Compounds were tested using entry assays for HIV/EBOV GP, HIVpp, IAVpp, MARVpp, and VSVpp, as previously described (7, 8). Infections were performed in 96-well plates by adding diluted HIV/EBOV GP, HIVpp, IAVpp, MARVpp, or VSVpp to 5 × 103 indicated cells per well in the presence and absence of the test compounds. The mixtures were then incubated for 72 hours at 37°C to screen the compound libraries. Luciferase activity, which reflected the number of pseudoparticles in the host cells, was measured 3 days after infection using the Bright-Glo reagent (Promega) and an FB15 luminometer (Berthold Detection Systems) according to the manufacturer’s protocol. Test compounds were serially diluted to a final concentration of 1 μM in 1% DMSO, and the maximum activity (100% of the control) and background were derived from the control wells containing DMSO alone or from uninfected wells, respectively. The individual signals in each of the compound test wells were then divided by the average signals of the control values (wells lacking inhibitor) after subtracting the background, and the results were multiplied by 100 to determine the percent activity. The corresponding inhibition values were calculated by subtracting this value from 100. The specificity of the compounds for inhibiting HIV/EBOV GP was determined by evaluating the inhibition of IAVpp and VSVpp infection in parallel. Each sample was tested in duplicate, and all the experiments were repeated at least three times.

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