SYNTHESIS OF AMINO ACID AND PEPTIDE DERIVATIVES OF C60 FULLERENE AND THEIR ANTIVIRAL ACTIVITY AGAINST HEPATITIS C VIRUS

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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Identification of Three Interferon-Inducible Cellular Enzymes That Inhibit the Replication of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.02113-07 ◽

2007 ◽

Vol 82 (4) ◽

pp. 1665-1678 ◽

Cited By ~ 196

Author(s):

Dong Jiang ◽

Haitao Guo ◽

Chunxiao Xu ◽

Jinhong Chang ◽

Baohua Gu ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Protein Kinase ◽

Antiviral Activity ◽

Underlying Mechanism ◽

Hcv Infection ◽

Antiviral Effects ◽

Huh7 Cells ◽

Aromatic Amino Acid Residue

ABSTRACT Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis and is currently treated with alpha interferon (IFN-α)-based therapies. However, the underlying mechanism of IFN-α therapy remains to be elucidated. To identify the cellular proteins that mediate the antiviral effects of IFN-α, we created a HEK293-based cell culture system to inducibly express individual interferon-stimulated genes (ISGs) and determined their antiviral effects against HCV. By screening 29 ISGs that are induced in Huh7 cells by IFN-α and/or up-regulated in HCV-infected livers, we discovered that viperin, ISG20, and double-stranded RNA-dependent protein kinase (PKR) noncytolytically inhibited the replication of HCV replicons. Mechanistically, inhibition of HCV replication by ISG20 and PKR depends on their 3′-5′ exonuclease and protein kinase activities, respectively. Moreover, our work, for the first time, provides strong evidence suggesting that viperin is a putative radical S-adenosyl-l-methionine (SAM) enzyme. In addition to demonstrating that the antiviral activity of viperin depends on its radical SAM domain, which contains conserved motifs to coordinate [4Fe-4S] cluster and cofactor SAM and is essential for its enzymatic activity, mutagenesis studies also revealed that viperin requires an aromatic amino acid residue at its C terminus for proper antiviral function. Furthermore, although the N-terminal 70 amino acid residues of viperin are not absolutely required, deletion of this region significantly compromises its antiviral activity against HCV. Our findings suggest that viperin represents a novel antiviral pathway that works together with other antiviral proteins, such as ISG20 and PKR, to mediate the IFN response against HCV infection.

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Reduction of the infectivity of hepatitis C virus pseudoparticles by incorporation of misfolded glycoproteins induced by glucosidase inhibitors

Journal of General Virology ◽

10.1099/vir.0.82465-0 ◽

2007 ◽

Vol 88 (4) ◽

pp. 1133-1143 ◽

Cited By ~ 44

Author(s):

Cynthia Chapel ◽

Céline Garcia ◽

Birke Bartosch ◽

Philippe Roingeard ◽

Nicole Zitzmann ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Activity ◽

Diarrhea Virus ◽

E2 Glycoprotein ◽

Glucosidase Inhibitors ◽

Culture Model ◽

Viral Glycoproteins ◽

Viral Diarrhea Virus ◽

Derivatives Of

Folding and assembly into complexes of some viral glycoproteins are exquisitely sensitive to endoplasmic reticulum (ER) α-glucosidase inhibition, which prevents the trimming of glucose from N-linked glycans. Derivatives of deoxynojirimycin (DNJ) iminosugars, which are potent α-glucosidase inhibitors, were shown to have antiviral activity against bovine viral diarrhea virus, a pestivirus related to hepatitis C virus (HCV). The aim of this study was to determine whether these inhibitors would affect HCV infectivity and to provide novel insights on their mechanism of action. The overall antiviral activity of glucosidase inhibitors was shown by using the two most relevant models currently available: the cell-culture model enabling complete replication of the HCV JFH1 strain in Huh7.5 cells, and infectious HCV pseudotyped particles (HCVpp) produced in HEK-293T cells that display functional E1–E2 glycoprotein complexes. By using the latter model, it is shown that the inhibition of α-glucosidases by iminosugars results in the misfolding and misassembly of HCV glycoprotein pre-budding complexes. This inhibition of the assembly of E1–E2 in the ER of transfected HEK-293T cells leads to a reduction in the incorporation of E1–E2 complexes into HCVpp. More importantly, it is demonstrated that the infectivity of HCVpp that are released under treatment is reduced and that this reduction in infectivity is due to the incorporation of misfolded envelope glycoproteins in secreted particles. These properties suggest the potential usefulness of DNJ derivatives in combating HCV infection.

