Dengue Virus Protease Research Articles

Machine-Learning Approach to Identify Potential Dengue Virus Protease Inhibitors: A Computational Perspective.

The global prevalence of dengue virus (DENV), a widespread flavivirus, has led to varied epidemiological impacts, economic burdens, and health consequences. The alarming increase in infections is exacerbated by the absence of approved antiviral agents against the DENV. Within flaviviruses, the NS3/NS2B serine protease plays a pivotal role in processing the viral polyprotein into distinct components, making it an attractive target for antiviral drug development. In this study, machine-learning (ML) techniques were employed to build predictive models for the screening of a library containing 32,000 protease inhibitors. Utilizing GNINA for structure-based virtual screening, the top potential candidates underwent a subsequent evaluation of their absorption, distribution, metabolism, excretion, and toxicity properties. Selected compounds were subjected to molecular dynamics simulations and binding free energy calculations via MM/GBSA. The results suggest that comp530 possesses binding potential to DENV protease as a noncovalent inhibitor with multiple positions for chemical substitutions, presenting opportunities for optimizing their selectivity and specificity. However, other compounds predicted via ML models may still provide a promising start for covalent inhibitors.

In Vitro Cleavage Assay to Characterize DENV NS2B3 Antagonism of cGAS.

cGAS is a key cytosolic dsDNA receptor that senses viral infection and elicits interferon production through the cGAS-cGAMP-STING axis. cGAS is activated by dsDNA from viral and bacterial origins as well as dsDNA leaked from damaged mitochondria and nucleus. Eventually, cGAS activation launches the cell into an antiviral state to restrict the replication of both DNA and RNA viruses. Throughout the long co-evolution, viruses devise many strategies to evade cGAS detection or suppress cGAS activation. We recently reported that the Dengue virus protease NS2B3 proteolytically cleaves human cGAS in its N-terminal region, effectively reducing cGAS binding to DNA and consequent production of the second messenger cGAMP. Several other RNA viruses likely adopt the cleavage strategy. Here, we describe a protocol for the purification of recombinant human cGAS and Dengue NS2B3 protease, as well as the in vitro cleavage assay.

Predicting repurposed drugs targeting the NS3 protease of dengue virus using machine learning-based QSAR, molecular docking, and molecular dynamics simulations

ABSTRACT Dengue fever, prevalent in Southeast Asian countries, currently lacks effective pharmaceutical interventions for virus replication control. This study employs a strategy that combines machine learning (ML)-based quantitative-structure-activity relationship (QSAR), molecular docking, and molecular dynamics simulations to discover potential inhibitors of the NS3 protease of the dengue virus. We used nine molecular fingerprints from PaDEL to extract features from the NS3 protease dataset of dengue virus type 2 in the ChEMBL database. Feature selection was achieved through the low variance threshold, F-Score, and recursive feature elimination (RFE) methods. Our investigation employed three ML models – support vector machine (SVM), random forest (RF), and extreme gradient boosting (XGBoost) – for classifier development. Our SVM model, combined with SVM-RFE, had the best accuracy (0.866) and ROC_AUC (0.964) in the testing set. We identified potent inhibitors on the basis of the optimal classifier probabilities and docking binding affinities. SHAP and LIME analyses highlighted the significant molecular fingerprints (e.g. ExtFP69, ExtFP362, ExtFP576) involved in NS3 protease inhibitory activity. Molecular dynamics simulations indicated that amphotericin B exhibited the highest binding energy of −212 kJ/mol and formed a hydrogen bond with the critical residue Ser196. This approach enhances NS3 protease inhibitor identification and expedites the discovery of dengue therapeutics.

