Characterization of the <i>in vitro</i> anti-EV-A71 activity of MADAL385.

(A) Structural formula of MADAL385. (B) Concentration dependent inhibition of EV-A71 BrCr replication. RD cells were infected with EV-A71 BrCr (MOI = 0.1) for 1h in presence of the compound, after which the inoculum was removed, and cells were treated with serial dilutions of MADAL385. After 3 days, viral progeny was quantified by end-point titration. Statistical analysis was performed using the one-way ANOVA test; ****p(C) Antiviral activity of MADAL385 in the context of mCherry-EV-A71 reporter virus infection (MOI = 0.1). Images were taken with a fluorescence microscope at 24h post infection. (D) Binding (left panel) and internalization (right panel) inhibition assays. The graph represents the RNA levels relative to the untreated-infected group (VC). Statistical analysis was performed using the one-way ANOVA test; ***p(E) Thermo-stability assay. EV-A71 viruses, in the presence/absence of compounds (at a concentration of 50-fold in vitro EC50: 15 μM MADAL385, 150 μM suramin and 15 μM pirodavir), were subjected to heat-inactivation at the indicated temperature for 2 min, after which the samples were cooled down on ice. Viral titers were quantified by end-point titration. Pirodavir and suramin were used as reference compounds. Statistical analysis was performed using the two-way ANOVA test in reference to the Pirodavir treament; *p<0.05, ****p<0.0001. Error bars represent the mean ± standard deviation (SD) of at least 2 independent experiments with three replicates.