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Hepatitis C virus (HCV) RNA is synthesized by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by pre-formed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPOR) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPOS) viruses (e.g. H77S.3 and N.2). In luciferase assays, LPOS HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPOR HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNA-dependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPOS H77S.3 and the LPOR H77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. Mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPOS and LPOR viruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) variants characterized by genomic deletions in the structural protein region have been sporadically detected in liver and serum of hepatitis C patients. These defective genomes are capable of autonomous RNA replication and are packaged into infectious viral particles in cells co-infected with the wild-type virus. The prevalence of such forms in the chronically HCV-infected population and the impact on the severity of liver disease or treatment outcome are currently unknown. In order to determine the prevalence of HCV defective variants and to study their association with clinical characteristics, a screening campaign was performed on pre-therapy serum samples from a well-characterized cohort of previously untreated genotype 1 HCV-infected patients who received treatment with PEG-IFNα and RBV. 132 subjects were successfully analyzed for the presence of defective species exploiting a long-distance nested PCR assay. HCV forms with deletions predominantly affecting E1, E2 and p7 proteins were found in a surprising high fraction of the subjects (25/132, 19%). Their presence was associated with patient older age, higher viral load and increased necroinflammatory activity in the liver. While the presence of circulating HCV carrying deletions in the E1-p7 region did not appear to significantly influence sustained virological response rates to PEG-IFNα/RBV, our study indicates that the presence of these subgenomic HCV mutants could be associated with virological relapse in patients who did not have detectable viremia at the end of the treatment. [ABSTRACT FROM AUTHOR]

4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase α (PI4KIIIα) has recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4- anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIα and, to a lesser extent, PI4KIIIβ. In Huh7.5 cells, PI4KIIIα is responsible for the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9, indicating that this agent inhibits PI4KIIIα also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi membrane, suggesting that the PI4KIIIβ isoform was not significantly inhibited under our experimental conditions. Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously observed upon silencing PI4KIIIα by RNA interference. In light of our findings, we propose that the antiviral effect of 4- anilino quinazoline compounds is mediated by the inhibition of PI4KIIIα and the consequent depletion of PI4P required for the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane. This observation implies that HCV - by recruiting PI4KIIIa in the RNA replication complex - hijacks PI4P metabolism, ultimately resulting in a markedly altered subcellular distribution of the PI4KIIIα product. [ABSTRACT FROM AUTHOR]

Abstract: The HCV NS3 protease and NS5B polymerase play essential roles in the replication of the hepatitis C virus (HCV). Following the successful paradigm established for HIV protease and reverse transcriptase inhibitors, these enzymes have been elected as targets for the development of small molecule HCV inhibitors. By combining the power of high-throughput screening with rational, knowledge-based drug discovery, a number of competitive inhibitors of the NS3 protease as well as nucleoside and non-nucleoside inhibitors of the NS5B polymerase have been identified and some have now entered clinical trials. In this article we review recent progress in the discovery and development of small molecule inhibitors of these two essential viral enzymes as they are advancing in the clinic. [Copyright &y& Elsevier]

Hepatitis C virus (HCV) will continue to be a serious global health threat for many years to come because of the chronic nature of the infection, its high prevalence and the significant morbidity of the resulting disease. Recently, a small number of molecules have produced encouraging results in proof-of-concept clinical trials. At the same time, preclinical evidence is accumulating that development of resistance will eventually limit the efficacy of new drugs. Thus, combinations of multiple agents will be required to treat chronic HCV infection. [ABSTRACT FROM AUTHOR]

The treatment of chronic disease caused by the hepatitis C virus (HCV) is an unmet clinical need, since current therapy is only partially effective and limited by undesirable side effects. The viral serine protease and the RNA-dependent RNA polymerase are the best-studied targets for the development of novel therapeutic agents. These enzymes have been extensively characterized at the biochemical and structural level and thus used to set up screening assays for the identification of selective inhibitors. These efforts lead to the discovery of several classes of compounds with potential antiviral activity. The hepatitis C virus does not replicate in the laboratory. The formidable challenge posed by the difficulty of developing cell-based assays and preclinical animal systems has been partially overcome with several alternative approaches. The development of new assays permitted the optimization of enzyme inhibitors leading eventually to molecules with the desired drug-like properties, the most advanced of which are being considered for clinical trials. [Copyright &y& Elsevier]

