Identification of hepatitis C virus inhibitors targeting different aspects of infection using a cell-based assay

Abstract

With 2 to 3% of the worldwide population chronically infected, hepatitis C virus (HCV) infection continues to be a major health care burden. Unfortunately, current interferon-based treatment options are not effective in all patients and are associated with significant side effects. Consequently, there is an ongoing need to identify and develop new anti-HCV therapies. Toward this goal, we previously developed a cell-based HCV infection assay for antiviral compound screening based on a low-multiplicity-of-infection approach that uniquely allows for the identification of antiviral compounds that target cell culture-derived HCV (HCVcc) at any step of the viral infection cycle. Using this assay, here we report the screening of the NCI Diversity Set II library, containing 1,974 synthesized chemical compounds, and the identification of compounds with specific anti-HCV activity. In combination with toxicity counterscreening, we identified 30 hits from the compound library, 13 of which showed reproducible and dose-dependent inhibition of HCV with mean therapeutic indices (50% cytotoxic concentration [CC(50)]/50% effective concentration [EC(50)]) of greater than 6. Using HCV pseudotype and replicon systems of multiple HCV genotypes, as well as infectious HCVcc-based assembly and secretion analysis, we determined that different compounds within this group of candidate inhibitors target different steps of viral infection. The compounds identified not only will serve as biological probes to study and further dissect the biology of viral infection but also should facilitate the development of new anti-HCV therapeutic treatments.

Fig 1

Primary and secondary compound screen. (A) Experimental design. (B) Primary screen. DMSO-Huh7 cells were infected with HCV at 0.05 FFU/cell and treated with diluent alone, IFN-β (100 U/ml), or NSC compounds (50 μM). Compounds were added at 0, 2, and 4 days p.i. At day 6 p.i., cultures were assayed for NS3 protein levels by FRET. RFU values were blank and background corrected, and data are presented as a percentage of the maximum RFU values determined in diluent-treated HCVcc-infected cultures (100% maximum infectivity). The Z′ equation was used to measure the distance between the standard deviations for the positive (diluent-treated) and negative (IFN-β-treated) controls of the assay. (C) Secondary screen. DMSO-Huh7 cells were infected with HCV at 0.05 FFU/cell and treated with diluent alone or with the top 100 active NSC compounds (50 μM) identified in the primary screen. Compounds were added at 0, 2, and 4 days p.i. At day 6 p.i., culture supernatants were collected and analyzed for cytotoxicity, and cultures were lysed and assayed for NS3 protein levels by FRET. Corrected FRET RFU values from both the primary (black bars) and secondary (gray bars) screens are presented as a percentage of RFU values determined in diluent-treated HCVcc-infected cultures (100% maximum infectivity). Relative light unit values determined from the cytotoxicity screen were blank corrected and are presented as a percentage of RLU values determined in Triton X-100-treated samples (100% maximum cytotoxicity). Samples with RLU values of >25% were considered cytotoxic.

Fig 2

Chemical structures of candidate NSC compounds selected for mechanism-of-action analyses. Compounds are grouped by clusters 1 through 8.

Fig 3

Screening for HCV entry inhibitors. (A) DMSO-Huh7 cells were pretreated with diluent, anti-CD81 antibody (36 μg/ml), or the indicated NSC compounds (25 μM) for 1.5 h prior to infection with equal amounts of JFHpp or VSVGpp. Relative light units (RLU) were determined at 72 h p.i. and blank corrected and are graphed as the average percentage of entry achieved in three separate experiments ± standard deviation (SD) for JFHpp (green bars) and VSVGpp (blue bars) in antibody-treated or compound-treated cultures compared to diluent-treated control cultures (100% maximum entry). (B to D) The dose-response inhibition of HCVpp genotype 2a (JFHpp), 1a (H77pp), or 1b (Con1pp) was determined for the indicated NSC compounds. DMSO-Huh7 cells were pretreated with diluent or NSC 119886 (B), NSC 143101 (C), or NSC 311152 (D) at concentrations ranging from 50 μM to 0.1 μM for 1.5 h prior to infection with equal amounts of HCVpp (genotype 2a [JFHpp], 1a [H77pp], or 1b [Con1pp]) or VSVGpp. HCVpp entry RLU determined at 72 h p.i. relative to diluent-treated cells was calculated by subtracting background and mock-control RLU values and then normalizing for VSVGpp luciferase activity. Results are graphed as a percentage of entry achieved in two separate experiments ± SD in compound-treated cultures compared to diluent-treated control cultures (100% maximum HCVpp entry).

Fig 4

Screening of HCV RNA replication inhibitors. Huh7 cells harboring subgenomic replicons of genotype 2a, 1a, or 1b were treated with NSC 92896 (A), NSC 119886 (B), or NSC 308848 (C) at concentrations ranging from 50 μM to 0.1 μM for 6 days. Following treatment, intracellular RNA was isolated, HCV RNA levels were determined by RT-qPCR, and data were normalized to GAPDH. Results are graphed as the average percentage of HCV RNA copies/μg of total RNA in compound-treated cultures compared to diluent-treated (0 μM) cultures in two separate experiments ± SD.

Fig 5

Screening of HCVcc egress inhibitors. DMSO-Huh7 cells chronically infected with HCVcc were treated with indicated NSC compounds at concentrations ranging from 50 μM to 0.1 μM for 6 days. Following treatment, HCVcc-containing supernatant was collected and HCVcc infectivity titers were determined by limiting dilution analysis of HCV focus formation on naïve Huh7 cells. RNA was also extracted for quantification of intracellular RNA levels by RT-qPCR. Results are graphed as a percentage of GAPDH-normalized HCV RNA copies/μg of total RNA (black bars) or HCVcc infectivity titers (FFU/ml) (gray bars) determined in compound-treated cultures compared to diluent-treated (0 μM) cultures.