Orthoflaviviral Inhibitors in Clinical Trials, Preclinical In Vivo Efficacy Targeting NS2B-NS3 and Cellular Antiviral Activity via Competitive Protease Inhibition.

Orthoflaviviruses, including zika (ZIKV), West Nile (WNV), and dengue (DENV) virus, induce severely debilitating infections and contribute significantly to the global disease burden, yet no clinically approved antiviral treatments exist. This review offers a comprehensive analysis of small-molecule drug development targeting orthoflaviviral infections, with a focus on NS2B-NS3 inhibition. We systematically examined clinical trials, preclinical efficacy studies, and modes of action for various viral replication inhibitors, emphasizing allosteric and orthosteric drugs inhibiting NS2B-NS3 protease with in vivo efficacy and in vitro-tested competitive NS2B-NS3 inhibitors with cellular efficacy. Our findings revealed that several compounds with in vivo preclinical efficacy failed to show clinical antiviral efficacy. NS3-NS4B inhibitors, such as JNJ-64281802 and EYU688, show promise, recently entering clinical trials, underscoring the importance of developing novel viral replication inhibitors targeting viral machinery. To date, the only NS2B-NS3 inhibitor that has undergone clinical trials is doxycycline, however, its mechanism of action and clinical efficacy as viral growth inhibitor require additional investigation. SYC-1307, an allosteric inhibitor, exhibits high in vivo efficacy, while temoporfin and methylene blue represent promising orthosteric non-competitive inhibitors. Compound 71, a competitive NS2B-NS3 inhibitor, emerges as a leading preclinical candidate due to its high cellular antiviral efficacy, minimal cytotoxicity, and favorable in vitro pharmacokinetic parameters. Challenges remain in developing competitive NS2B-NS3 inhibitors, including appropriate biochemical inhibition assays as well as the selectivity and conformational flexibility of the protease, complicating effective antiviral treatment design.

Harnessing Natural Resources for Advancements in Dengue Virus Treatment.

Dengue fever, caused by the Dengue virus (DENV) and transmitted by Aedes aegypti mosquitoes, has become endemic in over 100 countries. Despite considerable research, there is a lack of specific drugs for clinical use against dengue. Hence, further exploration to identify anti-- dengue compounds is essential. In recent years, natural products have gained attention for their antiviral properties. Plant-based medicines are particularly appealing due to their safety and low toxicity. This review summarizes natural compounds with potential antiviral activity against DENV, highlighting their mechanisms of action. Various compounds, from traditional herbal remedies to novel plant isolates, show promise against dengue, targeting crucial viral proteins like the envelope protein, proteases, and RNA polymerase. Exploring natural sources of antiviral agents against dengue is crucial. These compounds offer hope for effective treatments and mitigating dengue's global impact.

An empirical evaluation of link quality utilization in ETX routing for VANETs.

Routing in vehicular ad hoc networks (VANETs) enables vehicles to communicate for safety and non-safety applications. However, there are limitations in wireless communication that can degrade VANET performance, so it is crucial to optimize the operation of routing protocols to address this. Various routing protocols employed the expected transmission count (ETX) in their operation as one way to achieve the required efficiency and robustness. ETX is used to estimate link quality for improved route selection. While some studies have evaluated the utilization of ETX in specific protocols, they lack a comprehensive analysis across protocols under varied network conditions. This research provides a comprehensive comparative evaluation of ETX-based routing protocols for VANETs using the nomadic community mobility model. It covers a foundational routing protocol, ad hoc on-demand distance vector (AODV), as well as newer variants that utilize ETX, lightweight ETX (LETX), and power-based light reverse ETX (PLR-ETX), which are referred to herein as AODV-ETX, AODV-LETX, and AODV-PLR, respectively. The protocols are thoroughly analyzed via ns-3 simulations under different traffic and mobility scenarios. Our evaluation model considers five performance parameters including throughput, routing overhead, end-to-end delay, packet loss, and underutilization ratio. The analysis provides insight into designing robust and adaptive ETX routing for VANET to better serve emerging intelligent transportation system applications through a better understanding of protocol performance under different network conditions. The key findings show that ETX-optimized routing can provide significant performance enhancements in terms of end-to-end delay, throughput, routing overhead, packet loss and underutilization ratio. The extensive simulations demonstrated that AODV-PLR outperforms its counterparts AODV-ETX and AODV-LETX and the foundational AODV routing protocol across the performance metrics.