Exploring the antiviral inhibitory activity of Niloticin against the NS2B/NS3 protease of Dengue virus (DENV2)

Dengue virus (DENV2) is the cause of dengue disease and a worldwide health problem. DENV2 replicates in the host cell using polyproteins such as NS3 protease in conjugation with NS2B cofactor, making NS3 protease a promising antiviral drug-target. This study investigated the efficacy of ‘Niloticin’ against NS2B/NS3-protease. In silico and in vitro analyses were performed which included interaction of niloticin with NS2B/NS3-protease, protein stability and flexibility, mutation effect, betweenness centrality of residues and analysis of cytotoxicity, protein expression and WNV NS3-protease activity. Similar like acyclovir, niloticin forms strong H-bonds and hydrophobic interactions with residues LEU149, ASN152, LYS74, GLY148 and ALA164. The stability of the niloticin-NS2B/NS3-protease complex was found to be stable compared to the apo NS2B/NS3-protease in structural deviation, PCA, compactness and FEL analysis. The IC50 value of niloticin was 0.14 μM in BHK cells based on in vitro cytotoxicity analysis and showed significant activity at 2.5 μM in a concentration-dependent manner. Western blotting and qRT-PCR analyses showed that niloticin reduced DENV2 protein transcription in a dose-dependent manner. Besides, niloticin confirmed the inhibition of NS3-protease by the SensoLyte 440 WNV protease detection kit. These promising results suggest that niloticin could be an effective antiviral drug against DENV2 and other flaviviruses.

Computational design and in vitro assay of lantadene-based novel inhibitors of NS3 protease of dengue virus

Abstract Dengue virus (DENV) infection is one of the diseases for which no drug is available for the treatment. The DENV NS2B-NS3 protease is considered to be the prime target for anti-dengue drug development because of its importance in the development of new virus subunits via DENV poly-protein breakdown. Pentacyclic triterpenoids (Lantadenes) from the weed Lantana camara L. and its semi-synthetic congeners have shown a wide array of biological activities in the last two decades. The virtual screening strategy was used on the library of 78 natural and semi-synthetic lantadenes to predict the potent antagonists for the NS2B-NS3 protease enzyme of DENV and their experimental validation by in vitro assay of lead molecules. In the in silico analysis of 78 triterpenoids, two lead molecules (−10.60 and −9.93 kcal/mol) were predicted to be inhibitors of protease (viral) when compared to its reference ligand 1,8-dihydroxy-4,5-dinitroanthraquinone (−5.377 kcal/mol). At the same time, binding affinity, pharmacokinetic, and toxicity profiling, along with molecular dynamics simulations, were studied. The in vitro viral infection inhibition assay inferred that lead molecule 62 exhibited a 60% and 45% reduction in DENV titers at 10 and 5 µM concentrations, respectively. The lead molecule 62 can further be optimized for its pharmacophore and has the potential to be developed as a drug-like molecule.

Phenolic compounds of Theobroma cacao L. show potential against dengue RdRp protease enzyme inhibition by In-silico docking, DFT study, MD simulation and MMGBSA calculation.

Currently, there is no antiviral medication for dengue, a potentially fatal tropical infectious illness spread by two mosquito species, Aedes aegypti and Aedes albopictus. The RdRp protease of dengue virus is a potential therapeutic target. This study focused on the in silico drug discovery of RdRp protease inhibitors. To assess the potential inhibitory activity of 29 phenolic acids from Theobroma cacao L. against DENV3-NS5 RdRp, a range of computational methods were employed. These included docking, drug-likeness analysis, ADMET prediction, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. The aim of these studies was to confirm the stability of the ligand-protein complex and the binding pose identified during the docking experiment. Twenty-one compounds were found to have possible inhibitory activities against DENV according to the docking data, and they had a binding affinity of ≥-37.417 kcal/mol for DENV3- enzyme as compared to the reference compound panduratin A. Additionally, the drug-likeness investigation produced four hit compounds that were subjected to ADMET screening to obtain the lead compound, catechin. Based on ELUMO, EHOMO, and band energy gap, the DFT calculations showed strong electronegetivity, favouravle global softness and chemical reactivity with considerable intra-molecular charge transfer between electron-donor to electron-acceptor groups for catechin. The MD simulation result also demonstrated favourable RMSD, RMSF, SASA and H-bonds in at the binding pocket of DENV3-NS5 RdRp for catechin as compared to panduratin A. According to the present findings, catechin showed high binding affinity and sufficient drug-like properties with the appropriate ADMET profiles. Moreover, DFT and MD studies further supported the drug-like action of catechin as a potential therapeutic candidate. Therefore, further in vitro and in vivo research on cocoa and its phytochemical catechin should be taken into consideration to develop as a potential DENV inhibitor.

