scholarly journals Preclinical Profile of VX-950, a Potent, Selective, and Orally Bioavailable Inhibitor of Hepatitis C Virus NS3-4A Serine Protease

2006 ◽  
Vol 50 (3) ◽  
pp. 899-909 ◽  
Author(s):  
Robert B. Perni ◽  
Susan J. Almquist ◽  
Randal A. Byrn ◽  
Gurudatt Chandorkar ◽  
Pravin R. Chaturvedi ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
Enzyme Inhibitor ◽  
Pharmacokinetic Profile ◽  
Hcv Rna ◽  
High Exposure ◽  
Genotype 1A ◽  
Hcv Protease ◽  
Orally Bioavailable

ABSTRACT VX-950 is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease, and it demonstrated excellent antiviral activity both in genotype 1b HCV replicon cells (50% inhibitory concentration [IC50] = 354 nM) and in human fetal hepatocytes infected with genotype 1a HCV-positive patient sera (IC50 = 280 nM). VX-950 forms a covalent but reversible complex with the genotype 1a HCV NS3-4A protease in a slow-on, slow-off process with a steady-state inhibition constant (Ki *) of 7 nM. Dissociation of the covalent enzyme-inhibitor complex of VX-950 and genotype 1a HCV protease has a half-life of almost an hour. A >4-log10 reduction in the HCV RNA levels was observed after a 2-week incubation of replicon cells with VX-950, with no rebound of viral RNA observed after withdrawal of the inhibitor. In several animal species, VX-950 exhibits a favorable pharmacokinetic profile with high exposure in the liver. In a recently developed HCV protease mouse model, VX-950 showed excellent inhibition of HCV NS3-4A protease activity in the liver. Therefore, the overall preclinical profile of VX-950 supports its candidacy as a novel oral therapy against hepatitis C.

scholarly journals NS3 protein of Hepatitis C virus associates with the tumour suppressor p53 and inhibits its function in an NS3 sequence-dependent manner

2006 ◽  
Vol 87 (6) ◽  
pp. 1703-1713 ◽  
Author(s):  
Lin Deng ◽  
Motoko Nagano-Fujii ◽  
Motofumi Tanaka ◽  
Yuki Nomura-Takigawa ◽  
Masanori Ikeda ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Subcellular Localization ◽  
Serine Protease ◽  
Protease Activity ◽  
Hcv Rna ◽  
Dependent Manner ◽  
Diffuse Type ◽  
Serine Protease Activity ◽  
Tumour Suppressor P53

The N-terminal 198 residues of NS3 (NS3-N) of Hepatitis C virus (HCV) subtype 1b obtained from 29 patients, as well as full-length NS3 (NS3-Full), were analysed for their subcellular localization, interaction with the tumour suppressor p53 and serine protease activity in the presence and absence of the viral cofactor NS4A. Based on the subcellular-localization patterns in the absence of NS4A, NS3-N sequences were classified into three groups, with each group exhibiting either dot-like, diffuse or a mixed type of localization. Chimeric NS3-Full sequences, each consisting of an individual NS3-N and a shared C-terminal sequence, showed the same localization patterns as those of the respective NS3-N. Site-directed mutagenesis experiments revealed that a single or a few amino acid substitutions at a particular position(s) of NS3-N altered the localization pattern. Interestingly, NS3 of the dot-like type, either NS3-N or NS3-Full, interacted with p53 more strongly than that of the diffuse type, in both the presence and the absence of NS4A. Moreover, NS3-N of the dot-like type suppressed trans-activating activity of p53 more strongly than that of the diffuse type. Serine protease activity did not differ significantly between the two types of NS3. In HCV RNA replicon-harbouring cells, physical interaction between NS3 and p53 was observed consistently and p53-mediated transcriptional activation was suppressed significantly compared with HCV RNA-negative control cells. Our results collectively suggest the possibility that NS3 plays an important role in the hepatocarcinogenesis of HCV by interacting differentially with p53 in an NS3 sequence-dependent manner.

scholarly journals Genetic Screen for Monitoring Hepatitis C Virus NS3 Serine Protease Activity

2003 ◽  
Vol 47 (5) ◽  
pp. 1760-1765 ◽  
Author(s):  
Miguel Angel Martinez ◽  
Bonaventura Clotet
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
Genetic System ◽  
Lytic Infection ◽  
Lambda Phage ◽  
Regulatory Circuit ◽  
Serine Protease Activity ◽  
Hcv Protease ◽  
Specific Inhibitors

ABSTRACT We have developed a genetic system to monitor the activity of the hepatitis C virus (HCV) NS3 serine protease. This genetic system is based on the bacteriophage lambda regulatory circuit where the viral repressor cI is specifically cleaved to initiate the switch from lysogeny to lytic infection. An HCV protease-specific target, NS5A-5B, was inserted into the lambda phage cI repressor. The target specificity of the HCV NS5A-5B repressor was evaluated by coexpression of this repressor with a β-galactosidase (βgal)-HCV NS32-181/421-34 protease construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI.HCV5AB-cro and the βgal-HCV NS32-181/421-34 protease constructs, lambda phage replicated up to 8,000-fold more efficiently than in cells that did not express the HCV NS32-181/421-34 protease. This simple, rapid, and highly specific assay can be used to monitor the activity of the HCV NS3 serine protease, and it has the potential to be used for screening specific inhibitors.