In Silico Screening of Phytocompounds from West African Traditional Medicine and Molecular Docking Targeting Dengue Virus Protein NS2B/NS3.

Traditional medicine offers a wide range of application for in silico study techniques. This drug research and development strategy is embryonic in the West African context, particularly in Burkina Faso, which is increasingly faced with emerging diseases such as dengue fever. Circulation of the 4 serotypes of this virus has been documented in the country. This study aims to evaluate the therapeutic potential of phytocompounds contained in the West African pharmacopoeia against dengue virus NS2B/NS3 protein, using computational methods integrating several software packages and databases. Based on a literature review, we identified 191 molecules from 30 plants known for their antiviral effects. Five met the inclusion criteria for molecular docking: patulin from calotropis procera, resiniferonol from Euphorbia poissonii, Securinol A from Flueggea virosa, Shikimic acid and Methyl gallate from Terminalia macroptera. The best binding scores were observed between resiniferonol and the serotypes 1, 2 and 4 NS2B/NS3 protease, with binding energies of -7.4 Kcal/mol, -6.8 Kcal/mol and -7.3 Kcal/mol respectively; while the NS2B/NS3 protease of serotype 3 had the best affinity for securinol A (-7 Kcal/mol). This study points the way to further research in computer aided drug design field and calls for multidisciplinary collaboration to promote West African medicinal plants against health challenges.

Exploring antiviral activity of Betanin and Glycine Betaine against dengue virus type-2 in transfected Hela cells.

Dengue virus (DENV) infection is a worldwide public health concern infecting approximately 400 million individuals and about 40,000 mortalities yearly. Despite this, no licensed or readily available antiviral medication is currently available specifically for DENV infection, and therapy is typically symptomatic. Therefore, the objective of the study was to investigate the antiviral activity of Beta vulgaris L. phytoconstituents against DENV-2 targeting NS3 protein. The antiviral activity of phytochemicals was examined through virtual ligand-based screening, antiviral inhibition and dosage response assays, western blotting analysis and MD simulations. We conducted toxicological, and pharmacokinetic analysis to assess plant-based natural compound's efficacy, safety, and non-toxic doses. Molecular docking and MD simulation results revealed that the nonstructural protein-3 (NS3) might prove as a funamental target for Betanin and Glycine Betaine against Dengue virus. Betanin and Glycine betaine were initially studied for their non-toxic doses in HeLa, CHO, and Vero cells via MTT assay. HeLa cells were transiently transfected with cloned vector pcDNA3.1/Zeo(+)/DENV-2 NS3 along with non-toxic doses (80 µM-10 µM) of selected phytochemicals. The dose-response assay illustrated downregulated expression of DENV-2 NS3 gene after administration of Betanin (IC50 = 4.35 µM) and Glycine Betaine (IC50 = 4.49 µM). Dose response analysis of Betanin (80 µM-10 µM) depicted the significant inhibition of NS3 protein expression as well. These results suggested downregulated expression of DENV-2 NS3 at mRNA and protein level portraying the DENV replication inhibition. Based on our study findings, NS3 protease is depicted as distinctive DENV-2 inhibitor target. We will channel our study further into in vitro characterization employing the mechanistic study to understand the role of host factors in anti-flavi therapeutic.

Immunization of recombinant NS3 protein (protease region) of dengue virus induces high levels of CTLA-4 and apoptosis in splenocytes of BALB/c mice.

DENV infection outcomes depend on the host's variable expression of immune receptors and mediators, leading to either resolution or exacerbation. While the NS3 protein is known to induce robust immune responses, the specific impact of its protease region epitopes remains unclear. This study investigated the effect of recombinant NS3 protease region proteins from all four DENV serotypes on splenocyte activation in BALB/c mice (n = 5/group). Mice were immunized with each protein, and their splenocytes were subsequently stimulated with homologous antigens. We measured the expression of costimulatory molecules (CD28, CD80, CD86, CD152) by flow cytometry, along with IL-2 production, CD25 expression, and examined the antigen-specific activation of CD4 + and CD8 + T cells. Additionally, the expression of IL-1, IL-10, and TGF-ß1 in splenocytes from immunized animals was assessed. Apoptosis was evaluated using Annexin V/PI staining and DNA fragmentation analysis. Stimulation of splenocytes from immunized mice triggered apoptosis (phosphatidylserine exposure and caspase 3/7 activation) and increased costimulatory molecule expression, particularly CD152. Low IL-2 production and low CD25 expression, as well as sustained expression of the IL-10 gene. These results suggest that these molecules might be involved in mechanisms by which the NS3 protein contributes to viral persistence and disease pathogenesis.

