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...

The hepatitis C virus (HCV) NS3/4A serine protease has been explored as a target for the inhibition of viral replication in preclinical models and in HCV-infected patients. TMC435350 is a highly specific and potent inhibitor of NS3/4A protease selected from a series of novel macrocyclic inhibitors. In biochemical assays using NS3/4A proteases of genotypes 1a and 1b, inhibition constants of 0.5 and 0.4 nM, respectively, were determined. TMC435350 inhibited HCV replication in a cellular assay (subgenomic 1b replicon) with a half-maximal effective concentration (EC 50 ) of 8 nM and a selectivity index of 5,875. The compound was synergistic with alpha interferon and an NS5B inhibitor in the replicon model and additive with ribavirin. In rats, TMC435350 was extensively distributed to the liver and intestinal tract (tissue/plasma area under the concentration-time curve ratios of >35), and the absolute bioavailability was 44% after a single oral administration. Compound concentrations detected in both plasma and liver at 8 h postdosing were above the EC 99 value measured in the replicon. In conclusion, given the selective and potent in vitro anti-HCV activity, the potential for combination with other anti-HCV agents, and the favorable pharmacokinetic profile, TMC435350 has been selected for clinical development.

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Resistance to hepatitis C virus (HCV) inhibitors targeting viral enzymes has been observed in in vitroChronic hepatitis C virus (HCV) infection can lead to liver fibrosis, cirrhosis, hepatocellular carcinoma, and ultimately liver failure. Approximately 170 million people worldwide are infected with HCV (54a). The current standard of care consists of pegylated alpha interferon (Peg-IFN) plus ribavirin (RBV), providing limited efficacy for genotype 1-infected patients, i.e., a sustained virological response (SVR) in 40 to 50% of the patients. Moreover, Peg-IFN/RBV therapy is associated with significant adverse events (9). Therefore, direct antiviral agents (DAA) (previously also known as "specifically targeted antiviral therapies for hepatitis C" or STAT-C) have been a major focus of drug discovery efforts over the last 2 decades. Several NS3/4A (protease), NS5A, and NS5B (polymerase) inhibitors either alone or in combination with Peg-IFN/RBV have recently shown potent antiviral effects in HCV-infected patients (22,36). However, viral resistance to these novel agents can occur rapidly when they are dosed as monotherapy (43,49).Because of the high mutation rate of the HCV polymerase (10 Ϫ3 to 10 Ϫ5 misincorporations per nucleotide copied [11]) and the high viral production rates in vivo (approximately 10 12 viruses per patient per day [37]), it can be assumed that HCV exists as a diverse population of nonidentical but closely related viral genomes, referred to as a quasispecies (10). A viral quasispecies is characterized by a dominant nucleotide sequence, called a master sequence, and a surrounding mutant spectrum, which can harbor minority subpopulations (42). Although in theory all single and double mutants are produced daily in an infected person (6, 40), it is important to note that mutation rates are not equally distributed over the entire genome and that additional factors, such as viral fitness and the replication environment, determine whether a mutation becomes fixed in a viral quasispecies population (12). The diversity of the viral variants present in an infected individual facilitates the adaptation of the quasispecies to external pressure, such as antiviral treatment, improving the survival chances of the population (53). The speed of such adaptation depends mainly on the turnover of the viral nucleic acid acting as a source of new viral genomes. Whereas in HIV the rapid turnover of infected CD4 ϩ T lymphocytes is responsible for the rapid turnover of nucleic acids, in HCV rapid turnover is explained by the short half-life (ϳ10 h) of HCV RNA strands in the hepatocyte (47). However, if mutation rates exceed a certain limit, called the error threshold, deleterious mutations will accumulate and the viral population will become extinct (4).Recent reports have demonstrated that mutations known to affect the activities of DAA compounds in vitro are present in some treatment-naive patients as either dominant or minority species

If the shoe fits: TMC435, a noncovalent small‐molecule inhibitor of the hepatitis C virus (HCV) NS3/NS4A protease, is currently undergoing clinical evaluation as an HCV therapeutic. In the crystal structure of the noncovalent NS3/NS4A protease–TMC435 complex the bound inhibitor exploits induced‐fit binding. The new structure is consistent with the emerging view of viral resistance to NS3/NS4A protease inhibitors.