Figures & data
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.
Table 1 Twenty-three active compounds that block Ebola VLP entry into HeLa cells.
Table 2 An additional 30 active compounds that block Ebola VLP entry in our assay at an IC50<10 µM and SI >10 plus three additional active compounds previously shown to inhibit Ebola virus infection.