Membrane Retention of West Nile Virus NS5 Depends on NS1 or NS3 for Enzymatic Activity.

West Nile virus (WNV) nonstructural protein 5 (NS5) possesses multiple enzymatic domains essential for viral RNA replication. During infection, NS5 predominantly localizes to unique replication organelles (ROs) at the rough endoplasmic reticulum (RER), known as vesicle packets (VPs) and convoluted membranes (CMs), with a portion of NS5 accumulating in the nucleus. NS5 is a soluble protein that must be in the VP, where its enzymatic activities are required for viral RNA synthesis. However, the mechanistic processes behind the recruitment of NS5 from the cytoplasm to the RER membrane remain unclear. Here, we utilize high-resolution confocal microscopy and sucrose density gradient ultracentrifugation to investigate whether the association of NS5 with other NS proteins contributes to its membrane recruitment and retention. We demonstrate that NS1 or NS3 partially influences the NS5 association with the membrane. We further demonstrate that processed NS5 is predominantly in the cytoplasm and nucleus, indicating that the processing of NS5 from the viral polyprotein does not contribute to its membrane localization. These observations suggest that other host or viral factors, such as the enwrapment of NS5 by the RO, may also be necessary for the complete membrane retention of NS5. Therefore, studies on the inhibitors that disrupt the membrane localization of WNV NS5 are warranted for antiviral drug development.

Correlation Between the Hepatitis C Virus NS3 Protein and CD30 Expression in Diffuse Large B-cell Lymphoma.

INTRODUCTION:  One of the etiologies of non-Hodgkin lymphoma (NHL) is chronic infection related to lymphoma pathogenesis, with a high prevalence of hepatitis C virus (HCV) infection seen. In determining the treatment and prognosis of NHL, cluster of differentiation 30 (CD30) immunohistochemical staining plays an important role. High levels of CD30 are found in patients with HCV infection. This study aimed to determine the prevalence of CD30 and HCV expression and its correlation with clinicopathological characteristics of Indonesian diffuse large B-cell lymphoma (DLBCL) patients. METHODS: A total of 86 formalin-fixed paraffin-embedded (FFPE) samples of DLBCL cases were collected over the course of two years from the Anatomical Pathology department at Dr. Sardjito General Hospital in the special region of Yogyakarta, Indonesia. Immunohistochemistry was performed to detect the two markers (CD30 and HCV). Chi-square tests were used to investigate the correlations between CD30 expression and clinicopathological features in DLBCL patients. RESULTS: The positivity rate of CD30 expression in 86 DLBCL samples was 25.6% (22/86) when using a 0% cut-off, and 7.0% (6/86) while using a 20% cutoff. The positivity rate of HCV expression in DLBCL samples was 34.9% (30/86). Positive CD30 expression, HCV expression and clinicopathological features (age, sex, Ann Arbor stage, extranodal involvement, and morphological variations) did not have statistically significant relationships (p>0.05). CONCLUSION: There was no statistically significant correlation between CD30 immunoreactivity (cut-off >0% or >20%) and HCV NS3 expression and clinicopathological features (age, sex, Ann Arbor stage, extranodal involvement, lactate dehydrogenase, Eastern Cooperative Oncology Group status and morphological variants) in DLBCL.

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

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.

The GA-Hecate Peptide inhibits the ZIKV Replicative Cycle in Different Steps and can Inhibit the Flavivirus NS2B-NS3 Protease after Cell Infection.