scholarly journals In VitroandIn VivoAntiviral Activity and Resistance Profile of the Hepatitis C Virus NS3/4A Protease Inhibitor ABT-450

2014 ◽  
Vol 59 (2) ◽  
pp. 988-997 ◽  
Author(s):  
Tami Pilot-Matias ◽  
Rakesh Tripathi ◽  
Daniel Cohen ◽  
Isabelle Gaultier ◽  
Tatyana Dekhtyar ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Agents ◽  
Cross Resistance ◽  
Hcv Rna ◽  
Genotype 1 ◽  
Common Amino Acid ◽  
Direct Acting Antiviral ◽  
Genotype 1A

ABSTRACTThe development of direct-acting antiviral agents is a promising therapeutic advance in the treatment of hepatitis C virus (HCV) infection. However, rapid emergence of drug resistance can limit efficacy and lead to cross-resistance among members of the same drug class. ABT-450 is an efficacious inhibitor of HCV NS3/4A protease, with 50% effective concentration values of 1.0, 0.21, 5.3, 19, 0.09, and 0.69 nM against stable HCV replicons with NS3 protease from genotypes 1a, 1b, 2a, 3a, 4a, and 6a, respectively.In vitro, the most common amino acid variants selected by ABT-450 in genotype 1 were located in NS3 at positions 155, 156, and 168, with the D168Y variant conferring the highest level of resistance to ABT-450 in both genotype 1a and 1b replicons (219- and 337-fold, respectively). In a 3-day monotherapy study with HCV genotype 1-infected patients, ABT-450 was coadministered with ritonavir, a cytochrome P450 3A4 inhibitor shown previously to markedly increase peak, trough, and overall drug exposures of ABT-450. A mean maximum HCV RNA decline of 4.02 log10was observed at the end of the 3-day dosing period across all doses. The most common variants selected in these patients were R155K and D168V in genotype 1a and D168V in genotype 1b. However, selection of resistant variants was significantly reduced at the highest ABT-450 dose compared to lower doses. These findings were informative for the subsequent evaluation of ABT-450 in combination with additional drug classes in clinical trials in HCV-infected patients. (Study M11-602 is registered at ClinicalTrials.gov under registration no. NCT01074008.)

scholarly journals 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

2016 ◽  
Vol 60 (5) ◽  
pp. 2954-2964 ◽  
Author(s):  
Frederick C. Lahser ◽  
Karin Bystol ◽  
Stephanie Curry ◽  
Patricia McMonagle ◽  
Ellen Xia ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protease Inhibitor ◽  
Cross Resistance ◽  
Hcv Rna ◽  
Wild Type ◽  
Genetic Barrier ◽  
Potent Effect ◽  
Ns5a Inhibitor ◽  
Genotype 1A

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.

scholarly journals The Octadecyloxyethyl Ester of (S)-9-[3-Hydroxy-2-(Phosphonomethoxy) Propyl]Adenine Is a Potent and Selective Inhibitor of Hepatitis C Virus Replication in Genotype 1A, 1B, and 2A Replicons

2009 ◽  
Vol 53 (6) ◽  
pp. 2660-2662 ◽  
Author(s):  
David L. Wyles ◽  
Kelly A. Kaihara ◽  
Brent E. Korba ◽  
Robert T. Schooley ◽  
James R. Beadle ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Active Compound ◽  
Hcv Rna ◽  
Genotype 1A ◽  
B Virus ◽  
Immunodeficiency Virus

ABSTRACT The octadecyloxyethyl (ODE) and hexadecyloxypropyl (HDP) esters of (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) are potent inhibitors of orthopoxvirus, herpesvirus, human immunodeficiency virus type 1, and hepatitis B virus replication in vitro. HDP and ODE esters of (S)-HPMPA and (R)-HPMPA were evaluated for their activity in hepatitis C virus (HCV) replicon assays using luciferase (1B and 2A replicons) or RNA (1B) quantification. The ODE ester of (S)-HPMPA [ODE-(S)-HPMPA] was the most active compound, with 50% effective concentrations (EC50s) in the 0.69 to 1.31 μM range. HDP and ODE esters of (R)-HPMPA were severalfold less active, while (S)-HPMPA and (R)-HPMPA were inactive. In genotype 1A and 1B replicons analyzed by HCV RNA analysis, ODE-(S)-HPMPA was the most active compound, with EC50s of 1.8 and 2.1 μM, respectively.

scholarly journals In VitroActivity and Resistance Profile of Samatasvir, a Novel NS5A Replication Inhibitor of Hepatitis C Virus

2014 ◽  
Vol 58 (8) ◽  
pp. 4431-4442 ◽  
Author(s):  
J. P. Bilello ◽  
L. B. Lallos ◽  
J. F. McCarville ◽  
M. La Colla ◽  
I. Serra ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Agents ◽  
Resistance Profile ◽  
Direct Acting Antiviral ◽  
Genotype 1A ◽  
Hcv Protease ◽  
Chronic Hcv ◽  
Direct Acting

