NS5B Inhibitors Research Articles

First report on molecular docking analysis and drug resistance substitutions to approved HCV NS5A and NS5B inhibitors amongst Iranian patients

BackgroundNS5A and NS5B proteins of hepatitis C virus (HCV) are the main targets of compounds that directly inhibit HCV infections. However, the emergence of resistance-associated substitutions (RASs) may cause substantial reductions in susceptibility to inhibitors.MethodsViral load and genotyping were determined in eighty-seven naïve HCV-infected patients, and the amplified NS5A and NS5B regions were sequenced by Sanger sequencing. In addition, physicochemical properties, structural features, immune epitopes, and inhibitors-protein interactions of sequences were analyzed using several bioinformatics tools.ResultsSeveral amino acid residue changes were found in NS5A and NS5B proteins; however, we did not find any mutations related to resistance to the treatment in NS5B. Different phosphorylation and few glycosylation sites were assessed. Disulfide bonds were identified in both proteins that had a significant effect on the function and structure of HCV proteins. Applying reliable software to predict B-cell epitopes, 3 and 5 regions were found for NS5A and NS5B, respectively, representing a considerable potential to induce the humoral immune system. Docking analysis determined amino acids involved in the interaction of inhibitors and mentioned proteins may not decrease the drug efficiency.ConclusionsStrong interactions between inhibitors, NS5A and NS5B proteins and the lack of efficient drug resistance mutations in the analyzed sequences may confirm the remarkable ability of NS5A and NS5B inhibitors to control HCV infection amongst Iranian patients. The results of bioinformatics analysis could unveil all features of both proteins, which can be beneficial for further investigations on HCV drug resistance and designing novel vaccines.

StackHCV: a web-based integrative machine-learning framework for large-scale identification of hepatitis C virus NS5B inhibitors.

Fast and accurate identification of inhibitors with potency against HCV NS5B polymerase is currently a challenging task. As conventional experimental methods is the gold standard method for the design and development of new HCV inhibitors, they often require costly investment of time and resources. In this study, we develop a novel machine learning-based meta-predictor (termed StackHCV) for accurate and large-scale identification of HCV inhibitors. Unlike the existing method, which is based on single-feature-based approach, we first constructed a pool of various baseline models by employing a wide range of heterogeneous molecular fingerprints with five popular machine learning algorithms (k-nearest neighbor, multi-layer perceptron, partial least squares, random forest and support vectors machine). Secondly, we integrated these baseline models in order to develop the final meta-based model by means of the stacking strategy. Extensive benchmarking experiments showed that StackHCV achieved a more accurate and stable performance as compared to its constituent baseline models on the training dataset and also outperformed the existing predictor on the independent test dataset. To facilitate the high-throughput identification of HCV inhibitors, we built a web server that can be freely accessed at http://camt.pythonanywhere.com/StackHCV . It is expected that StackHCV could be a useful tool for fast and precise identification of potential drugs against HCV NS5B particularly for liver cancer therapy and other clinical applications.

A novel fixed-dose combination treatment for chronic hepatitis C, based on NS5A inhibitor fopitasvir and NS5B inhibitor sofosbuvir.

Drug resistance caused by the extreme genetic variability of zhe hepatitis C virus has rendered effective combinations of drugs indispensable in the treatment of chronic hepatitis C (CHC). Herein, we developed a fixed-dose combination (FDC) treatment containing the NS5B inhibitor sofosbuvir (SOF) and the NS5A inhibitor fopitasvir (FOP). Then the dissolution behavior of FOP in FOP/SOF FDC was improved by co-micronizing FOP with lactose. The enhanced dissolution rate of FOP in the FDC was in good agreement with the behavior of the FOP singledrug tablet. In addition, pharmacokinetic studies showed that both FOP and SOF in the FDC exhibited similar characteristics (area under the curve, Cmax, Tmax, and T1/2) as those of tablets containing FOP or SOF alone. These results revealed that the FOP/SOF FDC represents a potential therapeutic option for the treatment of CHC.

