Non-Nucleoside Inhibitors of Zika virus RNA-dependent RNA polymerase

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-Barre 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.