In Vitro Resistance Profile of the Hepatitis C Virus NS3/4A Protease Inhibitor TMC435

Abstract

TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC 50 s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to ϳ2,000-fold for those with the D168V or D168I mutation, compared to the EC 50 for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC 50 s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (<3-fold change in EC 50 s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.Hepatitis C is a blood-borne infection that can ultimately result in severe liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma (7). The chronic nature of the disease and the significant possibility of long-term liver damage have led to the current global health burden, with an estimated 180 million people being infected, of whom 130 million are chronic hepatitis C virus (HCV) carriers (54).The current standard-of-care therapy for HCV-infected patients consists of a combination of weekly injected pegylated alpha interferon (Peg-IFN-␣) and twice-daily oral ribavirin. Treatment of HCV genotype 1-infected patients with this regimen for 48 weeks has a limited success rate (a 40 to 50% sustained virological response [SVR]) and is associated with a wide range of side effects, including flu-like symptoms, anemia, and depression, leading to treatment discontinuation in a significant proportion of patients (31, 48). Therefore, specifically targeted antiviral therapies for hepatitis C (STAT-C) have been a major focus of drug discovery efforts. Treatments with several NS3/4A protease inhibitors and NS5A and NS5B polymerase inhibitors, alone or in combination with Peg-IFN-␣-ribavirin, have recently shown encouraging results in clinical trials (17,36).HCV NS3 is an essential, bifunctional, multidomain protein that possesses protease and RNA helicase activiti...

Development of a high-throughput pyrosequencing assay for monitoring temporal evolution and resistance associated variant emergence in the Hepatitis C virus protease coding-region

“…In patient B, the Q80L mutation is detectable via pyrosequencing at low levels pre-Faldeprevir therapy and becomes dominant post-therapy. Interestingly, Q80L mutations do not render replicons resistant to a range of PIs (Lenz et al, 2010) and are not implicated in Faldeprevir resistance in vitro (Lagace et al, 2012) or in vivo (Berger et al, 2013). Our data demonstrate the Q80L mutation is selectively neutral with respect to viral replication and does not confer cross-resistance to Boceprevir or Teleprevir.…”

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

“…Mutations at R155 or D168 would disrupt the electrostatic network and destabilize this packing thereby lowering the affinity of these macrocyclic drugs. This observation provides a structural rationale for the drug resistance mutations R155K, as previously proposed (19), and D168A/V, which both confer a selective advantage in vitro in the presence of ITMN-191 or TMC435 (26,30). In addition, the TMC435 complex reveals that R155 is stabilized by a hydrogen bond with Q80, which also mutates to confer resistance to TMC435 (30).…”

“…The protease inhibitors ITMN-191 (3M5L), TMC435 (3KEE) (23), and boceprevir (2OC8) (24) protrude extensively from the substrate envelope in regions that correlate with known sites of resistance mutations. Most notably, the P2 moieties of all three drugs protrude to contact A156 and R155, which mutate to confer high-level resistance against nearly all drugs reported in the literature (25)(26)(27)(28)(29)(30). These findings suggest that drug resistance results from a change in molecular recognition and imply that drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations altering inhibitor binding will simultaneously interfere with the binding of substrates.…”

“…This observation provides a structural rationale for the drug resistance mutations R155K, as previously proposed (19), and D168A/V, which both confer a selective advantage in vitro in the presence of ITMN-191 or TMC435 (26,30). In addition, the TMC435 complex reveals that R155 is stabilized by a hydrogen bond with Q80, which also mutates to confer resistance to TMC435 (30). Thus many of the primary drug resistance mutations can be explained by the disruption of atomic interactions involving the P2 functional groups of the drugs.…”

Naturally Occurring Resistance-Associated Variants of Hepatitis C Virus Protease Inhibitors in Poor Responders to Pegylated Interferon-Ribavirin

“…Algorithm 2 integrates more mutations and was extracted from the website Geno2Pheno (http://hcv.geno2pheno.org/), developed by the Max-Planck-Institut für Informatik, Saarbrücken, Germany. Algorithm 3 was restricted to mutations that confer a Ͼ3-fold shift in HCV replicon activity (V36M plus T54S, V55A, R155K, and A156T/V) and has been used in clinical trials with boceprevir (1,(17)(18)(19). Table 1 summarizes the considered mutations for these three algorithms.…”