Distribution of naturally -occurring NS5B resistance-associated substitutions in Egyptian patients with chronic Hepatitis C

NS5B polymerase inhibitors represent the cornerstone of the present treatment of Hepatitis C virus infection (HCV). Naturally occurring substitution mutations to NS5B inhibitors have been recorded. The current study intended to demonstrate possible natural direct acting antiviral (DAA)-mutations of the HCV NS5B region in HCV patients in Minia governorate, Egypt. Samples were collected from 27 treatment-naïve HCV patients and 8 non-responders. Out of 27 treatment-naïve patients, 17 NS5B sequences (amino acids 221-345) from treatment-naïve patients and one sample of non-responders were successfully amplified. Nucleotide sequences have been aligned, translated into amino acids, and compared to drug resistance mutations reported in the literature. NS5B amino acid sequence analysis ensures several novel NS5B mutations existence (more than 40 substitution mutations) that have not been previously documented to be correlated with a resistant phenotype. It was found that K304R (82.4%), E327D and P300T (76.5% each) substitutions were the most distributed in the tested samples, respectively. S282T, the major resistance mutation that induces high sofosbuvir-resistance level in addition to other reported mutations (L320F/C) and (C316Y/N) were not recognized. Q309R mutation is a ribavirin-associated resistance, which was recognized in one strain (5.9%) of genotype 1g sequences. Besides, one substitution mutation (E237G) was identified in the successfully amplified non-responder sample. Our study showed various combinations of mutations in the analyzed NS5B genes which could enhance the possibility of therapy failure in patients administered regimens including multiple DAA.

Mechanistic Studies of a Pd-Catalyzed Direct Arylation En Route to Beclabuvir: Dual Role of a Tetramethylammonium Cation and an Unusual Turnover-Limiting Step

Mechanistic investigations on the intramolecular direct arylation of an indole-containing aryl bromide en route to the HCV NS5B inhibitor beclabuvir are reported. A combination of stoichiometric, k...

Insight into the drug resistance mechanisms of GS-9669 caused by mutations of HCV NS5B polymerase via molecular simulation

GS-9669 is a non-nucleos(t)ide inhibitor (NNI) binding to the thumb site II of the Hepatitis C virus (HCV) NS5B polymerase and has advanced into phase II trials. To clarify the drug resistance mechanisms of GS-9669 caused by M423T/I/V, L419M, R422K, and I482L mutations of NS5B polymerase (GT1b) and the receptor-ligand interactions during the binding process, a series of molecular simulation methods including molecular dynamics (MD) simulations and adaptive steered molecular dynamics (ASMD) simulations were performed for the wild-type (WT) and six mutant NS5B/GS-9669 complexes. The calculated results indicate that the binding free energies of the mutant systems are less negative than that of the WT system, indicating that these mutations will indeed cause NS5B to produce different degrees of resistance to GS-9669. The mutation-induced drug resistances are mainly caused by the loss of binding affinities of Leu419 and Trp528 with GS-9669 or the formation of multiple solvent bridges. Moreover, the ASMD calculations show that GS-9669 binds to the thumb II sites of the seven NS5B polymerases in distinct pathways without any obvious energy barriers. Although the recognition methods and binding pathways are distinct, the binding processes of GS-9669 with the WT and mutant NS5B polymerases are basically controlled thermodynamically. This study clearly reveals the resistance mechanisms of GS-9669 caused by M423T/I/V, L419M, R422K, and I482L mutations of HCV NS5B polymerase and provides some valuable clues for further optimization and design of novel NS5B inhibitors.

