Discovery of a series of novel compounds with moderate anti-hepatitis C virus NS3 protease activity in vitro

NS3, a non-structural protein of the HCV (hepatitis C virus), contains a protease and a helicase domain and plays essential roles in the processing of the viral polyprotein, viral RNA replication and translation. LMP7 (low-molecular-mass protein 7), a component of the immunoproteasome, was identified as an NS3-binding protein from yeast two-hybrid screens, and this interaction was confirmed by in vitro binding and co-immunoprecipitation analysis. The minimal domain of interaction was defined to be between the pro-sequence region of LMP7 (amino acids 1–40) and the protease domain of NS3. To elucidate the biological importance of this interaction, we studied the effect of this interaction on NS3 protease activity and on LMP7 immunoproteasome activity. Recombinant LMP7 did not have any effect on NS3 protease activity in vitro. The peptidase activities of LMP7 immunoproteasomes, however, were markedly reduced when tested in a stable cell line containing a HCV subgenomic replicon. The down-regulation of proteasome peptidase activities could interfere with the processing of viral antigens for presentation by MHC class I molecules, and may thus protect HCV from host immune surveillance mechanisms to allow persistent infection by the virus.

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Preclinical Characterization of the Novel Hepatitis C Virus NS3 Protease Inhibitor GS-9451

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00487-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 647-653 ◽

Cited By ~ 25

Author(s):

Huiling Yang ◽

Margaret Robinson ◽

Amoreena C. Corsa ◽

Betty Peng ◽

Guofeng Cheng ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Activity ◽

Protease Inhibitor ◽

Hcv Infection ◽

Cross Resistance ◽

Protease Gene ◽

Ns5a Inhibitor ◽

Ns3 Protease

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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Preclinical Pharmacokinetics and In Vitro Metabolism of Asunaprevir (BMS-650032), a Potent Hepatitis C Virus NS3 Protease Inhibitor

Journal of Pharmaceutical Sciences ◽

10.1002/jps.24356 ◽

2015 ◽

Vol 104 (9) ◽

pp. 2813-2823 ◽

Cited By ~ 18

Author(s):

Kathleen W. Mosure ◽

Jay O. Knipe ◽

Marc Browning ◽

Vinod Arora ◽

Yue-Zhong Shu ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitor ◽

In Vitro Metabolism ◽

Preclinical Pharmacokinetics ◽

Ns3 Protease

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In VitroActivity of Simeprevir against Hepatitis C Virus Genotype 1 Clinical Isolates and Its Correlation with NS3 Sequence and Site-Directed Mutants

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01444-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7548-7557 ◽

Cited By ~ 16

Author(s):

Thierry Verbinnen ◽

Bart Fevery ◽

Leen Vijgen ◽

Tom Jacobs ◽

Sandra De Meyer ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Genotype 1 ◽

Low Level ◽

Fold Reduction ◽

Hcv Genotype 1 ◽

High Level ◽

Ns3 Protease ◽

Level Resistance

ABSTRACTSimeprevir (TMC435) is a once-daily, single-pill, oral hepatitis C virus (HCV) NS3 protease inhibitor approved for the treatment of chronic HCV infection. Phenotypic characterization of baseline isolates and isolates from HCV genotype 1-infected patients failing with a simeprevir-based regimen was performed using chimeric replicons carrying patient-derived NS3 protease sequences. Cutoff values differentiating between full susceptibility to simeprevir (≤2.0-fold reduction in simeprevir activity) and low-level versus high-level resistance (≥50-fold reduction in simeprevir activity) were determined. The median simeprevir fold change in the 50% effective concentration (FC) of pretreatment genotype 1a isolates, with and without Q80K, and genotype 1b isolates was 11, 0.9, and 0.4, respectively. Naturally occurring NS3 polymorphisms that reduced simeprevir activity, other than Q80K, were uncommon in the simeprevir studies and generally conferred low-level resistancein vitro. Although the proportion of patients with failure differed by HCV geno/subtype and/or presence of baseline Q80K, the level of simeprevir resistance observed at failure was similarly high irrespective of type of failure, HCV genotype 1 subtype, and presence or absence of baseline Q80K. At the end of the study, simeprevir activity against isolates that lost the emerging amino acid substitution returned to pretreatment values. Activity of simeprevir against clinical isolates and site-directed mutant replicons harboring the corresponding single or double amino acid substitutions correlated well, showing that simeprevir resistance can be attributed to these substitutions. In conclusion, pretreatment NS3 isolates were generally fully susceptible (FC, ≤2.0) or conferred low-level resistance to simeprevirin vitro(FC, >2.0 and <50). Treatment failure with a simeprevir-based regimen was associated with emergence of high-level-resistance variants (FC, ≥50).

