Amiloride is a competitive inhibitor of coxsackievirus B3 RNA polymerase

Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D ^pol conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol. 82:1465-1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D ^pol in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D ^pol by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg ²⁺. Docking analysis suggested a binding site for amiloride and EIPA in 3D ^pol, located in close proximity to one of the Mg ²⁺ ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.