In silico design and analysis of NS4B inhibitors against hepatitis C virus

The hepatitis C virus is a communicable disease that gradually harms the liver leading to cirrhosis and hepatocellular carcinoma. Important therapeutic interventions have been reached since the discovery of the disease. However, its resurgence urges the need for new approaches against this malady. The NS4B receptor is one of the important proteins for Hepatitis C Virus RNA replication that acts by mediating different viral properties. In this work, we opt to explore the relationships between the molecular structures of biologically tested NS4B inhibitors and their corresponding inhibitory activities to assist the design of novel and potent NS4B inhibitors. For that, a set of 115 indol-2-ylpyridine-3-sulfonamides (IPSA) compounds with inhibitory activity against NS4B is used. A hybrid genetic algorithm combined with multiple linear regressions (GA-MLR) was implemented to construct a predictive model. This model was further used and applied to a set of compounds that were generated based on a pharmacophore modeling study combined with virtual screening to identify structurally similar lead compounds. Multiple filtrations were implemented for selecting potent hits. The selected hits exhibited advantageous molecular features, allowing for favorable inhibitory activity against HCV. The results showed that 7 out of 1285 screened compounds, were selected as potent candidate hits where Zinc14822482 exhibits the best predicted potency and pharmacophore features. The predictive pharmacokinetic analysis further justified the compounds as potential hit molecules, prompting their recommendation for a confirmatory biological evaluation. We believe that our strategy could help in the design and screening of potential inhibitors in drug discovery. Communicated by Ramaswamy H. Sarma

Scalable synthesis of a tetrasubstituted 7-azabenzofuran as a key intermediate for a class of potent HCV NS5B inhibitors

Abstract A series of tetrasubstituted 7-azabenzofurans displays remarkable pan-genotype inhibition of HCV NS5B polymerase. One of them has been identified as a potential clinical candidate. Completely different from the original synthesis of a common and key intermediate for this class of compounds, two novel and improved synthetic approaches are developed. Almost every step of the synthesis has been optimized, including several important but not fully explored transformations, such as radical bromination with methyl pyridine and cyanide substitution with TMSCN. The 7-azabenzofuran core is prepared by an acylation-heterocyclization reaction, using acyl chloride as both the reactant and the activation reagent for the N-oxide substrate. Compared with the BMS synthesis, the overall yield has been tripled, and those harmful or cost-effective synthetic steps have been reduced. Furthermore, a transition-metal-free synthetic method for the construction of 3-cyano substituted 7-azabenzofurans is presented.

Dissection of two drug-targeted regions of Hepatitis C virus subtype 4a infecting Egyptian patients.

Recently, treatment of HCV infection has been improved after the development of direct acting antivirals (DAAs) which target different viral proteins (NS3-4A, NS5A and NS5B). The activity and effectiveness of these DAAs are affected by the presence of resistance associated substitutions (RASs). This study aimed to characterize HCV genotypes circulating among Egyptian HCV patients, to dissect the full sequences of HCV NS3-4A and NS5B regions, and to characterize RASs associated with NS3-4A and NS5B inhibitors in HCV treatment-naïve patients. Genotyping of 80 HCV samples from treatment-naïve patients was done using restriction fragment length polymorphism and phylogenetic analysis based on some full NS5B sequences. Results showed the prevalence of HCV subtype 4a. Twenty four new full sequences of NS3-4A and NS5B regions of subtype 4a were deposited in the GenBank database. In general, the substitutions associated with NS3-4A-targeting drugs were absent predicting possible responsiveness of Egyptian HCV patients to these drugs. In addition, the absence of amino acid substitutions associated with resistance to Sofosbuvir may predict good response to treatment with Sofosbuvir. Some amino acid substitutions associated with resistance to different classes of non-nucleoside inhibitors were detected. Further investigations on treated Egyptian HCV patients may evaluate the effectiveness of the massively used drugs. Many predicted T-cell-binding epitopes in NS3-4A and NS5B regions were found to be highly conserved in the currently studied isolates; a finding that might be important for HCV vaccine development. We demonstrated potential NS3 epitopes that could be used in engineering T cells against HCV epitopes.Electronic supplementary materialThe online version of this article (10.1007/s11262-020-01776-y) contains supplementary material, which is available to authorized users.

