Anti-native EBOV assays were conducted in biosafety level 4 at the U.S. Army Medical Research Institute of Infectious Diseases. HeLa and HFF cells were seeded into 96-well plates and exposed to native EBOV (Makona). HeLa cells were incubated with a serially diluted lead compound (10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01, 0.005, and 0 μM) or 1% DMSO 2 hours before the addition of the virus (MOI = 1.5). The viruses were added to the HFF cells at an MOI of 20, as measured in HeLa cells. The infection was stopped after 48 hours by fixing the cells with a formalin solution. Immunostaining was performed with an Alexa Fluor 488–conjugated anti-GP antibody to detect the infected cells. Images were acquired with the PE Opera confocal platform using a 10× objective and analyzed using Acapella software. Genedata software was used to calculate the percent infection from the GP staining data, and this value was then converted into percent inhibition. To evaluate the effect of lead compound on cell viability, CellTiter-Glo (Promega Corp., Madison, WI, USA) assay was conducted using a plate reader (Tecan Infinite M2000 PRO; Tecan Group Ltd., Mannedorf, Switzerland) (7). These experiments were independently repeated three times. Compound 3.47 was used as a positive control (12).

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