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1
Chatterji, Udayan, Jose A. Garcia-Rivera, James Baugh, Katarzyna Gawlik, Kelly A. Wong, Weidong Zhong, Clifford A. Brass, Nikolai V. Naoumov, and Philippe A. Gallay. "The Combination of Alisporivir plus an NS5A Inhibitor Provides Additive to Synergistic Anti-Hepatitis C Virus Activity without Detectable Cross-Resistance." Antimicrobial Agents and Chemotherapy 58, no. 6 (March 31, 2014): 3327–34. http://dx.doi.org/10.1128/aac.00016-14.
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ABSTRACTAlisporivir (ALV), a cyclophilin inhibitor, is a host-targeting antiviral (HTA) with multigenotypic anti-hepatitis C virus (HCV) activity and a high barrier to resistance. Recent advances have supported the concept of interferon (IFN)-free regimens to treat chronic hepatitis C. As the most advanced oral HTA, ALV with direct-acting antivirals (DAAs) represents an attractive drug combination for IFN-free therapy. In this study, we investigated whether particular DAAs exhibit additive, synergistic, or antagonistic effects when combined with ALV. Drug combinations of ALV with NS3 protease, NS5B polymerase, and NS5A inhibitors were investigated in HCV replicons from genotypes 1a, 1b, 2a, 3, and 4a (GT1a to -4a). Combinations of ALV with DAAs exerted an additive effect on GT1 and -4. A significant and specific synergistic effect was observed with ALV-NS5A inhibitor combination on GT2 and -3. Furthermore, ALV was fully active against DAA-resistant variants, and ALV-resistant variants were fully susceptible to DAAs. ALV blocks the contact between cyclophilin A and domain II of NS5A, and NS5A inhibitors target domain I of NS5A; our data suggest a molecular basis for the use of these two classes of inhibitors acting on two distinct domains of NS5A. These results providein vitroevidence that ALV with NS5A inhibitor combination represents an attractive strategy and a potentially effective IFN-free regimen for treatment of patients with chronic hepatitis C. Due to its high barrier and lack of cross-resistance, ALV could be a cornerstone drug partner for DAAs.
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Qiu, Dike, Julie A. Lemm, Donald R. O’Boyle, Jin-Hua Sun, Peter T. Nower, Van Nguyen, Lawrence G. Hamann, et al. "The effects of NS5A inhibitors on NS5A phosphorylation, polyprotein processing and localization." Journal of General Virology 92, no. 11 (November 1, 2011): 2502–11. http://dx.doi.org/10.1099/vir.0.034801-0.
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Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multi-functional protein that is expressed in basally phosphorylated (p56) and in hyperphosphorylated (p58) forms. NS5A phosphorylation has been implicated in regulating multiple aspects of HCV replication. We recently reported the identification of a class of compounds that potently inhibit HCV RNA replication by targeting NS5A. Although the precise mechanism of inhibition of these compounds is not well understood, one activity that has been described is their ability to block expression of the hyperphosphorylated form of NS5A. Here, we report that an NS5A inhibitor impaired hyperphosphorylation without affecting basal phosphorylation at the C-terminal region of NS5A. This inhibitor activity did not require NS5A domains II and III and was distinct from that of a cellular kinase inhibitor that also blocked NS5A hyperphosphorylation, results that are consistent with an inhibitor-binding site within the N-terminal region of NS5A. In addition, we observed that an NS5A inhibitor promoted the accumulation of an HCV polyprotein intermediate, suggesting that inhibitor binding to NS5A may occur prior to the completion of polyprotein processing. Finally, we observed that NS5A p56 and p58 separated into different membrane fractions during discontinuous sucrose gradient centrifugation, consistent with these NS5A phosphoforms performing distinct replication functions. The p58 localization pattern was disrupted by an NS5A inhibitor. Collectively, our results suggest that NS5A inhibitors probably impact several aspects of HCV expression and regulation. These findings may help to explain the exceptional potency of this class of HCV replication complex inhibitors.
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Kati, Warren, Gennadiy Koev, Michelle Irvin, Jill Beyer, Yaya Liu, Preethi Krishnan, Thomas Reisch, et al. "In VitroActivity and Resistance Profile of Dasabuvir, a Nonnucleoside Hepatitis C Virus Polymerase Inhibitor." Antimicrobial Agents and Chemotherapy 59, no. 3 (December 22, 2014): 1505–11. http://dx.doi.org/10.1128/aac.04619-14.
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ABSTRACTDasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.
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Bukhtiyarova, Marina, Christopher J. Rizzo, Charles A. Kettner, Bruce D. Korant, Helen T. Scarnati, and Robert W. King. "Inhibition of the Bovine Viral Diarrhoea Virus NS3 Serine Protease by a Boron-Modified Peptidyl Mimetic of its Natural Substrate." Antiviral Chemistry and Chemotherapy 12, no. 6 (December 2001): 367–73. http://dx.doi.org/10.1177/095632020101200607.
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Bovine viral diarrhoea virus (BVDV) is closely related to hepatitis C virus (HCV), and has been used as a surrogate virus in drug development for HCV infection. Similar to HCV, BVDV-encoded NS3 serine proteinase is responsible for multiple cleavages in the viral polyprotein, generating mature NS4A, NS4B, NS5A and NS5B proteins. NS3-dependent cleavage sites of BVDV contain a strictly conserved leucine at P1, and either serine or alanine at P1′. The full length BVDV NS3/4A serine protease has been cloned and expressed in bacterial cells. The enzyme has been purified from the soluble portion of Escherichia coli via a two-step purification procedure employing chromatography on heparin resin and gel filtration. The protease activity was characterized using in vitro translated BVDV NS4A/B and NS5A/B polyprotein substrates. A boronic acid analogue of the BVDV NS4A/NS4B cleavage site was synthesized and shown to be an efficient inhibitor of the NS3 serine protease in vitro. The compound, designated DPC-AB9144–00, inhibited approximately 75% of the NS3/4 activity at 10 μM with the NS4A/B substrate. However, no antiviral activity was detected with DPC-AB9144–00 in BVDV-infected Madin—Darby bovine kidney cells at concentrations as great as 90 μM, suggesting permeability or that other cellular-derived limitations were present.
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O'Boyle, Donald R., Peter T. Nower, Min Gao, Robert Fridell, Chunfu Wang, Piyasena Hewawasam, Omar Lopez, et al. "Synergistic Activity of Combined NS5A Inhibitors." Antimicrobial Agents and Chemotherapy 60, no. 3 (December 28, 2015): 1573–83. http://dx.doi.org/10.1128/aac.02639-15.
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Daclatasvir (DCV) is a first-in-class hepatitis C virus (HCV) nonstructural 5A replication complex inhibitor (NS5A RCI) that is clinically effective in interferon-free combinations with direct-acting antivirals (DAAs) targeting alternate HCV proteins. Recently, we reported NS5A RCI combinations that enhance HCV inhibitory potentialin vitro, defining a new class of HCV inhibitors termed NS5A synergists (J. Sun, D. R. O’Boyle II, R. A. Fridell, D. R. Langley, C. Wang, S. Roberts, P. Nower, B. M. Johnson F. Moulin, M. J. Nophsker, Y. Wang, M. Liu, K. Rigat, Y. Tu, P. Hewawasam, J. Kadow, N. A. Meanwell, M. Cockett, J. A. Lemm, M. Kramer, M. Belema, and M. Gao, Nature 527:245–248, 2015, doi:10.1038/nature15711). To extend the characterization of NS5A synergists, we tested new combinations of DCV and NS5A synergists against genotype (gt) 1 to 6 replicons and gt 1a, 2a, and 3a viruses. The kinetics of inhibition in HCV-infected cells treated with DCV, an NS5A synergist (NS5A-Syn), or a combination of DCV and NS5A-Syn were distinctive. Similar to activity observed clinically, DCV caused a multilog drop in HCV, followed by rebound due to the emergence of resistance. DCV–NS5A-Syn combinations were highly efficient at clearing cells of viruses, in line with the trend seen in replicon studies. The retreatment of resistant viruses that emerged using DCV monotherapy with DCV–NS5A-Syn resulted in a multilog drop and rebound in HCV similar to the initial decline and rebound observed with DCV alone on wild-type (WT) virus. A triple combination of DCV, NS5A-Syn, and a DAA targeting the NS3 or NS5B protein cleared the cells of viruses that are highly resistant to DCV. Our data support the observation that the cooperative interaction of DCV and NS5A-Syn potentiates both the genotype coverage and resistance barrier of DCV, offering an additional DAA option for combination therapy and tools for explorations of NS5A function.
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McCormick, Christopher J., David Brown, Stephen Griffin, Lisa Challinor, David J. Rowlands, and Mark Harris. "A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A." Journal of General Virology 87, no. 1 (January 1, 2006): 93–102. http://dx.doi.org/10.1099/vir.0.81180-0.
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Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.
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Garcia-Rivera, Jose A., Michael Bobardt, Udayan Chatterji, Sam Hopkins, Matthew A. Gregory, Barrie Wilkinson, Kai Lin, and Philippe A. Gallay. "Multiple Mutations in Hepatitis C Virus NS5A Domain II Are Required To Confer a Significant Level of Resistance to Alisporivir." Antimicrobial Agents and Chemotherapy 56, no. 10 (July 16, 2012): 5113–21. http://dx.doi.org/10.1128/aac.00919-12.