HCVpred: A web server for predicting the bioactivity of hepatitis C virus NS5B inhibitors.

Hepatitis C virus (HCV) is one of the major causes of liver disease affecting an estimated 170 million people culminating in 300,000 deaths from cirrhosis or liver cancer. NS5B is one of three potential therapeutic targets against HCV (i.e., the other two being NS3/4A and NS5A) that is central to viral replication. In this study, we developed a classification structure-activity relationship (CSAR) model for identifying substructures giving rise to anti-HCV activities among a set of 578 non-redundant compounds. NS5B inhibitors were described by a set of 12 fingerprint descriptors and predictive models were constructed from 100 independent data splits using the random forest algorithm. The modelability (MODI index) of the data set was determined to be robust with a value of 0.88 exceeding established threshold of 0.65. The predictive performance was deduced by the accuracy, sensitivity, specificity, and Matthews correlation coefficient, which was found to be statistically robust (i.e., the former three parameters afforded values in excess of 0.8 while the latter statistical parameter provided a value >0.7). An in-depth analysis of the top 20 important descriptors revealed that aromatic ring and alkyl side chains are important for NS5B inhibition. Finally, the predictive model is deployed as a publicly accessible HCVpred web server (available at http://codes.bio/hcvpred/) that would allow users to predict the biological activity as being active or inactive against HCV NS5B. Thus, the knowledge and web server presented herein can be used in the design of more potent and specific drugs against the HCV NS5B.

Hepatitis C virus genotypes 1–3 infections regulate lipogenic signaling and suppress cholesterol biosynthesis in hepatocytes

Patients with different hepatitis C virus (HCV) genotype infections are associated with varying metabolic disorders. Although alteration of lipid metabolism has been confirmed as a virus-induced metabolic derangement in chronic hepatitis C patients, the impact of various HCV genotypes on hepatic cholesterol metabolism remains elusive. In this study, we thus investigated the HCV genotype-specific lipogenic and cholesterol metabolism profiles in an invitro cell culture system. We first conducted HCV cell culture system (HCVcc) assays by infecting Huh7.5.1cells with multiple infection-competent HCV strains, including the genotype 2a JFH1 and JFH1-based intergenotypic recombinants 1b and 3a. We then examined the expression levels of various lipid and cholesterol-related genes. The data showed that infection with individual HCV genotypes exerted unique gene expression regulatory effects on lipoproteins and cholesterol metabolism genes. Of note, all HCV strains suppressed cholesterol biosynthesis in hepatocytes through downregulating the expression of HMG-CoA reductase (HMGCR) and farnesyl-diphosphate farnesyltransferase 1 (FDFT1) - two essential enzymes for cholesterol biosynthesis. These HCV-mediated inhibitory effects could be reversed by treatment with sofosbuvir, a pangenotypic NS5B inhibitor. In addition, overexpression of HCV genotype 1b, 2a or 3a core protein significantly suppressed HMGCR mRNA transcription and translation, thus diminished cellular cholesterol biosynthesis. Nonetheless, the core protein had no effect on FDFT1 expression. Although HCV infection regulates host lipid metabolism in a genotype-specific manner, its inhibition on hepatocellular cholesterogenic gene expression and total cholesterol biosynthesis is a common effect among HCV genotype 1b, 2a and 3a.

Prevalence of HCV resistance-associated substitutions among treatment-failure patients receiving direct-acting antiviral agents.

