Hepatitis C virus nonstructural protein 5A inhibitors: Novel target—Now for new trials and new treatment strategies

Authors


  • Potential conflict of interest: Nothing to report.

Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 2010;465:96-100. Available at www. nature.com (Reprinted with permission.)

Abstract

The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people. Current therapy relies upon a combination of pegylated interferon-alpha and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC50) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log10 reduction in mean viral load measured 24 h post-dose that was sustained for an additional 120 h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144 h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors.

Comment

We are on the verge of a new era in the treatment of patients with chronic hepatitis C, with the first direct acting antiviral agents expected to become available in 2011. The most advanced agents target the hepatitis C virus (HCV) replication machinery, including the nonstructural protein 3/4A (NS3/4A) protease inhibitors, the NS5B polymerase inhibitors (nucleoside (NI)/non-nucleoside (NNI) and the cyclophilin inhibitors. Two NS3/4A protease inhibitors, telaprevir and boceprevir, currently lead the charge,1–4 and phase 3 registration trials will be completed this year.

Important lessons have been learned. HCV exists as a quasispecies generated by the error-prone NS5B RNA-dependent RNA polymerase, and drug resistance variants are selected within days of direct antiviral therapy. It is clear that monotherapy will not be possible with either telaprevir or boceprevir and that combination with pegylated-IFN-α (pegIFN) plus ribavirin (RBV) will be necessary, at a minimum. In the future, it is hoped that IFN-sparing combination direct antiviral regimens may be developed. Lessons from human immunodeficiency virus and hepatitis B virus tell us that the ideal candidates for such strategies would ideally be potent and have a high genetic barrier to the development of resistant variants. Multiple classes of antiviral drugs, with multiple different mechanisms of action, will minimize the potential for cross-resistant variants. Indeed, it has recently been argued that IFN-sparing regimens will require drug combinations with a genetic barrier of four or more mutations.5 In this context, Gao and colleagues6 have recently identified an exciting new compound that potently suppresses HCV replication in vitro and in vivo, with a broad HCV genotype spectrum, by targeting the HCV NS5A protein.

The research team used a chemical genetics approach to screen >1 million compounds in the Bristol-Myers Squibb library for anti-HCV activity. Active inhibitors of the NS3 protease, NS3 helicase, and NS5B polymerase were strategically excluded. The iminothiazolidinone BMS-858 was identified as a weak but specific inhibitor of HCV RNA replication. Subsequent chemical refinement identified BMS-790052 as a compound more suitable for clinical development. BMS-790052 has demonstrated potent activity against all HCV genotypes tested in vitro, with mean half-maximal effective concentrations (EC50) ranging from 9 pM and 50 pM for the HCV replicon genotypes 1b and 1a, up to 146 pM for the genotype 3a replicon, making it the most potent inhibitor of HCV replication publicly identified to date. (The HCV genotype spectrum of activity is also impressive: protease and polymerase inhibitors have been relatively specific for genotype 1 HCV to date, although telaprevir has activity against genotype 2 but not 3 HCV strains7). BMS-790052 is also a potent inhibitor of the Japanese fulminant hepatitis-1 infectious virus (EC50 = 28 pM), perhaps a more biologically relevant experimental system. Replicon combination studies also demonstrated additive–synergistic activity of BMS-790052 with IFN and RBV, as well as inhibitors of the NS3 protease and NS5B polymerase (NI/NNI). Two lines of evidence supported NS5A inhibition as the mechanism of action: (1) resistance was mapped to the NS5A protein in vitro and (2) a direct interaction between BMS-790052 and NS5A was supported by coprecipitation studies.

The major resistant variants identified for HCV genotype 1b were L31V and Y93H. For genotype 1a, the major variants identified were M28T and Q30H/R in addition to mutations at residues L31 and Y93. Higher levels of resistance were associated with substitutions on the HCV genotype 1a background; however, the EC50 of the most resistant genotype 1a variant, L31V, was ∼20 nM, levels readily achievable in vivo. Although more detailed virological studies will be required, many of the resistant variants displayed reduced fitness in vitro. Importantly, resistance to NS3A protease/NS5B polymerase inhibitors has not been mapped to the NS5A region, suggesting that cross-resistance is unlikely. Substitutions in NS5A have been associated with resistance to the cyclophilin inhibitors, including cyclosporine A. The authors therefore used the replicon system to test the sensitivity of BMS-790052–resistant variants to cyclosporine A. In all cases, the EC50 values were similar to those observed using a wild-type 1a replicon, suggesting that the risk of cross-resistance is also low here. A low risk of cyclophilin inhibitor cross-resistance was further supported by data mapping the inhibitory activity of BMS-790052 to the first 100 amino acids of NS5A (domain I), whereas variants associated with resistance to the cyclophilin inhibitors have mapped more distally to the C-terminal region (domain II [amino acid residues 250-342] and III [amino acid residues 356-447]).8, 9

Proof-of-concept single ascending dose-finding studies of healthy volunteers and patients with chronic hepatitis C were then performed. The pharmacokinetics of the compound seemed favorable. BMS-790052 was orally bioavailable and the plasma half-life ranged from 10-14 hours, suggesting that once daily dosing will be feasible. In patients infected with genotype 1 HCV, mean HCV RNA declines of 1.8 (0.2-0.3), 3.2 (2.9-4.0), and 3.3 (2.7-3.6) log10 IU/mL were observed at 24 hours following single doses of 1, 10, and 100 mg of the drug (Fig. 1). In two patients treated with the highest dose of BMS-790052, prolonged viral suppression to near undetectable levels at 144 hours was observed. Further studies will be needed to more clearly define the safety and efficacy during longer term use and in combination strategies.

Figure 1.

A novel signaling network controls tumor cell migration and invasion in HCC. The micro-RNA miR-151 and its hosting gene FAK are located within the same chromosomal locus (on chromosome 8) which is often found amplified in human HCC. The protein FAK/PTK2 controls activation of GTPases like Rho, Rac, and Cdc42, which in turn promote tumor cell migration, invasiveness, and thus metastasis of HCC. The analogous biological effect is mediated by a distinct pathway, in which the miR-151 suppresses transcription of the molecule RhoGDIA, an inhibitor of Cdc42, Rac, and Rho GTPases.

The exact function of the NS5A protein remains unclear. It is not known to have enzymatic activity, yet it is necessary for viral replication and important for the production of infectious virus particles.10 Therefore, the exact mechanism of action of BMS-790052 is yet to be resolved. NS5A exists as dimers that are believed to form a functional oligomeric complex.11 The authors speculate that BMS-790052 disrupts the oligomerization of NS5A dimers, thereby directly or allosterically interfering with protein function.

In conclusion, Gao and colleagues used an unbiased high-throughput screening approach to identify a novel HCV drug target. They present experimental and early phase clinical data confirming antiviral effect of BMS-790052, a first-in-class NS5A inhibitor. The compound shows potent efficacy and activity against a broad range of HCV genotypes. NS5A inhibitors can now be considered a future third class of direct-acting antiviral agents for the treatment of HCV. Further studies using BMS-790052 in triple therapy regimens with pegIFN plus RBV are eagerly awaited. In the future, NS5A inhibitors may be valuable for the treatment of HCV protease/polymerase inhibitor resistance. IFN-sparing combination regimens using NS5A inhibitors with HCV protease and polymerase inhibitors should also be evaluated.

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