A Non-Active-Site Inhibitor with Selectivity for Zika Virus NS2B-NS3 Protease.

Flavivirus infection usually results in fever accompanied by headache, arthralgia, and, in some cases, rash. Although the symptoms are mild, full recovery can take several months. Flaviviruses encode seven nonstructural proteins that represent potential drug targets for this viral family. Focusing on the Zika virus NS2B-NS3 protease, we uncovered a unique inhibitor, MH1, composed of aminothiazolopyridine and benzofuran moieties. MH1 inhibits ZVP with a biochemical IC50 of 440 nM and effectively blocks cleavage of ZVP substrates in cells. Surprisingly, MH1 inhibits the other flaviviral proteases at least 18-fold more weakly. This same phenomenon was observed in assays of the viral cytopathic effect, where only Zika virus showed sensitivity to MH1. This selectivity was unexpected since flaviviral proteases have high similarity in sequence and protein structure. MH1 binds at an allosteric site, as demonstrated by its ability to stabilize ZVP synergistically with an active site inhibitor. To understand its selectivity, we constructed a series of hybrid proteases composed of select segments of ZVP, which is sensitive to MH1, and dengue virus protease, which is essentially insensitive to MH1. Our results suggest that MH1 binds to the NS3 protease domain, disrupting its interaction with NS2B. These interactions are essential for substrate binding and cleavage. In particular, the unique dynamic properties of NS2B from Zika seem to be required for the function of MH1. Insights into the mechanism of MH1 function will aid us in developing non-active-site-directed, pan-flaviviral inhibitors, by highlighting the importance of evaluating and considering the dynamics of the NS2B regions.

Potential inhibitor for dengue virus protease (NS2B-NS3): Computer-aided drug discovery

Potential inhibitor for dengue virus protease (NS2B-NS3): Computer-aided drug discovery

Analysis of Cleavage Activity of Dengue Virus Protease by Co-transfections.

The genome of the dengue virus codes for a single polypeptide that yields three structural and seven non-structural (NS) proteins upon post-translational modifications. Among them, NS protein-3 (NS3) possesses protease activity, involved in the processing of the self-polypeptide and in the cleavage of host proteins. Identification and analysis of such host proteins as substrates of this protease facilitate the development of specific drugs. In vitro cleavage analysis has been applied, which requires homogeneously purified components. However, the expression and purification of both S3 and erythroid differentiation regulatory factor 1 (EDRF1) are difficult and unsuccessful on many occasions. EDRF1 was identified as an interacting protein of dengue virus protease (NS3). The amino acid sequence analysis indicates the presence of NS3 cleavage sites in this protein. As EDRF1 is a high-molecular-weight (~138 kDa) protein, it is difficult to express and purify the complete protein. In this protocol, we clone the domain of the EDRF1 protein (C-terminal end) containing the cleavage site and the NS3 into two different eukaryotic expression vectors containing different tags. These recombinant vectors are co-transfected into mammalian cells. The cell lysate is subjected to SDS-PAGE followed by western blotting with anti-tag antibodies. Data suggest the disappearance of the EDRF1 band in the lane co-transfected along with NS3 protease but present in the lane transfected with only EDRF1, suggesting EDRF1 as a novel substrate of NS3 protease. This protocol is useful in identifying the substrates of viral-encoded proteases using ex vivo conditions. Further, this protocol can be used to screen anti-protease molecules. Key features • This protocol requires the cloning of protease and substrate into two different eukaryotic expression vectors with different tags. • Involves the transfection and co-transfection of both the above recombinant vectors individually and together. • Involves western blotting of the same PVDF membrane containing total proteins of the cell lysate with two different antibodies. • Does not require purified proteins for the analysis of cleavage of any suspected substrate by the protease.