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PCR-Based In Vitro Synthesis of Hepatitis C Virus NS3 Protease for Rapid Phenotypic Resistance Testing of Protease Inhibitors

Journal of Clinical Microbiology ◽

10.1128/jcm.03257-13 ◽

2014 ◽

Vol 52 (4) ◽

pp. 1139-1145 ◽

Cited By ~ 1

Author(s):

J. Qiao ◽

J. Yu ◽

H. Yang ◽

H. Wei

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitors ◽

Resistance Testing ◽

In Vitro Synthesis ◽

Phenotypic Resistance ◽

Ns3 Protease

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Inhibition of hepatitis C virus NS3 protease activity by product-based peptides is dependent on helicase domain

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/s0960-894x(00)00625-9 ◽

2001 ◽

Vol 11 (2) ◽

pp. 203-206 ◽

Cited By ~ 25

Author(s):

Anja Johansson ◽

Ina Hubatsch ◽

Eva Åkerblom ◽

Gunnar Lindeberg ◽

Susanne Winiwarter ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Activity ◽

Helicase Domain ◽

Ns3 Protease

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In VitroResistance Profile of the Hepatitis C Virus NS3 Protease Inhibitor BI 201335

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05166-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 569-572 ◽

Cited By ~ 41

Author(s):

Lisette Lagacé ◽

Peter W. White ◽

Christiane Bousquet ◽

Nathalie Dansereau ◽

Florence Dô ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitor ◽

Protease Inhibitors ◽

Alpha Interferon ◽

Phenotypic Characterization ◽

Genotype 1A ◽

Ns3 Protease

ABSTRACTThein vitroresistance profile of BI 201335 was evaluated through selection and characterization of variants in genotype 1a (GT 1a) and genotype 1b (GT 1b) replicons. NS3 R155K and D168V were the most frequently observed resistant variants. Phenotypic characterization of the mutants revealed shifts in sensitivity specific to BI 201335 that did not alter susceptibility to alpha interferon. In contrast to macrocyclic and covalent protease inhibitors, changes at V36, T54, F43, and Q80 did not confer resistance to BI 201335.

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Impact of Preexisting Hepatitis C Virus Genotype 6 NS3, NS5A, and NS5B Polymorphisms on theIn VitroPotency of Direct-Acting Antiviral Agents

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02205-18 ◽

2019 ◽

Vol 63 (4) ◽

Cited By ~ 2

Author(s):