BACKGROUND: Peptide drugs are advantageous because they are subject to rational design and exhibit highly diverse structures and broad biological activities. The NS2B-NS3 protein is a particularly promising flavivirus therapeutic target, with extensive research on the development of inhibitors as therapeutic candidates, and was used as a model in this work to determine the mechanism by which GA-Hecate inhibits ZIKV replication. OBJECTIVE: The present study aimed to evaluate the potential of GA-Hecate, a new antiviral developed by our group, against the Brazilian Zika virus and to evaluate the mechanism of action of this compound on the flavivirus NS2B-NS3 protein. METHODS: Solid-phase peptide Synthesis, High-Performance Liquid Chromatography, and Mass Spectrometry were used to obtain, purify, and characterize the synthesized compound. Real-time and enzymatic assays were used to determine the antiviral potential of GA-Hecate against ZIKV. RESULTS: The RT-qPCR results showed that GA-Hecate decreased the number of ZIKV RNA copies in the virucidal, pre-treatment, and post-entry assays, with 5- to 6-fold fewer RNA copies at the higher nontoxic concentration in Vero cells (HNTC: 10 µM) than in the control cells. Enzymatic and kinetic assays indicated that GA-Hecate acts as a competitive ZIKV NS2B-NS3 protease inhibitor with an IC50 of 32 nM and has activity against the yellow fever virus protease. CONCLUSION: The results highlight the antiviral potential of the GA-Hecate bioconjugate and open the door for the development of new antivirals.

Using Multiscale Molecular Modeling to Analyze Possible NS2b-NS3 Protease Inhibitors from Philippine Medicinal Plants.

Within the field of Philippine folkloric medicine, the utilization of indigenous plants like Euphorbia hirta (tawa-tawa), Carica papaya (papaya), and Psidium guajava (guava) as potential dengue remedies has gained attention. Yet, limited research exists on their comprehensive effects, particularly their anti-dengue activity. This study screened 2944 phytochemicals from various Philippine plants for anti-dengue activity. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling provided 1265 compounds demonstrating pharmacokinetic profiles suitable for human use. Molecular docking targeting the dengue virus NS2b-NS3 protease's catalytic triad (Asp 75, Ser 135, and His 51) identified ten ligands with higher docking scores than reference compounds idelalisib and nintedanib. Molecular dynamics simulations confirmed the stability of eight of these ligand-protease complexes. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) analysis highlighted six ligands, including veramiline (-80.682 kJ/mol), cyclobranol (-70.943 kJ/mol), chlorogenin (-63.279 kJ/mol), 25beta-Hydroxyverazine (-61.951 kJ/mol), etiolin (-59.923 kJ/mol), and ecliptalbine (-56.932 kJ/mol) with favorable binding energies, high oral bioavailability, and drug-like properties. This integration of traditional medical knowledge with advanced computational drug discovery methods paves new pathways for the development of treatments for dengue.

Deciphering the interaction surface between the West Nile virus NS3 and NS5 proteins.

West Nile virus (WNV) is the most prevalent mosquito-borne virus and the leading cause of viral encephalitis in the continental United States. It belongs to the family Flaviviridae which includes other important human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV) and Zika viruses (ZIKV). Despite several decades of research, no specific antiviral drugs are available to treat flavivirus infections. The present study characterizes the interaction between the WNV NS3 and NS5 proteins for the purpose of identifying hotspots in the protein-protein interaction which could be targeted for the development of antiviral therapeutics. We previously developed an interaction model in silico based on data available in the literature. Here, potential interacting residues on NS3 and NS5 were mutated in a WNV replicon, and seven mutations in the NS3 protein were found to drastically reduce viral replication. In addition to being well conserved among mosquito-borne flaviviruses, these residues are located on the protein's surface in two clusters which might be interesting new targets for future drug development.

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.

Generalized open-source workflows for atomistic molecular dynamics simulations of viral helicases.

Viral helicases are promising targets for the development of antiviral therapies. Given their vital function of unwinding double-stranded nucleic acids, inhibiting them blocks the viral replication cycle. Previous studies have elucidated key structural details of these helicases, including the location of substrate binding sites, flexible domains, and the discovery of potential inhibitors. Here we present a series of new Galaxy tools and workflows for performing and analyzing molecular dynamics simulations of viral helicases. We first validate them by demonstrating recapitulation of data from previous simulations of Zika (NS3) and SARS-CoV-2 (NSP13) helicases in apo and complex with inhibitors. We further demonstrate the utility and generalizability of these Galaxy workflows by applying them to new cases, proving their usefulness as a widely accessible method for exploring antiviral activity.