Expression of dengue virus and Zika virus NS2B-NS3pro constructs alter cellular fatty acids, but co-expression with a Zika virus virus-like particle is detrimental to virus-like particle expression

ObjectiveStudies have shown that Flavivirus infection remodels the host cell to favour viral replication. In particular, the host cell lipid profile is altered, and it has been proposed that this process alters membrane fluidity to allow wrapping of the outer structural proteins around the viral nucleocapsid. We investigated whether expression of the Zika virus (ZIKV) and dengue virus (DENV) protease induced alterations in the cellular lipid profile, and subsequently whether co-expression of these proteases with VLP constructs was able to improve VLP yield.ResultsOur results showed that both ZIKV and DENV proteases induced alterations in the lipid profile, but that both active and inactive proteases induced many of the same changes. Neither co-transfection of protease and VLP constructs nor bicistronic vectors allowing expression of both protease and VLP separated by a cell cleavable linker improved VLP yield, and indeed many of the constructs showed significantly reduced VLP production. Further work in developing improved VLP expression platforms is required.

Theoretical study of dengue virus protease NS3 and interaction with Bowman Birk-type inhibitors

Currently, dengue is considered one of the viral infections that most affect tropical areas in the world and is transmitted to humans by the Aedes aegypti mosquito. The objective of this study was to evaluate the interactions between the dengue virus NS3 protease and compounds derived from flavonoids and Bowman-Birk-like inhibitors. Initially, the 3D structures of the NS3 and NS3/NS2B proteases were evaluated, and the compounds to be studied were optimized using the Sybyl 7.3 and Gaussian 03 programs, respectively. Molecular docking was carried out using the FlexX and EHits 2.0 packages. The studied compounds exhibited a linear relationship between the experimental and theoretical inhibition constants for the NS3 enzyme. However, this behavior did not occur for the NS3/NS2B enzyme. Therefore, it is expected that the mimetic peptides (Bowman-Birk type) have a greater preference for the initial stage of viral replication. Among the inhibitors with the highest potential were the peptide mol 18r (Bowman-Birk type) and the flavonoid derivative panduratin2. Therefore, this study contributes to the design and computational screening of new dengue virus inhibitors using the NS3 enzyme as a template."

Differential localization of dengue virus protease affects cell homeostasis and triggers to thrombocytopenia

Thrombocytopenia is one of the symptoms of many virus infections which is the "hallmark" in the case of dengue virus. In this study, we show the differential localization of existing two forms of dengue virus protease, i.e., NS2BNS3 to the nucleus and NS3 to the nucleus and mitochondria. We also report a nuclear transcription factor, erythroid differentiation regulatory factor 1 (EDRF1), as the substrate for this protease. EDRF1 regulates the expression and activity of GATA1, which in turn controls spectrin synthesis. Both GATA1 and spectrins are required for platelet formation. On the other hand, we found that the mitochondrial activities will be damaged by NS3 localization which cleaves GrpEL1, a co-chaperone of mitochondrial Hsp70. Levels of both EDRF1 and GrpEL1 were found to deteriorate in dengue virus-infected clinical samples. Hence, we conclude that NS2BNS3-mediated EDRF1 cleavage and the NS3-led mitochondrial dysfunction account for thrombocytopenia.

N-sulfonyl peptide-hybrids as a new class of dengue virus protease inhibitors.

Dengue virus (DENV) from the Flaviviridae family causes an epidemic disease that seriously threatens human life. The viral serine protease NS2B-NS3 is a promising target for drug development against DENV and other flaviviruses. We here report the design, synthesis, and in-vitro characterization of potent peptidic inhibitors of DENV protease with a sulfonyl moiety as N-terminal cap, thereby creating sulfonamide-peptide hybrids. The in-vitro target affinities of some synthesized compounds were in the nanomolar range, with the most promising derivative reaching a Ki value of 78nM against DENV-2 protease. The synthesized compounds did not have relevant off-target activity nor cytotoxicity. The metabolic stability of compounds against rat liver microsomes and pancreatic enzymes was remarkable. In general, the integration of sulfonamide moieties at the N-terminus of peptidic inhibitors proved to be a promising and attractive strategy for further drug development against DENV infections.

