EC90 = 25.6 μM, and CC50 > 180 μM in the Huh 9-13 replicon system, thus providing a good starting point for further hit evolution.

Unlabelled: Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor highly expressed in the liver and modulating HDL metabolism. Hepatitis C virus (HCV) is able to directly interact with SR-BI and requires this receptor to efficiently enter into hepatocytes to establish productive infection. A complex interplay between lipoproteins, SR-BI and HCV envelope glycoproteins has been reported to take place during this process. SR-BI has been demonstrated to act during binding and postbinding steps of HCV entry. Although the SR-BI determinants involved in HCV binding have been partially characterized, the postbinding function of SR-BI remains largely unknown. To uncover the mechanistic role of SR-BI in viral initiation and dissemination, we generated a novel class of anti-SR-BI monoclonal antibodies that interfere with postbinding steps during the HCV entry process without interfering with HCV particle binding to the target cell surface. Using the novel class of antibodies and cell lines expressing murine and human SR-BI, we demonstrate that the postbinding function of SR-BI is of key impact for both initiation of HCV infection and viral dissemination. Interestingly, this postbinding function of SR-BI appears to be unrelated to HDL interaction but to be directly linked to its lipid transfer function., Conclusion: Taken together, our results uncover a crucial role of the SR-BI postbinding function for initiation and maintenance of viral HCV infection that does not require receptor-E2/HDL interactions. The dissection of the molecular mechanisms of SR-BI-mediated HCV entry opens a novel perspective for the design of entry inhibitors interfering specifically with the proviral function of SR-BI., (Copyright © 2012 American Association for the Study of Liver Diseases.)

The current standard of care (SOC) for hepatitis C virus (HCV) genotype 1-infected patients consists of telaprevir or boceprevir in addition to pegylated interferon and ribavirin treatment. Other selective inhibitors of HCV replication are being developed. Drug pressure may, depending on the class of inhibitors, (rapidly) select for drug-resistant variants. Here we describe four different approaches to select in vitro for drug-resistant HCV subgenomic replicons.

The major targets for direct-acting antivirals (DAAs) are the NS3/4A protease, the NS5A protein, and the NS5B polymerase. The latter enzyme offers several target sites: the catalytic domain for nucleoside/nucleotide analogs and different allosteric sites for non-nucleoside inhibitors. Two protease inhibitors have already been approved and more than 40 new NS3/4A, NS5A, or NS5B inhibitors are in development pipeline. Not only these agents can achieve very high cure rates when combined with PEG-IFN and RBV, but have also started to provide promising results when combined in IFN-free, all-oral combinations. In addition to the more canonical drug targets, new alternative viral targets for small molecule drug development are emerging, such as p7 or NS4B. Current research is focusing on defining the most efficacious DAA combination regimens, i.e., those which provide the highest rates of viral eradication, broadest spectrum of action, minimal or no clinical resistance, shortest treatment duration, and good tolerability.

Objectives: Drug-resistant minority viral variants can pre-exist in the viral quasispecies of chronically infected hepatitis C virus (HCV) patients and can emerge gradually upon drug treatment. When heterogeneous clinical samples are tested for drug susceptibility in a chimeric replicon-based phenotyping assay, biphasic dose-response curves may be observed. The effect of drug-resistant minority viral variants on the biphasic phenotype of mixtures was assessed in detail., Methods: Susceptibility of mutant/wild-type mixtures containing minorities of NS3 mutants with different replication capacities and susceptibilities to protease inhibitors were tested in a transient replicon assay. The contribution of both variants in the mixture to the overall replication level was described with an E(max) model., Results: The 90% and 99% effective concentrations (EC(90) and EC(99), respectively) provide a more accurate measure of the susceptibility of the population than the determination of EC(50) values. Reduced susceptibility at the EC(50) level correlated with the replication capacity of the NS3 mutant in the mixture. Using replication-enhanced mutant/wild-type mixtures demonstrated that the relative difference between the replication capacity of the variants present in the mixture results in biphasic dose-response curves. Modelling revealed that in mixtures containing wild-type and resistant variants with low replication capacity, the contributions of the wild-type variants are higher than expected from the replication level of the replicons transfected alone., Conclusions: Differences in the replication capacity of variants present in HCV replicon-based phenotype assays can lead to biphasic dose-response curves. Using EC(90) or EC(99) values increases the sensitivity of the assay to minor variants.

