Non-nucleoside Inhibitors of Zika Virus RNA-Dependent RNA Polymerase

Abstract

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.

Novel Nonnucleoside Inhibitors of Zika Virus Polymerase Identified through the Screening of an Open Library of Antikinetoplastid Compounds

“…One of them is that they must be administered in prodrug forms to be converted into the active-form triphosphate inside the cells, thus complicating their mode of action. Alternatively, nonnucleoside analogue inhibitors (NNIs) that act on allosteric regulation sites constitute an interesting therapeutic option, either alone or searching for combined therapies with NIs ( 29 – 31 ). Here, we have employed our recently developed high-throughput screening platform to evaluate the potential inhibitory effect of different compounds against the ZIKV NS5 RdRp domain.…”

“…This behavior is compatible with both inhibitors binding to the same allosteric regulatory site. In fact, previous studies have described other ZIKV allosteric inhibitors that bind near the active site ( 16 ) and identified at least an allosteric pocket (N-pocket) in the NS5 RdRp domain of DENV ( 35 ) that is also conserved in ZIKV ( 30 ), with which it shares 68.1% of identity. However, we have no experimental data to support whether that this allosteric pocket constitutes the target of these compounds, and further work should be performed to clearly identify the binding site of these inhibitors.…”

Amino Acid Substitutions in NS5 Contribute Differentially to Tembusu Virus Attenuation in Ducklings and Cell Cultures

“…NS5, the largest (905 amino acids) and most conserved flavivirus protein, contains three independent domains: N-terminal methyltransferase (MTase) domain, which is involved in RNA capping and prevents host recognition as well as promotes translation [ 21 , 22 , 23 ]; C-terminal RdRp domain, which is responsible for de novo RNA synthesis [ 24 , 25 ]; and the short Linker in between, which regulates the cross talk between RdRp and MTase dmoains [ 26 ]. The critical roles played by NS5 in viral replication and interferon antagonism make it a prime target for antiviral therapy and vaccine development [ 27 , 28 ].…”

Targeting the RdRp of Emerging RNA Viruses: The Structure-Based Drug Design Challenge

“…The most potent compound tested so far on ZIKA RdRp had an IC 50 value of 7.3 µM in cell-free assays and showed inhibition of ZIKV replication in vitro with an EC 50 value of 24.3 µM. However, crystallographic structures of four compounds binding the RdRp in the N-pocket helped to clarify their binding mode and highlighted the difference between the RdRp of ZIKV and DENV, information that may become essential for the design of specific small molecule inhibitors [125]. In HCV, DENV, and ZIKV RdRps, this site is located at the palm/thumb subdomains interface, and it appears to be a promising target for broad-spectrum antivirals [126].…”