NS5 RNA-dependent RNA Polymerase Research Articles

Molecular networking-based mass spectral identification of Brucea javanica (L.) Merr. metabolites and their selective binding affinities for dengue virus enzymes

Brucea javanica, a valued traditional medicinal plant in Malaysia, known for its fever-treating properties yet remains underexplored for its potential antiviral properties against dengue. This study aims to simultaneously identify chemical classes and metabolites within B. javanica using molecular networking (MN), by Global Natural Product Social (GNPS), and SIRIUS in silico annotation. Liquid chromatography-mass spectrometry (LC-MS2)-based MN explores chemical diversity across four plant parts (leaves, roots, fruits, and stem bark), revealing diverse metabolites such as tryptophan-derived alkaloids, terpenoids, and octadecadenoids. Simultaneous LC-MS2 and MN analyses reveal a discriminative capacity for individual plant components, with roots accumulating tryptophan alkaloids, fruits concentrating quassinoids, leaves containing fusidanes, and stem bark primarily characterised by simple indoles. Subsequently, extracts were evaluated for dengue antiviral activity using adenosine triphosphate (ATP) and plaque assays, indicates potent efficacy in the dichloromethane (DCM) extract from roots (EC50 = 0.3 μg/mL, SI = 10). Molecular docking analysis of two major compounds; canthin-6-one (264) and 1-hydroxy-11-methoxycanthin-6-one (275) showed potential binding interactions with active sites of NS5 RNA-dependent RNA polymerase (RdRp) of dengue virus (DENV) protein. Subsequent in vitro evaluation revealed compounds 264 and 275 had a promising dengue antiviral activity with SI value of 63 and 1.85. These identified metabolites emerge as potential candidates for further evaluation in dengue antiviral activities.

A jingmenvirus RNA-dependent RNA polymerase structurally resembles the flavivirus counterpart but with different features at the initiation phase.

Jingmenviruses are a category of emerging segmented viruses that have garnered global attention in recent years, and are close relatives of the flaviviruses in the Flaviviridae family. One of their genome segments encodes NSP1 homologous to flavivirus NS5. NSP1 comprises both the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRP) modules playing essential roles in viral genome replication and capping. Here we solved a 1.8-Å resolution crystal structure of the NSP1 RdRP module from Jingmen tick virus (JMTV), the type species of jingmenviruses. The structure highly resembles flavivirus NS5 RdRP despite a sequence identity less than 30%. NSP1 RdRP enzymatic properties were dissected in a comparative setting with several representative Flaviviridae RdRPs included. Our data indicate that JMTV NSP1 produces characteristic 3-mer abortive products similar to the hepatitis C virus RdRP, and exhibits the highest preference of terminal initiation and shorter-primer usage. Unlike flavivirus NS5, JMTV RdRP may require the MTase for optimal transition from initiation to elongation, as an MTase-less NSP1 construct produced more 4-5-mer intermediate products than the full-length protein. Taken together, this work consolidates the evolutionary relationship between the jingmenvirus group and the Flaviviridae family, providing a basis to the further understanding of their viral replication/transcription process.

LGP2 directly interacts with flavivirus NS5 RNA-dependent RNA polymerase and downregulates its pre-elongation activities.

LGP2 is a RIG-I-like receptor (RLR) known to bind and recognize the intermediate double-stranded RNA (dsRNA) during virus infection and to induce type-I interferon (IFN)-related antiviral innate immune responses. Here, we find that LGP2 inhibits Zika virus (ZIKV) and tick-borne encephalitis virus (TBEV) replication independent of IFN induction. Co-immunoprecipitation (Co-IP) and confocal immunofluorescence data suggest that LGP2 likely colocalizes with the replication complex (RC) of ZIKV by interacting with viral RNA-dependent RNA polymerase (RdRP) NS5. We further verify that the regulatory domain (RD) of LGP2 directly interacts with RdRP of NS5 by biolayer interferometry assay. Data from in vitro RdRP assays indicate that LGP2 may inhibit polymerase activities of NS5 at pre-elongation but not elongation stages, while an RNA-binding-defective LGP2 mutant can still inhibit RdRP activities and virus replication. Taken together, our work suggests that LGP2 can inhibit flavivirus replication through direct interaction with NS5 protein and downregulates its polymerase pre-elongation activities, demonstrating a distinct role of LGP2 beyond its function in innate immune responses.