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ABSTRACTAlisporivir is the most advanced host-targeting antiviral cyclophilin (Cyp) inhibitor in phase III studies and has demonstrated a great deal of promise in decreasing hepatitis C virus (HCV) viremia in infected patients. In an attempt to further elucidate the mechanism of action of alisporivir, HCV replicons resistant to the drug were selected. Interestingly, mutations constantly arose in domain II of NS5A. To demonstrate that these mutations are responsible for drug resistance, they were reintroduced into the parental HCV genome, and the resulting mutant viruses were tested for replication in the presence of alisporivir or in the absence of the alisporivir target, CypA. We also examined the effect of the mutations on NS5A binding to itself (oligomerization), CypA, RNA, and NS5B. Importantly, the mutations did not affect any of these interactions. Moreover, the mutations did not preserve NS5A-CypA interactions from alisporivir rupture. NS5A mutations alone render HCV only slightly resistant to alisporivir. In sharp contrast, when multiple NS5A mutations are combined, significant resistance was observed. The introduction of multiple mutations in NS5A significantly restored viral replication in CypA knockdown cells. Interestingly, the combination of NS5A mutations renders HCV resistant to all classes of Cyp inhibitors. This study suggests that a combination of multiple mutations in domain II of NS5A rather than a single mutation is required to render HCV significantly and universally resistant to Cyp inhibitors. This in accordance within vivodata that suggest that alisporivir is associated with a low potential for development of viral resistance.
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Lemm, Julie A., Donald O'Boyle, Mengping Liu, Peter T. Nower, Richard Colonno, Milind S. Deshpande, Lawrence B. Snyder, et al. "Identification of Hepatitis C Virus NS5A Inhibitors." Journal of Virology 84, no. 1 (October 7, 2009): 482–91. http://dx.doi.org/10.1128/jvi.01360-09.
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ABSTRACT Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was ∼5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.
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Wong, Mun-Teng, and Steve S. Chen. "Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation." Journal of Virology 90, no. 20 (August 3, 2016): 9075–95. http://dx.doi.org/10.1128/jvi.00960-16.
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ABSTRACTHepatitis C virus (HCV) infection reorganizes cellular membranes to create an active viral replication site named the membranous web (MW). The role that human choline kinase-α (hCKα) plays in HCV replication remains elusive. Here, we first showed that hCKα activity, not the CDP-choline pathway, promoted viral RNA replication. Confocal microscopy and subcellular fractionation of HCV-infected cells revealed that a small fraction of hCKα colocalized with the viral replication complex (RC) on the endoplasmic reticulum (ER) and that HCV infection increased hCKα localization to the ER. In the pTM-NS3-NS5B model, NS3-NS5B expression increased the localization of the wild-type, not the inactive D288A mutant, hCKα on the ER, and hCKα activity was required for effective trafficking of hCKα and NS5A to the ER. Coimmunoprecipitation showed that hCKα was recruited onto the viral RC presumably through its binding to NS5A domain 1 (D1). hCKα silencing or treatment with CK37, an hCKα activity inhibitor, abolished HCV-induced MW formation. In addition, hCKα depletion hindered NS5A localization on the ER, interfered with NS5A and NS5B colocalization, and mitigated NS5A-NS5B interactions but had no apparent effect on NS5A-NS4B and NS4B-NS5B interactions. Nevertheless, hCKα activity was not essential for the binding of NS5A to hCKα or NS5B. These findings demonstrate that hCKα forms a complex with NS5A and that hCKα activity enhances the targeting of the complex to the ER, where hCKα protein, not activity, mediates NS5A binding to NS5B, thereby promoting functional membranous viral RC assembly and viral RNA replication.IMPORTANCEHCV infection reorganizes the cellular membrane to create an active viral replication site named the membranous web (MW). Here, we report that human choline kinase-α (hCKα) acts as an essential host factor for HCV RNA replication. A fraction of hCKα colocalizes with the viral replication complex (RC) on the endoplasmic reticulum (ER) in HCV-infected cells. NS3-NS5B expression increases ER localization of wild-type, but not D288A mutant, hCKα, and hCKα activity facilitates the transport of itself and NS5A to the ER. Silencing or inactivation of hCKα abrogates MW formation. Moreover, hCKα is recruited by NS5A independent of hCKα activity, presumably through binding to NS5A D1. hCKα activity then mediates the ER targeting of the hCKα-NS5A complex. On the ER membrane, hCKα protein,per se, induces NS5A binding to NS5B, thereby promoting membranous RC formation and viral RNA replication. Our study may benefit the development of hCKα-targeted anti-HCV therapeutics.
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Liu, Dandan, Juan Ji, Tanya P. Ndongwe, Eleftherios Michailidis, Charles M. Rice, Robert Ralston, and Stefan G. Sarafianos. "Fast Hepatitis C Virus RNA Elimination and NS5A Redistribution by NS5A Inhibitors Studied by a Multiplex Assay Approach." Antimicrobial Agents and Chemotherapy 59, no. 6 (April 6, 2015): 3482–92. http://dx.doi.org/10.1128/aac.00223-15.
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ABSTRACTWhile earlier therapeutic strategies for the treatment of hepatitis C virus (HCV) infection relied exclusively on interferon (IFN) and ribavirin (RBV), four direct-acting antiviral agents (DAAs) have now been approved, aiming for an interferon-free strategy with a short treatment duration and fewer side effects. To facilitate studies on the mechanism of action (MOA) and efficacy of DAAs, we established a multiplex assay approach, which employs flow cytometry, aGaussialuciferase reporter system, Western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), a limited dilution assay (50% tissue culture infectious dose [TCID50]), and an image profiling assay that follows the NS5A redistribution in response to drug treatment. We used this approach to compare the relative potency of various DAAs and the kinetics of their antiviral effects as a potential preclinical measure of their potential clinical utility. We evaluated the NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). In terms of kinetics, our data demonstrate that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, LDV has a more pronounced effect than DCV on the viral replication, assembly, and infectivity of released virus. Our approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations.
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Reghellin, V., L. Donnici, S. Fenu, V. Berno, V. Calabrese, M. Pagani, S. Abrignani, F. Peri, R. De Francesco, and P. Neddermann. "NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes." Antimicrobial Agents and Chemotherapy 58, no. 12 (September 15, 2014): 7128–40. http://dx.doi.org/10.1128/aac.03293-14.
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ABSTRACTThe hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIIIα complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIIIα-targeting inhibitor. In addition, both the NS5A and PI4KIIIα classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIIIα. Because of the similarities between the effects induced by treatment with PI4KIIIα or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIIIα complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIIIα complex.
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Simicic, Petra, Anamarija Slovic, Leona Radmanic, Adriana Vince, and Snjezana Zidovec Lepej. "Molecular Epidemiology and Baseline Resistance of Hepatitis C Virus to Direct Acting Antivirals in Croatia." Pathogens 11, no. 7 (July 19, 2022): 808. http://dx.doi.org/10.3390/pathogens11070808.
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Molecular epidemiology of hepatitis C virus (HCV) is exceptionally complex due to the highly diverse HCV genome. Genetic diversity, transmission dynamics, and epidemic history of the most common HCV genotypes were inferred by population sequencing of the HCV NS3, NS5A, and NS5B region followed by phylogenetic and phylodynamic analysis. The results of this research suggest high overall prevalence of baseline NS3 resistance associate substitutions (RAS) (33.0%), moderate prevalence of NS5A RAS (13.7%), and low prevalence of nucleoside inhibitor NS5B RAS (8.3%). Prevalence of RAS significantly differed according to HCV genotype, with the highest prevalence of baseline resistance to NS3 inhibitors and NS5A inhibitors observed in HCV subtype 1a (68.8%) and subtype 1b (21.3%), respectively. Phylogenetic tree reconstructions showed two distinct clades within the subtype 1a, clade I (62.4%) and clade II (37.6%). NS3 RAS were preferentially associated with clade I. Phylogenetic analysis demonstrated that 27 (9.0%) HCV sequences had a presumed epidemiological link with another sequence and classified into 13 transmission pairs or clusters which were predominantly comprised of subtype 3a viruses and commonly detected among intravenous drug users (IDU). Phylodynamic analyses highlighted an exponential increase in subtype 1a and 3a effective population size in the late 20th century, which is a period associated with an explosive increase in the number of IDU in Croatia.
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Kumthip, Kattareeya, Pattranuch Chusri, Nikolaus Jilg, Lei Zhao, Dahlene N. Fusco, Hong Zhao, Kaku Goto, et al. "Hepatitis C Virus NS5A Disrupts STAT1 Phosphorylation and Suppresses Type I Interferon Signaling." Journal of Virology 86, no. 16 (June 6, 2012): 8581–91. http://dx.doi.org/10.1128/jvi.00533-12.
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Responses to alpha interferon (IFN-α)-based treatment are dependent on both host and viral factors and vary markedly among patients infected with different hepatitis C virus (HCV) genotypes (GTs). Patients infected with GT3 viruses consistently respond better to IFN treatment than do patients infected with GT1 viruses. The mechanisms underlying this difference are not well understood. In this study, we sought to determine the effects of HCV NS5A proteins from different genotypes on IFN signaling. We found that the overexpression of either GT1 or GT3 NS5A proteins significantly inhibited IFN-induced IFN-stimulated response element (ISRE) signaling, phosphorylated STAT1 (P-STAT1) levels, and IFN-stimulated gene (ISG) expression compared to controls. GT1 NS5A protein expression exhibited stronger inhibitory effects on IFN signaling than did GT3 NS5A protein expression. Furthermore, GT1 NS5A bound to STAT1 with a higher affinity than did GT3 NS5A. Domain mapping revealed that the C-terminal region of NS5A conferred these inhibitory effects on IFN signaling. The overexpression of HCV NS5A increased HCV replication levels in JFH1-infected cells through the further reduction of levels of P-STAT1, ISRE signaling, and downstream ISG responses. We demonstrated that the overexpression of GT1 NS5A proteins resulted in less IFN responsiveness than did the expression of GT3 NS5A proteins through stronger binding to STAT1. We confirmed that GT1 NS5A proteins exerted stronger IFN signaling inhibition than did GT3 NS5A proteins in an infectious recombinant JFH1 virus. The potent antiviral NS5A inhibitor BMS-790052 did not block NS5A-mediated IFN signaling suppression in an overexpression model, suggesting that NS5A's contributions to replication are independent of its subversive action on IFN. We propose a model in which the binding of the C-terminal region of NS5A to STAT1 leads to decreased levels of P-STAT1, ISRE signaling, and ISG transcription and, ultimately, to preferential GT1 resistance to IFN treatment.