Dengue Virus dependence on glucokinase activity and glycolysis Confers Sensitivity to NAD(H) biosynthesis inhibitors.

Viruses have developed sophisticated strategies to control metabolic activity of infected cells in order to supply replication machinery with energy and metabolites. Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue fever, is no exception. Previous reports have documented DENV interactions with metabolic pathways and shown in particular that glycolysis is increased in DENV-infected cells. However, underlying molecular mechanisms are still poorly characterized and dependence of DENV on this pathway has not been investigated in details yet. Here, we identified an interaction between the non-structural protein 3 (NS3) of DENV and glucokinase regulator protein (GCKR), a host protein that inhibits the liver-specific hexokinase GCK. NS3 expression was found to increase glucose consumption and lactate secretion in hepatic cell line expressing GCK. Interestingly, we observed that GCKR interaction with GCK decreases DENV replication, indicating the dependence of DENV to GCK activity and supporting the role of NS3 as an inhibitor of GCKR function. Accordingly, in the same cells, DENV replication both induces and depends on glycolysis. By targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), we decreased cellular glycolytic activity and inhibited DENV replication in hepatic cells. Infection of primary organotypic liver cultures (OLiC) from hamsters was also inhibited by 6-AN. Altogether, our results show that DENV has evolved strategies to control glycolysis in the liver, which could account for hepatic dysfunctions associated to infection. Besides, our findings suggest that lowering intracellular availability of NAD(H) could be a valuable therapeutic strategy to control glycolysis and inhibit DENV replication in the liver.

Caching Policy in Low Earth Orbit Satellite Mega-Constellation Information-Centric Networking for Internet of Things.

Information-Centric Networking (ICN) is the emerging next-generation internet paradigm. The Low Earth Orbit (LEO) satellite mega-constellation based on ICN can achieve seamless global coverage and provide excellent support for Internet of Things (IoT) services. Additionally, in-network caching, typically characteristic of ICN, plays a paramount role in network performance. Therefore, the in-network caching policy is one of the hotspot problems. Especially, compared to caching traditional internet content, in-networking caching IoT content is more challenging, since the IoT content lifetime is small and transient. In this paper, firstly, the framework of the LEO satellite mega-constellation Information-Centric Networking for IoT (LEO-SMC-ICN-IoT) is proposed. Then, introducing the concept of "viscosity", the proposed Caching Algorithm based on the Random Forest (CARF) policy of satellite nodes combines both content popularity prediction and satellite nodes location prediction, for achieving good cache matching between the satellite nodes and content. And using the matching rule, the Random Forest (RF) algorithm is adopted to predict the matching relationship among satellite nodes and content for guiding the deployment of caches. Especially, the content is cached in advance at the future satellite to maintain communication with the current ground segment at the time of satellite switchover. Additionally, the policy considers both the IoT content lifetime and the freshness. Finally, a simulation platform with LEO satellite mega-constellation based on ICN is developed in Network Simulator 3 (NS-3). The simulation results show that the proposed caching policy compared with the Leave Copy Everywhere (LCE), the opportunistic (OPP), the Leave Copy down (LCD), and the probabilistic algorithm which caches each content with probability 0.5 (prob 0.5) yield a significant performance improvement, such as the average number of hops, i.e., delay, cache hit rate, and throughput.

Identification of novel and potential inhibitors against the dengue virus NS2B/NS3 protease using virtual screening and biomolecular simulations.

Approximately 3.9 billion individuals are vulnerable to dengue infection, a prevalent cause of tropical diseases worldwide. Currently, no drugs are available for preventing or treating Flavivirus diseases, including Dengue, West Nile, and the more recent Zika virus. The highly conserved Flavivirus NS2B-NS3 protease, crucial for viral replication, is a promising therapeutic target. This study employed in-silico methodologies to identify novel and potentially effective anti-dengue small molecules. A pharmacophore model was constructed using an experimentally validated NS2B-NS3 inhibitor, with the Gunner Henry score confirming the model's validity. The Natural Product Activity and Species Source (NPASS) database was screened using the validated pharmacophore model, yielding a total of 60 hits against the NS2B-NS3 protease. Furthermore, the docking finding reveals that our newly identified compounds from the NPASS database have enhanced binding affinities and established significant interactions with allosteric residues of the target protein. MD simulation and post-MD analysis further validated this finding. The free binding energy was computed in terms of MM-GBSA analysis, with the total binding energy for compound 1 (-57.3 ± 2.8 and - 52.9 ± 1.9 replica 1 and 2) indicating a stronger binding affinity for the target protein. Overall, this computational study identified these compounds as potential hit molecules, and these findings can open up a new avenue to explore and develop inhibitors against Dengue virus infection.