Fiona McPhee ◽

Joseph Ueland ◽

Vincent Vellucci ◽

Scott Bowden ◽

William Sievert ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Agents ◽

Virologic Response ◽

Virus Genotype ◽

Direct Acting Antiviral ◽

East And Southeast Asia ◽

Direct Acting ◽

Ns3 Protease

ABSTRACTHCV genotype 6 (GT-6) is found predominantly in East and Southeast Asia. Clinical studies have focused on patients infected with hepatitis C virus (HCV) GT-6a, where high sustained virologic response (SVR) rates to direct-acting antivirals (DAAs) have been achieved. However, GT-6 is highly diverse, with 29 reported subtypes. We explored the diversity of GT-6 polymorphisms at residues associated with DAA resistance, their impact on DAAin vitropotency when evaluated in a GT-6a consensus replicon, and their association with specific GT-6 subtypes. GT-6 sequences from 25 patient-derived samples and 105 sequences from the U.S. HCV database were compared, and substitutions at resistance-associated residue positions were phenotyped against different DAAs. Preexisting resistance-associated substitutions (RASs) to NS3 protease (A156V and D168E) and NS5B nucleotide (L159F and S282C) inhibitors were rare (<4%). Preexisting RASs to NS5A inhibitors were common, especially at L28 (A/F/G/M/T/V) and R30 (E/N/S).In vitrosusceptibilities of NS5A-L28A and -L28T were dramatically reduced against all tested NS5A drugs (90% effective concentration [EC90] range, 119 to 2,032 nM) compared with susceptibilities against a GT-6a consensus replicon (EC90range, 0.1 to 19 nM). These L28 RASs preexisted in combination with R30S (EC90[L28A-R30S] of ≥720 nM or EC90[L28T-R30S] of ≥128 nM against tested DAAs) or as L28T-L31I (EC90[tested DAAs] of >5,000 nM) and were detected in evaluated GT-6b and -6f sequences. NS5A-L28A-R30A, observed in GT-6r, did not replicate. In conclusion, HCV GT-6b, GT-6f, and GT-6r sequences harbored highly resistant RASs to all evaluated NS5A drugs. Therefore, monitoring SVR in patients infected with these GT-6 subtypes treated with NS5A drug-containing regimens is suggested to confirm any association between noted NS5A polymorphisms and treatment failure.

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Hyperphosphorylation of the Hepatitis C Virus NS5A Protein Requires an Active NS3 Protease, NS4A, NS4B, and NS5A Encoded on the Same Polyprotein

Journal of Virology ◽

10.1128/jvi.73.12.9984-9991.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 9984-9991 ◽

Cited By ~ 90

Author(s):

Petra Neddermann ◽

Angelica Clementi ◽

Raffaele De Francesco

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Activity ◽

Nonstructural Protein ◽

Kinetic Studies ◽

Stable Complex ◽

Polyprotein Processing ◽

Total Inhibition ◽

Necessary And Sufficient ◽

Ns3 Protease

ABSTRACT The nonstructural protein NS5A of hepatitis c virus (HCV) has been demonstrated to be a phosphoprotein with an apparent molecular mass of 56 kDa. In the presence of other viral proteins, p56 is converted into a slower-migrating form of NS5A (p58) by additional phosphorylation events. In this report, we show that the presence of NS3, NS4A, and NS4B together with NS5A is necessary and sufficient for the generation of the hyperphosphorylated form of NS5A (p58) and that all proteins must be encoded on the same polyprotein (in cis). Kinetic studies of NS5A synthesis and pulse-chase experiments demonstrate that fully processed NS5A is the substrate for the formation of p58 and that p56 is converted to p58. To investigate the role of NS3 in NS5A hyperphosphorylation, point and deletion mutations were introduced into NS3 in the context of a polyprotein containing the proteins from NS3 to NS5A. Mutation of the catalytic serine residue into alanine abolished protease activity of NS3 and resulted in total inhibition of NS5A hyperphosphorylation, even if polyprotein processing was allowed by addition of NS3 and NS4A in trans. The same result was obtained by deletion of the first 10 or 28 N-terminal amino acids of NS3, which are known to be important for the formation of a stable complex between NS3 and its cofactor NS4A. These data suggest that the formation of p58 is closely connected to HCV polyprotein processing events. Additional data obtained with NS3 containing the 34 C-terminal residues of NS2 provide evidence that in addition to NS3 protease activity the authentic N-terminal sequence is required for NS5A hyperphosphorylation.

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