Background & Aims: Worldwide, about 180 million people are chronically infected with the hepatitis C virus (HCV). Current in vitro culture systems for HCV depend chiefly on human hepatoma cell lines. Although primary human hepatocytes support HCV infection in vitro, and immunodeficient mice repopulated with human hepatocytes support HCV infection in vivo, these models are limited because of shortage of human livers to isolate hepatocytes. Therefore, there is significant interest in the establishment from of a HCV culture system in human stem cell-derived hepatocyte-like cells., Methods: Human embryonic stem cell (hESC)-derived hepatocytes were infected with HCV in the presence or absence of direct acting antivirals. After inoculation, replication of HCV was analyzed extensively., Results: We demonstrate that hESC-derived hepatocytes can be infected with the HCV JFH1 genotype 2a, resulting in the production of viral RNA in the stem cell progeny. Viral replication is inhibited by a non-nucleoside HCV polymerase-inhibitor (HCV-796), a cyclophilin binding molecule (Debio 025-Alisporivir) and the protease inhibitor VX-950 (Telaprevir). Stem cell-derived hepatocytes produced, for more than 10 days, the HCV core protein as well as virions that were capable of re-infecting hepatoma cells., Conclusions: Hepatocytes derived from hESC support the complete HCV replication cycle (including the production of infectious virus), and viral replication in these cells is efficiently inhibited by selective inhibitors of HCV replication., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Several novel 1,2,4-triazole and imidazole L-ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7 and 8) derivatives were prepared and evaluated for their inhibitory activity against hepatitis C virus (HCV) replication and human tumour cell proliferation. Compounds 6 and 9 exerted the most pronounced cytostatic effects in all tumour cell lines tested, and were highly selective for human T-cell acute lymphoblastic leukaemia cells (CEM/0) with IC(50)s of 10 ± 4 and 7.3 ± 0.1 μM, respectively. Unlike compound 9, compound 6 showed no toxicity in human diploid fibroblasts. One of the possible mechanisms of action of compound 6 accounting for observed cytostatic activity towards haematological malignancies might be inhibition of inosine monophosphate dehydrogenase (IMPDH) activity, a key enzyme of de novo purine nucleotide biosynthesis providing the cells with precursors for DNA and RNA synthesis indispensable for cell growth and division, which has emerged as an important target for antileukemic therapy. In addition, this compound proved to be the most potent inhibitor of the hepatitis C virus replication as well. However, observed antiviral effect was most likely associated with the effect that the compound exerted on the host cell rather than with selective effect on the replication of the virus itself. In conclusion, results of this study put forward compound 6 as a potential novel antitumor agent (IMPDH inhibitor) for treating leukaemia. Its significant biological activity and low toxicity in human diploid fibroblasts encourage further development of this compound as a lead., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Hepatitis C virus (HCV) infection has been recognized as the major cause of liver failure that can lead to hepatocellular carcinoma. Among all the HCV proteins, NS5B polymerase represents a leading target for drug discovery strategies. Herein, we describe our initial research efforts towards the identification of new chemotypes as allosteric NS5B inhibitors. In particular, the design, synthesis, in vitro anti-NS5B and in cellulo anti-HCV evaluation of a series of 1-oxo-1H-pyrido[2,1-b][1,3]benzothiazole-4-carboxylate derivatives are reported. Some of the newly synthesized compounds showed an IC(50) ranging from 11 to 23 μM, and molecular modeling and biochemical studies suggested that the thumb domain could be the target site for this new class of NS5B inhibitors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase.

