The Inhibition of NS2B/NS3 Protease: A New Therapeutic Opportunity to Treat Dengue and Zika Virus Infection.

In the global pandemic scenario, dengue and zika viruses (DENV and ZIKV, respectively), both mosquito-borne members of the flaviviridae family, represent a serious health problem, and considering the absence of specific antiviral drugs and available vaccines, there is a dire need to identify new targets to treat these types of viral infections. Within this drug discovery process, the protease NS2B/NS3 is considered the primary target for the development of novel anti-flavivirus drugs. The NS2B/NS3 is a serine protease that has a dual function both in the viral replication process and in the elusion of the innate immunity. To date, two main classes of NS2B/NS3 of DENV and ZIKV protease inhibitors have been discovered: those that bind to the orthosteric site and those that act at the allosteric site. Therefore, this perspective article aims to discuss the main features of the use of the most potent NS2B/NS3 inhibitors and their impact at the social level.

Development of a sensitive microplate assay for characterizing RNA methyltransferase activity: Implications for epitranscriptomics and drug development.

RNA methylation is a ubiquitous post-transcriptional modification found in diverse RNA classes and is a critical regulator of gene expression. In this study, we used Zika virus RNA methyltransferase (MTase) to develop a highly sensitive microplate assay that uses a biotinylated RNA substrate and radiolabeled AdoMet coenzyme. The assay is fast, highly reproducible, exhibits linear progress-curve kinetics under multiple turnover conditions, has high sensitivity in competitive inhibition assays, and significantly lower background levels compared with the currently used method. Using our newly developed microplate assay, we observed no significant difference in the catalytic constants of the full-length nonstructural protein 5 enzyme and the truncated MTase domain. These data suggest that, unlike the Zika virus RNA-dependent RNA polymerase activity, the MTase activity is unaffected by RNA-dependent RNA polymerase-MTase interdomain interaction. Given its quantitative nature and accuracy, this method can be used to characterize various RNA MTases, and, therefore, significantly contribute to the field of epitranscriptomics and drug development against infectious diseases.

Drug repurposing toward the inhibition of RNA-dependent RNA polymerase of various flaviviruses through computational study.

Numerous pathogens affecting human is present in the flavivirus family namely west nile, dengue, yellow fever, and zika which involves in development of global burden and distressing the environment economically. Till date, no approved drugs are available for targeting these viruses. The threat which urged the identification of small molecules for the inhibition of these viruses is the spreading of serious viral diseases. The recent outbreak of zika and dengue infections postured a solemn risk to worldwide public well-being. RNA-dependent RNA polymerase (RdRp) is the supreme adaptable enzymes of all the RNA viruses which is responsible for the replication and transcription of genome among the structural and nonstructural proteins of flaviviruses. It is understood that the RdRp of the flaviviruses are similar stating that the japanese encephalitis and west nile shares 70% identity with zika whereas the dengue serotype 2 and 3 shares the identity of 76% and 81%, respectively. In this study, we investigated the binding site of four flaviviral RdRp and provided insights into various interaction of the molecules using the computational approach. Our study helps in recognizing the potent compounds that could inhibit the viral protein as a common inhibitor. Additionally, with the conformational stability analysis, we proposed the possible mechanism of inhibition of the identified common small molecule toward RdRp of flavivirus. Finally, this study could be an initiative for the identification of common inhibitors and can be explored further for understanding the mechanism of action through in vitro studies for the study on efficacy.

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.

Structure-Based Optimization and Characterization of Macrocyclic Zika Virus NS2B-NS3 Protease Inhibitors.

Zika virus (ZIKV) is a human pathogenic arbovirus. So far, neither a specific treatment nor a vaccination against ZIKV infections has been approved. Starting from our previously described lead structure, a series of 29 new macrocyclic inhibitors of the Zika virus protease containing different linker motifs have been synthesized. By selecting hydrophobic d-amino acids as part of the linker, numerous inhibitors with Ki values < 5 nM were obtained. For 12 inhibitors, crystal structures in complex with the ZIKV protease up to 1.30 Å resolution were determined, which contribute to the understanding of the observed structure-activity relationship (SAR). In immunofluorescence assays, an antiviral effect was observed for compound 26 containing a d-homocyclohexylalanine residue in its linker segment. Due to its excellent selectivity profile and low cytotoxicity, this inhibitor scaffold could be a suitable starting point for the development of peptidic drugs against the Zika virus and related flaviviruses.

