A cell protection screen reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle

Abstract

The hepatitis C virus (HCV) life cycle involves multiple steps, but most current drug candidates target only viral replication. The inability to systematically discover inhibitors targeting multiple steps of the HCV life cycle has hampered antiviral development. We present a simple screen for HCV antivirals based on the alleviation of HCV-mediated cytopathic effect in an engineered cell line-n4mBid. This approach obviates the need for a secondary screen to avoid cytotoxic false-positive hits. Application of our screen to 1280 compounds, many in clinical trials or approved for therapeutic use, yielded >200 hits. Of the 55 leading hits, 47 inhibited one or more aspects of the HCV life cycle by >40%. Six compounds blocked HCV entry to levels similar to an antibody (JS-81) targeting the HCV entry receptor CD81. Seven hits inhibited HCV replication and/or infectious virus production by >100-fold, with one (quinidine) inhibiting infectious virus production by 450-fold relative to HCV replication levels. This approach is simple and inexpensive and should enable the rapid discovery of new classes of HCV life cycle inhibitors.

Fig. 1.

A n4mBid cell protection anti-HCV drug screen reports the presence of inhibitors targeting multiple stages of the HCV life cycle. (A) Schematic of screen. (B) Effect of known anti-HCV molecules on rescuing n4mBid cells from the HCV-induced cytopathic effect. (Upper) Drug dose–responses reporting n4mBid cell viability in the presence of HCV infection. (Lower) Drug cytotoxicity in the absence of HCV infection. (i and v) HCV replication inhibitors 2′CMA (•), VX-950 (▲), and cyclosporin A (■). (ii and vi) HCV entry-blocking anti-human CD81 mAb, JS-81. (iii and vii) The HCV assembly/release inhibitor naringenin. (iv and viii) IFNα-2a. Cell viability is presented as a percentage of mock-treated cells in the absence of both HCV infection and inhibitors. Values are the mean ± SD of two independent experiments carried out in duplicate.

Fig. 2.

Leading six HCV entry inhibitors from small-molecule library screen. (A) Chemical structures. (B) Dose-dependent effects on entry of H77 HCVpp (shaded bars) and VSV-Gpp (open bars) into Huh-7.5 cells. The anti-CD81 antibody JS-81 (1 μg/mL) was included as a positive control for each dose–response. Drug cytotoxicity in the absence of pseudoparticle transduction (solid circles) was determined by CellTiter-Glo assay. Values are expressed as a percentage of 0.1% DMSO-treated cells.

Fig. 3.

Leading inhibitors of HCV replication and infectious virus production from small-molecule library screen. (A) Intracellular HCV RNA levels and supernatant infectivity 96 h after electroporation of Huh-7.5 cells with the genomic RNA of Jc1 HCV and treatment with 15 leading replication/infectious virus production inhibitor hits at the concentrations (SDC) indicated in Table 1. Intracellular HCV RNA levels and supernatant infectivity were determined by quantitative RT–PCR and a cell-viability–adjusted median tissue culture infectious dose (TCID₅₀)/mL assay, respectively. Note that a value is not shown for the supernatant infectivity of TTNPB-treated cells because no cells were infected at any of the serial dilutions of this supernatant. For inhibitor cytotoxicity determination, cell viabilities of Huh-7.5 cells electroporated with carrier (water) before inhibitor treatment were measured by CellTiter-Glo assay. Each point is the mean ± SD of at least three independent experiments. (B) Quinidine dose-dependently inhibits infectious HCV production in Huh-7.5 cells. Virus replication and supernatant infectivity were determined by a Gluc reporter HCV-based assay. Cytotoxicity to Huh-7.5 cells electroporated with only carrier was determined as in A. Each point is the mean ± SD of two independent experiments carried out in duplicate. Values in A and B are expressed as a percentage of 0.1% DMSO-treated cells.