Hepatitis C virus (HCV) drug development has resulted in treatment regimens composed of interferon‐free, all‐oral combinations of direct‐acting antivirals. While the new regimens are potent and highly efficacious, the full clinical impact of HCV drug resistance, its implications for retreatment, and the potential role of baseline resistance testing remain critical research and clinical questions. In this report, we discuss the viral proteins targeted by HCV direct‐acting antivirals and summarize clinically relevant resistance data for compounds that have been approved or are currently in phase 3 clinical trials. Conclusion: This report provides a comprehensive, systematic review of all resistance information available from sponsors’ trials as a tool to inform the HCV drug development field. (Hepatology 2015;62:1623–1632)

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Background & Aims All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus (HCV) genotype 1a cell culture-infectious virus H77S.3. Methods We performed detailed kinetic analyses of specific steps in the HCV life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production. Results Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared to protease or polymerase inhibitors. By 24 hrs after addition of an NS5A inhibitor, polyprotein synthesis was reduced less than 50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hrs. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing HCV particles, indicating a block in virus assembly. Conclusions DAAs that target NS5A rapidly inhibit intracellular assembly of gentoype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against pre-formed replicase, thereby resulting in slow shut-off of viral RNA synthesis.

Elbasvir is an investigational NS5A inhibitor with in vitro activity against multiple HCV genotypes. Antiviral activity of elbasvir was measured in replicons derived from wild-type or resistant variants of genotypes 1a, 1b, and 3. The barrier to resistance was assessed by the number of resistant colonies selected by exposure to various elbasvir concentrations. In a phase 1b dose-escalating study, virologic responses were determined in 48 noncirrhotic adult men with chronic genotype 1 or 3 infections randomized to placebo or elbasvir from 5 to 50 mg (genotype 1) or 10 to 100 mg (genotype 3) once daily for 5 days. The NS5A gene was sequenced from plasma specimens obtained before, during, and after treatment. Elbasvir suppressed the emergence of resistanceassociated variants (RAVs) in vitro in a dose-dependent manner. Variants selected by exposure to high elbasvir concentrations typically encoded multiple amino acid substitutions (most commonly involving loci 30, 31, and 93), conferring high-level elbasvir resistance. In the monotherapy study, patients with genotype 1b had greater reductions in HCV RNA levels than patients with genotype 1a at all elbasvir doses; responses in patients with genotype 3 were generally less pronounced than for genotype 1, particularly at lower elbasvir doses. M28T, Q30R, L31V, and Y93H in genotype 1a, L31V and Y93H in genotype 1b, and A30K, L31F, and Y93H in genotype 3 were the predominant RAVs selected by elbasvir monotherapy. Virologic findings in patients were consistent with the preclinical observations. NS5A-RAVs emerged most often at amino acid positions 28, 30, 31, and 93 in both the laboratory and clinical trial. (The MK-8742 P002 trial has been registered at ClinicalTrials.gov under identifier NCT01532973.)

Hepatitis C virus~HCV! nonstructural protein 3~NS3! has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A~NS4A! to form a stable complex. Previous attempts to produce the NS30NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single-chain form of the NS30NS4A complex~His-NS4A 21-32 -GSGS-NS3 3-631 ! was constructed in which the NS4A core peptide is fused to the N-terminus of the NS3 protease domain as previously described~Taremi et al., 1998!. This protein contains a histidine tagged NS4A peptide~a.a. 21-32! fused to the full-length NS3~a.a. 3-631! through a flexible tetra amino acid linker. The recombinant protein was expressed to high levels in Escherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities. The single-chain recombinant NS3-NS4A protein possesses physiological properties equivalent to those of the NS30NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA. Comparison of the helicase activity of the single-chain recombinant NS3-NS4A with that of the full-length NS3~without NS4A! and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity.