Figure 3.

MBX2254 and MBX2270 inhibit a late stage of Ebola virus (EBOV) entry. A, Single-cycle time-of-addition experiment was performed with Ebola pseudotype virus (HIV/EBOV-GP) to determine the stage of EBOV entry blocked by MBX2254 and MBX2270, using A549 cells as described in “Methods” section. MBX2254 (10 µmol/L) and MBX2270 (30 µmol/L) were added for 1 hour before infection (−1 hour), 1 hour during adsorption (0 hour), and 2 hours and 12 hours after infection. Inhibition of HIV/EBOV-GP pseudotype infection was detected as a reduced luciferase signal. Error bars indicate standard deviations. B, MBX2254 and MBX2270 do not act as receptor antagonists. A549 cells were pretreated with MBX2254 (10 µmol/L) and MBX2270 (30 µmol/L) at 0°C for 1 hour. After incubation, the compounds were removed and cells were washed with ice-cold phosphate-buffered saline and infected with HIV/EBOV-GP at 0°C. After 1 hour of infection, unadsorbed viruses were washed with phosphate-buffered saline and incubated for 72 hours at 37°C. Inhibition of HIV/EBOV-GP pseudotype infection was detected as a reduced luciferase signal. Error bars indicate standard deviations. C, Inhibitory activity against cathepsin B (CatB) in vitro. MBX2254 and MBX2270 were evaluated, as described in “Methods” section, for their effects on CatB. E64, a pancaspase inhibitor, was included as a positive control for the assay. Compound 7, another EBOV entry inhibitor (reported elsewhere) that does not modulate CatB, was used as negative control. D, Endosomal pH on exposure to MBX2254 and MBX2270. A549 cells were preincubated in the presence of MBX2254 and MBX2270. After incubation, cells were fixed, stained with LysoTracker Red, and then viewed and photographed with a confocal microscope. Compound 3.47, a known EBOV entry inhibitor that does not modulate endosomal pH, was used as a negative control, and 10 mmol/L ammonium chloride (NH₄Cl) was used as a positive control for inhibition of acidification.