Allosteric inhibitors of Coxsackie virus A24 RNA polymerase

Abstract

Coxsackie virus A24 (CVA24), a causative agent of acute hemorrhagic conjunctivitis, is a prototype of enterovirus (EV) species C. The RNA polymerase (3Dpol) of CVA24 can uridylylate the viral peptide linked to the genome (VPg) from distantly related EV, and is thus, a good model for studying this reaction. Once UMP is bound, VPgpU primes RNA elongation. Structural and mutation data have identified a conserved binding surface for VPg on the RNA polymerase (3Dpol), located about 20Å from the active site. Here, computational docking of over 60,000 small compounds was used to select those with the lowest (best) specific binding energies (BE) for this allosteric site. Compounds with varying structures and low BE were assayed for their effect on formation of VPgU by CVA24-3Dpol. Two compounds with the lowest specific BE for the site inhibited both uridylylation and formation of VPgpolyU at 10–20 µM. These small molecules can be used to probe the role of this allosteric site in polymerase function, and may be the basis for novel antiviral compounds.

Repurposing approved drugs on the pathway to novel therapies

“…A good deal of time can be lost to these nonspecific aggregators, which can have misleading activity in a variety of different assays at low micromolar concentrations . Mitroxantrone even turned up in a preliminary screen to identify inhibitors of uridylyation of the peptide linked to the genome, VPg, to VPgpU by the coxsackie virus A24 polymerase in the author's group. Several different assays were required to show it precipitated the RNA substrate, rather than specifically inhibiting the polymerase.…”

“…Even molecules suggested to be specific, for example, those with high affinity for a G‐protein coupled receptor, have been found to bind completely unrelated molecules, such as phosphodiesterases, whereby the binding sites can be very different . Screening a large compound library by docking found many molecules selected to bind to a specific site in fact bound preferentially to a different one when given a larger region of the protein surface to choose from …”