A small-molecule inhibitor of hepatitis C virus infectivity

Abstract

One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. We report here the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs with low-nanomolar 50% effective concentrations. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of noninfectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its prefusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.

FIG 1

Chemical structure of HCV infectivity inhibitor 1 (HCV II-1), also known as GS-563253 or [(4R,7S)-2,2,2-trifluoroethyl 2-methyl-5-oxo-7-phenyl-1,4,5,6,7,8-hexahydro-[4,5′-biquinoline]-3-carboxylate].

FIG 2

Characterization of intracellular HCV particles during HCV II-1 treatment. Equilibrium density gradient centrifugation was performed as indicated in Materials and Methods. In all graphs, the solid red line represents HCV infectivity, the blue line represents HCV RNA levels, and the black line represents the average buoyant density. The data shown represent the averages of three assays, and error bars represent the standard deviations. (A) Equilibrium density gradient centrifugation of the intracellular material from an HCV-infected cell culture treated with 0.2% DMSO for 72 h. (B) Equilibrium density gradient centrifugation of the intracellular material from an HCV-infected cell culture treated with 5× EC₅₀ (530 nM) of the NS3-4A protease inhibitor BILN-2061 for 72 h. (C) Equilibrium density gradient centrifugation of the intracellular material from an HCV-infected cell culture treated with 5× EC₅₀ (200 nM) of HCV II-1 dissolved in DMSO for 72 h.

FIG 3

Characterization of extracellular HCV particles during HCV II-1 treatment. Equilibrium density gradient centrifugation was performed as indicated in Materials and Methods. In all graphs, the solid red line represents HCV infectivity, the blue line represents HCV RNA levels, and the black line represents the average buoyant density. The data shown represent the averages of three assays, and the error bars represent the standard deviations. (A) Equilibrium density gradient centrifugation of the extracellular material from an HCV-infected cell culture treated with 0.2% DMSO for 72 h. (B) Equilibrium density gradient centrifugation of the extracellular material from an HCV-infected cell culture treated with 5× EC₅₀ (530 nM) of the NS3-4A protease inhibitor BILN-2061 for 72 h. (C) Equilibrium density gradient centrifugation of the extracellular material from an HCV-infected cell culture treated with 5× EC₅₀ (200 nM) of HCV II-1 for 72 h.

FIG 4

Inhibition of HCV infectivity by HCV II-1 in a cell-free assay. HCV infectious stability in cell culture medium at 37°C during treatment with various concentrations of HCV II-1 (0.2% DMSO [solid circles and solid line], 0.5× EC₅₀ = 20 nM [solid squares and dashed line], and 1.0× EC₅₀ = 40 nM [solid diamonds and solid line]). The data shown represent the averages of three assays, and the error bars represent the standard deviations.

FIG 5

Characterization of HCV particles during HCV II-1 and RNase A treatment. Equilibrium density gradient centrifugation was performed as indicated in Materials and Methods. In all graphs, the solid red line represents HCV infectivity, the blue line represents HCV RNA levels, and the black line represents the average buoyant density. The data shown represent the averages of three assays, and the error bars represent the standard deviations. (A) HCV was treated with final concentrations of 0.2% DMSO plus 50 μg of RNase A/ml at 37°C for 1 h in cell culture medium before equilibrium density gradient centrifugation. (B) HCV was treated with final concentrations of 0.2% DMSO plus 5% Triton X-100 plus 50 μg of RNase A/ml for 1 h at 37°C before equilibrium density gradient centrifugation. (C) HCV was treated with final concentrations of 800 nM (20× EC₅₀) HCV II-1 plus 50 μg of RNase A/ml for 1 h at 37°C before equilibrium density gradient centrifugation.

FIG 6

Inhibition of HCV infectivity by HCV II-1 is not reversible by dialysis or ultrafiltration. (A) HCV infectivity after 1 h of incubation with either DMSO or 5× EC₅₀ HCV II-1. (B) HCV infectivity after 1 h of incubation with either DMSO or 5× EC₅₀ HCV II-1, followed by 24 h of dialysis or 1 h of ultrafiltration. The data shown represent the averages of three assays, and the error bars represent standard deviations.

FIG 7

Kinetics of entry inhibition by HCV II-1. (A and B) Inhibition of GT1b/2a and GT2a HCV entry into cells by DMSO, HCV II-1, anti-CD81 Ab, bafilomycin A1 (BafA1), and EI-1 (GT1b/2a case only) during viral attachment at 4°C (“attachment”), during viral uptake into cells at 37°C (“postattachment”), or during both stages (“continuous”). All inhibitors were used at 5× EC₅₀. DMSO was used at 0.2%, HCV II-1 was used at 400 nM, anti-CD81 Ab was used at 2.5 μg/ml, bafilomycin A1 (BafA1) was used at 15 nM, and EI-1 was used at 170 nM final concentrations. These data represent the average of three assays, and error bars represent the standard deviations. (B) Time course of HCV(2a) entry during the postattachment stage with 5× EC₅₀ HCV II-1 (solid circles), anti-CD81 Ab (pierced squares), or BafA1 treatment (pierced circles). The times of half-maximal inhibition (t_1/2) were 150 ± 24 min for HCV II-1, 63 ± 12 min for anti-CD81 Ab, and 158 ± 20 min for BafA1. These data represent the averages of three assays, and the error bars represent the standard deviations.

FIG 8

Reduction of extracellular HCV by HCV II-1 alone and in combination with BILN-2061 or daclatasvir. (A) HCV(2a) persistently infected cellular monolayers were treated with 5× EC₅₀ concentrations of HCV II-1 (200 nM), NS3-4A protease inhibitor BILN-2061 (530 nM), or NS5A inhibitor daclatasvir (0.1 nM) for 20 days (see Materials and Methods). The HCV levels were normalized relative to the level of the DMSO control at each time point. These data are the averages of three assays, and the error bars represent standard deviations. The results for DMSO (solid circles), HCV II-1 (pierced circles), BILN-2061 (solid squares), daclatasvir (pierced squares), HCV II-1/BILN-2061 (solid diamonds), HCV II-1/daclatasvir (pierced diamonds), and BILN-2061/daclatasvir (solid hexagons) are indicated. (B) Photos of the HCV(2a)-infected cultures after 20 days of various inhibitor treatments. The yellow cells are infected (i.e., stained with anti-NS5A Ab as previously described [23]). The pluses signify an approximate quantification of the percentages of infected cells (see Materials and Methods).