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We report on the potent and selective in vitro antiviral activity of 2'-C-methylcytidine (2'-C-MetCyt) against foot-and-mouth disease virus (FMDV). FMDV belongs to the Picornaviridae and has the potential to cause devastating epidemics in livestock. The 50% and 90% effective concentrations (EC50 and EC90) for inhibition of the FMDV-induced cytopathic effect (CPE) formation were 6.4+/-3.8 and 10.8+/-5.4 microM. Comparable EC50 values for inhibition of viral RNA synthesis were observed. Treatment of FMDV-infected BHK-21 cells with 77 microM 2'-C-MetCyt resulted in a (1.6-3.2)x10(3)-fold reduction of infectious virus yield. Time-of-drug addition experiments suggest that 2'-C-MetCyt interacts with viral replication at a time point that coincides with the onset of intracellular viral RNA synthesis. In contrast to emergency vaccination, a potent and selective antiviral agent may provide almost immediate (prophylactic/therapeutic) protection against infection and thus constitute an important alternative/supplementary option to contain outbreaks such as those caused by FMDV.

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.

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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 3Dpol, the RNA-dependent RNA polymerase. 3Dpol 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 3Dpol 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 3Dpol, 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 3Dpol that can serve as a starting point for antiviral drug design.

Citation: van der Linden L, Vives-Adrin L, Selisko B, Ferrer-Orta C, Liu X, Lanke K, et al. (2015) 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. PLoS Pathog 11(3): e1004733.

The family Picornaviridae contains 12 genera, and includes many human and animal pathogens (reviewed in [1]). Among these is the genus Enterovirus which contains four human enterovirus species (HEV-A, -B, -C, -D), three human rhinovirus species (HRV-A, -B, -C), simian enterovirus, bovine enterovirus, and porcine enterovirus. The HEV species include poliovirus (PV), coxsackievirus (CV), echovirus, and several numbered enteroviruses (EV). PV is the cause of poliomyelitis, which can lead to acute flaccid paralysis. Enterovirus 71, a major cause of hand-foot-and-mouth disease, is also frequently associated with flaccid paralysis and is a growing concern due to major epidemics in Southeast Asia. Coxsackieviruses are the main cause of viral meningitis, conjunctivitis, herpangina, myocarditis, and pancreatitis. HRV infections manifest themselves in most cases as the relatively mild common cold, but can cause serious exacerbations in patients with asthma or chronic obstructive pulmonary disease (COPD). Other well-known picornavirus genera are Hepatovirus, which contains hepatitis A virus, Aphthovirus, which contains foot-and-mouth disease virus (FMDV), and Cardiovirus, which includes encephalomyocarditis virus (EMCV), Theiler's murine encephalomyelitis virus, and the recently discovered Saffold virus (SAFV), which, unlike the other cardioviruses, is a human-tropic virus.

Currently, the toolbox to control picornavirus infections consists solely of vaccines against PV, hepatitis A virus, and FMDV. Prevention of diseases caused by the non-polio enteroviruses through vaccination seems unachievable given the great number of serotypes, with about 30 coxsackieviruses, 30 echoviruses, 50 numbered enteroviruses, and more than 150 rhinoviruses [2]. With this in mind, current efforts are aimed at developing antiviral compounds with broad-spectrum activity, targeting a wide range of viruses within a genus or ideally even multiple genera. No specific antiviral drugs have yet been clinically approved for the treatment of enteroviruses or any other picornavirus.

We recently described a series of 5-nitro-2-phenoxybenzonitriles that inhibit in vitro enterovirus replication [23]. Further optimization of this class of molecules led to the identification of 2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile), hereafter referred to as GPC-N114 (Fig. 1A), with potent and selective in vitro antiviral activity against CVB3. This small molecule inhibits CVB3 replication in multicycle CPE-reduction antiviral assay with a 50% effective concentration (EC50) of 0.15 0.02 M (Table 1).

(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 M (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 M 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 M GPC-N114, 2 mM GuHCl, or 80 M 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.

To study its spectrum of activity, GPC-N114 was evaluated in similar multicycle antiviral assays with representatives of the different HEV and HRV species, as well as several other picornaviruses. All enteroviruses and rhinoviruses included in this study were found to be sensitive to the inhibitory effect of GPC-N114, with EC50 values ranging from 0.1 M to 1.7 M (Fig. 1B and Table 1). GPC-N114 also inhibited the replication of EMCV (strain mengovirus), a member of the genus Cardiovirus (EC50 = 5.4 0.49 M). By contrast, FMDV and equine rhinitis A virus (ERAV), two members of the genus Aphthovirus, proved insensitive to its inhibitory effect (tested with concentrations up to 25 M and 100 M, respectively).

To evaluate the antiviral effect of GPC-N114 in a single round of replication, CVB3 and EMCV, as representatives of the enterovirus and cardiovirus genus, were incubated in the presence of different concentrations of the compound and virus titers were determined at 8 h post infection (p.i.). Cell viability assays performed in parallel showed that GPC-N114 was not toxic at these concentrations (S1A Fig.). For CVB3, the maximal inhibitory effect was obtained with concentrations of 3 M and higher, although replication was not fully abrogated (Fig. 1C). Replication of EMCV was fully inhibited at concentrations of 10 M or higher (Fig. 1C). Therefore, a concentration of 10 M was selected for all further cellular assays. Detailed analysis of the kinetics of CVB3 and EMCV replication at this concentration showed that GPC-N114 strongly delayed virus replication (Fig. 1D and S1B Fig.). Comparable results were observed for a panel of other enteroviruses (EV71, CVA21, HRV2, and HRV14) and cardioviruses (EMCV and SAFV) (Fig. 1E and S1B Fig.). Again, no antiviral activity was observed against the aphthovirus ERAV (Fig. 1E). 006ab0faaa