Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect

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 (EC(50)) 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 log(10) 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.

Figure 1

Structures of the iminothiazolidinone BMS-858, BMS-790052, the biotin-tagged HCV NS5A inhibitor 1 and its inactive stereoisomer 2.

Figure 2. The active inhibitor 1 binds to genotype 1b HCV NS5A whereas the inactive stereoisomer 2 does not.

Genotype 1b replicon cells were exposed to either 1 or 2 for approximately 18 h before the cells were lysed. A portion of the supernatant was set aside to serve as input control for immunoblot analysis (lanes 1 and 2) while the remainder was mixed with streptavidin–agarose beads and incubated for several hours. Bound proteins were detected by immunoblotting with primary antibodies directed to HCV NS5A. Lanes 1 and 3 depict the results of experiments with the active inhibitor compound 1 whereas lanes 2 and 4 depict the results with the inactive stereoisomer 2. PowerPoint slide

Figure 3. Mean plasma concentration–time profile (time 0–72 h) of BMS-790052 after single oral administration of 1–200 mg of drug to healthy subjects.

In a double-blind, placebo-controlled, sequential, single ascending-dose study, eight male or female subjects were randomized within each dose panel (1, 10, 25, 50, 100 and 200 mg) to drug or placebo in a ratio of 3:1. BMS-790052 or placebo was administered in the fasted state. The plasma samples obtained at various times were analysed for BMS-790052 by a validated liquid chromatography tandem mass spectrometry assay. Pharmacokinetic parameter values for individual subjects were derived by non-compartmental methods by a validated pharmacokinetic analysis programme. PBA EC₉₀, protein-binding-adjusted EC₉₀ for the individual genotype in a replicon assay. Error bars, standard deviation. PowerPoint slide

Figure 4. Mean change in log₁₀ HCV RNA with 90% confidence intervals after administration of single oral doses of BMS-790052 to HCV-infected patients.

In a double-blind, placebo-controlled, sequential, single ascending-dose study, six subjects were randomized within each dose panel (1, 10, 100 mg) to drug or placebo in a ratio of 5:1. BMS-790052 or placebo was administered in the fasted state. Owing to a dosing error, all six subjects received BMS-790052 in the 1 mg panel. One subject in the 10 mg panel withdrew from the study 8 h after administration of the study drug for non-drug-related reasons; HCV RNA data from the subject are included up until the subject withdrew. PowerPoint slide