Non-nucleoside inhibitors of the RNA-dependent RNA polymerase of the hepatitis C virus that are based on a benzimidazole or indole scaffold have been reported to interact with thumb domain 1 of the enzyme. Escape mutants that confer in vitro resistance to these inhibitors map to amino acids P495, P496 or V499. We here report a novel resistance mutation (T389S/A) that was identified following resistance selection with the benzimidazole non-nucleoside polymerase inhibitor JT-16 in HCV Con-1 subgenomic replicon (genotype 1b). This JT-16 resistant replicon retained wild-type susceptibility to protease inhibitors and nucleoside polymerase inhibitors. Replicons that carry mutations T389A and T389S have moderate levels of resistance to JT-16 (7- and 13-fold, respectively). Mutation P495A is associated with high-level (44-fold) resistance. Surprisingly, this previously reported 'key' mutation for benzimidazole resistance, P495A, was detected in only 15% of the resistant population. Furthermore, the replication fitness of the T389S mutant was significantly higher than that of the P495A mutant. By means of molecular modeling a structural hypothesis was formulated to explain the emergence of the T389S/A mutation in the JT-16 resistant replicon. Our data demonstrate that low-level resistant, but fit, variants can develop during in vitro resistance selection with the benzimidazole inhibitor JT-16. Moreover, different substitutions to the benzimidazole scaffold can affect the (pattern of) resistance mutations that emerge during resistance selection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background & Aims: To delay or prevent the selection of HCV drug-resistant variants, combination therapy will be needed. Our aim was to determine the antiviral efficacy of various combinations of non-nucleoside polymerase inhibitors (NNI) (that have a different allosteric binding site) and the barrier towards resistance development of such combinations., Methods: Short-term antiviral combination assays were performed in a checkerboard format. Resistance selection experiments employing HCV replicons were performed using two different protocols: (i) a short-term treatment with fixed concentrations and (ii) a long-term treatment with increasing concentrations., Results: All pair-wise combinations of NNI resulted in an additive antiviral effect in short-term antiviral assays. Combination treatment of two NNIs markedly reduced or even prevented the emergence of double resistant colonies. However, double and even triple NNI-resistant variants emerged readily when relatively low starting concentrations were used in a long-term selection protocol. Genotyping confirmed the presence of the previously published resistance mutations. For some NNI, different signature mutations appeared depending on the other NNI in the particular combination. Remarkably, variants that were selected to be resistant to three different classes of NNIs [a thiophene carboxylic acid (TCA), a benzimidazole (JT-16), and a benzofuran (HCV-796)] proved resistant to yet a fourth class of NNIs (benzothiadiazines)., Conclusions: Double and even triple NNI-resistant HCV replicons can be readily selected with a stepwise resistance selection protocol. Depending on the particular combination, different signature mutations may arise for some NNI. Resistance to three classes of NNI resulted in resistance to yet a fourth class., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Roughly 20 years after the discovery of the hepatitis C virus (HCV), and 10 years after the launch of the current standard of care (SOC) therapy, i.e. the combination of pegylated interferon-alpha and ribavirin, antiviral treatment of chronic hepatitis C is at the dawn of a new era. The current SOC will be combined with a direct acting antiviral (DAA), i.e. either the HCV NS3 protease inhibitor Telaprevir or Boceprevir. Combinations of DAAs may have the potential to completely cure chronic HCV infection. Clinical data suggest that ribavirin may remain at least for some time, an important component even in combinations of different DAAs. Ironically, and much in contrast to the DAAs, the precise mechanism(s) by which ribavirin exerts its anti-HCV activity in infected patients still waits to be unravelled. Here we review the current views on the mechanism of action of ribavirin against chronic infections with HCV. Concerted efforts of modern pharmacogenetics, novel insights into innate immunity and contributions from molecular virology will hopefully allow deciphering the precise mechanism(s) that are at the basis of the antiviral effect of this nucleoside analogue. Such insights may help design improved strategies to fight chronic infections with HCV., (Copyright © 2011 Elsevier B.V. All rights reserved.)

GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the β-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the β-hairpin in the thumb subdomain.

Hepatitis C virus (HCV) inhibitors include direct-acting antivirals (DAAs) such as NS3 serine protease inhibitors, nucleoside and nonnucleoside polymerase inhibitors, and host-targeting antivirals (HTAs) such as cyclophilin inhibitors that have been developed in recent years. Drug-resistant HCV variants have been reported both in vitro and in the clinical setting for most classes of drugs. We report a comparative study in which the genetic barrier to drug resistance of a representative selection of these inhibitors is evaluated employing a number of resistance selection protocols. The NS3 protease inhibitors VX-950 and BILN 2061, the nucleoside polymerase inhibitor 2'-C-methylcytidine, three nonnucleoside polymerase inhibitors (thiophene carboxylic acid, benzimidazole, and benzothiadiazine), and DEB025 were included. For each drug and passage in the selection process, the phenotype and genotype of the drug-resistant replicon were determined. For a number of molecules (BILN 2061 and nonnucleoside inhibitors), drug-resistant variants were readily selected when wild-type replicon-containing cells were directly cultured in the presence of high concentrations of the inhibitor. Resistance to DEB025 could be selected only following a lengthy stepwise selection procedure. For some DAAs, the signature mutations that emerged under inhibitor pressure differed depending on the selection protocol that was employed. Replication fitness of resistant mutants revealed that the C445F mutation in the RNA-dependent RNA polymerase can restore loss of fitness caused by a number of unfit resistance mutations. These data provide important insights into the various pathways leading to drug resistance and allow a direct comparison of the genetic barriers of various HCV drugs.

Objectives: Some 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., Methods: Anti-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., Results: Several 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., Conclusions: Semi-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.

About 3% of world's population is infected by the hepatitis C virus (HCV), for which prophylactic vaccine is not available yet. Nowadays, pegylated interferon-α and ribavirin are commonly used to treat HCV; unfortunately these drugs often produce significant side effects. Upon the desperate need of anti-HCV drugs, a plan to establish a new compound library was set that included leads with high antiviral activity, good hydrophilicity, yet low toxicity. Accordingly, 26 new conjugated compounds were synthesized through the chemical coupling of various 9-(β-D-ribofuranosyl)purine-8-thiones with 3-(chloromethyl)coumarins bearing various substituents. A -SCH(2)- unit was used to link the coumarin and the purine moieties. The three hydroxyl groups at the 2'-, 3'-, and 5'-positions were selectively protected with an acyl or acetal group in these coumarin-purine ribofuranosides. Their anti-HCV and cytostatic determination assays were performed, and the structure-activity relationship was established. Three conjugates in the family of 8-(coumarin-3'-yl)methylthio-9-(β-D-ribofuranos-1''-yl)purine possessed an appealing ability to inhibit HCV replication with EC(50) between 5.5 and 6.6 μM and EC(90) of ∼20 μM. These data in the new compound library provide clues for the future in the development of anti-HCV leads for viral eradication.