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.

scholarly journals Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture

2003 ◽  
Vol 77 (5) ◽  
pp. 3181-3190 ◽  
Author(s):  
Keril J. Blight ◽  
Jane A. McKeating ◽  
Joseph Marcotrigiano ◽  
Charles M. Rice
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Replication ◽  
Full Length ◽  
Hcv Rna ◽  
Metabolic Labeling ◽  
Virus Genotype ◽  
Adaptive Mutations ◽  
Genotype 1A

ABSTRACT Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.

scholarly journals Activity of a Potent Hepatitis C Virus Polymerase Inhibitor in the Chimpanzee Model

2007 ◽  
Vol 51 (12) ◽  
pp. 4290-4296 ◽  
Author(s):  
Chih-Ming Chen ◽  
Yupeng He ◽  
Liangjun Lu ◽  
Hock Ben Lim ◽  
Rakesh L. Tripathi ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Viral Load ◽  
Hepatitis C ◽  
Half Life ◽  
Nonstructural Protein ◽  
Hcv Rna ◽  
Antiviral Efficacy ◽  
Genotype 1A ◽  
Genotype 1B ◽  
Infected Chimpanzee

ABSTRACT A-837093 is a potent and specific nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase. It possesses nanomolar potencies in both enzymatic and replicon-based cell culture assays. In rats and dogs this compound demonstrated an oral plasma half-life of greater than 7 h, and its bioavailability was >60%. In monkeys it had a half-life of 1.9 h and 15% bioavailability. Its antiviral efficacy was evaluated in two chimpanzees infected with HCV in a proof-of-concept study. The design included oral dosing of 30 mg per kg of body weight twice a day for 14 days, followed by a 14-day posttreatment observation. Maximum viral load reductions of 1.4 and 2.5 log10 copies RNA/ml for genotype 1a- and 1b-infected chimpanzees, respectively, were observed within 2 days after the initiation of treatment. After this initial drop in the viral load, a rebound of plasma HCV RNA was observed in the genotype 1b-infected chimpanzee, while the genotype 1a-infected chimpanzee experienced a partial rebound that lasted throughout the treatment period. Clonal analysis of NS5B gene sequences derived from the plasma of A-837093-treated chimpanzees revealed the presence of several mutations associated with resistance to A-837093, including Y448H, G554D, and D559G in the genotype 1a-infected chimpanzee and C316Y and G554D in the genotype 1b-infected chimpanzee. The identification of resistance-associated mutations in both chimpanzees is consistent with the findings of in vitro selection studies, in which many of the same mutations were selected. These findings validate the antiviral efficacy and resistance development of benzothiadiazine HCV polymerase inhibitors in vivo.

scholarly journals Baseline Hepatitis C Virus (HCV) NS3 Polymorphisms and Their Impact on Treatment Response in Clinical Studies of the HCV NS3 Protease Inhibitor Faldaprevir

2013 ◽  
Vol 58 (2) ◽  
pp. 698-705 ◽  
Author(s):  
Kristi L. Berger ◽  
Ibtissem Triki ◽  
Mireille Cartier ◽  
Martin Marquis ◽  
Marie-Josée Massariol ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Protease Inhibitor ◽  
Clinical Studies ◽  
Phase 2 ◽  
Genotype 1A ◽  
Treatment Naïve ◽  
Hcv Protease

ABSTRACTA challenge to the treatment of chronic hepatitis C with direct-acting antivirals is the emergence of drug-resistant hepatitis C virus (HCV) variants. HCV with preexisting polymorphisms that are associated with resistance to NS3/4A protease inhibitors have been detected in patients with chronic hepatitis C. We performed a comprehensive pooled analysis from phase 1b and phase 2 clinical studies of the HCV protease inhibitor faldaprevir to assess the population frequency of baseline protease inhibitor resistance-associated NS3 polymorphisms and their impact on response to faldaprevir treatment. A total of 980 baseline NS3 sequences were obtained (543 genotype 1b and 437 genotype 1a sequences). Substitutions associated with faldaprevir resistance (at amino acid positions 155 and 168) were rare (<1% of sequences) and did not compromise treatment response: in a phase 2 study in treatment-naive patients, six patients had faldaprevir resistance-associated polymorphisms at baseline, of whom five completed faldaprevir-based treatment and all five achieved a sustained virologic response 24 weeks after the end of treatment (SVR24). Among 13 clinically relevant amino acid positions associated with HCV protease resistance, the greatest heterogeneity was seen at NS3 codons 132 and 170 in genotype 1b, and the most common baseline substitution in genotype 1a was Q80K (99/437 [23%]). The presence of the Q80K variant did not reduce response rates to faldaprevir-based treatment. Across the three phase 2 studies, there was no significant difference in SVR24 rates between patients with genotype 1a Q80K HCV and those without Q80K HCV, whether treatment experienced (17% compared to 26%;P= 0.47) or treatment naive (62% compared to 66%;P= 0.72).

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