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Krishnan, Preethi, Rakesh Tripathi, Gretja Schnell, Thomas Reisch, Jill Beyer, Michelle Irvin, Wangang Xie, et al. "Resistance Analysis of Baseline and Treatment-Emergent Variants in Hepatitis C Virus Genotype 1 in the AVIATOR Study with Paritaprevir-Ritonavir, Ombitasvir, and Dasabuvir." Antimicrobial Agents and Chemotherapy 59, no. 9 (June 22, 2015): 5445–54. http://dx.doi.org/10.1128/aac.00998-15.
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ABSTRACTAVIATOR, a phase 2 clinical trial, evaluated ritonavir-boosted paritaprevir (a protease inhibitor), ombitasvir (an NS5A inhibitor), and dasabuvir (a nonnucleoside polymerase inhibitor) (the three-drug [3D] regimen) with or without ribavirin (RBV) for 8, 12, or 24 weeks in 406 HCV genotype 1 (GT1)-infected patients. The rate of sustained virologic response 24 weeks after treatment ranged from 88% to 100% across the arms of the 3D regimen with or without RBV; 20 GT1a-infected patients and 1 GT1b-infected patient experienced virologic failure (5.2%). Baseline resistance-conferring variants in NS3 were rare. M28V in GT1a and Y93H in GT1b were the most prevalent preexisting variants in NS5A, and C316N in GT1b and S556G in both GT1a and GT1b were the most prevalent variants in NS5B. Interestingly, all the GT1a sequences encoding M28V in NS5A were from the United States, while GT1b sequences encoding C316N and S556G in NS5B were predominant in the European Union. Variants preexisting at baseline had no significant impact on treatment outcome. The most prevalent treatment-emergent resistance-associated variants (RAVs) in GT1a were R155K and D168V in NS3, M28T and Q30R in NS5A, and S556G in NS5B. The single GT1b-infected patient experiencing virologic failure had no RAVs in any target. A paritaprevir-ritonavir dose of 150/100 mg was more efficacious in suppressing R155K in NS3 than a 100/100-mg dose. In patients who failed after receiving 12 or more weeks of treatment, RAVs were selected in all 3 targets, while most patients who relapsed after 8 weeks of treatment did so without any detectable RAVs. Results from this study guided the selection of the optimal treatment regimen, treatment duration, and paritaprevir dose for further development of the 3D regimen. (This study has been registered at ClinicalTrials.gov under registration number NCT01464827.)
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Ng, Teresa, Tami Pilot-Matias, Rakesh Tripathi, Gretja Schnell, Preethi Krishnan, Thomas Reisch, Jill Beyer, et al. "Resistance Analysis of a 3-Day Monotherapy Study with Glecaprevir or Pibrentasvir in Patients with Chronic Hepatitis C Virus Genotype 1 Infection." Viruses 10, no. 9 (August 28, 2018): 462. http://dx.doi.org/10.3390/v10090462.
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Glecaprevir (an NS3/4A protease inhibitor) and pibrentasvir (an NS5A inhibitor) are potent and pangenotypic hepatitis C virus (HCV) direct-acting antivirals. This report describes the baseline polymorphisms and treatment-emergent substitutions in NS3 or NS5A detected in samples from HCV genotype 1-infected patients receiving 3-day monotherapy of glecaprevir or pibrentasvir, respectively. None of the NS3 polymorphisms detected in the 47 baseline samples collected prior to glecaprevir monotherapy conferred reduced susceptibility to glecaprevir. The NS3 A156T substitution, which conferred resistance to glecaprevir but had low replication efficiency, emerged in one genotype 1a-infected patient among the 35 patients with available post-baseline sequence data. Baseline NS5A polymorphisms were detected in 12 of 40 patients prior to pibrentasvir monotherapy; most polymorphisms were single-position NS5A amino acid substitutions that did not confer resistance to pibrentasvir. Among the 19 patients with available post-baseline NS5A sequence data, 3 had treatment-emergent NS5A substitutions during pibrentasvir monotherapy. All treatment-emergent NS5A substitutions were linked multiple-position, almost exclusively double-position, substitutions that conferred resistance to pibrentasvir. Replicons engineered with these double-position substitutions had low replication efficiency. In conclusion, resistance-conferring substitutions emerged in a small number of genotype 1-infected patients during glecaprevir or pibrentasvir monotherapy; unlike other NS5A inhibitors, pibrentasvir did not select single-position NS5A substitutions during monotherapy.
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Yau, Alan Hoi Lun, and Eric M. Yoshida. "Hepatitis C Drugs: The End of the Pegylated Interferon Era and the Emergence of All-Oral, Interferon-Free Antiviral Regimens: A Concise Review." Canadian Journal of Gastroenterology and Hepatology 28, no. 8 (2014): 445–51. http://dx.doi.org/10.1155/2014/549624.
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Between 2001 and 2011, the standard of care for chronic hepatitis C virus (HCV) infection was a combination of pegylated interferon (PEGIFN) and ribavirin (RBV). In May 2011, boceprevir and telaprevir, two first-generation NS3/4A protease inhibitors, were approved in combination with PEG-IFN and RBV for 24 to 48 weeks in hepatitis C virus genotype 1 infections. In December 2013, simeprevir, a second-generation NS3/4A protease inhibitor, was approved for use with PEG-IFN and RBV for 12 weeks in genotype 1, while sofosbuvir, a NS5B nucleotide polymerase inhibitor, was approved for use with PEG-IFN and RBV for 12 weeks in genotypes 1 and 4, as well as with RBV alone for 12 weeks in genotype 2 and for 24 weeks in genotype 3. Sofosbuvir combined with simeprevir or an NS5A replication complex inhibitor (ledipasvir or daclatasvir) with or without RBV for 12 weeks in genotype 1 resulted in a sustained virological response >90%, irrespective of previous treatment history or presence of cirrhosis. Similarly impressive sustained virological response rates have been shown with ABT-450/r (ritonavir-boosted NS3/4A protease inhibitor)-based regimens in combination with other direct-acting antiviral agent(s) with or without RBV for 12 weeks in genotype 1. The optimal all-oral interferon-free antiviral regimen likely entails a combination of an NS5B nucleotide polymerase inhibitor with either a second-generation NS3/4A protease inhibitor or an NS5A replication complex inhibitor with or without RBV. Further research is needed to determine the role of resistance testing, clarify the optimal follow-up duration post-treatment, and evaluate the antiviral efficacy and safety in difficult-to-cure patient populations.
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Friborg, Jacques, Steven Levine, Chaoqun Chen, Amy K. Sheaffer, Susan Chaniewski, Stacey Voss, Julie A. Lemm, and Fiona McPhee. "Combinations of Lambda Interferon with Direct-Acting Antiviral Agents Are Highly Efficient in Suppressing Hepatitis C Virus Replication." Antimicrobial Agents and Chemotherapy 57, no. 3 (December 28, 2012): 1312–22. http://dx.doi.org/10.1128/aac.02239-12.
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ABSTRACTThe clinical efficacy of a pegylated form of human lambda 1 interferon (IFN-λ1; also referred to herein as lambda) has been demonstrated in patients chronically infected with hepatitis C virus (HCV) representing genotypes 1 through 4. In these proof-of-concept studies, lambda showed an improved safety profile compared to the pegylated form of alfa interferon (referred to herein as alfa). In the study described in this report, an assessment of thein vitroantiviral activity of type III IFNs toward different HCV replicons revealed that the unpegylated recombinant form of IFN-λ1 (rIFN-λ1) exerted the most robust effect, while rIFN-λ3 exhibited greater activity than rIFN-λ2. More importantly, cross-resistance to rIFN-λ1 was not observed in replicon cell lines known to have reduced susceptibility to investigational direct-acting antiviral (DAA) agents targeting the essential HCV nonstructural protein NS3, NS5A, or NS5B. When combined with either rIFN-α, the NS3 protease inhibitor (NS3 PI) asunaprevir (ASV), the NS5A replication complex inhibitor (NS5A RCI) daclatasvir (DCV), or the NS5B polymerase site I inhibitor (NS5B I) BMS-791325, rIFN-λ1 displayed a mixture of additive and synergistic effects. In three-drug combination studies, inclusion of lambda with ASV and DCV also yielded additive to synergistic effects. In line with these observations, it was demonstrated that a regimen that used a combination of rIFN-λ1 with one or two DAAs was superior to an IFN-free regimen in clearing HCV RNA in genotype 1a cell lines representing wild-type and NS3 protease inhibitor-resistant sequences. Overall, these data support further clinical development of lambda as part of alternative combination treatments with DAAs for patients chronically infected with HCV.
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Lemm, Julie A., Mengping Liu, Robert G. Gentles, Min Ding, Stacey Voss, Lenore A. Pelosi, Ying-Kai Wang, et al. "Preclinical Characterization of BMS-791325, an Allosteric Inhibitor of Hepatitis C Virus NS5B Polymerase." Antimicrobial Agents and Chemotherapy 58, no. 6 (April 14, 2014): 3485–95. http://dx.doi.org/10.1128/aac.02495-13.
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ABSTRACTBMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC50) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC50s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC50, 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC50s of >4 μM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC50. Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposuresin vivoin several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥10-fold above the inhibitor EC50s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.
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Pelosi, Lenore A., Stacey Voss, Mengping Liu, Min Gao, and Julie A. Lemm. "Effect on Hepatitis C Virus Replication of Combinations of Direct-Acting Antivirals, Including NS5A Inhibitor Daclatasvir." Antimicrobial Agents and Chemotherapy 56, no. 10 (July 30, 2012): 5230–39. http://dx.doi.org/10.1128/aac.01209-12.