Preparation of several triazoloacyclic nucleoside phosphonates is described. The key step of the synthesis involves a copper(I)-catalysed azide-alkyne 1,3-dipolar cycloaddition between azidoalkylphosphonates and propargylated nucleobases. The antiviral properties of these new analogues have been evaluated and revealed interesting potencies., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Resistance to hepatitis C virus (HCV) inhibitors targeting viral enzymes has been observed in in vitro replicon studies and during clinical trials. The factors determining the emergence of resistance and the changes in the viral quasispecies population under selective pressure are not fully understood. To assess the dynamics of variants emerging in vitro under various selective pressures with TMC380765, a potent macrocyclic HCV NS3/4A protease inhibitor, HCV genotype 1b replicon-containing cells were cultured in the presence of a low, high, or stepwise-increasing TMC380765 concentration(s). HCV replicon RNA from representative samples thus obtained was analyzed using (i) population, (ii) clonal, and (iii) 454 deep sequencing technologies. Depending on the concentration of TMC380765, distinct mutational patterns emerged. In particular, culturing with low concentrations resulted in the selection of low-level resistance mutations (F43S and A156G), whereas high concentrations resulted in the selection of high-level resistance mutations (A156V, D168V, and D168A). Clonal and 454 deep sequencing analysis of the replicon RNA allowed the identification of low-frequency preexisting mutations possibly contributing to the mutational pattern that emerged. Stepwise-increasing TMC380765 concentrations resulted in the emergence and disappearance of multiple replicon variants in response to the changing selection pressure. Moreover, two different codons for the wild-type amino acids were observed at certain NS3 positions within one population of replicons, which may contribute to the emerging mutational patterns. Deep sequencing technologies enabled the study of minority variants present in the HCV quasispecies population present at baseline and during antiviral drug pressure, giving new insights into the dynamics of resistance acquisition by HCV.

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025(res) replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025(res) replicons. Unlike WT, DEB025(res) replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025(res) replicon. NMR titration experiments with peptides derived from the WT or the DEB025(res) domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

Background & Aims: Hepatitis C virus (HCV) establishes chronic infections in 3% of the world's population. Infection leads to progressive liver disease; hepatocytes are the major site of viral replication in vivo. However, chronic infection is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. We therefore screened a series of neural and brain-derived cell lines for their ability to support HCV entry and replication., Methods: We used a panel of neural-derived cell lines, HCV pseudoparticles (HCVpp), and an infectious, HCV JFH-1 cell-culture system (HCVcc) to assess viral tropism., Results: Two independently derived neuroepithelioma cell lines (SK-N-MC and SK-PN-DW) permitted HCVpp entry. In contrast, several neuroblastoma, glioma, and astrocytoma cell lines were refractory to HCVpp infection. HCVcc infected the neuroepithelioma cell lines and established a productive infection. Permissive neuroepithelioma cells expressed CD81, scavenger receptor BI (SR-BI), and the tight junction proteins Claudin-1 (CLDN1) and occludin, whereas nonpermissive neural cell lines lacked CLDN1 and, in some cases, SR-BI. HCVpp infection of the neuroepithelioma cells was neutralized by antibodies to CD81, SR-BI, CLDN1, and HCV E2. Furthermore, anti-CD81, interferon, and the anti-NS3 protease inhibitor VX-950 significantly reduced HCVcc infection of neuroepithelioma and hepatoma cells., Conclusions: Neuroepithelioma-derived cell lines express functional receptors that support HCV entry at levels comparable to those of hepatoma cells. HCV infection in vitro is not restricted to hepatic-derived cells, so HCV might infect cells of the CNS in vivo., (Copyright © 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Phosphoramidate dinucleosides named "GC 3'-OH" series, carrying various phosphoramidate linkages have been previously reported as hepatitis C virus (HCV) inhibitors. To enhance the efficacy of these dinucleotides, we synthesized a novel "GC 3'-H" series as potential chain terminators. We showed that their inhibition potency is strongly increased by the introduction of novel neutral and bis-negatively charged phosphoramidate side chains. Their inhibitory effect on HCV NS5B polymerase was evaluated in vitro and in HCV subgenomic replicon containing Huh-6 cells. As expected, 3'-H compounds are more potent than their 3'-OH counterparts to inhibit HCV polymerase activity. The most potent inhibitor, a 5'-phosphorylated dinucleotide bearing a bis-negatively charged amino side chain (7), exhibits an IC(50) value of 8 μM in vitro and EC(50) value of 2.6 μM in the HCV subgenomic replicon system. A molecular structure model is presented to propose an interpretation of the gain afforded by the of 3'-H-cytidine modification.