SAR evolution towards potent C-terminal carboxamide peptide inhibitors of Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) is a member of the Flaviviridae family that can cause neurological disorders and congenital malformations. The NS2B-NS3 viral serine protease is an attractive target for the development of new antiviral agents against ZIKV. We report here a SAR study on a series of substrate-like linear tripeptides that inhibit in a non-covalent manner the NS2B-NS3 protease. Optimization of the residues at positions P1, P2, P3 and of the N-terminal and C-terminal portions of the tripeptide allowed the identification of inhibitors with sub-micromolar potency with phenylglycine as arginine-mimicking group and benzylamide as C-terminal fragment. Further SAR exploration and application of these structural changes to a series of peptides having a 4-substituted phenylglycine residue at the P1 position led to potent compounds showing double digit nanomolar inhibition of the Zika protease (IC50 = 30 nM) with high selectivity against trypsin-like proteases and the proteases of other flavivirus, such as Dengue 2 virus (DEN2V) and West Nile virus (WNV).

NS2B-NS3 protease inhibitors as promising compounds in the development of antivirals against Zika virus: A systematic review.

Zika virus (ZIKV) infections are associated with severe neurological complications and are a global public health concern. There are no approved vaccines or antiviral drugs to inhibit ZIKV replication. NS2B-NS3 protease (NS2B-NS3 pro), which is essential for viral replication, is a promising molecular target for anti-ZIKV drugs. We conducted a systematic review to identify compounds with promising effects against ZIKV; we discussed their pharmacodynamic and pharmacophoric characteristics. The online search, performed using the PubMed/MEDLINE and SCOPUS databases, yielded 56 articles; seven relevant studies that reported nine promising compounds with inhibitory activity against ZIKV NS2B-NS3 pro were selected. Of these, five (niclosamide, nitazoxanide, bromocriptine, temoporfin, and novobiocin) are currently available on the market and have been tested for off-label use against ZIKV. The 50% inhibitory concentration values of these compounds for the inhibition of NS2B-NS3 pro ranged at 0.38-21.6 µM; most compounds exhibited noncompetitive inhibition (66%). All compounds that could inhibit the NS2B-NS3 pro complex showed potent in vitro anti-ZIKV activity with a 50% effective concentration ranging 0.024-50 µM. The 50% cytotoxic concentration of the compounds assayed using A549, Vero, and WRL-69 cell lines ranged at 0.6-1388.02 µM and the selectivity index was 3.07-1698. This review summarizes the most promising antiviral agents against ZIKV that have inhibitory activity against viral proteases.

The Screening of Phytochemicals Against NS5 Polymerase to Treat Zika Virus Infection: Integrated Computational Based Approach.

BACKGROUND: The recent Zika Virus (ZIKV) outbreak provides a spur for new, efficient, and safe anti-Zika Virus agents. RNA-dependent RNA polymerase (RdRp) is critical amongst the seven non-structural proteins for viral replication and considered an attractive drug target. METHODS: In this study, molecular docking approach was used to rationally screen the library of 5000 phytochemicals to find inhibitors against NS5 RdRp. LigX tool was used to analyze the 2D plots of receptor-ligand interactions. The top-ranked compounds were then subjected to in-silico pharmacokinetic study. RESULTS: The compounds namely Polydatin, Dihydrogenistin, Liquiritin, Rhapontin and Cichoriin were successfully bound inside the pocket of NS5 RdRp. Polydatin was the leading phytochemical that showed high docking score -18.71 (kcal/mol) and bonding interaction at the active-site of NS5 RdRp. They were subjected to analyze drug-like properties that further reinforced their validation and showed that they have more capability to attach with the receptor as compared to SOFOSBUVIR control drug. MD simulation of the top two complexes was performed and the simulated complexes showed stability and ligands were kept within the bonding pocket. CONCLUSION: The study might facilitate the development of a natural and cost-effective drug against ZIKV. Further validation, however, is necessary to confirm its effectiveness and its biocompatibility.