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Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00363-17 ◽

2017 ◽

Vol 61 (7) ◽

Cited By ~ 14

Author(s):

Ernest Asante-Appiah ◽

Stephanie Curry ◽

Patricia McMonagle ◽

Paul Ingravallo ◽

Robert Chase ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Antiviral Activity ◽

De Novo ◽

Clinical Isolates ◽

Amino Acid Substitutions ◽

Resistance Selection ◽

Direct Acting ◽

Nm Range

ABSTRACT Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents—grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor—in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC50) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir. The activity profiles of grazoprevir and elbasvir supported the testing of the direct-acting antivirals in clinical studies.

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In Vitro Antiviral Activity and Resistance Profile of the Next-Generation Hepatitis C Virus NS3/4A Protease Inhibitor Glecaprevir

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01620-17 ◽

2017 ◽

Vol 62 (1) ◽

Cited By ~ 32

Author(s):

Teresa I. Ng ◽

Rakesh Tripathi ◽

Thomas Reisch ◽

Liangjun Lu ◽

Timothy Middleton ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Antiviral Activity ◽

Protease Inhibitor ◽

Amino Acid Position ◽

Next Generation ◽

Genotype 3 ◽

Hcv Genotypes

ABSTRACTGlecaprevir (formerly ABT-493) is a novel hepatitis C virus (HCV) NS3/4A protease inhibitor (PI) with pangenotypic activity. It inhibited the enzymatic activity of purified NS3/4A proteases from HCV genotypes 1 to 6in vitro(half-maximal [50%] inhibitory concentration = 3.5 to 11.3 nM) and the replication of stable HCV subgenomic replicons containing proteases from genotypes 1 to 6 (50% effective concentration [EC50] = 0.21 to 4.6 nM). Glecaprevir had a median EC50of 0.30 nM (range, 0.05 to 3.8 nM) for HCV replicons containing proteases from 40 samples from patients infected with HCV genotypes 1 to 5. Importantly, glecaprevir was active against the protease from genotype 3, the most-difficult-to-treat HCV genotype, in both enzymatic and replicon assays demonstrating comparable activity against the other HCV genotypes. In drug-resistant colony selection studies, glecaprevir generally selected substitutions at NS3 amino acid position A156 in replicons containing proteases from genotypes 1a, 1b, 2a, 2b, 3a, and 4a and substitutions at position D/Q168 in replicons containing proteases from genotypes 3a, 5a, and 6a. Although the substitutions A156T and A156V in NS3 of genotype 1 reduced susceptibility to glecaprevir, replicons with these substitutions demonstrated a low replication efficiencyin vitro. Glecaprevir is active against HCV with most of the common NS3 amino acid substitutions that are associated with reduced susceptibility to other currently approved HCV PIs, including those at positions 155 and 168. Combination of glecaprevir with HCV inhibitors with other mechanisms of action resulted in additive or synergistic antiviral activity. In summary, glecaprevir is a next-generation HCV PI with potent pangenotypic activity and a high barrier to the development of resistance.