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ABSTRACTThree hepatitis C virus (HCV) inhibitors, asunaprevir (ASV; BMS-650032), daclatasvir (DCV; BMS-790052), and BMS-791325, each targeting a different nonstructural protein of the virus (NS3, NS5A, and NS5B, respectively), have independently demonstrated encouraging preclinical profiles and are currently undergoing clinical evaluation. Since drug-resistant variants have rapidly developed in response to monotherapy with almost all direct-acting antiviral agents (DAAs) for HCV, the need for combination therapies to effectively eradicate the virus from infected patients is clear. These studies demonstrated the additive-synergistic effects on replicon inhibition and clearance of combining NS3 protease or NS5B RNA polymerase inhibitors with the first-in-class, NS5A replication complex inhibitor daclatasvir (DCV) and reveal new resistance pathways for combinations of two small-molecule inhibitors that differ from those that develop during monotherapy. The results suggest that under a specific selective pressure, a balance must be reached in the fitness costs of substitutions in one target gene when substitutions are also present in another target gene. Further synergies and additional novel resistance substitutions were observed during triple-combination treatment relative to dual-drug therapy, indicating that, in combination, HCV inhibitors can exert cross-target influences on resistance development. Enhanced synergies in replicon inhibition and a reduced frequency of resistance together lend strong support to the utility of combinations of DAAs for the treatment of HCV, and the identification of altered resistance profiles during combination treatment provides useful information for monitoring resistance in the clinic.
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Hikita, Hayato, and Tetsuo Takehara. "NS5A-P32 Deletion in Hepatitis C Genotype 1b Infection is the Most Refractory Treatment-Mediated Amino Acid Change Exhibiting Resistance to all NS5A Inhibitors." Seminars in Liver Disease 40, no. 02 (December 13, 2019): 143–53. http://dx.doi.org/10.1055/s-0039-3402001.
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AbstractNS5A-P32 deletion (P32del) is a resistance-associated amino acid change that has recently gained popularity in direct-acting antiviral treatment for chronic hepatitis C. Although not yet detected in naive patients, it appears in 5 to 10% of hepatitis C genotype 1b patients who fail to respond to daclatasvir/asunaprevir and sofosbuvir/ledipasvir treatments. In contrast to signature resistance-associated substitutions, such as substitutions at the NS5A-L31 and NS5A-Y93 positions, it shows complete resistance to all NS5A inhibitors in replicon and cell culture. Studies of humanized liver mice suggest that P32del retains good replication fitness and requires two classes of antivirals, except NS5A inhibitors, to be suppressed effectively. Patients with the P32del virus do not respond to glecaprevir/pibrentasvir but do respond to sofosbuvir/velpatasvir/voxilaprevir, presumably to sofosbuvir + glecaprevir/pibrentasvir, and at least partially to sofosbuvir/velpatasvir + ribavirin. Attention should be given to P32del in patients who experience failure with any NS5A inhibitor, especially those with genotype 1b infection.
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Hopkins, Sam, Michael Bobardt, Udayan Chatterji, Jose A. Garcia-Rivera, Precious Lim, and Philippe A. Gallay. "The Cyclophilin Inhibitor SCY-635 Disrupts Hepatitis C Virus NS5A-Cyclophilin A Complexes." Antimicrobial Agents and Chemotherapy 56, no. 7 (May 14, 2012): 3888–97. http://dx.doi.org/10.1128/aac.00693-12.
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ABSTRACTThe nonimmunosuppressive cyclophilin (Cyp) inhibitor SCY-635 blocks hepatitis C virus (HCV) replication bothin vitroandin vivoand represents a novel potent anti-HCV agent. However, its mechanism of action remains to be fully elucidated. A growing body of evidence suggests that cyclophilin A (CypA) is absolutely necessary for HCV replication and that the HCV nonstructural 5A (NS5A) protein serves as a main viral ligand for CypA. In this study, we examined the effect of SCY-635 on HCV replication. Specifically, we asked whether SCY-635 blocks HCV replication by targeting CypA-NS5A interactions. We also investigated the possibility that HCV can escape SCY-635 selection pressure and whether this resistance influences either CypA-NS5A interactions or the dependence of HCV on CypA. We found not only that SCY-635 efficiently inhibits HCV replication, but it is sufficient alone to clear HCV replicon-containing cells. We found that SCY-635 prevents CypA-NS5A interactions in a dose-dependent manner. SCY-635 prevents the contact between CypA and NS5A derived from genotypes 1 to 3. Together, these data suggest that NS5A-CypA interactions control HCV replication and that SCY-635 blocks viral replication by preventing the formation of these complexes. We also found that NS5A mutant proteins found in SCY-635-resistant HCV replicons behave similarly to wild-type NS5A in terms of both CypA binding and SCY-635-mediated dissociation and inhibition of CypA binding. However, the NS5A mutations found in SCY-635-resistant HCV replicons rescued viral replication in CypA-knockdown cells, suggesting that the NS5A mutations, which arosein vitrounder SCY-635 selection, do not alter the binding affinity of CypA for NS5A. These specific mutations in NS5A eliminate the dependence of HCV RNA replication on the expression of host CypA
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McPhee, Fiona, Amy K. Sheaffer, Jacques Friborg, Dennis Hernandez, Paul Falk, Guangzhi Zhai, Steven Levine, et al. "Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032)." Antimicrobial Agents and Chemotherapy 56, no. 10 (August 6, 2012): 5387–96. http://dx.doi.org/10.1128/aac.01186-12.
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ABSTRACTAsunaprevir (ASV; BMS-650032) is a hepatitis C virus (HCV) NS3 protease inhibitor that has demonstrated efficacy in patients chronically infected with HCV genotype 1 when combined with alfa interferon and/or the NS5A replication complex inhibitor daclatasvir. ASV competitively binds to the NS3/4A protease complex, withKivalues of 0.4 and 0.24 nM against recombinant enzymes representing genotypes 1a (H77) and 1b (J4L6S), respectively. Selectivity was demonstrated by the absence of any significant activity against the closely related GB virus-B NS3 protease and a panel of human serine or cysteine proteases. In cell culture, ASV inhibited replication of HCV replicons representing genotypes 1 and 4, with 50% effective concentrations (EC50s) ranging from 1 to 4 nM, and had weaker activity against genotypes 2 and 3 (EC50, 67 to 1,162 nM). Selectivity was again demonstrated by the absence of activity (EC50, >12 μM) against a panel of other RNA viruses. ASV exhibited additive or synergistic activity in combination studies with alfa interferon, ribavirin, and/or inhibitors specifically targeting NS5A or NS5B. Plasma and tissue exposuresin vivoin several animal species indicated that ASV displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC50s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients.
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Svitkin, Yuri V., Arnim Pause, Marcelo Lopez-Lastra, Sandra Perreault, and Nahum Sonenberg. "Complete Translation of the Hepatitis C Virus Genome In Vitro: Membranes Play a Critical Role in the Maturation of All Virus Proteins except for NS3." Journal of Virology 79, no. 11 (June 1, 2005): 6868–81. http://dx.doi.org/10.1128/jvi.79.11.6868-6881.2005.
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ABSTRACT We developed an in vitro translation extract from Krebs-2 cells that translates the entire open reading frame of the hepatitis C virus (HCV) strain H77 and properly processes the viral protein precursors when supplemented with canine microsomal membranes (CMMs). Translation of the C-terminal portion of the viral polyprotein in this system is documented by the synthesis of NS5B. Evidence for posttranslational modification of the viral proteins, the N-terminal glycosylation of E1 and the E2 precursor (E2-p7), and phosphorylation of NS5A is presented. With the exception of NS3, efficient generation of all virus-specific proteins is CMM dependent. A time course of the appearance of HCV products indicates that the viral polyprotein is cleaved cotranslationally. A competitive inhibitor of the NS3 protease inhibited accumulation of NS3, NS4B, NS5A, and NS5B, but not that of NS2 or structural proteins. CMMs also stabilized HCV mRNA during translation. Finally, the formyl-[35S]methionyl moiety of the initiator tRNAMet was incorporated exclusively into the core protein portion of the polyprotein, demonstrating that translation initiation in this system occurs with high fidelity.
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Schnell, Gretja, Rakesh Tripathi, Jill Beyer, Thomas Reisch, Preethi Krishnan, Liangjun Lu, Tatyana Dekhtyar, et al. "Hepatitis C Virus Genotype 4 Resistance and Subtype Demographic Characterization of Patients Treated with Ombitasvir plus Paritaprevir/Ritonavir." Antimicrobial Agents and Chemotherapy 59, no. 11 (August 17, 2015): 6807–15. http://dx.doi.org/10.1128/aac.01229-15.
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ABSTRACTHepatitis C virus (HCV) genotype 4 (GT4) is genetically diverse, with 17 confirmed subtypes, and comprises approximately 13% of infections worldwide. In this study, we identified GT4 subtypes by phylogenetic analysis, assessed differences in patient demographics across GT4 subtypes, examined baseline sequence variability among subtypes and the potential impact on treatment outcome, and analyzed the development of viral resistance in patients who received a regimen of ombitasvir (nonstructural protein 5A [NS5A] inhibitor) plus ritonavir-boosted paritaprevir (NS3/4A inhibitor) with or without ribavirin (RBV) for the treatment of HCV GT4 infection. Phylogenetic analysis of HCV NS3/4A, NS5A, and NS5B nucleotide sequences identified 7 subtypes (4a, 4b, 4c, 4d, 4f, 4g/4k, and 4o) among 132 patient samples. Subtype prevalence varied by country, and the distributions of patient birth cohort and race were significantly different across GT4 subtypes 4a, 4d, and non-4a/4d. Baseline amino acid variability was detected in NS5A across GT4 subtypes but had no impact on treatment outcome. Three patients experienced virologic failure and were infected with subtype 4d, and the predominant resistance-associated variants at the time of failure were D168V in NS3 and L28V in NS5A. Overall, high response rates were observed among patients infected with 7 HCV GT4 subtypes, with no impact of baseline variants on treatment outcome. GT4 subtype distribution in this study differed based on patient demographics and geography.
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Krishnan, Preethi, Jill Beyer, Neeta Mistry, Gennadiy Koev, Thomas Reisch, David DeGoey, Warren Kati, et al. "In VitroandIn VivoAntiviral Activity and Resistance Profile of Ombitasvir, an Inhibitor of Hepatitis C Virus NS5A." Antimicrobial Agents and Chemotherapy 59, no. 2 (December 1, 2014): 979–87. http://dx.doi.org/10.1128/aac.04226-14.