Template requirements of Zika RNA polymerase during in vitro RNA synthesis from the 3'-end of virus minus-strand RNA.

As ZIKV continues to spread, many "unknowns" remain and research is needed to advance the understanding of this important pathogen. Viral RNA dependent-RNA polymerases (RdRp) are validated targets for inhibitors of the replication of several viruses. Several studies have set up in vitro enzymatic assays of the RdRp of the Zika virus for testing of candidate inhibitors. While most of these studies use short synthetic polymers, we have shown in a previous work that the Zika polymerase domain is capable of a de novo synthesis of the viral genome using the natural viral RNA as template. Here we have studied the role of the sequences at the 3'end of the minus-strand RNA in the initiation of the RNA synthesis by the Zika isolated RdRp. Our results strongly suggest that the region containing the 105 first nucleotides from the 3' end of the minus-strand RNA is important for initiation of the positive RNA synthesis. This indicates that this region displays all the primary and secondary structures to be efficiently recognized by the recombinant RdRp in vitro. Moreover, we show that the 46 nucleotides are sufficient to initiate RNA synthesis. In addition, the ZIKV polymerase domain poorly replicated the RNA of other RNA viruses and appeared highly selective for its own RNA.

Peptide-Bismuth Bicycles: In Situ Access to Stable Constrained Peptides with Superior Bioactivity.

Constrained peptides are promising next-generation therapeutics. We report here a fundamentally new strategy for the facile generation of bicyclic peptides using linear precursor peptides with three cysteine residues and a non-toxic trivalent bismuth(III) salt. Peptide-bismuth bicycles form instantaneously at physiological pH, are stable in aqueous solution for many weeks, and much more resistant to proteolysis than their linear precursors. The strategy allows the in situ generation of bicyclic ligands for biochemical screening assays. We demonstrate this for two screening campaigns targeting the proteases from Zika and West Nile viruses, revealing a new lead compound that displayed inhibition constants of 23 and 150 nM, respectively. Bicyclic peptides are up to 130 times more active and 19 times more proteolytically stable than their linear analogs without bismuth.

Optimal flexibility of the linker region of Zika virus NS5 methyltransferase-polymerase is critical for virus replication.

The flavivirus NS5 protein contains an N-terminal methyl-transferase (MTase) connected through a flexible linker with a C-terminal RNA-dependent RNA-polymerase (RdRp) domain, that work cooperatively to replicate and methylate the viral genome. In this study we probed the importance of an evolutionary-conserved hydrophobic residue (Val266) located at the start of the ten-residue interdomain linker of Zika virus (ZIKV) NS5. In flavivirus NS5 crystal structures, the start of the linker forms a 310 helix when NS5 adopts a compact conformation, but becomes disordered or extended in open conformations. Using reverse genetics system, we either introduced rigidity in the linker through mutation to a proline or flexibility through a glycine mutation at position 266. ZIKV NS5 Val 266 to Pro mutation was lethal for viral RNA replication while the Gly mutation was severely attenuated. Serial passaging of cell culture supernatant derived from C6/36 mosquito cells transfected with mutant ZIKV RNA showed that the attenuation can be rescued. Next generation deep sequencing revealed four single nucleotide polymorphisms that occur with an allele frequency >98%. The single non-synonymous NS5 mutation Glu419 to Lys is adjacent to RdRp motif G at the tip of the fingers subdomain, while the remaining three are synonymous variants at nucleotide positions 1403, 4403 and 6653 in the genome. Reverse engineering the changes into the ZIKV NS5/Val266Gly background followed by serial passaging revealed that residue 266 is under strong positive selection to revert back to Val. The interaction of the specific conformation of the NS5 linker with Val at position 266 and the RNA binding motif G region may present a potential strategy for allosteric antiviral drug development.

Chikungunya nsP4 homology modeling reveals a common motif with Zika and Dengue RNA polymerases as a potential therapeutic target.

