Amiloride Is a Competitive Inhibitor of Coxsackievirus B3 RNA Polymerase

Abstract

). 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.The family Picornaviridae is a family of positive-sense RNA viruses which contains numerous human pathogens, causing poliomyelitis, myocarditis, meningitis, hepatitis, the common cold, and other diseases. The viral genomic RNA is ϳ7,500 nucleotides (nt) long and contains a 22-amino-acid peptide, VPg, covalently linked to the 5Ј end and a poly(A) tail at the 3Ј end. Genome replication occurs via synthesis of a complementary, negative-sense RNA strand, catalyzed by the viral RNA-dependent RNA polymerase, 3D pol , in association with a number of viral and host proteins. It is a complex process taking place in membrane-associated replication complexes in the cytoplasm of infected cells (reviewed in references 5, 7, and 23). The synthesis of both complementary and genomic RNA strands is initiated by attachment of two UMP nucleotides to a tyrosine residue of VPg, resulting in the production of VPgpUpU. VPg uridylylation requires a template. In the case of genomic strand synthesis, an internal stem-loop in the genomic RNA strand (cis-acting replication element [CRE]) is used as a template, with subsequent translocation of VPg-pUpU to the 3Ј end of the complementary strand and its elongation into a full-length genomic strand (9,15,16,26). The complementary strand synthesis does not absolutely depend on CRE-it can also be templated by the poly(A) tail of the genomic strand (9,15,16,26).VPg uridylylation and RNA elongation have been reproduced successfully in vitro by use of purified components. VPg uridylylation assays require 3D pol , VPg, CRE or poly(A), UTP, and Mg 2ϩ or Mn 2ϩ (19,20), with CRE-templated reaction stimulated by viral proteins 3CD or 3C (18, 19), whereas an elongation assay mix contains an RNA primer instead of VPg (21).Coxsackievirus B3 (CVB3) is a picornavirus responsible for 14 to 32% of human myocarditis cases (1). Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) inhibit CVB3 propagation in cell culture by inhibiting viral genome replication (11). Two amino acid substitutions in 3D pol (S299T and A372V) confer partial resistance of the virus to the compounds, suggesting that amiloride analogues may act as inhibitors of CVB3 3D pol (11). Here we show that amiloride and EIPA inhibit VPg uridylylation and RNA elongation by CVB3 3D pol in vitro, acting as competitive inhibitors with respect to...

Inhibition of Enterovirus 71 by Adenosine Analog NITD008

“…Our findings agree with a previous report that resistance mutations against amiloride (an inhibitor of the 3D polymerase of coxsackievirus B3) were mapped to both the 3A and 3D proteins, although the contribution of the 3A mutation (I54L) to the resistance phenotype was not experimentally determined in that study (36). Amiloride was shown to directly inhibit the polymerase activity of coxsackievirus B3 by suppressing VPg uridylylation and RNA elongation (37).…”

Cell-Based High-Throughput Screening Assay Identifies 2′,2′-Difluoro-2′-deoxycytidine Gemcitabine as a Potential Antipoliovirus Agent

“…8 Three pipeline candidate drugs targeting PV capsid protein, Pleconaril (WIN63843) 9 (https://clinicaltrials.gov/show/NCT00031512), Pocapavir (V-073) 10,11 (http://adisinsight.springer.com/drugs/800007750), and Vapendavir (BTA-798) 12 (http://investors.aviragentherapeutics.com/releasedetail.cfm?releaseid=899451), were evaluated in phase II clinical trials, but no clinical study report or future development plan has been released. Although Ribavirin, 13,14 Amiloride, 15 and compound GPC-N114 16 were shown to have detectable antiviral activities by action on PV RNA polymerase, these antiviral agents have not moved on to clinical trials. Thus, there is rationale to continue to identify and characterize new antipolioviral agents for ultimate development of therapeutic drugs against PV infections.…”

Amiloride inhibits the initiation of Coxsackievirus and poliovirus RNA replication by inhibiting VPg uridylylation

“…This was shown to be an indirect mutagenic effect that was mediated by an increase in the intracellular concentration of Mg +2 and Mn +2 , which affected the fidelity of the viral polymerase (Levi et al, 2010). The effect of amiloride on single-nucleotide (AMP) incorporation was investigated in assays containing purified CVB3 3D pol and a 10-nucleotide, self-annealing, RNA primer-template (SSU) (Gazina et al, 2011). A small inhibitory effect on nucleotide incorporation (<9%) was observed in these reactions when amiloride was added with ATP in the presence of Mg +2 .…”