Zika virus (ZIKV) remains a potentially significant public health concern because it can cause teratogenic effects such as microcephaly in newborns and neurological disease like Guillain-Barré syndrome. Together with efforts to develop a vaccine, the discovery of antiviral molecules is important to control ZIKV infections and to prevent its most severe symptoms. Here we report the development of small non-nucleoside inhibitors (NNIs) of ZIKV RNA-dependent RNA polymerase (RdRp) activity. These NNIs target an allosteric pocket (“N-pocket”) located next to a putative hinge region between the thumb and the palm subdomains, that was originally described for dengue virus (DENV) RdRp. We first tested DENV RdRp N-pocket inhibitors against ZIKV RdRp, introduced chemical modifications into these molecules and assessed their potency using both enzymatic and cell-based assays. The most potent compound has an IC50 value of 7.3 μM and inhibits ZIKV replication in a cell-based assay with an EC50 value of 24.3 μM. Importantly we report four high-resolution crystal structures detailing how these NNIs insert into the N-pocket of ZIKV RdRp. Our observations point to subtle differences in the size, shape, chemical environment and hydration of the N-pocket from ZIKV RdRp compared to DENV RdRp, that are crucial for the design of improved antiviral inhibitors against ZIKV. IMPORTANCE Zika virus belongs to the flavivirus family that comprises several important human pathogens. There is currently neither an approved vaccine nor antiviral drugs available to prevent infection by ZIKV. The NS5 polymerase, which is responsible for replicating the viral RNA genome, represents one of the most promising targets for antiviral drug development. Starting from compounds recently developed against dengue virus NS5, we designed and synthetized inhibitors targeting the Zika virus NS5. We showed that these novel compounds inhibit viral replication by targeting the polymerase activity. High-resolution X-ray crystallographic structures of protein-inhibitor complexes demonstrate specific binding to an allosteric site within the polymerase called the N-pocket. This work paves the way for future structure-based design of potent compounds specifically targeting the ZIKV RNA polymerase activity.

Flavivirus nonstructural protein 5 (NS5) contains an N-terminal methyltransferase (MTase) domain and a C-terminal polymerase (RNA-dependent RNA polymerase [RdRp]) domain fused through a 9-amino-acid linker. While the individual NS5 domains are structurally conserved, in the full-length protein, their relative orientations fall into two classes: the NS5 proteins from Japanese encephalitis virus (JEV) and Zika virus (ZIKV) adopt one conformation, while the NS5 protein from dengue virus serotype 3 (DENV3) adopts another. Here, we report a crystallographic structure of NS5 from DENV2 in a conformation similar to the extended one seen in JEV and ZIKV NS5 crystal structures. Replacement of the DENV2 NS5 linker with DENV1, DENV3, DENV4, JEV, and ZIKV NS5 linkers had modest or minimal effects on in vitro DENV2 MTase and RdRp activities. Heterotypic DENV NS5 linkers attenuated DENV2 replicon growth in cells, while the JEV and ZIKV NS5 linkers abolished replication. Thus, the JEV and ZIKV linkers likely hindered essential DENV2 NS5 interactions with other viral or host proteins within the virus replicative complex. Overall, this work sheds light on the dynamics of the multifunctional flavivirus NS5 protein and its interdomain linker. Targeting the NS5 linker is a possible strategy for producing attenuated flavivirus strains for vaccine design. IMPORTANCE Flaviviruses include important human pathogens, such as dengue virus and Zika virus. NS5 is a nonstructural protein essential for flavivirus RNA replication with dual MTase and RdRp enzyme activities and thus constitutes a major drug target. Insights into NS5 structure, dynamics, and evolution should inform the development of antiviral inhibitors and vaccine design. We found that NS5 from DENV2 can adopt a conformation resembling that of NS5 from JEV and ZIKV. Replacement of the DENV2 NS5 linker with the JEV and ZIKV NS5 linkers abolished DENV2 replication in cells, without significantly impacting in vitro DENV2 NS5 enzymatic activities. We propose that heterotypic flavivirus NS5 linkers impede DENV2 NS5 protein-protein interactions that are essential for virus replication.

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