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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.

Hydroboration of [(1 R* ,2 R* ,4 R* )-7-oxabicyclo[2.2.1]hept-5-en-2-yl]methyl benzoate ( 5 ), which was prepared by Diels–Alder reaction of furan with acrolein and subsequent reduction and benzoylation of the Diels–Alder product, afforded [(1 R* ,2 S* ,4 S* ,6 S* )-6-hydroxy-7-oxabicyclo[2.2.1]heptan-2-yl]methyl benzoate ( 6 ) and [(1 R* ,2 R* ,4 R* ,5 S* )-5-hydroxy-7-oxabicyclo[2.2.1]heptan-2-yl]methyl benzoate ( 7 ). The key intermediates, [(1 R* ,2 S* ,4 S* ,6 R* )-6-hydroxy-7-oxabicyclo[2.2.1]heptan-2-yl]methyl benzoate ( 10 ) and [(1 R* ,2 R* ,4 R* ,5 R* )-5-hydroxy-7-oxabicyclo[2.2.1]heptan-2-yl]methyl benzoate ( 11 ), were prepared from 6 and 7 , respectively, by oxidation with pyridinium dichromate and subsequent reduction of the thus obtained ketones. The Mitsunobu reaction of 10 and 11 with 6-chloropurine and subsequent reductive deprotection with diisobutylaluminium hydride afforded 6-chloropurine derivatives, which were converted to other purine analogues. Thymine analogues were prepared by Mitsunobu reaction of 10 and 11 with 3-benzoyl-5-methylpyrimidine-2,4(1 H ,3 H )-dione and subsequent methanolysis. The target compounds were tested for the activity against Coxsackie virus.

(1 R* ,2 R* ,3 R* ,4 S* )-7-Oxabicyclo[2.2.1]hept-5-ene-2,3-dimethanol ( 10 ) and (1 R* ,2 R* ,3 R* ,4 S* )-bicyclo[2.2.2]oct-5-ene-2,3-dimethanol ( 14 ), which were prepared by the Diels–Alder reaction and subsequent reduction with lithium aluminium hydride, were treated with benzyl azidoformate to give benzyl N -[(1 R* ,2 R* ,3 S* ,6 S* ,7 S* ,9 S* )-9-(hydroxymethyl)-4,8-dioxatricyclo[4.2.1.0 3,7 ]nonan-2-yl]carbamate ( 11 ) and benzyl N -[(1 R* ,2 R* ,3 R* ,6 R* ,7 S* ,10 S* )-10-(hydroxymethyl)-4-oxatricyclo[4.3.1.0 3,7 ]decan-2-yl]carbamate ( 15 ). Hydrogenolysis of carbamates 11 or 15 afforded (1 R* ,2 R* ,3 S* ,6 S* ,7 S* ,9 S* )-2-amino-4,8-dioxatricyclo[4.2.1.0 3,7 ]nonane-9-methanol ( 12 ) or (1 R* ,2 R* ,3 R* ,6 R* ,7 S* ,10 S* )-2-amino-4-oxatricyclo[4.3.1.0 3,7 ]decane-10-methanol ( 16 ). The amines 12 and 16 were transformed to thymine and purine nucleoside analogues. The target compounds were tested for the activity against Coxsackie virus.

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.

Novel class of the carbocyclic nucleosides based on bicyclo[2.2.1]heptene/heptane was prepared by two approaches. Thymine analogues were synthesized starting from methyl (1 R *,4 S *)-bicyclo[2.2.1]hepta-2,5-diene-2-carboxylate 1 by Michael addition of the thymine salt to the double bond as the key step. The yield and ratio of the isomers of this reaction depended on the used base (DBU, K 2 CO 3 ). Purine nucleoside analogues were synthesized by the linear synthesis, the purine nucleobase was build-up on the amino group. The amino groups ( exo / endo configuration) were introduced to the scaffold by the Curtius rearrangement. Norbornene analogues were converted to saturated and cis -hydroxylated nucleoside derivatives. [(1 R *,2 S *,3 S *,4 S *)-3-(6-Chloro-9 H -purin-9-yl)bicyclo[2.2.1]hept-5-en-2-yl]methanol ( 13a ) and [(1 R *,2 R *,3 R *,4 S *)-3-(6-chloro-9 H -purin-9-yl)bicyclo[2.2.1]hept-5-en-2-yl]methanol ( 13b ) showed moderate activity against Coxsackie virus CVB3.

Enterovirus A71 (EV-A71) is one of the main causative agents of hand-foot-and-mouth disease and is occasionally associated with severe neurological complications. EV-A71 pathophysiology is poorly understood due to the lack of small animal models that robustly support viral replication in relevant organs/tissues. Here, we show that adult severe combined immune-deficient (SCID) mice can serve as an EV-A71 infection model to study neurotropic determinants and viral tropism. Mice inoculated intraperitoneally with an EV-A71 clinical isolate had an initial infection of the lung compartment, followed by neuroinvasion and infection of (motor)neurons, resulting in slowly progressing paralysis of the limbs. We identified a substitution (V135I) in the capsid protein VP2 as a key requirement for neurotropism. This substitution was also present in a mouse-adapted variant, obtained by passaging the clinical isolate in the brain of one-day-old mice, and induced exclusive neuropathology and rapid paralysis, confirming its role in neurotropism. Finally, we showed that this residue enhances the capacity of EV-A71 to use mouse PSGL1 for viral entry. Our data reveal that EV-A71 initially disseminates to the lung and identify viral and host determinants that define the neurotropic character of EV-A71, pointing to a hitherto understudied role of PSGL1 in EV-A71 tropism and neuropathology.