Development of Zika Virus Mini-Replicon Based Single-Round Infectious Particles as Gene Delivery Vehicles.

Zika virus (ZIKV) is a type of RNA virus that belongs to the Flaviviridae family. We have reported the construction of a DNA-launched replicon of the Asian-lineage Natal RGN strain and the production of single-round infectious particles (SRIPs) via the combination of prM/E virus-like particles with the replicon. The main objective of the study was to engineer the ZIKV replicon as mammalian expression vectors and evaluate the potential of ZIKV mini-replicon-based SRIPs as delivery vehicles for heterologous gene expression in vitro and in vivo. The mini-replicons contained various genetic elements, including NS4B, an NS5 methyltransferase (MTase) domain, and an NS5 RNA-dependent RNA polymerase (RdRp) domain. Among these mini-replicons, only ZIKV mini-replicons 2 and 3, which contained the full NS5 and NS4B-NS5 genetic elements, respectively, exhibited the expression of reporters (green fluorescent protein (GFP) and cyan fluorescent protein-yellow fluorescent fusion protein (CYP)) and generated self-replicating RNAs. When the mini-replicons were transfected into the cells expressing ZIKV prM/E, this led to the production of ZIKV mini-replicon-based SRIPs. ZIKV mini-replicon 3 SRIPs showed a significantly higher yield titer and a greater abundance of self-replicating replicon RNAs when compared to ZIKV mini-replicon 2 SRIPs. Additionally, there were disparities in the dynamics of CYP expression and cytotoxic effects observed in various infected cell types between ZIKV mini-replicon 2-CYP and 3-CYP SRIPs. In particular, ZIKV mini-replicon 3-CYP SRIPs led to a substantial decrease in the survival rates of infected cells at a MOI of 2. An in vivo gene expression assay indicated that hACE2 expression was detected in the lung and brain tissues of mice following the intravenous administration of ZIKV mini-replicon 3-hACE2 SRIPs. Overall, this study highlights the potential of ZIKV mini-replicon-based SRIPs as promising vehicles for gene delivery applications in vitro and in vivo.

Identification of Zika virus NS2B-NS3 protease and NS5 polymerase inhibitors by structure-based virtual screening of FDA-approved drugs

Zika virus (ZIKV) is a mosquito-borne human flavivirus responsible that causing emergency outbreaks in Brazil. ZIKV is suspected of causing Guillain-Barre syndrome in adults and microcephaly. The NS2B-NS3 protease and NS5 RNA-dependent RNA polymerase (RdRp), central to ZIKV multiplication, have been identified as attractive molecular targets for drugs. We performed a structure-based virtual screening of 2,659 FDA-approved small molecule drugs in the DrugBank database using AutoDock Vina in PyRx v0.8. Accordingly, 15 potential drugs were selected as ZIKV inhibitors because of their high values (binding affinity - binding energy) and we analyzed the molecular interactions between the active site amino acids and the compounds. Among these drugs, tamsulosin was found to interact most efficiently with NS2B/NS3 protease, as indicated by the lowest binding energy value (−8.27 kJ/mol), the highest binding affinity (−5.7 Kcal/mol), and formed H-bonds with amino acid residues TYRB130, SERB135, TYRB150. Furthermore, biotin was found to interact most efficiently with NS5 RdRp with a binding energy of −150.624 kJ/mol, a binding affinity of −5.6 Kcal/mol, and formed H-bonds with the amino acid residues ASPA665 and ASPA540. In vitro, in vivo, and clinical studies are needed to demonstrate anti-ZIKV safety and the efficacy of these FDA-approved drug candidates. Communicated by Ramaswamy H. Sarma