Direct-acting antiviral (DAA) failure, which is mainly associated with the selection of resistance-associated substitutions (RASs), is not rare in HCV treatment. RAS data collected from published literature and RAS prevalence were integrated using meta-analysis. DAA-failure-associated RASs were identified by comparing their prevalence between DAA-failure and DAA-naïve patients. Prevalences of emerging RASs that occurred during treatment were also estimated. A total of 2932 DAA-naïve patients and 1466 DAA-failure patients were included. Significant differences in the prevalence of RASs were found in 76 scenarios that involved 34 RASs (11 in NS3, 18 in NS5A and 5 in NS5B), 4 genotypes (GTs) (GT1a, GT1b and GT3-4) and 14 DAAs (6 NS3 protease inhibitors [PIs], 6 NS5A inhibitors and 2 NS5B inhibitors). For NS3, the DAA-failure-associated RASs included V36L, Y56H, Q80K/R, R155K, A156T and D168A/E/L/T/V/Y. Substitutions at R155 and D168 were dominant for most NS3 PIs. For NS5A, DAA-failure-associated RASs included K24R, Q30R, L31M, and P32L in GT1a; R30Q/H, L31F/I/M/V, P58S, and Y93H in GT1b; A30K, L31M and Y93H in GT3; and M31V and Y93H in GT4. Y93H was the most prevalent RAS for NS5A inhibitors. DAA-failure-associated RASs were found at only five positions in NS5B. The majority of DAA-failure patients relapsed. A significant difference was detected for only four RAS sites between relapse patients and nonresponse/breakthrough patients. The RAS prevalence in DAA-failure patients varied among the HCV GTs and DAA regimens. The identified treatment-selected resistance patterns for broadly used DAA regimens will enable the selection of optimized retreatment options.

Multi-step parallel synthesis enabled optimization of benzofuran derivatives as pan-genotypic non-nucleoside inhibitors of HCV NS5B

In a lead optimization effort towards NS5B NNI inhibitors, two multi-step parallel libraries were designed and successfully synthesized. Through this effort we discovered compound 9B, which achieved rigorous and delicate balance of inhibition across the common genotypes and mutants with <10 nM potency. In addition, the bicyclic compounds 9B exhibited improved FASSIF solubility over the tetracyclic compound MK-8876. This strategic approach demonstrated that, even within limited reaction scope, multi-step parallel libraries could provide access to more complex chemical space. This expedient access facilitates diverse, purpose-driven optimization of SAR and physicochemical properties.

Discovery of 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs for delivering nucleoside HCV NS5B polymerase inhibitors

Our HCV research program investigated novel 2′-dihalogenated nucleoside HCV polymerase inhibitors and identified compound 1, a 5′-phosphoramidate prodrug of 2′-deoxy-2′-α-bromo-β-chloro uridine. Although 1 had a favorable in vitro activity profile in HCV replicons, oral dosing in dog resulted in low levels of the active 5′-triphosphate (TP) in liver. Metabolism studies using human hepatocytes provided a simple assay for screening alternative phosphoramidate prodrug analogs. Compounds that produced high TP concentrations in hepatocytes were tested in dog liver biopsy studies. This method identified 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrug 14, which provided 100-fold higher TP concentrations in dog liver in comparison to 1 (4 and 24 h after 5 mg/kg oral dose).

Progress of antiviral therapy for hepatitis C virus-related decompensated cirrhosis

Direct-acting antiviral agents (DAAs) are the main antiviral therapeutics for hepatitis C virus-related decompensated stage cirrhosis. DAAs of NS3/4A protease inhibitors use is not recommended for patients with decompensated cirrhosis due to characteristics of DAAs metabolism in liver. The recent guidelines have recommended sofosbuvir (SOF)-based plan including pan-genotype plan of sofosbuvir(SOF)/velpatasvir (VEL), sofosbuvir combined with daclatasvir (DCV), genotype 1,4,5,6 specific plan of sofosbuvir (SOF) / ledipasvir (LDV) for 24 weeks or above in combination with ribavirin for 12 weeks because NS5B and NS5A inhibitors has no obvious effect on CYP450 enzyme system and achievement of sustained virological response (SVR) rates at 12/24 weeks is achievable in 88% ~ 100%, and liver reserve function improves in 42% ~ 53% of patients. Furthermore, approximately 15.5% ~ 49% of patients waiting for liver transplantation after treatment with DAAs do not require liver transplantation for short-term and 10.3% ~19.2% of patients receiving SOF/LDV, and SOF combined with DCV not needed liver transplantation. Thus, the clinical application of DAAs provides a safe and reliable antiviral treatment plan for hepatitis C virus-related decompensated stage cirrhosis.

