In Vitro Resistance Studies of Hepatitis C Virus Serine Protease Inhibitors, VX-950 and BILN 2061

Abstract

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3⅐4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3⅐4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3⅐4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. It is estimated that 170 million patients worldwide and about 1% of the population in developed countries are chronically infected with hepatitis C virus (HCV) 1 (1). The majority of acute HCV infections become chronic, some of which progress toward liver cirrhosis or hepatocellular carcinoma (2, 3). The current standard of care is pegylated interferon ␣ in combination with ribavirin, which has a sustained viral response rate of 40 -50% in genotype 1 HCV-infected patients, which accounts for the majority of the hepatitis C population in the United States and Japan, and of 80 -90% in patients infected with genotype 2 or 3 HCV (4, 5) (for a review, see Ref. 6). Thus, more effective therapeutic drugs with fewer side effects and shorter treatment durations are needed for patients infected with HCV.HCV is an enveloped, single-stranded RNA virus with a 9.6-kb positive-polarity genome, which encodes a polyprotein precursor of about 3,000 amino acids. The HCV polyprotein is proteolytically processed by cellular and HCV proteases into at least 10 distinct products, in the order of NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (for a review, see Ref. 7). NS3 serine protease and helicase as well as NS5B RNA-dependent RNA polymerase are believed to be components of a replication complex responsible for viral RNA replication and have been shown to be essential for the HCV replication in chimpanzees (8). These HCV enzymes have been the major targets for the development of HCV-specific therapeutics during the past decade (for a review, see Ref. 9). However, successful discovery of a new HCV-specific drug candidate has been hampered by the lack of a robust, reproducible infectious virus cell culture system. The development of a HCV replicon system by Lohmann et al. (10) and subsequent optimization by several laboratories (11, 12) has enabled quantitative evaluation of the antiviral potency of HCV inhibitors.The HCV NS3⅐4A protease is responsible for cleavage at four sites within the HCV polyprotein to generate the N termini of the NS4A, NS4B, NS5A, and NS5B proteins (13-17). It has been shown that the central region (amino acids 21-30) of the 54-residue NS4A protein is essentia...

The non-immunosuppressive cyclosporin DEBIO-025 is a potent inhibitor of hepatitis C virus replicationin vitro

“…A selectivity index (CC 50 /EC 50 ) of approximately 900 was calculated for DEBIO-025, whereas this value was approximately 40 for CsA. For comparative reasons, the antiviral activity of both a nucleoside and a non-nucleoside HCV polymerase inhibitor, 2Ј-C-methyl-cytidine (24), 25 and an HCV protease inhibitor (VX-950) [26][27][28] was determined in Huh 5-2 cells. The EC 50 values for these three compounds were, respectively, 0.1 Ϯ 0.06 g/mL, 0.66 Ϯ 0.09 g/mL, and 0.4 Ϯ 0.06 g/mL.…”

“…The preparation of DEBIO-025 was based on the synthetic strategy for D-MeAla 3 -EtXaa 4 -cyclosporin analogs described previously. 23 CsA was purchased from Fluka Chemie GmbH (9471 Buchs, Switzerland), 2Ј-C-methyl-Cytidine, 24 25 and VX-950 [26][27][28] were synthesized by standard methods.…”

Antiviral activity of telaprevir (VX-950) and peginterferon alfa-2a in patients with hepatitis C

“…In HCV replicon studies, these viral variants remained sensitive to interferon. 9 In patients receiving telaprevir and peginterferon alfa-2a, the only evidence of viral selection was detection of the A156T variant in 1 patient at day 8 and the V36A variant in another patient at the 1-week follow-up visit. Despite the presence of these variants, the patients' HCV RNA levels continued to decline and remained undetectable at 12 weeks after the end of dosing, suggesting that peginterferon alfa and ribavirin or the immune response were able to suppress the remaining virus.…”

“…When given as monotherapy, peginterferon alfa has limited efficacy against chronic hepatitis C. 6,7 In HCV replicon studies, interferon and telaprevir had synergistic effects in reducing HCV RNA, 8 and viral variants with decreased sensitivity to telaprevir remained sensitive to interferon. 9 Based on these data and the different antiviral mechanisms of action of telaprevir and interferon, it was hypothesized that the combination of telaprevir and peginterferon alfa would result in greater antiviral activity than either telaprevir or peginterferon alfa alone and would reduce the incidence of viral breakthrough.…”

Virologic Escape during Danoprevir (ITMN-191/RG7227) Monotherapy Is Hepatitis C Virus Subtype Dependent and Associated with R155K Substitution

“…In vitro and in vivo studies indicate that overlapping but distinct sets of NS3/4A variants are associated with reduced susceptibility to PIs (18,23,30,38,44,49). In genotype 1a, resistance against both linear and macrocyclic PIs is principally achieved by substitution at position R155.…”