Structures of dengue virus RNA replicase complexes

Dengue is a mosquito-borne viral infection caused by dengue virus (DENV), a member of the flaviviruses. The DENV genome is a 5'-capped positive-sense RNA with a unique 5'-stem-loop structure (SLA), which is essential for RNA replication and 5' capping. The virus-encoded proteins NS5 and NS3 are responsible for viral genome replication, but the structural basis by which they cooperatively conduct the required tasks has remained unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of SLA-bound NS5 (PC), NS3-bound PC (PC-NS3), and an RNA-elongating NS5-NS3 complex (EC). While SLA bridges the NS5 methyltransferase and RNA-dependent RNA polymerase domains in PC, the NS3 helicase domain displaces it in elongation complex (EC). The SLA- and NS3-binding sites overlap with that of human STAT2. These structures illuminate the key steps in DENV genome replication, namely, SLA-dependent replication initiation, processive RNA elongation, and 5' capping of the nascent genomic RNA, thereby providing foundations to combat flaviviruses.

Investigating the Mechanism of Action of Anti-Dengue Compounds as Potential Binders of Zika Virus RNA-Dependent RNA Polymerase.

The World Health Organization (WHO) has designated the Zika virus (ZIKV) as a significant risk to the general public's health. Currently, there are no vaccinations or medications available to treat or prevent infection with the Zika virus. Thus, it is urgently required to develop a highly efficient therapeutic molecule. In the presented study, a computationally intensive search was carried out to identify potent compounds that have the potential to bind and block the activity of ZIKV NS5 RNA-dependent RNA polymerase (RdRp). The anti-dengue chemical library was subjected to high-throughput virtual screening and MM/GBSA analysis in order to rate the potential candidates. The top three compounds were then chosen. According to the MM/GBSA analysis, compound 127042987 from the database had the highest binding affinity to the protein with a minimum binding free energy of -77.16 kcal/mole. Compound 127042987 had the most stable RMSD trend and the greatest number of hydrogen bond interactions when these chemical complexes were evaluated further under a 100 ns molecular dynamics simulation. Compound 127042987 displayed the best binding free energy (GBind) of -96.50 kcal/mol, surpassing the native ligand binding energy (-66.17 kcal/mole). Thereafter, an MM/GBSA binding free energy study was conducted to validate the stability of selected chemical complexes. Overall, this study illustrated that compound 127042987 showed preferred binding free energies, suggesting a possible inhibitory mechanism against ZIKV-RdRp. As per this study, it was proposed that compound 127042987 could be used as a therapeutic option to prevent Zika virus infection. These compounds need to be tested in experiments for further validation.

Natural Compounds as Non-Nucleoside Inhibitors of Zika Virus Polymerase through Integration of In Silico and In Vitro Approaches.

Although the past epidemic of Zika virus (ZIKV) resulted in severe neurological consequences for infected infants and adults, there are still no approved drugs to treat ZIKV infection. In this study, we applied computational approaches to screen an in-house database of 77 natural and semi-synthetic compounds against ZIKV NS5 RNA-dependent RNA-polymerase (NS5 RdRp), an essential protein for viral RNA elongation during the replication process. For this purpose, we integrated computational approaches such as binding-site conservation, chemical space analysis and molecular docking. As a result, we prioritized nine virtual hits for experimental evaluation. Enzymatic assays confirmed that pedalitin and quercetin inhibited ZIKV NS5 RdRp with IC50 values of 4.1 and 0.5 µM, respectively. Moreover, pedalitin also displayed antiviral activity on ZIKV infection with an EC50 of 19.28 µM cell-based assays, with low toxicity in Vero cells (CC50 = 83.66 µM) and selectivity index of 4.34. These results demonstrate the potential of the natural compounds pedalitin and quercetin as candidates for structural optimization studies towards the discovery of new anti-ZIKV drug candidates.

Inhibitory Potential of Chromene Derivatives on Structural and Non-Structural Proteins of Dengue Virus.

Dengue fever is a mosquito-borne viral disease that has become a serious health issue across the globe. It is caused by a virus of the Flaviviridae family, and it comprises five different serotypes (DENV-1 to DENV-5). As there is no specific medicine or effective vaccine for controlling dengue fever, there is an urgent need to develop potential inhibitors against it. Traditionally, various natural products have been used to manage dengue fever and its co-morbid conditions. A detailed analysis of these plants revealed the presence of various chromene derivatives as the major phytochemicals. Inspired by these observations, authors have critically analyzed the anti-dengue virus potential of various 4H chromene derivatives. Further, in silico, in vitro, and in vivo reports of these scaffolds against the dengue virus are detailed in the present manuscript. These analogues exerted their activity by interfering with various stages of viral entry, assembly, and replications. Moreover, these analogues mainly target envelope protein, NS2B-NS3 protease, and NS5 RNA-dependent RNA polymerase, etc. Overall, chromene-containing analogues exerted a potent activity against the dengue virus and the present review will be helpful for the further exploration of these scaffolds for the development of novel antiviral drug candidates.