Exploring allosteric hits of the NS2B-NS3 protease of DENV2 by structure-guided screening

Despite the rising number of cases and increasing global disease burden, there is no definitive therapy against dengue to date, which necessitates the urgent discovery of inhibitors against the virus. The NS2B-NS3 serine protease of the dengue virus (DENV) catalyses polyprotein cleavage and is a potential target for drug discovery. The protease possesses a potentially druggable allosteric site, and the binding of inhibitors to this site locks the protease in an inactive conformation. The allosteric site is a potential druggable target for drug discovery against flaviviruses. This study aimed to identify serotype-specific hits against the allosteric site in the NS2B-NS3 protease of DENV serotype 2 (DENV2) from the Enamine, Selleck, and ChemDiv antiviral libraries. The prepared libraries were screened using a redocking and rescoring-based strategy with Glide SP and Glide XP, and the hitlist was initially screened by comparing their docking scores with that of reported allosteric inhibitors, myricetin and curcumin. The hitlist was subsequently screened by comparing the molecular mechanics with generalised Born and surface area solvation (MM-GBSA) energy with that of the standards. Ten hits were finally selected by virtual screening, and the stability of the hit-receptor complexes was determined with 100 ns molecular dynamics (MD) simulations in an explicit solvent. Trajectory visualisation and analyses of the RMSD and RMSF values revealed that three hits, including two catechins, remained stably bound to the allosteric binding site throughout the production run. Hit-receptor interaction analyses revealed that the hits formed highly stable interactions with Glu 88, Trp 89, Leu 149, Ile 165, and Asn 167, and MM-GBSA energy analysis revealed that the three hits had high binding affinity to the allosteric site. The findings obtained herein can aid in identifying novel serotype-specific inhibitors of DENV protease in future.

The Dengue virus protease NS2B3 cleaves cyclic GMP-AMP synthase to suppress cGAS activation

Dengue virus (DENV) is one of the most prevalent mosquito-transmitted human viruses that causes significant morbidity and mortality worldwide. To persist in the cell and consequently cause disease, DENV is evolved with mechanisms to suppress the induction of type I interferons by antagonizing cGAS-STING signaling. Using recombinant proteins and invitro cleavage assays, we have shown that the DENV protease NS2B3 is capable of cleaving cGAS in the N-terminal region without disrupting the C-terminal catalytic center. This generates two major cleavage products: cleavage product N-terminal (CP-N) and cleavage product C-terminal (CP-C). We observed reduction in DNA-binding affinity of CP-C as compared to full-length cGAS. Reduction in DNA-binding affinity is also correlated with the decrease in enzymatic activity of CP-C. CP-N, on the other hand, has almost comparable DNA-binding ability as that of the full-length cGAS. In fact, CP-N competitively inhibits cyclic GMP-AMP production by both full-length cGAS and CP-C. We hypothesize that high DNA-binding affinity of CP-N enables it to sequester the DNA from CP-C and noncleaved full-length cGAS and thus reduces the rate of enzyme activation and cyclic GMP-AMP synthesis. Furthermore, we found that NS2B3 physically interacts with full-length cGAS and CP-C, laying the basis for their shuttling to and eventual degradation in the autophagosome. Overall, our study highlights a multifaceted and effective strategy by which an RNA virus antagonizes cGAS-STING signaling which may be useful for the design of antivirals targeting viral proteases.

