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

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 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol 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 3Dpol 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.status: publishe

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

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 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol 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 3Dpol 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

Data collection and refinement statistics.

<p>† Rwork = ∑hkl ||Fobs(hkl)|—|Fcalc(hkl)|| / ∑hkl |Fobs(hkl)|, where Fobs and Fcalc are the structure factors, deduced from measured intensities and calculated from the model, respectively.</p><p>‡ Rfree = as for Rwork but for 5% of the total reflections chosen at random and omitted from refinement.</p><p>Data collection and refinement statistics.</p

Characterization of the inhibitory mechanism of GPC-N114 on CVB3 3D^pol.

<p>(A) GPC-N114 does not compete with UTP. The experiment was performed for CVB3 3D^pol as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004733#ppat.1004733.g002" target="_blank">Fig. 2E</a> for EMCV 3D^pol. (B) GPC-N114 is more efficient when added to the reaction mix before the template-primer. Order-of-addition experiments were performed by measuring CVB3 3D^pol elongation activity when GPC-N114 was added to the reaction mix before or after poly(rA)/dT15. A typical of three independent experiments is presented.</p

The binding site of GPC-N114 on CVB3 3D^pol.

<p>(A) Ribbon diagram of the CVB3 3D^pol structure (green). The inhibitor bound to the bottom of the template channel is shown in stick representation in yellow. (B) Interaction network between GPC-N114 and its binding pocket of CVB3 3D^pol represented by surfaces. The polymerase residues in direct contact with the inhibitor are shown with carbon atoms in green and explicitly labeled. Hydrogen bonds are depicted as dashed lines. (C) Top down cross-sectional view of the molecular surface of CVB3 3D^pol with the electrostatic potential, represented in blue and red for positive and negative charges, respectively. The trajectory of the RNA template (green) is compared with the position of the GPC-N114 inhibitor (yellow). The inset box shows a close up of the position of the inhibitor that is coincident with that of the template acceptor nucleotide. The predicted position of the RNA template was determined using the FMDV 3D^pol–RNA complex (PDB entry 1WNE) as a guide.</p

Effects of mutations M300V and I304V on the GPC-N114 binding site in EMCV 3D^pol.

<p>(A) Surface representation of the putative GPC-N114 binding pocket in EMCV 3D^pol (slate blue) with the modeled inhibitor depicted in atom color (carbon atoms in yellow). (B-D) Environment of 300 and 304 residues on EMCV wt and mutants 3D^pol. Motifs A and C are represented in transparent grey and motif B in solid color: (B) grey (wt), (C) cyan (M300V) and (D) red (I304V). Residues interacting with motif B α-helix are shown as sticks colored by atom. The GPC-N114 binding site on CVB3 3D^pol is represented as a yellow surface.</p

GPC-N114 inhibits picornavirus replication.

<p>(A) Structural formula of GPC-N114. (B) Representative dose-response curves of multicycle CPE-reduction assays for CVB3, PV1, and EV71. CPE was quantified by MTS assay at 3 d p.i. and is expressed as percentage of uninfected, untreated controls. (C, D) Antiviral activity of GPC-N114 against CVB3 and EMCV. BGM cells were infected with CVB3 (left panels) or EMCV (right panels) at an MOI of 0.1. Immediately after infection, GPC-N114 was added at the indicated concentrations (C) or at 10 μM (D). The enterovirus inhibitor guanidine hydrochloride (GuHCl) and the cardiovirus inhibitor dipyridamole were included as controls. Virus titers were determined by endpoint titration after 8 h (C) or at the indicated times p.i. (D). Experiments were performed in triplicate and mean values ± SD are depicted. (E) Antiviral activity of GPC-N114 against a range of picornaviruses. Cells were infected with the indicated viruses at an MOI of 0.5 after which 10 μM GPC-N114 was added. Virus titers were determined at 8 h p.i. Experiments were performed in triplicate and mean values ± SD are depicted. (F) GPC-N114 inhibits viral RNA replication. RNA of subgenomic replicons of CVB3 or EMCV was transfected into BGM cells. Subsequently, cells were treated either with 0.1% DMSO, 10 μM GPC-N114, 2 mM GuHCl, or 80 μM dipyridamole. Firefly luciferase levels were determined 2, 4, 6, and 8 h after RNA transfection to assess the level of replication and translation. Experiments were performed in triplicate and mean values ± SD are depicted.</p

Antiviral activity of GPC-N114.

<p>Shown are mean values calculated from at least three experiments ± SD. The cytotoxicity values (CC₅₀) were for Hela H cells 8.54 ± 0.42 μM, for Hela R19 cells 7.07 ± 0.38 μM, and for RD, BGM, and Vero cells >100 μM</p><p>Antiviral activity of GPC-N114.</p