Novel S-aryltriazole acyclonucleosides were designed as structural analogs based on the previously identified antiviral aryltriazole acyclonucleosides in our laboratories. These S-aryltriazole nucleosides were synthesized in excellent yields via S(N)Ar-mediated S-arylation, followed by subsequent ammonolysis. X-ray structural analysis revealed special structural feature brought by the S-linkage, which may represent an unfavorable factor contributing to the lack of anti-HCV activity for this family of triazole nucleosides., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Unlabelled: Numerous anti-hepatitis C virus (HCV) drugs targeting either the viral nonstructural 3 (NS3) protease or NS5B polymerase are currently in clinical testing. However, rapid resistance development is a major problem and optimal therapy will clearly require a combination of multiple mechanisms of action. Cyclosporine A (CsA) and its nonimmunosuppressant derivatives are among the more promising drugs under development. Based on work with subgenomic HCV replicons it has been thought that they act as NS5B-inhibitors. In this study we show that CsA inhibits replication of full-length HCV Japanese Fulminant Hepatitis (JFH1) genomes about 10-fold more efficiently than subgenomic replicons. This effect is dependent on the presence of NS2 in the viral polyprotein and mediated through cellular cyclophilin A. NS2 is either an additional target for CsA-dependent inhibition or modulates the antiviral activity against NS3 to NS5B proteins. CsA is thus the first anti-HCV drug shown to act through NS2., Conclusion: CsA inhibits replication of JFH1 full-length genomes much more efficiently than subgenomic replicons by targeting cleavage at the NS2/NS3 junction and possibly other nonreplication lifecycle steps.

Hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B constitutes a target of choice for the development of anti-HCV drugs. Although many small molecules have been identified as allosteric inhibitors of NS5B, very few are active in clinical applications. We have screened 17,000 compounds in an enzymatic assay involving the purified NS5B in order to increase the therapeutic arsenal. We hoped to shed some light on the precise mechanism of RNA synthesis. We succeeded in isolating a series of 21 original inhibitors of the RNA synthesis by NS5B. Four of these non-nucleoside inhibitors (NNIs) could be mapped to the known binding site called 'B' as judged by the decrease in their inhibition potency when assayed with a 'B' site mutant, M423T NS5B. Incidentally, our in silico model pointed to Y477 as a key residue for inhibitor binding. In vitro, Y477F mutant loses its sensitivity to the newly discovered inhibitors but is unable to extend primers during the elongation phase. Our results demonstrate that elements of the 'B' site are involved in the conformational changes required in the switch between the different RNA synthesis steps and that compounds targeting this site could lock the enzyme in its initiation phase.

Beta-2'-C-methyl purines (1, 2) are known inhibitors of hepatitis C virus (HCV). We herein report the synthesis, biological and enzymatic evaluation of their 5'-phosphoramidate ProTides. Described herein are seven l-alanine phosphoramidate derivatives with variations to the amino acid ester. The 1-naphthyl phosphoramidate of beta-2'-methylguanosine containing the benzyl ester (20) was the most active at 0.12microM, an 84-fold of increase in activity compared to the parent nucleoside (2) with no increase of cytotoxicity. The carboxypeptidase mediated hydrolysis of several ProTides showed a predictive correlation with their activity versus HCV in replicon.

