Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs

Abstract

In light of the current outbreak of Ebola virus disease, there is an urgent need to develop effective therapeutics to treat Ebola infection, and drug repurposing screening is a potentially rapid approach for identifying such therapeutics. We developed a biosafety level 2 (BSL-2) 1536-well plate assay to screen for entry inhibitors of Ebola virus-like particles (VLPs) containing the glycoprotein (GP) and the matrix VP40 protein fused to a beta-lactamase reporter protein and applied this assay for a rapid drug repurposing screen of Food and Drug Administration (FDA)-approved drugs. We report here the identification of 53 drugs with activity of blocking Ebola VLP entry into cells. These 53 active compounds can be divided into categories including microtubule inhibitors, estrogen receptor modulators, antihistamines, antipsychotics, pump/channel antagonists, and anticancer/antibiotics. Several of these compounds, including microtubule inhibitors and estrogen receptor modulators, had previously been reported to be active in BSL-4 infectious Ebola virus replication assays and in animal model studies. Our assay represents a robust, effective and rapid high-throughput screen for the identification of lead compounds in drug development for the treatment of Ebola virus infection.

Keywords: Antipsychotics; Ebola virus; Ebola virus glycoprotein; VP40; drug repurposing screen; estrogen receptor modulator; microtubule inhibitor; virus entry.

Figure 1

Schematic representation of the Ebola VLP entry assay and compound screening method using this assay. (A) Ebola VLPs contain Ebola GP and the VP40 protein fused to a beta-lactamase (Bla) reporter. HeLa cells are loaded with the beta-lactamase substrate CCF2-AM, which results in green fluorescence. After VLP loading into cells, Bla hydrolyzes the substrate CCF2-AM, disrupting the fluorescence resonance energy transfer (FRET) in the substrate and thus causing blue fluorescence. The ratio of blue/green fluorescence intensities represents the activity of Bla inside cells. (B) Flow chart of compound screening in 1536-well plates using Ebola VLP entry assay. (C) Scatter plot of the results from a DMSO plate screening. The wells in columns 1 and 2 of the 1536-well assay plates contained HeLa cells as a control (0% response); Ebola VLPs were added to all other wells (positive control, 100% response). The signal-to-basal ratio (S/B) in this plate was 2.5-fold, with a CV of 11% and a Z′ factor of 0.62.

Figure 2

Concentration-response curves of the inhibition of Ebola VLP entry by 23 identified active compounds. These 23 compounds with confirmed anti-Ebola virus entry activity can be divided into six categories: (A) microtubule inhibitors, (B) estrogen receptor modulators, (C) antihistamines, (D) antipsychotics, (E) pump/channel antagonists, and (F) anticancer/antibiotics. The data indicate the mean±SD.

Figure 3

Images of the inhibition of Ebola VLP entry into HeLa cells by representative compounds that block Ebola VLP entry into host cells using a high content assay. Vinorelbine, Vincristine, Vinblastine, and Colchicine concentration-dependently blocked the blue fluorescence representing VLP entry into cells. Maraviroc, an HIV entry blocker, did not show any effect in this assay and served as a negative control. A 20× objective was used to capture the images.