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ABSTRACTOmbitasvir (ABT-267) is a hepatitis C virus (HCV) NS5A inhibitor with picomolar potency, pan-genotypic activity, and 50% effective concentrations (EC50s) of 0.82 to 19.3 pM against HCV genotypes 1 to 5 and 366 pM against genotype 6a. Ombitasvir retained these levels of potency against a panel of 69 genotype 1 to 6 chimeric replicons containing the NS5A gene derived from HCV-infected patients, despite the existence of natural sequence diversity within NS5A.In vitroresistance selection identified variants that conferred resistance to ombitasvir in the HCV NS5A gene at amino acid positions 28, 30, 31, 58, and 93 in genotypes 1 to 6. Ombitasvir was evaluatedin vivoin a 3-day monotherapy study in 12 HCV genotype 1-infected patients at 5, 25, 50, or 200 mg dosed once daily. All patients in the study were HCV genotype 1a infected and were without preexisting resistant variants at baseline as determined by clonal sequencing. Decreases in HCV RNA up to 3.1 log10IU/ml were observed. Resistance-associated variants at position 28, 30, or 93 in NS5A were detected in patient samples 48 hours after the first dose. Clonal sequencing analysis indicated that wild-type virus was largely suppressed by ombitasvir during 3-day monotherapy, and at doses higher than 5 mg, resistant variant M28V was also suppressed. Ombitasvir was well tolerated at all doses, and there were no serious or severe adverse events. These data support clinical development of ombitasvir in combination with inhibitors targeting HCV NS3/4A protease (ABT-450 with ritonavir) and HCV NS5B polymerase (ABT-333, dasabuvir) for the treatment of chronic HCV genotype 1 infection. (Study M12-116 is registered at ClinicalTrials.gov under registration no. NCT01181427.)
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Hamdy, Jehad, Nouran Emadeldin, Mostafa Hamed, Efseveia Frakolaki, Sotirios Katsamakas, Niki Vassilaki, Grigoris Zoidis, Anna Hirsch, Mohammad Abdel-Halim, and Ashraf Abadi. "Design and Synthesis of Novel Bis-Imidazolyl Phenyl Butadiyne Derivatives as HCV NS5A Inhibitors." Pharmaceuticals 15, no. 5 (May 20, 2022): 632. http://dx.doi.org/10.3390/ph15050632.
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In today’s global plan to completely eradicate hepatitis C virus (HCV), the essential list of medications used for HCV treatment are direct-acting antivirals (DAAs), as interferon-sparing regimens have become the standard-of-care (SOC) treatment. HCV nonstructural protein 5A (NS5A) inhibitors are a very common component of these regimens. Food and Drug Administration (FDA)-approved NS5A inhibitors, although very potent, do not have the same potency against all eight genotypes of HCV. Therefore, this study aims to synthesize NS5A inhibitor analogues with high potency pan-genotypic activity and high metabolic stability. Starting from an NS5A inhibitor scaffold previously identified by our research group, we made several modifications. Two series of compounds were created to test the effect of changing the length and spatial conformation (para-para vs. meta-meta-positioned bis-imidazole-proline-carbamate), replacing amide groups in the linker with imidazole groups, as well as different end-cap compositions and sizes. The frontrunner inhibits genotype 1b (Con1) replicon, with an EC50 value in the picomolar range, and showed high genotypic coverage with nanomolar range EC50 values against four more genotypes. This together with its high metabolic stability (t½ > 120 min) makes it a potential preclinical candidate.
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Gottwein, Judith M., Sanne B. Jensen, Yi-Ping Li, Lubna Ghanem, Troels K. H. Scheel, Stéphanie B. N. Serre, Lotte Mikkelsen, and Jens Bukh. "Combination Treatment with Hepatitis C Virus Protease and NS5A Inhibitors Is Effective against Recombinant Genotype 1a, 2a, and 3a Viruses." Antimicrobial Agents and Chemotherapy 57, no. 3 (December 28, 2012): 1291–303. http://dx.doi.org/10.1128/aac.02164-12.
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ABSTRACTWith the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants relying only on the JFH1 NS3 helicase, NS5B, and the 3′ untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ∼4 log10focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reportedin vivo. In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a, and 3a viruses were suppressed by combination treatment with relatively low concentrations.
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de Salazar, Adolfo, Julia Dietz, Velia Chiara di Maio, Johannes Vermehren, Stefania Paolucci, Beat Müllhaupt, Nicola Coppola, et al. "Prevalence of resistance-associated substitutions and retreatment of patients failing a glecaprevir/pibrentasvir regimen." Journal of Antimicrobial Chemotherapy 75, no. 11 (August 8, 2020): 3349–58. http://dx.doi.org/10.1093/jac/dkaa304.
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Abstract Objectives To investigate resistance-associated substitutions (RASs) as well as retreatment efficacies in a large cohort of European patients with failure of glecaprevir/pibrentasvir. Methods Patients were identified from three European Resistance Reference centres in Spain, Italy and Germany. Sequencing of NS3, NS5A and NS5B was conducted and substitutions associated with resistance to direct antiviral agents were analysed. Clinical and virological parameters were documented retrospectively and retreatment efficacies were evaluated. Results We evaluated 90 glecaprevir/pibrentasvir failures [3a (n = 36), 1a (n = 23), 2a/2c (n = 20), 1b (n = 10) and 4d (n = 1)]. Ten patients were cirrhotic, two had previous exposure to PEG-interferon and seven were coinfected with HIV; 80 had been treated for 8 weeks. Overall, 31 patients (34.4%) failed glecaprevir/pibrentasvir without any NS3 or NS5A RASs, 62.4% (53/85) showed RASs in NS5A, 15.6% (13/83) in NS3 and 10% (9/90) in both NS5A and NS3. Infection with HCV genotypes 1a and 3a was associated with a higher prevalence of NS5A RASs. Patients harbouring two (n = 34) or more (n = 8) RASs in NS5A were frequent. Retreatment was initiated in 56 patients, almost all (n = 52) with sofosbuvir/velpatasvir/voxilaprevir. The overall sustained virological response rate was 97.8% in patients with end-of-follow-up data available. Conclusions One-third of patients failed glecaprevir/pibrentasvir without resistance. RASs in NS5A were more prevalent than in NS3 and were frequently observed as dual and triple combination patterns, with a high impact on NS5A inhibitor activity, particularly in genotypes 1a and 3a. Retreatment of glecaprevir/pibrentasvir failures with sofosbuvir/velpatasvir/voxilaprevir achieved viral suppression across all genotypes.
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Bartels, Doug J., James C. Sullivan, Eileen Z. Zhang, Ann M. Tigges, Jennifer L. Dorrian, Sandra De Meyer, Darin Takemoto, et al. "Hepatitis C Virus Variants with Decreased Sensitivity to Direct-Acting Antivirals (DAAs) Were Rarely Observed in DAA-Naive Patients prior to Treatment." Journal of Virology 87, no. 3 (November 14, 2012): 1544–53. http://dx.doi.org/10.1128/jvi.02294-12.
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ABSTRACTThe prevalence of naturally occurring hepatitis C virus (HCV) variants that are less sensitive to direct-acting antiviral (DAA) inhibitors has not been fully characterized. We used population sequence analysis to assess the frequency of such variants in plasma samples from 3,447 DAA-naive patients with genotype 1 HCV. In general, HCV variants with lower-level resistance (3- to 25-fold increased 50% inhibitor concentration [IC50]) to telaprevir were observed as the dominant species in 0 to 3% of patients, depending on the specific variant, whereas higher-level resistant variants (>25-fold-increased IC50) were not observed. Specific variants resistant to NS5A inhibitors were predominant in up to 6% of patients. Most variants resistant to nucleo(s/t)ide active-site NS5B polymerase inhibitors were not observed, whereas variants resistant to non-nucleoside allosteric inhibitors were observed in up to 18% of patients. The presence of DAA-resistant variants in NS5A, NS5B, or NS3 (including telaprevir-resistant variants), in baseline samples of treatment-naive patients receiving a telaprevir-based regimen in phase 3 studies did not affect the sustained viral response (SVR). Treatment-naive patients with viral populations containing the telaprevir-resistant variants NS3 V36M, T54S, or R155K at baseline achieved a 74% SVR rate, whereas patients with no resistant variants detected prior to treatment achieved a 76% SVR rate. The effect of specific resistant variant frequency on response to various DAA treatments in different patient populations, including interferon nonresponders, should be further studied.
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Malandris, Konstantinos, Georgios Kalopitas, Eleni Theocharidou, and Georgios Germanidis. "The Role of RASs /RVs in the Current Management of HCV." Viruses 13, no. 10 (October 18, 2021): 2096. http://dx.doi.org/10.3390/v13102096.
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The approval of combination therapies with direct-acting antiviral (DAA) regimens has led to significant progress in the field of hepatitis C virus (HCV) treatment. Although most patients treated with these agents achieve a virological cure, resistance to DAAs is a major issue. The rapid emergence of resistance-associated substitutions (RASs), in particular in the context of incomplete drug pressure, has an impact on sustained virological response (SVR) rates. Several RASs in NS3, NS5A and NS5B have been linked with reduced susceptibility to DAAs. RAS vary based on HCV characteristics and the different drug classes. DAA-resistant HCV variant haplotypes (RVs) are dominant in cases of virological failure. Viruses with resistance to NS3-4A protease inhibitors are only detected in the peripheral blood in a time frame ranging from weeks to months following completion of treatment, whereas NS5A inhibitor-resistant viruses may persist for years. Novel agents have been developed that demonstrate promising results in DAA-experienced patients. The recent approval of broad-spectrum drug combinations with a high genetic barrier to resistance and antiviral potency may overcome the problem of resistance.
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Yang, Huiling, Margaret Robinson, Amoreena C. Corsa, Betty Peng, Guofeng Cheng, Yang Tian, Yujin Wang, et al. "Preclinical Characterization of the Novel Hepatitis C Virus NS3 Protease Inhibitor GS-9451." Antimicrobial Agents and Chemotherapy 58, no. 2 (August 12, 2013): 647–53. http://dx.doi.org/10.1128/aac.00487-13.