Direct-acting antiviral interactions with opioids, alcohol or illicit drugs of abuse in HCV-infected patients.

The hepatitis C virus (HCV) prevalence is extremely high in patients who consume and inject illicit drugs. Concerns about poor adherence and fear of interaction with drugs of abuse could constitute further disincentive for treatment initiation in these patients. We discussed the pharmacokinetics (PKs) and pharmacodynamics (PD) of currently prescribed direct antiviral agents (NSA5 inhibitors: daclatasvir, elbasvir, ledipasvir, pibrentasvir, velpatasvir; NS5B inhibitor: sofosbuvir; NS3/4A protease inhibitors: glecaprevir, grazoprevir, voxilaprevir) and most common substances of abuse (opioids: buprenorphine, fentanyl, heroin, methadone, morphine, oxycodone; stimulants: amphetamines, cathinones, cocaine; cannabinoids; ethanol). Overall, most direct-acting antivirals (DAAs) are substrates and inhibitors of the transmembrane transporter P-glycoprotein (P-gp), and several of them are metabolized by cytochrome P450 enzymes. Clinically relevant interactions are associated with P-gp and CYP3A modulators. Most substances of abuse are eliminated by Phase I and Phase II metabolizing enzymes, but none of them are either major inhibitors or inducers. PK studies did not show any relevant interactions between DAA and methadone or buprenorphine. Based on pharmacological considerations, neither efficacy loss nor adverse drug event associated with detrimental interaction are expected with opioids, stimulants, cannabinoids and ethanol. In summary, our literature review shows that the interaction potential of DAA with most opioids and illicit drugs is limited and should not be a hurdle to the initiate DAA.

Preclinical evaluation of Amphihevir, a first-in-class clinical Hepatitis C virus NS4B inhibitor.

Amphihevir, a benzofuran derivative, is the first reported NS4B inhibitor that has advanced to clinical trials (currently in Phase Ib). Here, we report the results of a preclinical study of its potency, toxicity, selectivity, DMPK, and safety profiles. Amphihevir displayed good antiviral activities against genotype 1a (EC50=0.34 nM) and genotype 1b (EC50=1.97 nM) replicons and evident cytotoxicity in twelve strains of cell lines derived from animals and humans. Amphihevir was found to be inactive against other viruses, human kinases, and GPCRs, which implies its good selectivity. A 9-day long-term treatment of genotype 1b replicon with Amphihevir resulted in a 3.8 Log10 decline of the hepatitis C viral RNA at a concentration of 25×EC90 Drug resistance screening showed that mutations occurred at H94, F98, and V105 of NS4B, which mediated the resistance to Amphihevir. This result suggests that NS4B is the main target of Amphihevir. There was no cross-resistances between Amphihevir and NS5A, NS3/4A, and NS5B inhibitors, suggesting that Amphihevir on combination of other anti- hepatitis C virus drugs could treat hepatitis C, as proven by studies of Amphihevir and other hepatitis C virus inhibitors. Pharmacokinetic studies demonstrated that Amphihevir has good oral bioavailability and appropriate T1/2 in rats and dogs, thereby supporting its use once per day. Finally, Amphihevir showed good safety profiles in rats and dogs. The results shed light on the use of Amphihevir as a potential treatment option for chronic hepatitis C patients.

Design, Synthesis, and Biological Evaluation of Substituted 4,6-Dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione Analogues as Potent NS4B Inhibitors for the Treatment of Dengue Virus Infection.