In this article, the author discusses introduction of direct-acting antivirals (DAA) treatments for chronic hepatitis C. Topics discussed include discovery of Nonstructural protein 5A (NS5A) inhibitors by random screening using the hepatitis C virus (HCV) replicon system, sequencing of replicons resistant to Daclatasvir (DCV), and multiple aspects of the HCV life cycle.

Presents a homology model that predicts the components of the zinc binding site of the serine protease domain of NS3 protein of hepatitis C virus. Need of zinc for enzymatic activity; Properties of the metal-substituted NS3 proteinases.

Abstract: The hepatitis C virus (HCV) nonstructural 5A (NS5A) phosphoprotein has been intensely studied due to its ability to subvert the host interferon-induced antiviral response. However, more recent studies suggest that it may also play an important regulatory role in HCV RNA replication as well as modulate host intracellular signaling pathways. Phosphorylation of NS5A appears to be a highly regulated process and several cellular protein kinases responsible for NS5A phosphorylation have been identified in vitro. Studies utilizing the HCV replicon cell culture system have suggested a provocative role for the differential phosphorylation of NS5A in the regulation of viral RNA replication through its association with the viral replication complex, including several host cell factors. Importantly, recent in vivo data linking loss of NS5A hyperphosphorylation to non-productive HCV replication in the chimpanzee model have provided high validation for targeting the cellular kinases involved, particularly the kinases responsible for NS5A phosphorylation, for antiviral therapeutic intervention. Understanding the process of NS5A phosphorylation and the definite identification of the culprit cellular protein kinase(s) will shed light on the mechanisms of HCV RNA replication and/or pathogenesis. [Copyright &y& Elsevier]

Naturally occurring hepatitis C virus (HCV) subgenomic RNAs have been found in several HCV patients. These subgenomic deletion mutants, mostly lacking the genes encoding envelope glycoproteins, were found in both liver and serum, where their relatively high abundance suggests that they are capable of autonomous replication and can be packaged and secreted in viral particles, presumably harboring the envelope proteins from wild type virus coinfecting the same cell. We recapitulated some of these natural subgenomic deletions in the context of the isolate JFH-1 and confirmed these hypotheses in vitro. In Huh-7.5 cells, these deletion-containing genomes show robust replication and can be efficiently trans-packaged and infect naïve Huh-7.5 cells when cotransfected with the full-length wild-type J6/JFH genome. The genome structure of these natural subgenomic deletion mutants was dissected, and the maintenance of both core and NS2 regions was proven to be significant for efficient replication and trans-packaging. To further explore the requirements needed to achieve trans-complementation, we provided different combinations of structural proteins in trans. Optimal trans-complementation was obtained when fragments of the polyprotein encompassing core to p7 or E1 to NS2 were expressed. Finally, we generated a stable helper cell line, constitutively expressing the structural proteins from core to p7, which efficiently supports trans-complementation of a subgenomic deletion encompassing amino acids 284 to 732. This cell line can produce and be infected by defective particles, thus representing a powerful tool to investigate the life cycle and relevance of natural HCV subgenomic deletion mutants in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Hepatitis C virus (HCV) exists in six major genotypes. Compared with the 1b enzyme, genotype 2b HCV polymerase exhibits a more than 100-fold reduction in sensitivity to the indole-N-acetamide class of non-nucleoside inhibitors. These compounds have been shown to bind in a pocket occupied by helix A of the mobile Λ1 loop in the apoenzyme. The three-dimensional structure of the HCV polymerase from genotype 2b was determined to 1.9-Å resolution and compared with the genotype 1b enzyme. This structural analysis suggests that genotypic variants result in a different shape of the inhibitor binding site. Mutants of the inhibitor binding pocket were generated in a 1b enzyme and evaluated for their binding affinity and sensitivity to inhibition by indole-N-acetamides. Most of the point mutants showed little variation in activity and IC50, with the exception of 15- and 7-fold increases in IC50 for Leu392Ile and Val494Ala mutants (1b→2b), respectively. Furthermore, a 1b replicon with 20-fold resistance to this class of inhibitors was selected and shown to contain the Leu392Ile mutation. Chimeric enzymes, where the 2b fingertip Λ1 loop, pocket or both replaced the corresponding regions of the 1b enzyme, were also generated. The fingertip chimera retained 1b-like inhibitor binding affinity, whereas the other two chimeric constructs and the 2b enzyme displayed between 50- and 100-fold reduction in binding affinity. Together, these data suggest that differences in the amino acid composition and shape of the indole-N-acetamide binding pocket are responsible for the resistance of the 2b polymerase to this class of inhibitors. [Copyright &y& Elsevier]

