High-Throughput Minigenome System for Identifying Small-Molecule Inhibitors of Ebola Virus Replication.
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.
Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York 10029, United States.
Megan R
MR
Edwards
Department of Pathology, Department of Microbiology and Immunology, and Galveston National Laboratory, University of Texas Medical Branch at Galveston , Galveston, Texas 77555, United States.
Colette
C
Pietzsch
Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York 10029, United States.
Thibaut
T
Vausselin
Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York 10029, United States.
Megan L
ML
Shaw
Department of Pathology, Department of Microbiology and Immunology, and Galveston National Laboratory, University of Texas Medical Branch at Galveston , Galveston, Texas 77555, United States.
Alexander
A
Bukreyev
Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York 10029, United States.
Christopher F
CF
Basler
Ebola virus; RNA polymerase; antiviral; filovirus; high-throughput screen; translation