The RNA Template Channel of the RNA-Dependent RNA Polymerase as a Target for Development of Antiviral Therapy of Multiple Genera within a Virus Family

Abstract

The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3D ^pol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3D ^pol, (ii) had reduced activity against EMCV 3D ^pol with the resistance mutations, and (iii) was most efficient in inhibiting 3D ^pol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3D ^pol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3D ^pol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.

Author Summary

Virus replication relies on multiplication of viral genomes by viral polymerases. For enteroviruses, a large group of clinically important human pathogens for which no antiviral therapy is available, this function is performed by 3D ^pol, the RNA-dependent RNA polymerase. 3D ^pol is therefore an attractive target for novel antiviral strategies. Most polymerase inhibitors identified today are nucleoside analogs, a class of compounds that exert broad-spectrum activity but often suffer from off-target effects. Non-nucleoside inhibitors on the other hand, in general have a more narrow spectrum of activity and are more prone to resistance development because in most cases they bind the surface of the enzyme which is less conserved and structurally more flexible. In this study, we present the identification of GPC-N114 as a non-nucleoside inhibitor of 3D ^pol with broad-spectrum antiviral activity against both enteroviruses and cardioviruses, which also belong to the picornavirus family. Remarkably, it acts by targeting the RNA template-primer binding site in the core of 3D ^pol, making GPC-N114 the first anti-picornaviral compound with this mechanism of action. Thus, the characterization of GPC-N114 has led to the identification of a novel drug-binding pocket in 3D ^pol that can serve as a starting point for antiviral drug design.