Summary: We present the first structure of a noncovalent inhibitor bound to the protease domain of hepatitis C virus NS3 protein (NS3p), solved by NMR. The inhibitor exploits interactions with the S′ region of NS3p to form a long-lived complex, although the absence of negative charges strongly reduces the association rate. The inhibitor stabilizes the N-terminal domain of NS3p and the substrate-binding site, and correctly aligns catalytic His-Asp residues. These actions were previously attributed exclusively to the cofactor NS4A, which interacts with the N-terminal domain of the NS3p and functions as an activator in vivo. The structure of the inhibitor/NS3p complex is very similar to that of the NS3p–NS4A complex, showing that binding of the NS4A cofactor is not the only event leading to a stable active-site conformation. [Copyright &y& Elsevier]

The hepatitis C virus encodes a single polyprotein that is processed by host and viral proteases to yield at least 10 mature viral proteins. The nonstructural (NS) protein 5A is a phosphoprotein, and experimental data indicate that the phosphorylation state of NS5A is important for the outcome of viral RNA replication. We were able to identify kinase inhibitors that specifically inhibit the formation of the hyperphosphorylated form of NS5A (p58) in cells, These kinase inhibitors were used for inhibitor affinity chromatography in order to identify the cellular targets of these compounds. The kinases casein kinase I (CKI), p38 MAPK, CIT (Citron Rho-interacting kinase), GAK, JNK2, PKA, RSK1/2, and RIPK2 were identified in the high affinity binding fractions of two NSSA hyperphosphorylation inhibitors (NS5A-p58-i). Even though these kinases are targets of the NS5A-p58-i, the only kinase showing an effect on NS5A hyperphosphorylation was confirmed to be CKI-α. Although this finding does not exclude the possibility that other kinase(s) might be involved in basal or regulatory phosphorylation of NS5A, we show here that NS5A is a direct substrate of CKI-α. Moreover, in vitro phosphorylation of NS5A by CKI-α resulted for the first time in the production of basal and hyperphosphorylated forms resembling those produced in cells. In vitro kinase reactions performed with NS5A peptides show that Ser-2204 is a preferred substrate residue for CKI-α after pre-phosphorylation of Ser-2201. [ABSTRACT FROM AUTHOR]

The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic intervention against HCV. We have deter- mined the crystal structures of the HCV polymerase complexed with two indole-based allosteric inhibitors at 2.3- and 2.4-Å resolution. The structures show that these inhibitors bind to a site on the surface of the thumb domain. A cyclohexyl and phenyl ring substituents, bridged by an indole moiety, fill two closely spaced pockets, whereas a carboxylate substituent forms a salt bridge with an exposed arginine side chain. Interestingly, in the apoenzyme, the inhibitor binding site is occupied by a small a-helix at the tip of the N-terminal loop that connects the fingers and thumb domains. Thus, these molecules inhibit the enzyme by preventing formation of intramolecular contacts between these two domains and consequently precluding their coordinated movements during RNA synthesis. Our structures identify a novel mechanism by which a new class of allosteric inhibitors inhibits the HCV polymerase and open the way to the development of novel antiviral agents against this clinically relevant human pathogen. [ABSTRACT FROM AUTHOR]

Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase (s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors. [ABSTRACT FROM AUTHOR]

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhihitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors. [ABSTRACT FROM AUTHOR]

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant to the inhibitors. Interestingly, proline 495 lies in a recently identified noncatalytic GTPbinding site, thus validating it as a potential allosteric site that can be targeted by small-molecule inhibitors of HCV polymerase. [ABSTRACT FROM AUTHOR]

The urgent need for efficacious drugs to treat chronic hepatitis C virus (HCV) infection requires a concerted effort to develop inhibitors specific for virally encoded enzymes. We demonstrate that 2'-C-methyl ribonucleosides are efficient chain-terminating inhibitors of HCV genome replication. Characterization of drug-resistant HCV replicons defined a single S282T mutation within the active site of the viral polymerase that conferred loss of sensitivity to structurally related compounds in both replicon and isolated polymerase assays. Biochemical analyses demonstrated that resistance at the level of the enzyme results from a combination of reduced affinity of the mutant polymerase for the drug and an increased ability to extend the incorporated nucleoside analog. Importantly, the combination of these agents with interferon-α results in synergistic inhibition of HCV genome replication in cell culture. Furthermore, 2'-C-methyl-substituted ribonucleosides also inhibited replication of genetically related viruses such as bovine diarrhea virus, yellow fever, and West African Nile viruses. These observations, together with the finding that 2'-C-methyl-guanosine in particular has a favorable pharmacological profile, suggest that this class of compounds may have broad utility in the treatment of HCV and other flavivirus infections. [ABSTRACT FROM AUTHOR]

Studies the inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs. Inhibition of NS5B enzyme activity; Gel-based incorporation assay; Inhibition of DNA polymerases; Intracellular metabolism to the active triphosphate.

The difluoromethyl group was designed by computational chemistry methods as a mimetic of the canonical P1 cysteine thiol for inhibitors of the hepatitis C virus NS3 protease. This modification led to the development of competitive, non-covalent inhibitor 4 (Ki 30 nM) and reversible covalent inhibitors (6, Ki 0.5 nM; and 8 Ki* 10 pM). [Copyright &y& Elsevier]

Presents a study which based the design of inhibitors with nanomolar potencies of hepatitis C virus (HCV) NS3 proteinase on eglin c. What HCV infection leads to; Information on the prevalence of HCV; Method used for the study; Results of the study.

States that in the absence of a cure for hepatitis C virus (HCV), there is a search for inhibitors of the virally encoded protease NS3. Information on HCV; Role of HCV in the onset of hepatocellular carcinoma; Function of NS3; Methods employed to conduct study on inhibitors for NS3; How these inhibitors were obtained; Implications of study's results.

States that the nonstructural protein NS3 of the hepatitis C virus (HCV), harbors a serine protease domain that is responsible for most of the processing events of the nonstructural region of the polyprotein. How inhibition of NS3 affects the disease caused by HCV; Methodology used to conduct inhibition process; Identification of an important determinant for product binding; Discussion on findings.

Hepatitis C virus (HCV) has been the subject of intensive studies for nearly two decades. Nevertheless, some aspects of the virus life cycle are still a mystery. The HCV nonstructural protein 5A (NS5A) has been shown to be a modulator of cellular processes possibly required for the establishment of viral persistence. NS5A is heavily phosphorylated, and a switch between a basally phosphorylated form of NS5A (p56) and a hyperphosphorylated form of NS5A (p58) seems to play a pivotal role in regulating HCV replication. Using kinase inhibitors that specifically inhibit the formation of NS5A-p58 in cells, we identified the CKI kinase family as a target. NS5A-p58 increased upon overexpression of CKI-α, CKI-δ, and CKI-ε, whereas the RNA interference of only CKI-α reduced NS5A hyperphosphorylation. Rescue of inhibition of NS5A-p58 was achieved by CKI-α overexpression, and we demonstrated that the CKI-α isoform is targeted by NS5A hyperphosphorylation inhibitors in living cells. Finally, we showed that down-regulation of CKI-α attenuates HCV RNA replication. [ABSTRACT FROM AUTHOR]