A series of substituted 4,6-dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione analogues were synthesized and evaluated as potent dengue virus inhibitors. Throughout a structure-activity relationship exploration on the amide of the indolone moiety, a wide range of substitutions were found to be well tolerated for chemical optimization at this position. Among these compounds, 15 (JMX0254) displayed the most potent and broad inhibitory activities, effective against DENV-1 to -3 with EC50 values of 0.78, 0.16, and 0.035 μM, respectively, while compounds 16, 21, 27-29, 47, and 70 exhibited relatively moderate to high activities with low micromolar to nanomolar potency against all four serotypes. The biotinylated compound 73 enriched NS4B protein from cell lysates in pull-down studies, and the findings together with the mutation investigations further validated dengue NS4B protein as the target of this class of compounds. More importantly, compound 15 exhibited good in vivo pharmacokinetic properties and efficacy in the A129 mouse model, indicating its therapeutic potential against the dengue virus infection as a drug candidate for further preclinical development.

Sofosbuvir susceptibility of genotype 1 to 6 HCV from DAA-naïve subjects

High sequence diversity of HCV may lead to variation in susceptibility to antiviral agents amongst different genotypes and subtypes of the virus. We assessed the susceptibility to sofosbuvir of chimeric replicons carrying the full length NS5B coding region from 479 HCV infected, treatment-naïve patients, including 15 subtypes in 6 genotypes. NS5B replicon vectors with subtype 1b, subtype 4a and subtype 6a backbone were modified to support testing of patient samples. We also evaluated sofosbuvir susceptibility in a panel of 331 replicons containing engineered NS5B inhibitor resistance-associated substitutions. The mean 50% effective sofosbuvir concentration (EC50) amongst different genotypes ranged from 32 (subtype 2a) to 130 nM (genotype 4); while some variation in susceptibility amongst patient isolates was observed, the 95th percentile for any genotype did not exceed 189 nM. Levels of resistance to sofosbuvir in replicons containing S282T were between 2.4 and 18 fold-change in EC50; no other single NS5B resistance-associated substitution demonstrated reduced sofosbuvir susceptibility. These data suggest that S282T is the only known substitution that confers detectable resistance to sofosbuvir in vitro. Sofosbuvir displayed potent antiviral activity across a diverse range of NS5B mutants and HCV clinical isolates in multiple subtypes of genotypes 1 to 6.

Effects of resistance-associated variants in genotype 2hepatitis C virus on viral replication and susceptibility to antihepatitis C virus drugs.

Development of direct-acting antivirals (DAAs) has made antihepatitis C virus (HCV) treatment highly safe and effective. However, the emergence of resistant-associated variants (RAVs) after failure of DAA therapy affects retreatment outcomes. In particular, genotype 1 HCV with P32 deletion has been reported to be highly resistant to all approved non-structural protein (NS)5A inhibitors. However, analysis of RAVs in genotype 2 HCV has been limited. Accordingly, in this study, we evaluated the roles of genotype 2 HCV variants in antiviral drug efficacy. We utilized HCV-2b/2a (JFH-1) chimeric virus (genotype 2a), which replicates more robustly than JFH-1. We constructed various genotype 2a JFH-1-based HCV cell culture systems with NS3 (D168E), NS5A (F28S, F28S/M31I, P32 deletion, and Y93H), and NS5B (S282T) RAVs and analyzed the replication ability and sensitivity to various anti-HCV reagents. Genotype 2a-based HCV with NS5A-P32 deletion could not replicate even in long-term cultures. Genotype 2a-based HCV with NS5A-F28S/M31I showed significantly higher replication ability than the wild-type strain, and replication could not be suppressed, even with high concentrations of NS5A inhibitors, including pibrentasvir and velpatasvir (<1000-10 000 fold-resistance compared with the wild-type strain). However, genotype 2a-based HCV with NA5A-F28S/M31I was sensitive to HCV protease inhibitor, NS5B inhibitor, interferon-α, and ribavirin. Genotype 2a-based HCV with NS5B-S282T was resistant to sofosbuvir, but was highly sensitive to ribavirin compared with the control. When undertaking retreatment for genotype 2a HCV-infected patients who fail to respond to DAAs, the optimized retreatment should be chosen according to the sensitivity of the emerging RAVs to anti-HCV drugs.