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ABSTRACTGS-9451 is a selective hepatitis C virus (HCV) NS3 protease inhibitor in development for the treatment of genotype 1 (GT1) HCV infection. Key preclinical properties of GS-9451, includingin vitroantiviral activity, selectivity, cross-resistance, and combination activity, as well as pharmacokinetic properties, were determined. In multiple GT1a and GT1b replicon cell lines, GS-9451 had mean 50% effective concentrations (EC50s) of 13 and 5.4 nM, respectively, with minimal cytotoxicity; similar potency was observed in chimeric replicons encoding the NS3 protease gene of GT1 clinical isolates. GS-9451 was less active in GT2a replicon cells (EC50= 316 nM). Additive to synergisticin vitroantiviral activity was observed when GS-9451 was combined with other agents, including alpha interferon, ribavirin, and the polymerase inhibitors GS-6620 and tegobuvir (GS-9190), as well as the NS5A inhibitor ledipasvir (GS-5885). GS-9451 retained wild-type activity against multiple classes of NS5B and NS5A inhibitor resistance mutations. GS-9451 was stable in hepatic microsomes and hepatocytes from human and three other tested species. Systemic clearance was low in dogs and monkeys but high in rats. GS-9451 showed good oral bioavailability in all three species tested. In rats, GS-9451 levels were ∼40-fold higher in liver than plasma after intravenous dosing, and elimination of GS-9451 was primarily through biliary excretion. Together, these results are consistent with the antiviral activity observed in a recent phase 1b study. The results ofin vitrocross-resistance and combination antiviral assays support the ongoing development of GS-9451 in combination with other agents for the treatment of chronic HCV infection.
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Lemm, Julie A., John E. Leet, Donald R. O'Boyle, Jeffrey L. Romine, Xiaohua Stella Huang, Daniel R. Schroeder, Jeffrey Alberts, et al. "Discovery of Potent Hepatitis C Virus NS5A Inhibitors with Dimeric Structures." Antimicrobial Agents and Chemotherapy 55, no. 8 (May 16, 2011): 3795–802. http://dx.doi.org/10.1128/aac.00146-11.
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ABSTRACTThe exceptionalin vitropotency of the hepatitis C virus (HCV) NS5A inhibitor BMS-790052 has translated into anin vivoeffect in proof-of-concept clinical trials. Although the 50% effective concentration (EC50) of the initial lead, the thiazolidinone BMS-824, was ∼10 nM in the replicon assay, it underwent transformation to other inhibitory species after incubation in cell culture medium. The biological profile of BMS-824, including the EC50, the drug concentration required to reduce cell growth by 50% (CC50), and the resistance profile, however, remained unchanged, triggering an investigation to identify the biologically active species. High-performance liquid chromatography (HPLC) biogram fractionation of a sample of BMS-824 incubated in medium revealed that the most active fractions could readily be separated from the parental compound and retained the biological profile of BMS-824. From mass spectral and nuclear magnetic resonance data, the active species was determined to be a dimer of BMS-824 derived from an intermolecular radical-mediated reaction of the parent compound. Based upon an analysis of the structural elements of the dimer deemed necessary for anti-HCV activity, the stilbene derivative BMS-346 was synthesized. This compound exhibited excellent anti-HCV activity and showed a resistance profile similar to that of BMS-824, with changes in compound sensitivity mapped to the N terminus of NS5A. The N terminus of NS5A has been crystallized as a dimer, complementing the symmetry of BMS-346 and allowing a potential mode of inhibition of NS5A to be discussed. Identification of the stable, active pharmacophore associated with these NS5A inhibitors provided the foundation for the design of more potent inhibitors with broad genotype inhibition. This culminated in the identification of BMS-790052, a compound that preserves the symmetry discovered with BMS-346.
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Dvory-Sobol, H., D. Wyles, W. Ouyang, K. Chodavarapu, J. McNally, W. Cheng, S. Shafran, et al. "O059 : Long-term persistence of HCV NS5A variants after treatment with NS5A inhibitor ledipasvir." Journal of Hepatology 62 (April 2015): S221. http://dx.doi.org/10.1016/s0168-8278(15)30073-8.
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Wang, Chunfu, Lourdes Valera, Lingling Jia, Melissa J. Kirk, Min Gao, and Robert A. Fridell. "In VitroActivity of Daclatasvir on Hepatitis C Virus Genotype 3 NS5A." Antimicrobial Agents and Chemotherapy 57, no. 1 (October 22, 2012): 611–13. http://dx.doi.org/10.1128/aac.01874-12.
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ABSTRACTThe NS5A replication complex inhibitor daclatasvir (DCV; BMS-790052) inhibits hybrid replicons containing hepatitis C virus (HCV) genotype 3a (HCV3a) NS5A genes with 50% effective concentrations (EC50s) ranging from 120 to 870 pM. Selection studies with a hybrid HCV3a replicon identified NS5A residues 31 and 93 as sites for DCV-selected resistance. Our results support the potential use of DCV as a component in combination therapies for HCV3a chronic infection.
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Cheng, Guofeng, Yang Tian, Brian Doehle, Betty Peng, Amoreena Corsa, Yu-Jen Lee, Ruoyu Gong, et al. "In VitroAntiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir." Antimicrobial Agents and Chemotherapy 60, no. 3 (January 11, 2016): 1847–53. http://dx.doi.org/10.1128/aac.02524-15.
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Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, includingin vitropotency,in vitroresistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50values of 0.11 to 1.1 nM. LDV has relatively lessin vitroantiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50values of 16 to 530 nM.In vitroresistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment.In vitroantiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50values equivalent to those for the wild type.
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Lin, Hui-Mei, Jing-Chyi Wang, Han-Shu Hu, Pei-Shan Wu, Chi-Chen Yang, Chung-Pu Wu, Szu-Yuan Pu, et al. "Resistance Analysis and Characterization of a Thiazole Analogue, BP008, as a Potent Hepatitis C Virus NS5A Inhibitor." Antimicrobial Agents and Chemotherapy 56, no. 1 (October 17, 2011): 44–53. http://dx.doi.org/10.1128/aac.00599-11.
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ABSTRACTHepatitis 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 (EC50) 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.
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Kalamvoki, M., and P. Mavromara. "Calcium-Dependent Calpain Proteases Are Implicated in Processing of the Hepatitis C Virus NS5A Protein." Journal of Virology 78, no. 21 (November 1, 2004): 11865–78. http://dx.doi.org/10.1128/jvi.78.21.11865-11878.2004.
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ABSTRACT The nonstructural 5A (NS5A) protein of the hepatitis C virus (HCV) is a multifunctional phosphoprotein that is implicated in viral replication and HCV-mediated pathogenesis. We report here that the NS5A protein from the HCV genotype 1a is processed into shorter distinct forms when expressed in mammalian cells (Vero, HepG2, HuH-7, and WRL68) infected with an NS5A-expressing HSV-1-based amplicon vector or when transiently transfected with NS5A-expressing plasmids in the absence of exogenous apoptotic stimuli. Inhibitor studies combined with cell-free cleavage assays suggest that calcium-dependent calpain proteases, in addition to caspase-like proteases, are involved in NS5A processing. Interestingly, His-tagging experiments indicated that all the detectable NS5A-cleaved products are N-terminal forms of the protein. Additionally, immunofluorescence studies showed that, despite proteolytic cleavage, the NS5A protein exhibits a cytoplasm-perinuclear localization similar to that of the full-length protein. Thus, our results are consistent with recent data that demonstrated that NS5A is capable of perturbing intracellular calcium homeostasis and suggest that NS5A is both an inducer and a substrate of the calcium-dependent calpain protease(s). This may imply that cleavage of NS5A by calpain(s) could play a role in the modulation of NS5A function.
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Owens, Christopher M., Bradley B. Brasher, Alex Polemeropoulos, Michael H. J. Rhodin, Nicole McAllister, Kelly A. Wong, Christopher T. Jones, Lijuan Jiang, Kai Lin, and Yat Sun Or. "Preclinical and Clinical Resistance Profile of EDP-239, a Novel Hepatitis C Virus NS5A Inhibitor." Antimicrobial Agents and Chemotherapy 60, no. 10 (August 8, 2016): 6216–26. http://dx.doi.org/10.1128/aac.00815-16.
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ABSTRACTEDP-239, a potent and selective hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor developed for the treatment of HCV infection, has been investigatedin vitroandin vivo. This study sought to characterize genotypic changes in the HCV NS5A sequence of genotype 1 (GT1) replicons and to compare those changes to GT1 viral RNA mutations isolated from clinical trial patients. Resistance selection experimentsin vitrousing a subgenomic replicon identified resistance-associated mutations (RAMs) at GT1a NS5A amino acid positions 24, 28, 30, 31, and 93 that confer various degrees of resistance to EDP-239. Key RAMs were similarly identified in GT1b NS5A at amino acid positions 31 and 93. Mutations F36L in GT1a and A92V in GT1b do not confer resistance to EDP-239 individually but were found to enhance the resistance of GT1a K24R and GT1b Y93H. RAMs were identified in GT1 patients at baseline or after dosing with EDP-239 that were similar to those detectedin vitro. Baseline RAMs identified at NS5A position 93 in GT1, or positions 28 or 30 in GT1a only, correlated with a reduced treatment response. RAMs at additional positions were also detected and may have contributed to reduced EDP-239 efficacy. The most common GT1a and GT1b RAMs found to persist up to weeks 12, 24, or 48 were those at NS5A positions 28, 30, 31, 58 (GT1a only), and 93. Those RAMs persisting at the highest frequencies up to weeks 24 or 48 were L31M and Q30H/R for GT1a and L31M and Y93H for GT1b. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.)
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Lawitz, Eric J., William D. O'Riordan, Armen Asatryan, Bradley L. Freilich, Terry D. Box, J. Scott Overcash, Sandra Lovell, et al. "Potent Antiviral Activities of the Direct-Acting Antivirals ABT-493 and ABT-530 with Three-Day Monotherapy for Hepatitis C Virus Genotype 1 Infection." Antimicrobial Agents and Chemotherapy 60, no. 3 (December 28, 2015): 1546–55. http://dx.doi.org/10.1128/aac.02264-15.