Among the diseases transmitted by vectors, there are those caused by viruses named arboviruses (arthropod-borne viruses). In past years, viruses transmitted by mosquitoes have been of relevance in global health, such as Chikungunya (CHIKV), Dengue (DENV), and Zika (ZIKV), which have Aedes aegypti as a common vector, thus raising the possibility of multi-infection. Previous reports have described the general structure of RNA-dependent RNA polymerases termed right-hand fold, which is conserved in positive single-stranded RNA viruses. Here, we report a comparison between sequences and the computational structure of RNA-dependent RNA polymerases from CHIKV, DENV, and ZIKV and the conserved sites to be considered for the design of an antiviral drug against the three viruses. We show that the sequential identity between consensus sequences from CHIKV and DENV is 8.1% and the similarity is 15.1%; the identity between CHIKV and ZIKV is 9.3%, and the similarity is 16.6%; and the identity between DENV and ZIKV is 68.6%, and the similarity is 79.2%. Nevertheless, the structural alignment shows that the root-mean-square deviation (RMSD) measurement value in general structure comparison between CHIKV RdRp and ZIKV RdRp was 1.248 Å, RMSD between CHIKV RdRp and DENV RdRp was 1.070 Å, and RMSD between ZIKV RdRp and DENV RdRp was 1.106 Å. Despite the low identity and similarity of CHIKV sequence with DENV and ZIKV, we show that A, B, C, and E motifs are structurally well conserved. These structural similarities offer a window into drug design against these arboviruses giving clues about critical target sites.

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.

Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro.

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40-80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.

In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases.

The potential outcome of flavivirus and alphavirus co-infections is worrisome due to the development of severe diseases. Hundreds of millions of people worldwide live under the risk of infections caused by viruses like chikungunya virus (CHIKV, genus Alphavirus), dengue virus (DENV, genus Flavivirus), and zika virus (ZIKV, genus Flavivirus). So far, neither any drug exists against the infection by a single virus, nor against co-infection. The results described in our study demonstrate the inhibitory potential of two flavonoids derived from citrus plants: Hesperetin (HST) against NS2B/NS3pro of ZIKV and nsP2pro of CHIKV and, Hesperidin (HSD) against nsP2pro of CHIKV. The flavonoids are noncompetitive inhibitors and the determined IC50 values are in low µM range for HST against ZIKV NS2B/NS3pro (12.6 ± 1.3 µM) and against CHIKV nsP2pro (2.5 ± 0.4 µM). The IC50 for HSD against CHIKV nsP2pro was 7.1 ± 1.1 µM. The calculated ligand efficiencies for HST were > 0.3, which reflect its potential to be used as a lead compound. Docking and molecular dynamics simulations display the effect of HST and HSD on the protease 3D models of CHIKV and ZIKV. Conformational changes after ligand binding and their effect on the substrate-binding pocket of the proteases were investigated. Additionally, MTT assays demonstrated a very low cytotoxicity of both the molecules. Based on our results, we assume that HST comprise a chemical structure that serves as a starting point molecule to develop a potent inhibitor to combat CHIKV and ZIKV co-infections by inhibiting the virus proteases.

Synthesis, Structure-Activity Relationships, and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B-NS3 Protease.

Flaviviruses, including Zika, dengue, and West Nile viruses, are important human pathogens. The highly conserved NS2B-NS3 protease of Flavivirus is essential for viral replication and therefore a promising drug target. Through compound screening, followed by medicinal chemistry studies, a novel series of 2,5,6-trisubstituted pyrazine compounds are found to be potent, allosteric inhibitors of Zika virus protease (ZVpro) with IC50 values as low as 130 nM. Their structure-activity relationships are discussed. The ZVpro inhibitors also inhibit homologous proteases of dengue and West Nile viruses, and their inhibitory activities are correlated. The most potent compounds 47 and 103 potently inhibited Zika virus replication in cells with EC68 values of 300-600 nM and in a mouse model of Zika infection. These compounds represent novel pharmacological leads for drug development against Flavivirus infections.

Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses.