Influence of Zika virus 3′-end sequence and nonstructural protein evolution on the viral replication competence and virulence

ABSTRACT Zika virus (ZIKV) has been circulating in human networks over 70 years since its first appearance in Africa, yet little is known about whether the viral 3′-terminal sequence and nonstructural (NS) protein diverged genetically from ancient ZIKV have different effects on viral replication and virulence in currently prevailing Asian lineage ZIKV. Here we show, by a reverse genetics approach using an infectious cDNA clone for a consensus sequence (Con1) of ZIKV, which represents Asian ZIKV strains, and another clone derived from the MR766 strain isolated in Uganda, Africa in 1947, that the 3′-end sequence –UUUCU-3′ homogeneously present in MR766 genome and the –GUCU-3′ sequence strictly conserved in Asian ZIKV isolates are functionally equivalent in viral replication and gene expression. By gene swapping experiments using the two infectious cDNA clones, we show that the NS1–5 proteins of MR766 enhance replication competence of ZIKV Con1. The Con1, which was less virulent than MR766, acquired severe bilateral hindlimb paralysis when its NS1–5 genes were replaced by the counterparts of MR766 in type I interferon receptor (IFNAR1)-deficient A129 mice. Moreover, MR766 NS5 RNA-dependent RNA polymerase (RdRp) alone also rendered the Con1 virulent, despite there being no difference in RdRp activity between MR766 and Con1 NS5 proteins. By contrast, the Con1 derivatives expressing MR766 Nsps, like Con1, did not develop severe disease in wild-type mice treated with an IFNAR1 blocking antibody. Together, our findings uncover an unprecedented role for ZIKV NS proteins in determining viral pathogenicity in immunocompromised hosts.

Discovery of New Zika Protease and Polymerase Inhibitors through the Open Science Collaboration Project OpenZika.

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 (IC50 range from 0.61 to 17 μM). Two compounds thermally destabilized NS3hel and showed binding affinity in the micromolar range (Kd range from 9 to 35 μM). Moreover, the compounds LabMol-301 inhibited both NS5 RdRp and NS2B-NS3pro (IC50 of 0.8 and 7.4 μM, respectively) and LabMol-212 thermally destabilized the ZIKV NS3hel (Kd 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.

Repurposing of the antihistamine mebhydrolin napadisylate for treatment of Zika virus infection

Zika virus (ZIKV) infection can lead to severe neurological disorders and fetal defects, which has become a public health problem worldwide. However, effective clinical treatment for ZIKV infection was still arduous. Antihistamines are attractive candidates for drug repurposing due to their low price and widespread availability. Here we screened FDA-approved antihistamine drugs to obtain anti-ZIKV candidate compounds and identified mebhydrolin napadisylate (MHL) that exhibits the potent inhibition of ZIKV infection in various cell lines in a histamine H1 receptor-independent manner. Mechanistic studies suggest that MHL acts as a ZIKV NS5 RNA-dependent RNA polymerase (RdRp) inhibitor, supported by a structure–activity relationship (SAR) analysis showing the correlation between the inhibitory effect upon viral RNA synthesis and ZIKV infectivity. Furthermore, MHL was shown to bind ZIKV NS5 RdRp in vitro and predicted to interact with key residues at the active site of ZIKV NS5 RdRp by molecular docking analysis. Our data together suggest that MHL suppresses ZIKV infection through the inhibition of ZIKV NS5 RdRp activity. This study highlights that MHL might be a promising clinical anti-ZIKV therapeutic.