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ABT-493 is a hepatitis C virus (HCV) nonstructural (NS) protein 3/4A protease inhibitor, and ABT-530 is an HCV NS5A inhibitor. These direct-acting antivirals (DAAs) demonstrated potent antiviral activity against major HCV genotypes and high barriers to resistancein vitro. In this open-label dose-ranging trial, antiviral activity and safety were assessed during 3 days of monotherapy with ABT-493 or ABT-530 in treatment-naive adults with HCV genotype 1 infection, with or without compensated cirrhosis. The presence of baseline resistance-associated variants (RAVs) was also evaluated. The mean maximal decreases in HCV RNA levels from baseline were approximately 4 log10IU/ml for all ABT-493 doses ranging from 100 mg to 700 mg and for ABT-530 doses of ≥40 mg. There were no meaningful differences in viral load declines for patients with versus without compensated cirrhosis. Twenty-four (50%) of the baseline samples from patients treated with ABT-493 had RAVs to NS3/4A protease inhibitors. Among 40 patients treated with ABT-530, 6 (15%) carried baseline RAVs to NS5A inhibitors. Viral load declines in patients with single baseline NS5A RAVs were similar to those in patients without RAVs. One patient harbored baseline RAVs at 3 NS5A positions and appeared to have a slightly less robust viral load decline on day 3 of monotherapy. No serious or grade 3 (severe) or higher adverse events and no clinically relevant laboratory abnormalities were observed with either compound. ABT-493 and ABT-530 demonstrated potent antiviral activity and acceptable safety during 3-day monotherapy in patients with HCV genotype 1 infection, with or without compensated cirrhosis. Based on these results, phase II studies assessing the combination of these DAAs for the treatment of chronic HCV infection in patients with or without compensated cirrhosis have been initiated. (This study has been registered at ClinicalTrials.gov under registration no. NCT01995071.)
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Wang, Chunfu, Lingling Jia, Haichang Huang, Dike Qiu, Lourdes Valera, Xin Huang, Jin-Hua Sun, et al. "In VitroActivity of BMS-790052 on Hepatitis C Virus Genotype 4 NS5A." Antimicrobial Agents and Chemotherapy 56, no. 3 (December 27, 2011): 1588–90. http://dx.doi.org/10.1128/aac.06169-11.
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ABSTRACTThe antiviral profile of BMS-790052, a potent hepatitis C virus (HCV) replication complex inhibitor targeting nonstructural protein NS5A, is well characterized for HCV genotype-1. Here, we report that BMS-790052 inhibits hybrid replicons containing HCV genotype-4 NS5A genes with 50% effective concentrations (EC50s) ranging from 7 to 13 pM. NS5A residue 30 was an important site for BMS-790052-selected resistance in the hybrid replicons. Our results support the potential of BMS-790052 as a valuable component of combination therapy for HCV genotype-4 chronic infection.
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Bilello, J. P., L. B. Lallos, J. F. McCarville, M. La Colla, I. Serra, C. Chapron, J. M. Gillum, C. Pierra, D. N. Standring, and M. Seifer. "In VitroActivity and Resistance Profile of Samatasvir, a Novel NS5A Replication Inhibitor of Hepatitis C Virus." Antimicrobial Agents and Chemotherapy 58, no. 8 (May 27, 2014): 4431–42. http://dx.doi.org/10.1128/aac.02777-13.
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ABSTRACTThe hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is a clinically validated target for drugs designed to treat chronic HCV infection. This study evaluated thein vitroactivity, selectivity, and resistance profile of a novel anti-HCV compound, samatasvir (IDX719), alone and in combination with other antiviral agents. Samatasvir was effective and selective against infectious HCV and replicons, with 50% effective concentrations (EC50s) falling within a tight range of 2 to 24 pM in genotype 1 through 5 replicons and with a 10-fold EC50shift in the presence of 40% human serum in the genotype 1b replicon. The EC90/EC50ratio was low (2.6). A 50% cytotoxic concentration (CC50) of >100 μM provided a selectivity index of >5 × 107. Resistance selection experiments (with genotype 1a replicons) and testing against replicons bearing site-directed mutations (with genotype 1a and 1b replicons) identified NS5A amino acids 28, 30, 31, 32, and 93 as potential resistance loci, suggesting that samatasvir affects NS5A function. Samatasvir demonstrated an overall additive effect when combined with interferon alfa (IFN-α), ribavirin, representative HCV protease, and nonnucleoside polymerase inhibitors or the nucleotide prodrug IDX184. Samatasvir retained full activity in the presence of HIV and hepatitis B virus (HBV) antivirals and was not cross-resistant with HCV protease, nucleotide, and nonnucleoside polymerase inhibitor classes. Thus, samatasvir is a selective low-picomolar inhibitor of HCV replicationin vitroand is a promising candidate for future combination therapies with other direct-acting antiviral drugs in HCV-infected patients.
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Liu, Mengping, Maria Tuttle, Min Gao, and Julie A. Lemm. "Potency and Resistance Analysis of Hepatitis C Virus NS5B Polymerase Inhibitor BMS-791325 on All Major Genotypes." Antimicrobial Agents and Chemotherapy 58, no. 12 (September 29, 2014): 7416–23. http://dx.doi.org/10.1128/aac.03851-14.
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ABSTRACTBMS-791325 is a hepatitis C virus (HCV) inhibitor binding to the thumb domain of the NS5B RNA-dependent RNA polymerase. BMS-791325 is well characterized in genotype 1 (GT1) and exhibits good inhibitory activity (50% effective concentration [EC50], <10 nM) against hybrid replicons containing patient NS5B sequences from GT3a, -4a, and -5a while potency against GT2 is significantly reduced (J. A. Lemm et al., Antimicrob. Agents Chemother. 58:3485–3495, 2014, doi:http://dx.doi.org/10.1128/AAC.02495-13). BMS-791325 potency against GT6a hybrid replicons is more variable, with two of three hybrid clones having EC50s similar to that for GT1 while a third patient clone was ∼10 times less susceptible to BMS-791325. To characterize the resistance profile of BMS-791325 beyond GT1, curing studies were performed across GT1a and -3a to -6a and demonstrated that GT1a has the highest resistance barrier versus BMS-791325 while GT6a has the lowest. Selection of GT3 to -6 NS5B chimeric replicon cells at different concentrations of BMS-791325 revealed substitutions in the thumb domain of NS5B at residues 494 and 495 that conferred different levels of resistance to BMS-791325 but remained susceptible to NS5A or NS3 protease inhibitors. In addition, we demonstrate that the reduced potency of BMS-791325 against one GT6a patient is due to an A494 polymorphism present in ∼21% of sequences in the European HCV database. The results from this report suggest that BMS-791325 is a candidate for combination treatment of HCV GT3 to -6 chronic infections, and the resistance profiles identified will provide useful information for future clinical development.
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Krishnan, Preethi, Gretja Schnell, Rakesh Tripathi, Jill Beyer, Thomas Reisch, Xinyan Zhang, Carolyn Setze, et al. "Analysis of Hepatitis C Virus Genotype 1b Resistance Variants in Japanese Patients Treated with Paritaprevir-Ritonavir and Ombitasvir." Antimicrobial Agents and Chemotherapy 60, no. 2 (December 7, 2015): 1106–13. http://dx.doi.org/10.1128/aac.02606-15.
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ABSTRACTTreatment of HCV genotype 1b (GT1b)-infected Japanese patients with paritaprevir (NS3/4A inhibitor boosted with ritonavir) and ombitasvir (NS5A inhibitor) in studies M12-536 and GIFT-I demonstrated high sustained virologic response (SVR) rates. The virologic failure rate was 3% (13/436) across the two studies. Analyses were conducted to evaluate the impact of baseline resistance-associated variants (RAVs) on treatment outcome and the emergence and persistence of RAVs in patients experiencing virologic failure. Baseline paritaprevir resistance-conferring variants in NS3 were infrequent, while Y93H in NS5A was the most prevalent ombitasvir resistance-conferring variant at baseline. A comparison of baseline prevalence of polymorphisms in Japanese and western patients showed that Q80L and S122G in NS3 and L28M, R30Q, and Y93H in NS5A were significantly more prevalent in Japanese patients. In the GIFT-I study, the prevalence of Y93H in NS5A varied between 13% and 21% depending on the deep-sequencing detection threshold. Among patients with Y93H comprising <1%, 1 to 40%, or >40% of their preexisting viral population, the 24-week SVR (SVR24) rates were >99% (276/277), 93% (38/41), and 76% (25/33), respectively, indicating that the prevalence of Y93H within a patient's viral population is a good predictor of treatment response. The predominant RAVs at the time of virologic failure were D168A/V in NS3 and Y93H alone or in combination with other variants in NS5A. While levels of NS3 RAVs declined over time, NS5A RAVs persisted through posttreatment week 48. Results from these analyses are informative in understanding the resistance profile of an ombitasvir- plus paritaprevir/ritonavir-based regimen in Japanese GT1b-infected patients.
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Coito, Carlos, Deborah L. Diamond, Petra Neddermann, Marcus J. Korth, and Michael G. Katze. "High-Throughput Screening of the Yeast Kinome: Identification of Human Serine/Threonine Protein Kinases That Phosphorylate the Hepatitis C Virus NS5A Protein." Journal of Virology 78, no. 7 (April 1, 2004): 3502–13. http://dx.doi.org/10.1128/jvi.78.7.3502-3513.2004.
