The current Zika virus (ZIKV) outbreak became a global health threat of complex epidemiology and devastating neurological impacts, therefore requiring urgent efforts towards the development of novel efficacious and safe antiviral drugs. Due to its central role in RNA viral replication, the non-structural protein 5 (NS5) RNA-dependent RNA-polymerase (RdRp) is a prime target for drug discovery. Here we describe the crystal structure of the recombinant ZIKV NS5 RdRp domain at 1.9 Å resolution as a platform for structure-based drug design strategy. The overall structure is similar to other flaviviral homologues. However, the priming loop target site, which is suitable for non-nucleoside polymerase inhibitor design, shows significant differences in comparison with the dengue virus structures, including a tighter pocket and a modified local charge distribution.
The Zika virus (ZIKV) is a neurotropic arbovirus considered a global threat to public health. Although there have been several efforts in drug discovery projects for ZIKV in recent years, there are still no antiviral drugs approved to date. Here, we describe the results of a global collaborative crowdsourced open science project, the OpenZika project, from IBM's World Community Grid (WCG), which integrates different computational and experimental strategies for advancing a drug candidate for ZIKV. Initially, molecular docking protocols were developed to identify potential inhibitors of ZIKV NS5 RNA-dependent RNA polymerase (NS5 RdRp), NS3 protease (NS2B-NS3pro), and NS3 helicase (NS3hel). Then, a machine learning (ML) model was built to distinguish active vs inactive compounds for the cytoprotective effect against ZIKV infection. We performed three independent target-based virtual screening campaigns (NS5 RdRp, NS2B-NS3pro, and NS3hel), followed by predictions by the ML model and other filters, and prioritized a total of 61 compounds for further testing in enzymatic and phenotypic assays. This yielded five non-nucleoside compounds which showed inhibitory activity against ZIKV NS5 RdRp in enzymatic assays (IC 50 range from 0.61 to 17 μM). Two compounds thermally destabilized NS3hel and showed binding affinity in the micromolar range (K d range from 9 to 35 μM). Moreover, the compounds LabMol-301 inhibited both NS5 RdRp and NS2B-NS3pro (IC 50 of 0.8 and 7.4 μM, respectively) and LabMol-212 thermally destabilized the ZIKV NS3hel (K d of 35 μM). Both also protected cells from death induced by ZIKV infection in in vitro cell-based assays. However, while eight compounds (including LabMol-301 and LabMol-212) showed a cytoprotective effect and prevented ZIKV-induced cell death, agreeing with our ML model for prediction of this cytoprotective effect, no compound showed a direct antiviral effect against ZIKV. Thus, the new scaffolds discovered here are promising hits for future structural optimization and for advancing the discovery of further drug candidates for ZIKV. Furthermore, this work has demonstrated the importance of the integration of computational and experimental approaches, as well as the potential of large-scale collaborative networks to advance drug discovery projects for neglected diseases and emerging viruses, despite the lack of available direct antiviral activity and cytoprotective effect data, that reflects on the assertiveness of the computational predictions. The importance of these efforts rests with the need to be prepared for future viral epidemic and pandemic outbreaks.
SARS-CoV-2 is the causative agent of COVID-19. The highly conserved viral NSP15 endoribonuclease, NendoU, is a key enzyme involved in viral immune evasion, and a promising target for the development of new classes antivirals. Yet, the broad variety of recognition sequences, complex assembly and kinetics, and lack of structural complexes hamper the development of new competitive or allosteric inhibitors for this target. In here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index of 2. By using cryo-EM in distinct pH's combined with X-ray crystallography and structural analysis, we demonstrate the potential for NendoU to shift between open and closed states, and assembly in larger supramolecular entities, which might serve as a mechanism of self-regulation. Further, we report results from our large fragment screening campaign against NendoU, revealing multiple new allosteric sites for the development of new inhibitors.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.
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