Large-Scale Screening and Identification of Novel Ebola Virus and Marburg Virus Entry Inhibitors

Abstract

Filoviruses are highly infectious, and no FDA-approved drug therapy for filovirus infection is available. Most work to find a treatment has involved only a few strains of Ebola virus and testing of relatively small drug libraries or compounds that have shown efficacy against other virus types. Here we report the findings of a high-throughput screening of 319,855 small molecules from the Molecular Libraries Small Molecule Repository library for their activities against Marburg virus and Ebola virus. Nine of the most potent, novel compounds that blocked infection by both viruses were analyzed in detail for their mechanisms of action. The compounds inhibited known key steps in the Ebola virus infection mechanism by blocking either cell surface attachment, macropinocytosis-mediated uptake, or endosomal trafficking. To date, very few specific inhibitors of macropinocytosis have been reported. The 2 novel macropinocytosis inhibitors are more potent inhibitors of Ebola virus infection and less toxic than ethylisopropylamiloride, one commonly accepted macropinocytosis inhibitor. Each compound blocked infection of primary human macrophages, indicating their potential to be developed as new antifiloviral therapies.

FIG 1

Structural analysis of active compounds identified after counterscreens. (A) The relatedness of compounds that inhibited MARV GP pseudotype infection by >80% at 50 μM was evaluated by determining the structural similarity using PubChem criteria and the relationship stringency based upon Tanimoto coefficients. Compounds with Tanimoto scores of >0.8 (high stringency) were considered related and are shown as nodes connected in each network. Seventeen compounds (black nodes) inhibited infection of both wild-type MARV and wild-type EBOV at a 50% effective concentration of less than 30 μM. Of the 17 compounds, 9 were available for more detailed study and are numbered. (B) Structures of the 9 compounds indicated by their PubChem identification numbers.

FIG 2

Effect of compound treatment on binding of EBOV to the cell surface. (A) HeLa cells were pretreated with 50 μM of the indicated compound for 1 h and were then incubated with wild-type EBOV for 2.5 h. The cells were then fixed and stained without permeabilization using an EBOV GP-specific antibody followed by an Alexa Fluor 546-labeled secondary antibody (red). Cell bodies were stained using CellMask blue. 3D modeling of deconvolved image z-stacks was done using Imaris software. (B) The number of virus particles present on the cell surface was counted. (C) A second analysis of the number of cells that had no virus bound was also performed. Data are the averages ± SDs for at least 100 cells. All assays were performed 3 times with similar outcomes each time. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

Effect of compound treatment on macropinocytosis and uptake of EBOV into cells. (A) HeLa cells pretreated with 50 μM of the indicated compounds for 1 h, followed by incubation with fluorescently labeled dextran (molecular weight, 10,000; green) as a marker of macropinocytic uptake. Images of cells were captured, and the number of dextran-positive vesicles was counted. EIPA, a known inhibitor of macropinocytosis, was used to block uptake. The cell nucleus (intense blue) and cytoplasm (weak blue) were stained with CellMask blue. (B) The average number of vesicles per cell ± SD was determined for images of at least 50 cells. (C) Measurement of transferrin uptake. HeLa cells were serum starved for 4 h, followed by treatment with the indicated compounds for 1 h in serum-free medium. Treated cells were incubated with 25 μg/ml of transferrin conjugated to Alexa Fluor 488, unbound transferrin was washed off, and the cells were fixed in formalin. Fixed cells were imaged. (D) The fluorescence intensity of the cells after excitation at 488 nm was measured. The fluorescence of cells without transferrin treatment was used to determine the background signal. The percentage of transferrin-positive cells for each treatment was plotted. EIPA (25 μM), a specific inhibitor of macropinocytosis, and chlorpromazine (25 μM), an inhibitor of clathrin-mediated endocytosis and a known inhibitor of transferrin uptake, were used as controls. All data are represented as the means ± SDs for 3 replicates. (E) Cells were incubated with EBOV for 2.5 h in the presence of each of the indicated compounds, and then nonpermeabilized cells were stained for EBOV GP followed by staining with an Alexa Fluor 546-labeled secondary antibody (red). Cells were then permeabilized and the staining was repeated but an Alexa Fluor 488-labeled secondary antibody was used. Cell bodies (blue) were stained with CellMask blue. (F) Deconvolved image stacks were used to generate 3D models of cells with bound virus particles using Imaris software. Internalized virus particles (green, not red) and cell numbers were counted. The number of cells with 15 ± 5 virus particles (mean ± SD for the control) inside the cell cytoplasm was calculated as a measure of virus uptake efficiency. The average ± SD for more than 200 cells is shown. All assays were performed 3 times with similar outcomes. **, P < 0.01; ***, P < 0.001.

FIG 4

Effect of compound treatment on VLP trafficking. HeLa cells treated with 50 μM of the indicated compounds for 1 h were incubated with EBOV VLPs containing VP40-GFP for 2.5 h. Fixed cells were permeabilized and stained for EEA1 (an early endosome marker) and LAMP1 (a lysosome marker). 3D modeling of deconvolved image stacks was done using Imaris software. The portion of VLPs colocalized with EEA1 (A) or LAMP1 (B) is shown as the average ± SD for more than 200 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

Effect of compound treatment on interaction with endosomal acidification and cathepsin protease activity. (A) HeLa cells were pretreated with the indicated compounds at 50 μM for 1 h and then incubated for 1 h with acridine orange, a pH-sensitive fluorescent dye that accumulates in endosomes and which has a pH-sensitive fluorescent emission peak measured at 665 nm and a pH-independent emission peak measured at 530 nm. The ratio of the fluorescent emission at 665 nm to that at 530 nm was used to measure endosomal acidification relative to that for untreated cells and is given as the average ± SD for 3 independent measurements. (B) EBOV GP-pseudotyped VSV pseudovirions were incubated with cathepsin B in the presence or absence of 50 μM each inhibitor. E64 served as a positive control that blocked cathepsin B activity. Proteins from treated virions were separated and immunoblotted for the GP and VSV matrix protein. Ratios of quantitation of full-length GP (uncleaved) to matrix protein were generated. Shown are the mean values ± SDs relative to the values for pseudovirions not treated with cathepsin B.