Uncovering of Anti-dengue Molecules from Plants Prescribed for Dengue: A Computational Investigation

Dengue fever is a tropical disease spread worldwide, transmitted by the mosquito Aedes aegypti. It affects 100 million people worldwide every year and half a million cases of dengue hemorrhagic fever are registered. At present, it poses sever health burden as combined infections of COVID-19. Currently, as a combined infection with COVID-19, it is becoming a serious health burden. To identify the active molecule, Maestro V12.7 was used with different tools including LigPrep, Grid Generation, SiteMap, Glide XP Docking, Pharmachophores and MM-GBSA. The UNRESS tool was also used to assess the protein stability with this dengue protein. The docking result showed that all examined phytocomponents except berberine and -(+)-l-alliin had good docking scores of -8.577 (azadirachtin), -8.112 (curcumin), -7.348 (apigenin) and -6.028 (andrographolide). However, berberine and -(+)-l-alliin possessed good hydrogen-bonding interactions with RdRp. In addition, molecular dynamic simulations demonstrate that the complex of azadirachtin and dengue protein has a solid understanding of the precise interactions. As per the research results, the present research suggests that this is the first statement of azadirachtin against NS5 RNA-dependent RNA polymerase domain (RdRp), despite extensive research on this molecule in previous investigations. Furthermore, we anticipate that molecules such as curcumin, apigenin, and andrographolide would show beneficial effects while in vitro and in vivo studies are conducted on virally related objects. Since we performed ADMET and pharmacokinetic properties in this research, we feel that the phytochemicals of the screened anti-dengue molecules may not need to be evaluated for toxicological effects.

Effect of guggulsterone, a sterol identified in Commiphora gileadensis (Becham), on the dengue virus enzymes: Pharmacokinetics, molecular docking and molecular dynamics simulations studies

ObjectivesDengue infection is a serious public health problem in several regions of the world due to the lack of effective and appropriate therapy mainly for severe infection. Therefore, the development of agents with inhibitory properties targeting the dengue virus (DNV) replication is of utmost significance. MethodsGuggulsterone, a sterol reported ina local medicinal tree, Commiphora gileadensis, was investigated in silico for its inhibitory potency of dengue virus. Interaction between this guggulsterone and NS5 RNA dependent RNA polymerase, dengue methyltransferase, NS3 protease-helicase, and dengue virus type 2 envelope glycoprotein were determined using molecular docking and molecular dynamics simulations study. Guggulsterone's ADME and toxicity were predicted in silico as well. ResultsOur data revealed that guggulsterone has the lowest docking energy (−5.5 kcal/mol) with dengue NS5 RNA-dependent RNA polymerase. While the interaction of guggulsterone with dengue virus type 2 envelope glycoprotein exhibited the highest docking energy (−3.4 kcal/mol), and was the most stable complex during molecular dynamic simulation. Guggulsterone was predicted to be a probable inhibitor of dengue virus type 2 envelope glycoprotein. ADME prediction showed no violation of Lipinski and Veber rules of guggulsterone. The tested compound may inhibit CYP2C19 and CYP2C9 and cannot inhibit CYP1A2, CYP2D6, and CYP3A4. Guggulsterone was shown to have no hepatotoxicity, cytotoxicity, or mutagenicity effect. ConclusionsIt can be concluded from this study that guggulsterone may be applied as a natural compound for the prevention or treatment of dengue infections. More in vitro and in vivo testing is needed to validate the effectiveness of this natural compound.

Chalcones from Angelica keiskei (ashitaba) inhibit key Zika virus replication proteins

Zika virus (ZIKV) is a dangerous human pathogen and no antiviral drugs have been approved to date. The chalcones are a group of small molecules that are found in a number of different plants, including Angelica keiskei Koidzumi, also known as ashitaba. To examine chalcone anti-ZIKV activity, three chalcones, 4-hydroxyderricin (4HD), xanthoangelol (XA), and xanthoangelol-E (XA-E), were purified from a methanol-ethyl acetate extract from A. keiskei. Molecular and ensemble docking predicted that these chalcones would establish multiple interactions with residues in the catalytic and allosteric sites of ZIKV NS2B-NS3 protease, and in the allosteric site of the NS5 RNA-dependent RNA-polymerase (RdRp). Machine learning models also predicted 4HD, XA and XA-E as potential anti-ZIKV inhibitors. Enzymatic and kinetic assays confirmed chalcone inhibition of the ZIKV NS2B-NS3 protease allosteric site with IC50s from 18 to 50 µM. Activity assays also revealed that XA, but not 4HD or XA-E, inhibited the allosteric site of the RdRp, with an IC50 of 6.9 µM. Finally, we tested these chalcones for their anti-viral activity in vitro with Vero cells. 4HD and XA-E displayed anti-ZIKV activity with EC50 values of 6.6 and 22.0 µM, respectively, while XA displayed relatively weak anti-ZIKV activity with whole cells. With their simple structures and relative ease of modification, the chalcones represent attractive candidates for hit-to-lead optimization in the search of new anti-ZIKV therapeutics.

