New class of early-stage enterovirus inhibitors with a novel mechanism of action.

4-dimethylamino benzoic acid (compound 12, synonym: 4EDMAB) was identified as an in vitro inhibitor of Coxsackie virus B3 (CVB3) replication in CPE-based assays (EC50 of 9.1 ± 1.5 μM). Next, the activity of twenty-three analogues was assessed, their structure-activity relationship was deduced and a more potent analogue was identified (EC50 of 2.6 ± 0.5 μM). The antiviral activity of 4EDMAB was further confirmed by quantifying viral RNA yield. Time-of-drug-addition assay revealed that 4EDMAB exerts its antiviral activity at the early stages of virus replication. Six compound-resistant viruses were selected and genotyped and all the mutations appeared to be in the capsid protein VP1. Reverse engineering showed that single mutants Y75C, A88V, A98V, D133N and R219K were respectively 15-, 2-, 4-, 17- and 76-fold resistant to 4EDMAB. The compound protected both wild type (WT) CVB3 and the five resistant mutants from heat inactivation. The plaque size produced by the A88V, D133N and R219K mutants was smaller than that of WT and these mutants were also more heat-sensitive than WT in the absence of the compound. These findings suggest that these three mutations increase virion capsid flexibility and compensate for the stabilizing effects of 4EDMAB. Molecular modelling suggests that the compound binds to a small cavity in VP1, which is different from the hydrophobic pocket in the canyon where typical capsid binders (such as pleconaril) bind. Modelling studies also suggest a direct ionic interaction between the negatively charged carboxylic group of 4EDMAB and the positively charged guanidino group of arginine 219. Moreover, the in vitro combination of 4EDMAB and pleconaril resulted in synergistic antiviral effect. In conclusion, 4EDMAB is a novel early-stage inhibitor, which targets VP1 with a mechanism that is different from that of known capsid binders.