High-Throughput Minigenome System for Identifying Small-Molecule Inhibitors of Ebola Virus Replication.

Abstract

Ebola virus (EBOV), a member of the family Filoviridae, is a nonsegmented negative-sense RNA virus that causes severe, often lethal, disease in humans. EBOV RNA synthesis is carried out by a complex that includes several viral proteins. The function of this machinery is essential for viral gene expression and viral replication and is therefore a potential target for antivirals. We developed and optimized a high-throughput screening (HTS) assay based on an EBOV minigenome assay, which assesses the function of the polymerase complex. The assay is robust in 384-well format and displays a large signal to background ratio and high Z-factor values. We performed a pilot screen of 2080 bioactive compounds, identifying 31 hits (1.5% of the library) with >70% inhibition of EBOV minigenome activity. We further identified eight compounds with 50% inhibitory concentrations below their 50% cytotoxic concentrations, five of which had selectivity index (SI) values >10, suggesting specificity against the EBOV polymerase complex. These included an inhibitor of inosine monophosphate dehydrogenase, a target known to modulate the EBOV replication complex. They also included novel classes of inhibitors, including inhibitors of protein synthesis and hypoxia inducible factor-1. Five compounds were tested for their ability to inhibit replication of a recombinant EBOV that expresses GFP (EBOV-GFP), and four inhibited EBOV-GFP growth at sub-cytotoxic concentrations. These data demonstrate the utility of the HTS minigenome assay for drug discovery and suggest potential directions for antifiloviral drug development.