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ABSTRACT The hepatitis C virus NS5A protein plays a critical role in virus replication, conferring interferon resistance to the virus through perturbation of multiple intracellular signaling pathways. Since NS5A is a phosphoprotein, it is of considerable interest to understand the role of phosphorylation in NS5A function. In this report, we investigated the phosphorylation of NS5A by taking advantage of 119 glutathione S-transferase-tagged protein kinases purified from Saccharomyces cerevisiae to perform a global screening of yeast kinases capable of phosphorylating NS5A in vitro. A database BLAST search was subsequently performed by using the sequences of the yeast kinases that phosphorylated NS5A in order to identify human kinases with the highest sequence homologies. Subsequent in vitro kinase assays and phosphopeptide mapping studies confirmed that several of the homologous human protein kinases were capable of phosphorylating NS5A. In vivo phosphopeptide mapping revealed phosphopeptides common to those generated in vitro by AKT, p70S6K, MEK1, and MKK6, suggesting that these kinases may phosphorylate NS5A in mammalian cells. Significantly, rapamycin, an inhibitor commonly used to investigate the mTOR/p70S6K pathway, reduced the in vivo phosphorylation of specific NS5A phosphopeptides, strongly suggesting that p70S6 kinase and potentially related members of this group phosphorylate NS5A inside the cell. Curiously, certain of these kinases also play a major role in mRNA translation and antiapoptotic pathways, some of which are already known to be regulated by NS5A. The findings presented here demonstrate the use of high-throughput screening of the yeast kinome to facilitate the major task of identifying human NS5A protein kinases for further characterization of phosphorylation events in vivo. Our results suggest that this novel approach may be generally applicable to the screening of other protein biochemical activities by mechanistic class.
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QADRI, Ishtiaq, Mieko IWAHASHI, Juan M. CAPASSO, Matthew W. HOPKEN, Sonia FLORES, Jerome SCHAACK, and Francis R. SIMON. "Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP-1." Biochemical Journal 378, no. 3 (March 15, 2004): 919–28. http://dx.doi.org/10.1042/bj20031587.
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Activation of cellular kinases and transcription factors mediates the early phase of the cellular response to chemically or biologically induced stress. In the present study we investigated the oxidant/antioxidant balance in Huh-7 cells expressing the HCV (hepatitis C virus) subgenomic replicon, and observed a 5-fold increase in oxidative stress during HCV replication. We used MnSOD (manganese-superoxide dismutase) as an indicator of the cellular antioxidant response, and found that its activity, protein levels and promoter activity were significantly increased, whereas Cu/ZnSOD was not affected. The oxidative stress-induced protein kinases p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) were activated in the HCV repliconcontaining cells and in Huh-7 cells transduced with Ad-NS5A [a recombinant adenovirus encoding NS5A (non-structural protein 5A)], coupled with a 4–5-fold increase in AP-1 (activator protein-1) DNA binding. Ava.1 cells, which encode a replication-defective HCV replicon, showed no significant changes in MnSOD, p38 MAPK or JNK activity. The AP-1 inhibitors dithiothreitol and N-acetylcysteine, as well as a dominant negative AP-1 mutant, significantly reduced AP-1 activation, demonstrating that this activation is oxidative stress-related. Exogenous NS5A had no effect on AP-1 activation in vitro, suggesting that NS5A acts at the upstream targets of AP-1 involving p38 MAPK and JNK signalling cascades. AP-1-dependent gene expression was increased in HCV subgenomic replicon-expressing Huh-7 cells. MnSOD activation was blocked by inhibitors of JNK (JNKI1) and p38 MAPK (SB203580), but not by an ERK (extracellular-signal-regulated kinase) inhibitor (U0126), in HCV-replicating and Ad-NS5A-transduced cells. Our results demonstrate that cellular responses to oxidative stress in HCV subgenomic replicon-expressing and Ad-NS5A-transduced cells are regulated by two distinct signalling pathways involving p38 MAPK and JNK via AP-1 that is linked to increased oxidative stress and therefore to an increased antioxidant MnSOD response.
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He, Yupeng, Seng-Lai Tan, Semih U. Tareen, Sangeetha Vijaysri, Jeffrey O. Langland, Bertram L. Jacobs, and Michael G. Katze. "Regulation of mRNA Translation and Cellular Signaling by Hepatitis C Virus Nonstructural Protein NS5A." Journal of Virology 75, no. 11 (June 1, 2001): 5090–98. http://dx.doi.org/10.1128/jvi.75.11.5090-5098.2001.
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ABSTRACT The NS5A nonstructural protein of hepatitis C virus (HCV) has been shown to inhibit the cellular interferon (IFN)-induced protein kinase R (PKR). PKR mediates the host IFN-induced antiviral response at least in part by inhibiting mRNA translation initiation through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). We thus examined the effect of NS5A inhibition of PKR on mRNA translation within the context of virus infection by using a recombinant vaccinia virus (VV)-based assay. The VV E3L protein is a potent inhibitor of PKR. Accordingly, infection of IFN-pretreated HeLa S3 cells with an E3L-deficient VV (VVΔE3L) resulted in increased phosphorylation levels of both PKR and eIF2α. IFN-pretreated cells infected with VV in which the E3L locus was replaced with theNS5A gene (VVNS5A) displayed diminished phosphorylation of PKR and eIF2α in a transient manner. We also observed an increase in activation of p38 mitogen-activated protein kinase in IFN-pretreated cells infected with VVΔE3L, consistent with reports that p38 lies downstream of the PKR pathway. Furthermore, these cells exhibited increased phosphorylation of the cap-binding initiation factor 4E (eIF4E), which is downstream of the p38 pathway. Importantly, these effects were reduced in cells infected with VVNS5A. NS5A was also found to inhibit activation of the p38-eIF4E pathway in epidermal growth factor-treated cells stably expressing NS5A. NS5A-induced inhibition of eIF2α and eIF4E phosphorylation may exert counteracting effects on mRNA translation. Indeed, IFN-pretreated cells infected with VVNS5A exhibited a partial and transient restoration of cellular and viral mRNA translation compared with IFN-pretreated cells infected with VVΔE3L. Taken together, these results support the role of NS5A as a PKR inhibitor and suggest a potential mechanism by which HCV might maintain global mRNA translation rate during early virus infection while favoring cap-independent translation of HCV mRNA during late infection.
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Bailly, François, Pierre Pradat, Victor Virlogeux, and Fabien Zoulim. "Antiviral Therapy in Patients with Hepatitis C Virus-Induced Cirrhosis." Digestive Diseases 33, no. 4 (2015): 613–23. http://dx.doi.org/10.1159/000375359.
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Background: Opportunities to treat infection with hepatitis C virus (HCV) are evolving rapidly. From the introduction of interferon (IFN)-α monotherapy in the early 1990s to the approval of telaprevir- and boceprevir-based triple therapies with pegylated (PEG)-IFN-α and ribavirin (RBV) in 2011, the chances of curing patients infected with HCV genotype 1 have improved dramatically to reach approximately 70%. Significant further improvements that may cure virtually all HCV patients with an all-oral, IFN-free regimen are becoming progressively available. Key Messages: Historically, a PEG-IFN/RBV combination therapy of patients with liver cirrhosis was associated with lower virological rates and a worse safety profile. The advent of the first protease inhibitor-based triple therapy was long expected, but the promise fell rapidly because of the numerous side effects and the requirement for intensive clinical management in cirrhotic patients. The newer direct-acting antivirals (DAAs) target the viral polymerase with either nucleos(t)ide analogues or nonnucleosidic inhibitors, the viral protease and the viral NS5A protein. Several clinical trials have now shown that a combination of sofosbuvir (nucleosidic polymerase inhibitor) with daclatasvir or ledipasvir (NS5A inhibitors), or sofosbuvir with simeprevir (protease inhibitor), or a combination of ABT-450 (protease inhibitor) with ritonavir (ABT-450/r), the nonnucleosidic polymerase inhibitor ABT-333 and the NS5A inhibitor ABT-267, can achieve a sustained virological response in up to 95% of naive patients or previously treated patients, even in those who failed prior treatment with first-generation protease inhibitors. The best treatment regimens enable the achievement of comparable results even in cirrhotics, while other regimens still require RBV or a longer treatment duration to achieve optimal results. This improved risk/benefit ratio justifies early access programs of IFN-free regimens for cirrhotic patients. The remaining difficult-to-treat patients are cirrhotics infected with HCV genotype 3 and those with decompensated cirrhosis, for whom novel DAA combinations should be evaluated in clinical trials. Conclusions: As new DAAs are becoming available in early access treatment programs, treatment strategy studies are being performed to optimize treatment regimens with respect to the choice of DAAs and treatment duration, based on viral genotypes, prior treatment response and the presence of liver cirrhosis. In the near future, this should allow: (i) a decrease in the complications of HCV-induced cirrhosis, (ii) liver transplantations to be performed in virally cured patients, and (iii) the rescue of patients in the worst clinical situation (decompensated cirrhosis and HCV recurrence on liver graft).
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Plaza, Zulema, Vincent Soriano, Eugenia Vispo, Maria del Mar Gonzalez, Pablo Barreiro, Eduardo Seclén, and Eva Poveda. "Prevalence of natural polymorphisms at the HCV NS5A gene associated with resistance to daclatasvir, an NS5A inhibitor." Antiviral Therapy 17, no. 5 (2012): 921–26. http://dx.doi.org/10.3851/imp2091.
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Wong, Kelly A., Angela Worth, Ross Martin, Evguenia Svarovskaia, Diana M. Brainard, Eric Lawitz, Michael D. Miller, and Hongmei Mo. "Characterization of Hepatitis C Virus Resistance from a Multiple-Dose Clinical Trial of the Novel NS5A Inhibitor GS-5885." Antimicrobial Agents and Chemotherapy 57, no. 12 (July 22, 2013): 6333–40. http://dx.doi.org/10.1128/aac.02193-12.
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ABSTRACTGS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log10HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at ≥3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30- and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilitiesin vitro. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT01193478.)
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Lahser, Frederick C., Karin Bystol, Stephanie Curry, Patricia McMonagle, Ellen Xia, Paul Ingravallo, Robert Chase, et al. "The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons." Antimicrobial Agents and Chemotherapy 60, no. 5 (February 29, 2016): 2954–64. http://dx.doi.org/10.1128/aac.00051-16.
Full textAbstract:
ABSTRACTThe selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for chronic HCV infection.