Blind docking of 4-Amino-7-Chloroquinoline analogs as potential dengue virus protease inhibitor using CB Dock a web server

Currently, there is no approved drug to combat dengue. Various quinoline derivatives are known for potential antimalarial, antiviral activities, etc. In the present work docking between 4-Amino-7-Chloroquinoline analogs was performed with dengue virus NS2B/NS3 protease using CB dock, a web server. Lys74, Ile165, Val147, Asn152, Asn167, Trp83 and Leu149 amino acid residues were found to be in contact with designed 4-Amino-7-Chloroquinoline analogs. Different modes of binding like hydrogen bonding, hydrophobic interactions, etc with designed compounds improve potential anti-dengue characteristics in silico. ADME results are in acceptable range.

A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease.

Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The KD value observed is in accordance with the IC50 determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.

Allosteric quinoxaline-based inhibitors of the flavivirus NS2B/NS3 protease

Viruses from the Flavivirus genus infect millions of people worldwide and cause severe diseases, including recent epidemics of dengue virus (DENV), and Zika virus (ZIKV). There is currently no antiviral treatment against flavivirus infections, despite considerable efforts to develop inhibitors against essential viral enzymes including NS2B/NS3 protease. Targeting the flavivirus NS2B/NS3 protease proved to be challenging because of the conformational dynamics, topology, and electrostatic properties of the active site. Here, we report the identification of quinoxaline-based allosteric inhibitors by fragment-based drug discovery approach as a promising new drug-like scaffold to target the NS2B/NS3 protease. Enzymatic assays and mutational analysis of the allosteric site in ZIKV NS2B/NS3 protease support noncompetitive inhibition mechanism as well as engineered DENV protease construct indicating the compounds likely compete with the NS2B cofactor for binding to the protease domain. Furthermore, antiviral activity confirmed the therapeutic potential of this new inhibitor scaffold.

Discovery of potent benzoxaborole inhibitors against SARS-CoV-2 main and dengue virus proteases

The RNA viruses SARS-CoV-2 and dengue pose a major threat to human health worldwide and their proteases (Mpro; NS2B/NS3) are considered as promising targets for drug development. We present the synthesis and biological evaluation of novel benzoxaborole inhibitors of these two proteases. The most active compound achieves single-digit micromolar activity against SARS-CoV-2 Mpro in a biochemical assay. The most active substance against dengue NS2B/NS3 protease has submicromolar activity in cells (EC50 0.54 μM) and inhibits DENV-2 replication in cell culture. Most benzoxaboroles had no relevant cytotoxicity or significant off-target inhibition. Furthermore, the class demonstrated passive membrane penetration and stability against the evaluated proteases. This compound class may contribute to the development of antiviral agents with activity against DENV or SARS-CoV-2.

Characterization of an Allosteric Pocket in Zika Virus NS2B-NS3 Protease

The NS2B-NS3 protease from Zika virus (ZIKV NS2B-NS3pro) cleaves the viral polyprotein, being essential for its replication and a therapeutic target. Inhibitors that target the active site of ZIKV NS2B-NS3pro have been developed, but they tend to have unfavorable pharmacokinetic properties due to their highly positive charge. Thus, the characterization of allosteric sites in this protease provides new strategies for inhibitor development. Here, we characterized a new allosteric pocket in ZIKV NS2B-NS3pro, analogous to the one previously described for the dengue virus protease. Molecular dynamics simulations indicate the presence of cavities around the residue Ala125, sampling protein conformations in which they are connected to the active site. This link between the residue Ala125 and the active site residues was reinforced by correlation network analysis. To experimentally verify the existence of this allosteric mechanism, we expressed and purified the Ala125Cys mutant of ZIKV NS2B-NS3pro and demonstrated that this variant is inhibited by the thiol-containing chemical probes 5,5'-dithiobis-(2-nitrobenzoic acid) and aldrithiol, which do not affect the activity of the wild-type protein. Inhibition of the mutant protein is reversed by the addition of strong reducing agents, supporting the involvement of Cys125 in covalent bond formation and enzyme inhibition. Together, our results provide experimental evidence for an allosteric pocket in ZIKV NS2B-NS3pro, in the region around Ala125, and computational insights on the structural connection between this region and the enzyme active site.