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 linen4mBid. 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.cell death | drug | high throughput | rescue | virus assembly H epatitis C virus (HCV) infection is a major public health problem, putting~180 million infected individuals worldwide (3% of the world's population) at risk for developing cirrhosis, hepatocellular carcinoma, and liver failure. Chronic hepatitis C is a leading cause for liver transplantation in the Western world. The current standard IFN-based therapy is costly, time-consuming, riddled with serious side effects, and cures only ∼50% of patients infected with the most common genotype. Although it is anticipated that small molecules that effectively inhibit the HCV life cycle can complement or even replace IFN therapy, no anti-HCV drugs have yet been approved for human use. Efforts to develop HCV antivirals mostly target only viral replication due to widespread dependence on the HCV replicon model (1-4), which effectively reproduces the RNA replication aspects of the HCV life cycle but does not allow screens to identify inhibitors of entry or infectious virus assembly and release.The identification of a genotype 2a isolate of HCV that reproduces the entire viral life cycle in cell culture (5-7) promised to accelerate the discovery of antivirals targeting all aspects of HCV growth in a more true-to-life model system. Several recent reports describe systems that can in principle screen for inhibitors targeting different steps of the HCV life cycle via the prevention of the spread of cell culture-derived hepatitis C virus (HCVcc) infection in a cell monolayer (8-11). Very recently, one such screen based on a colorimetric readout following immunostaining with an anti-E2 antibody identified inhibitors of multiple stages of the HCV life cycle (12). Although effective, this approach is labor-intensive and not readily amenable to high-throughput application, requiring antibodies and 12 washing steps in addition to a secondary screen to assess drug ...

Cell Culture Systems for Hepatitis C Virus

“…Upon HCV infection and cleavage of the NS3/4A protease SEAP is released into the cell culture supernatant. Cell lines were also engineered to express the pro-apoptotic factor n4mBid, where NS3-dependent cleavage and activation led to an easily measurable cytopathic effect (Chockalingam, Simeon et al 2010). Although HCV infects mainly hepatocytes, there is evidence for the existence of non-hepatic reservoirs suggesting that the virus might have a broader cell tropism.…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Reporter viruses make such assays adaptable to high-throughput screening, with obvious advantages for antiviral research [76,126,[129][130][131]. Figure 4 illustrates a possible assay setup based on a dual reporter system, typically the Gaussia/Renilla and firefly luciferases.…”

Introduction

“…Additionally, in the course of this study, antiviral activities of 6 against hepatitis C virus (HCV) and simian immunodeficiency virus (SIV) were reported [34,35]. Dual infection with HIV and HCV confers a higher rate of viral resistance and higher susceptibility to death compared with solely HIV-infected patients [36].…”