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Genotypic and Phenotypic Analyses of Hepatitis C Virus from Patients Treated with JTK-853 in a Three-Day Monotherapy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01432-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 436-444 ◽

Cited By ~ 1

Author(s):

Naoki Ogura ◽

Yukiyo Toyonaga ◽

Izuru Ando ◽

Kunihiro Hirahara ◽

Tsutomu Shibata ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Viral Resistance ◽

Hcv Rna ◽

Amino Acid Substitutions ◽

Sequencing Analysis ◽

Resistance Mutations ◽

In The Wild

ABSTRACTJTK-853, a palm site-binding NS5B nonnucleoside polymerase inhibitor, shows antiviral activityin vitroand in hepatitis C virus (HCV)-infected patients. Here, we report the results of genotypic and phenotypic analyses of resistant variants in 24 HCV genotype 1-infected patients who received JTK-853 (800, 1,200, or 1,600 mg twice daily or 1,200 mg three times daily) in a 3-day monotherapy. Viral resistance in NS5B was investigated using HCV RNA isolated from serum specimens from the patients. At the end of treatment (EOT) with JTK-853, the amino acid substitutions M414T (methionine [M] in position 414 at baseline was replaced with threonine [T] at EOT), C445R (cysteine [C] in position 445 at baseline was replaced with arginine [R] at EOT), Y448C/H (tyrosine [Y] in position 448 at baseline was replaced with cysteine [C] or histidine [H] at EOT), and L466F (leucine [L] in position 466 at baseline was replaced with phenylalanine [F] at EOT), which are known to be typical resistant variants of nonnucleoside polymerase inhibitors, were observed in a clonal sequencing analysis. These substitutions were also selected by a treatment with JTK-853in vitro, and the 50% effective concentration of JTK-853 in the M414T-, C445F-, Y448H-, and L466V-harboring replicons attenuated the susceptibility by 44-, 5-, 6-, and 21-fold, respectively, compared with that in the wild-type replicon (Con1). These findings suggest that amino acid substitutions of M414T, C445R, Y448C/H, and L466F are thought to be viral resistance mutations in HCV-infected patients receiving JTK-853 in a 3-day monotherapy.

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The Effect of Long-term Supplementation With Branched-chain Amino Acid Granules in Patients With Hepatitis C Virus-related Hepatocellular Carcinoma After Radiofrequency Thermal Ablation

Journal of Clinical Gastroenterology ◽

10.1097/mcg.0b013e31826be9ad ◽

2013 ◽

Vol 47 (4) ◽

pp. 359-366 ◽

Cited By ~ 25

Author(s):

Hiroki Nishikawa ◽

Yukio Osaki ◽

Eriko Iguchi ◽

Yorimitsu Koshikawa ◽

Soichiro Ako ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Thermal Ablation ◽

Radiofrequency Thermal Ablation ◽

Branched Chain Amino Acid ◽

Branched Chain

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P760 HIGH-THROUGHPUT AND ULTRASENSITIVE NEXT-GENERATION PCR ASSAY FOR THE QUANTIFICATION OF THE HEPATITIS C VIRUS MUTATION AT CORE AMINO ACID 70

Journal of Hepatology ◽

10.1016/s0168-8278(14)60921-1 ◽

2014 ◽

Vol 60 (1) ◽

pp. S324

Author(s):

M. Mukaide ◽

M. Sugiyama ◽

M. Korenaga ◽

K. Murata ◽

T. Kanto ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

High Throughput ◽

Next Generation ◽

Pcr Assay

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Prospective Characterization of Full-Length Hepatitis C Virus NS5A Quasispecies during Induction and Combination Antiviral Therapy

Journal of Virology ◽

10.1128/jvi.74.19.9028-9038.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 9028-9038 ◽

Cited By ~ 86

Author(s):

J.-B. Nousbaum ◽

S. J. Polyak ◽

S. C. Ray ◽

D. G. Sullivan ◽

A. M. Larson ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Antiviral Therapy ◽

Variable Region ◽

Response To Therapy ◽

Full Length ◽

Variable Regions ◽

C Terminus

ABSTRACT The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR237–276) sequence associated with IFN resistance was not found, although the presence of Ala245 within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P < 0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P < 0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.

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