Insights on Dengue and Zika NS5 RNA-dependent RNA polymerase (RdRp) inhibitors.

Over recent years, many outbreaks caused by (re)emerging RNA viruses have been reported worldwide, including life-threatening Flaviviruses, such as Dengue (DENV) and Zika (ZIKV). Currently, there is only one licensed vaccine against Dengue, Dengvaxia®. However, its administration is not recommended for children under nine years. Still, there are no specific inhibitors available to treat these infectious diseases. Among the flaviviral proteins, NS5 RNA-dependent RNA polymerase (RdRp) is a metalloenzyme essential for viral replication, suggesting that it is a promising macromolecular target since it has no human homolog. Nowadays, several NS5 RdRp inhibitors have been reported, while none inhibitors are currently in clinical development. In this context, this review constitutes a comprehensive work focused on RdRp inhibitors from natural, synthetic, and even repurposing sources. Furthermore, their main aspects associated with the structure-activity relationship (SAR), proposed mechanisms of action, computational studies, and other topics will be discussed in detail.

Inhibitory potential of plant-derived metabolites against Zika virus: a computational-aided approach

BackgroundThe Zika virus (ZIKV) has caused a heavy concern all over the world because of its high infectivity and mortality rate. Still, there's no specific drug or preventive medication to treat ZIKV infection despite comprehensive analysis by the researchers. PurposeThis study was designed to demonstrate the efficacy of some plant-derived bioactive compounds against ZIKV by using both structure and ligand-based virtual screening methods. MethodsA total of 35 plant metabolites were screened on the basis of their reported pharmacological activities against ZIKV NS2B-NS3 protease (5LC0), envelope protein (5JHM), capsid protein (5YGH) and NS5 RNA-dependent RNA polymerase protein (5U04) employing a molecular docking approach. Furthermore, the ADMET properties along with structural similar drug compounds as well as the probable drug targets were also predicted for our screened four metabolites i.e., chicoric acid, luteone, reserpine and rosmarinic acid. ResultsResults showed that our four screened metabolites i.e., chicoric acid, luteone, reserpine and rosmarinic acid provide the highest binding affinity to target ZIKV proteins. The Crucial binding sites, as well as drug surface hotspots were unraveled for viral targeted protein. The analysis of ADME properties exhibited that none of the compounds had side effects that would reduce their drug-like properties. As compared, the toxicity pattern analysis has unmasked the non-toxic essence of screened drug candidates. The root-mean-square deviation (RMSD) values of ligand-macromolecule complexes were 2 Å apart envelop protein-chicoric acid, although the root-mean-square fluctuation (RMSF) values showed normal atomic fluctuations within the molecular dynamics analysis except envelop protein-chicoric acid. Most of the targets of the tested compounds lie in enzymes such as protease, hydrolase and phosphatase. Besides, the study of drug structural similarity showed several structural analogs from the drugbank such as isoformononetin (DB04202), deserpidine (DB01089) and rescinnamine (DB01180) etc. and these analogs could even be a choice to combat ZIKV. ConclusionThese results may lead to the development of effective anti-ZIKV natural medicines. Further in vivo trials are strongly recommended.

Two RNA Tunnel Inhibitors Bind in Highly Conserved Sites in Dengue Virus NS5 Polymerase: Structural and Functional Studies.