The most frequent fetal birth defect associated with prenatal Zika virus (ZIKV) infection is brain calcification, which in turn may potentially affect neurological development in infants. Understanding the mechanism could inform the development of potential therapies against prenatal ZIKV brain calcification. In perivascular cells, bone morphogenetic protein (BMP) is an osteogenic factor that undergoes maturation to activate osteogenesis and calcification. Here, we show that ZIKV infection of cultivated primary human brain pericytes triggers BMP2 maturation, leading to osteogenic gene expression and calcification. We observed extensive calcification near ZIKV+ pericytes of fetal human brain specimens and in vertically transmitted ZIKV+ human signal transducer and activator of transcription 2-knockin mouse pup brains. ZIKV infection of primary pericytes stimulated BMP2 maturation, inducing osteogenic gene expression and calcification that were completely blocked by anti-BMP2/4 neutralizing antibody. Not only did ZIKV NS3 expression alone induce BMP2 maturation, osteogenic gene expression and calcification, but purified NS3 protease also effectively cleaved pro-BMP2 in vitro to generate biologically active mature BMP2. These findings highlight ZIKV-induced calcification where the NS3 protease subverts the BMP2-mediated osteogenic signalling pathway to trigger brain calcification.

Structure-based screening and validation of bioactive compounds as Zika virus methyltransferase (MTase) inhibitors through first-principle density functional theory, classical molecular simulation and QM/MM affinity estimation.

Recent Zika virus (ZIKV) outbreak and association with human diseases such as neurological disorders have raised global health concerns. However, in the absence of an approved anti-ZIKV drug has generated urgency for the drug development against ZIKV infection. Here, structure-based virtual screening of 8589 bioactive compounds, screened at the substrate-binding site of ZIKV nonstructural 5 (NS5)-based structure N-terminal methyltransferase (MTase) domain followed by ADMET (absorption, distribution, metabolism, excretion and toxicity) profiling concluded the four potential lead inhibitors, i.e. (4-acetylamino-benzenesulfonylamino)-acetic acid (F3342-0450), 3-(5-methylfuran-2-yl)-N-(4-sulfamoylphenyl)propanamide (F1736-0142), 8-(2-hydroxy-ethylamino)-1,3-dimethyl-7-(3-methyl-benzyl)-3,7-dihydro-purine-2,6-dione (F0886-0080) and N-[4-(aminosulfonyl)phenyl]-2,3-dihydro-1,4-benzodioxine-2-carboxamide (F0451-2187). Collectively, extra precision docking and Density Functional Theory(DFT) calculations studies identified the F3342-0450 molecule, having strong interactions on the active site of MTase, further supported by molecular dynamics simulation, binding affinity and hybrid QM/MM calculations, suggest a new drug molecule for the antiviral drug development against ZIKV infection. Communicated by Ramaswamy H. Sarma.

Zika virus protease induces caspase-independent pyroptotic cell death by directly cleaving gasdermin D.

The association of Zika virus (ZIKV) infection with a congenital malformation in fetuses, neurological, and other systemic complications in adults have brought significant global health emergency. ZIKV targets nerve cells in the brain and causes cell death, such as pyroptosis, leading to neuroinflammation. Here we described a novel mechanism of pyroptosis caused by ZIKV protease. We found that ZIKV protease directly cleaved the GSDMD into N-terminal fragment (1-249) leading to pyroptosis in a caspase-independent manner, suggesting a direct mechanism of ZIKV-induced cell death and subsequent inflammation. Our findings might shed new light to explore the pathogenesis of ZIKV infections where ZIKV protease might be a suitable target for the development of antiviral agents.

Zika virus NS4A N-Terminal region (1-48) acts as a cofactor for inducing NTPase activity of NS3 helicase but not NS3 protease.

Among Flaviviridae, in West Nile virus (WNV) and Hepatitis C virus (HCV), the non-structural protein NS4A modulates the NTPase activity of viral helicases during nucleic acid unwinding through its N-terminal disordered residues (1-50). In HCV, the acidic NS4A also serves as a cofactor for regulating the NS3 protease activity. However, in case of Zika virus (ZIKV), the role of NS4A and its impact on activities of NS3 helicase and protease is not known. In order to elucidate the role of NS4A, we checked the NTPase activity of NS3 helicase and protease activity of NS3 protease in presence of NS4A N-terminal region (residues 1-48) peptide. Our enzyme kinetics results together with binding experiment clearly demonstrate that NS3 helicase in presence of NS4A peptide increased the rate of ATP hydrolysis whereas the protease activity of NS3 protease was not affected. Therefore, like WNV and HCV, our results establish a role of ZIKV NS4A being a cofactor for modulating the NTPase activity of ZIKV NS3 helicase.