Unlabelled: Statins are 3-hydroxyl-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors used for the treatment of hypercholesterolemia. It was recently reported that statins inhibit in vitro hepatitis C virus (HCV) RNA replication. We here report that, of five statins studied, mevastatin and simvastatin exhibit the strongest in vitro anti-HCV activity, lovastatin and fluvastatin have moderate inhibitory effects, and pravastatin is devoid of an antiviral effect. A combination of statins with interferon-alpha (IFN-alpha) or HCV nonstructural (NS)5B polymerase or NS3 protease inhibitors results in an additive antiviral activity in short-term (3 days) antiviral assays. Neither statins, at a concentration of five-fold their median effective concentration (EC(50)) value, nor polymerase, protease inhibitors, or IFN-alpha, at concentrations 10- or 20-fold their EC(50) value, were able to clear cells from their replicon following four or six consecutive passages of antiviral pressure. However, the combination of HCV polymerase or protease inhibitors with mevastatin or simvastatin resulted in an efficient clearance of the cultures from their replicon. In colony formation experiments, mevastatin reduced the frequency or prevented the selection of HCV replicons resistant to the nonnucleoside inhibitor HCV-796., Conclusion: A combination of specific HCV inhibitors with statins may result in a more profound antiviral effect and may delay or prevent the development of resistance to such inhibitors.

Herein we report a successful application of a computer-aided design approach to identify a novel HCV helicase inhibitor. A de novo drug design methodology was used to generate an initial set of structures that could potentially bind to a putative binding site. Further structure refinement was carried out through docking a series of focused virtual libraries. The most promising compound was synthesised and it exhibited a submicromolar inhibition of the HCV helicase.

We report the synthesis and structure-activity relationship (SAR) of a large series of acridones and acridone-fragment derivatives designed on the basis of the selective antihepatitis C virus (HCV) activity shown by acridone 2, previously studied as a potential antibovine viral diarrhea virus (BVDV) compound. The evaluation of their ability to inhibit the HCV replication in Huh-5-2 cells led to the identification of new, selective inhibitors. This indicates that the acridone skeleton, when properly functionalized, is a suitable scaffold to obtain potential anti-HCV agents. Interestingly, during identification of possible cellular and viral targets, it was discovered that compound 23 exerts inhibitory activity on the HCV NS3 helicase, a very promising target for the development of anti-HCV drugs.

Background/aims: Following lead optimization, a set of substituted imidazopyridines was identified as potent and selective inhibitors of in vitro HCV replication. The particular characteristics of one of the most potent compounds in this series (5-[[3-(4-chlorophenyl)-5-isoxazolyl]methyl]-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine or GS-327073), were studied., Methods: Antiviral activity of GS-327073 was evaluated in HCV subgenomic replicons (genotypes 1b, 1a and 2a), in the JFH1 (genotype 2a) infectious system and against replicons resistant to various selective HCV inhibitors. Combination studies of GS-327073 with other selective HCV inhibitors were performed., Results: Fifty percent effective concentrations for inhibition of HCV subgenomic 1b replicon replication ranged between 2 and 50 nM and were 100-fold higher for HCV genotype 2a virus. The 50% cytostatic concentrations were > or = 17 microM, thus resulting in selectivity indices of > or = 340. GS-327073 retained wild-type activity against HCV replicons that were resistant to either HCV protease inhibitors or several polymerase inhibitors. GS-327073, when combined with either interferon alpha, ribavirin, a nucleoside polymerase or a protease inhibitor resulted in overall additive antiviral activity. Combinations containing GS-327073 proved highly effective in clearing hepatoma cells from HCV., Conclusions: GS-327073 is a potent in vitro inhibitor of HCV replication either alone or in combination with other selective HCV inhibitors.

A series of novel 2'-C-methylribonucleosides, involving 5-iodo and 5-alkynyl uridine analogues as well as related bicyclic furano- and pyrrolo[2,3-d]pyrimidinone compounds, has been synthesized and evaluated for their inhibitory effect on replication of the hepatitis C virus (HCV). The new nucleoside analogues did not show meaningful anti-HCV activity.

For establishment of the structure-activity relationship, 19 heterobicycle-coumarin conjugated compounds with the -SCH(2)- linker were synthesized and found to possess significant antiviral activities. Prominent examples included imidazopyridine-coumarin 12c, purine-coumarin 12d, and benzoxazole-coumarin 14c, which inhibited HCV replication at an EC(50) of 6.8, 2.0, and 12 microM, respectively. The heteroatoms in bicycles and the substituent effect on coumarin played essential roles.