Dengue virus (DENV) NS5 RNA-dependent RNA polymerase (RdRp), an important drug target, synthesizes viral RNA and is essential for viral replication. While a number of allosteric inhibitors have been reported for hepatitis C virus RdRp, few have been described for DENV RdRp. Following a diverse compound screening campaign and a rigorous hit-to-lead flowchart combining biochemical and biophysical approaches, two DENV RdRp nonnucleoside inhibitors were identified and characterized. These inhibitors show low- to high-micromolar inhibition in DENV RNA polymerization and cell-based assays. X-ray crystallography reveals that they bind in the enzyme RNA template tunnel. One compound (NITD-434) induced an allosteric pocket at the junction of the fingers and palm subdomains by displacing residue V603 in motif B. Binding of another compound (NITD-640) ordered the fingers loop preceding the F motif, close to the RNA template entrance. Most of the amino acid residues that interacted with these compounds are highly conserved in flaviviruses. Both sites are important for polymerase de novo initiation and elongation activities and essential for viral replication. This work provides evidence that the RNA tunnel in DENV RdRp offers interesting target sites for inhibition.IMPORTANCE Dengue virus (DENV), an important arthropod-transmitted human pathogen that causes a spectrum of diseases, has spread dramatically worldwide in recent years. Despite extensive efforts, the only commercial vaccine does not provide adequate protection to naive individuals. DENV NS5 polymerase is a promising drug target, as exemplified by the development of successful commercial drugs against hepatitis C virus (HCV) polymerase and HIV-1 reverse transcriptase. High-throughput screening of compound libraries against this enzyme enabled the discovery of inhibitors that induced binding sites in the RNA template channel. Characterizations by biochemical, biophysical, and reverse genetics approaches provide a better understanding of the biological relevance of these allosteric sites and the way forward to design more-potent inhibitors.

Development of antibody-based assays for high throughput discovery and mechanistic study of antiviral agents against yellow fever virus

Despite the availability of a highly effective yellow fever virus (YFV) vaccine, outbreaks of yellow fever frequently occur in Africa and South America with significant mortality, highlighting the pressing need for antiviral drugs to manage future outbreaks. To support the discovery and development of antiviral drugs against YFV, we characterized a panel of rabbit polyclonal antibodies against the three YFV structural proteins and five non-structural proteins and demonstrated these antibody reagents in conjunction with viral RNA metabolic labeling, double-stranded RNA staining and membrane floatation assays as powerful tools for investigating YFV polyprotein processing, replication complex formation, viral RNA synthesis and high throughput discovery of antiviral drugs. Specifically, the proteolytic processing of the viral polyprotein can be analyzed by Western blot assays. The predominant nuclear localization of NS5 protein as well as the relationship between intracellular viral non-structural protein distribution and foci of YFV RNA replication can be revealed by immunofluorescence staining and membrane flotation assays. Using an antibody against YFV NS4B protein as an example, in-cell western and high-content imaging assays have been developed for high throughput discovery of antiviral agents. A synergistic antiviral effect of an YFV NS4B-targeting antiviral agent BDAA and a NS5 RNA-dependent RNA polymerase inhibitor (Sofosbuvir) was also demonstrated with the high-content imaging assay. Apparently, the antibody-based assays established herein not only facilitate the discovery and development of antiviral agents against YFV, but also provide valuable tools to dissect the molecular mechanism by which the antiviral agents inhibit YFV replication.

Pyridobenzothiazolones Exert Potent Anti-Dengue Activity by Hampering Multiple Functions of NS5 Polymerase.

Treatment of dengue virus (DENV) and other flavivirus infections is an unmet medical need. The highly conserved flaviviral NS5 RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target that interacts with NS3 and viral RNA within the replication complex assembly. Biochemical and cell-based evidence indicate that targeting cavity B may lead to dual RdRp and NS5-NS3 interaction inhibitors. By ligand-based design around 1H-pyrido[2,1-b][1,3]benzothiazol-1-one (PBTZ) 1, we identified new potent and selective DENV inhibitors that exert dual inhibition of NS5 RdRp and NS3-NS5 interaction, likely through binding cavity B. Resistance studies with compound 4 generated sequence variants in the 3'-untranslated region of RNA while further biochemical experiments demonstrated its ability to block also RNA-NS5 interaction, required for correct RNA synthesis in cells. These findings shed light on the potential mechanism of action for this class of compounds, underlying how PBTZs are very promising lead candidates for further evaluation.