Identification of hepatitis C virus NS5A inhibitors

Abstract

Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was approximately 5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.

FIG. 1.

Chemical structures of compounds used in this study. (A) Structure of screen hit BMS-858. (B) Structure of library compound BMS-824. (C) Structure of BMS-665.

FIG. 2.

Resistance to BMS-858 in cells transfected with RNA from wild-type or 858R cells. Naive Huh-7 cells were electroporated with total cellular RNA containing 2 ng HCV RNA isolated from wild-type (A) or 858R (B) cell lines and incubated with or without compound. HCV RNA levels were determined at various times posttransfection (pt).

FIG. 3.

Transient replication of wild-type and Y93H HCV genomes. Huh-7 cells were transfected with wild-type (WT) or Y93H replicon RNA and incubated in the presence or absence of 2 μM PI or 1 μM BMS-824. Luciferase activities (relative light units [RLU]) were determined with lysates of cells harvested 72 h after transfection. The 4-h value was used to correct for differences in transfection efficiencies. This graph represents data from one of two experiments giving similar results.

FIG. 4.

Schematic depicting the nonstructural region of HCV replicon RNAs and alignment of the N-terminal 100 amino acids of genotype 1a and 1b NS5A. (A) Genotype 1a and 1b sequences are shown, with the genotype 1a sequence being represented by a hatched fill pattern, and the positions of the nonstructural proteins are indicated. The nomenclature used for each construct is shown on the left, and the EC₅₀ of BMS-824 or BMS-665 for each construct is indicated at the right. nd, not determined. (B) The genotype 1a sequence is derived from the H77 strain, and genotype 1b is Con1. Two dots indicate identical amino acid residues between genotypes 1a and 1b.

FIG. 5.

Effect of compound on p58 production. DNAs encoding the wild-type (wt)-, Y93H-, or D168V-containing 1-377 1b HCV replicons were expressed in a vaccinia virus transient expression system treated with either DMSO (no cmpd) or a titration of BMS-824 (0.05 to 0.002 μM) or NS3 PI (0.1 to 0.01 μM). NS5A (Y93H) and NS3 (D168V) mutants were treated with 0.05 μM BMS-824 and 0.1 μM PI, respectively. Cell lysates were separated by SDS-PAGE on 8% gels, and NS5A proteins were identified by Western immunoblotting using an anti-NS5A antibody. NS5A-specific bands, both p56 and p58, were quantified by phosphorimaging.

FIG. 6.

Inhibitor effect on genotype 1a/1b NS5A hybrids. DNAs encoding various HCV replicons were expressed in a vaccinia virus transient expression system treated with either DMSO (−) or 0.1 μM BMS-824 (+). Cell lysates were separated by SDS-PAGE on 8% gels, and NS5A proteins were identified by Western immunoblotting using an anti-NS5A antibody. Cmpd, compound.

FIG. 7.

Blocking NS5A hyperphosphorylation. (A) Cells transiently expressing the HCV replicon were [³³P]orthophosphate labeled in the presence or absence of BMS-824, and extracts were immunoprecipitated with antibody to NS5A. Precipitated samples were then incubated in phosphatase buffer with or without CIP or BMS-824. A lysate from mock-transfected cells (no DNA) that had been infected only with vaccinia virus was included as a control. Samples treated with BMS-824 during labeling are indicated at the top of the gel. (B) Cells transiently expressing the HCV replicon were treated with or without BMS-824 and harvested at various times posttransfection. Western immunoblotting was performed on cell extracts by using an NS5A-specific antibody. The times of harvest are indicated above the gel. (C) Cells transiently expressing the HCV replicon were labeled with [³⁵S]methionine for 30 min and chased in the presence (+) or absence (−) of either BMS-824 or PI (PI not shown). Cells were harvested at 4 h postlabeling and immunoprecipitated with NS5A-specific antibody.