Date Published: March 23, 2015
Publisher: Public Library of Science
Author(s): Lonneke van der Linden, Laia Vives-Adrián, Barbara Selisko, Cristina Ferrer-Orta, Xinran Liu, Kjerstin Lanke, Rachel Ulferts, Armando M. De Palma, Federica Tanchis, Nesya Goris, David Lefebvre, Kris De Clercq, Pieter Leyssen, Céline Lacroix, Gerhard Pürstinger, Bruno Coutard, Bruno Canard, David D. Boehr, Jamie J. Arnold, Craig E. Cameron, Nuria Verdaguer, Johan Neyts, Frank J. M. van Kuppeveld, Richard J. Kuhn.
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.
The family Picornaviridae contains 12 genera, and includes many human and animal pathogens (reviewed in ). 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.
There is a great need for broad-range antiviral compounds to treat infections with enteroviruses, which includes important human pathogens such as poliovirus, coxsackievirus, enterovirus 71, and rhinovirus. Here, we report a novel small molecule, GPC-N114 that exerts broad-spectrum anti-enterovirus activity and also inhibits members of the genus Cardiovirus, such as EMCV. Analysis of its mechanism of action revealed that GPC-N114 inhibits virus replication at the stage of RNA replication. GPC-N114-resistant enterovirus variants could not be obtained, but compound-resistant EMCV variants were readily selected in the presence of suboptimal concentration of GPC-N114. These variants were found to carry mutations in the viral RdRP, 3Dpol. Consistently, the in vitro elongation activity of CVB3 and EMCV 3Dpol was inhibited by GPC-N114, and the mutations identified in compound-resistant EMCV 3Dpol rendered this polymerase less susceptible to inhibition. GPC-N114 did not compete with incoming NTPs, but interfered with productive binding of the template-primer to 3Dpol in a primer elongation assay. This is in agreement with the crystallographic studies of the CVB3 3Dpol–GPC-N114 complex which revealed that the binding site of the compound is located at the junction of the palm and the fingers domains, and overlaps partially with the binding site of the template.