In Silico-Based Identification of Natural Inhibitors from Traditionally Used Medicinal Plants that can Inhibit Dengue Infection.

Dengue fever (DF) is an endemic disease that has become a public health concern around the globe. The NS3 protease-helicase enzyme is an important target for the development of antiviral drugs against DENV (dengue virus) due to its impact on viral replication. Inhibition of the activity of the NS3 protease-helicase enzyme complex significantly inhibits the infection associated with DENV. Unfortunately, there are no scientifically approved antiviral drugs for its prevention. However, this study has been developed to find natural bioactive molecules that can block the activity of the NS3 protease-helicase enzyme complex associated with DENV infection through molecular docking, MM-GBSA (molecular mechanics-generalized born surface area), and molecular dynamics (MD) simulations. Three hundred forty-two (342) compounds selected from twenty traditional medicinal plants were retrieved and screened against the NS3 protease-helicase protein by molecular docking and MM-GBSA studies, where the top six phytochemicals have been identified based on binding affinities. The six compounds were then subjected to pharmacokinetics and toxicity analysis, and we conducted molecular dynamics simulations on three protein-ligand complexes to validate their stability. Through computational analysis, this study revealed the potential of the two selected natural bioactive inhibitors (CID-440015 and CID-7424) as novel anti-dengue agents.

Discovery of azaleatin as a potential allosteric inhibitor for dengue NS2B-NS3 protease using in vitro and in silico studies.

The rise in dengue cases in tropical and sub-tropical areas has become a significant health concern. At present, there is no definitive cure for dengue fever, which underscores the importance of identifying potent inhibitors. Dengue NS2B-NS3 protease is the prime drug target due to its vital function for replication. Quercetin, a flavone, has anti-dengue virus properties but is limited by low bioavailability. Previous studies have shown that methoxy substitution in flavones improves bioavailability and metabolic stability. Azaleatin is a derivative of quercetin with a methoxy substitution at the C5 position, however its ability to inhibit dengue is unknown. In this study, azaleatin was investigated for its inhibition against dengue NS2B-NS3 protease using in vitro and in silico techniques. The fluorescence assay was used to determine the IC50 value and inhibition kinetics. The molecular interaction between azaleatin and NS2B-NS3 was studied using CB-Dock2 and AutoDock Vina. The complex's stability was then analysed using GROMACS. Besides, the ADMETlab 2.0 was utilized to predict pharmacokinetic of the azaleatin. Results showed that azaleatin inhibits dengue NS2B-NS3 protease non-competitively with a Ki of 26.82 µg/ml and an IC50 of 38 µg/ml. Molecular docking indicated binding of the azaleatin to the allosteric pocket of NS2B-NS3 with a docking score of -8.2 kcal/mol. Azaleatin was found stable in the pocket along 100 ns, supporting its inhibitory mode. The compound has favourable pharmacokinetic profiles and conformed to Lipinski's Rule of Five. Taken together, azaleatin inhibits NS2B-NS3 protease in a non-competitive mode, suggesting its potential as safer anti-dengue compound.Communicated by Ramaswamy H. Sarma.

Synthesis and structural characterization of new macrocyclic inhibitors of the Zika virus NS2B-NS3 protease.

Three new series of macrocyclic active site-directed inhibitors of the Zika virus (ZIKV) NS2B-NS3 protease were synthesized. First, attempts were made to replace the basic P3 lysine residue of our previously described inhibitors with uncharged and more hydrophobic residues. This provided numerous compounds with inhibition constants between 30 and 50 nM. A stronger reduction of the inhibitory potency was observed when the P2 lysine was replaced by neutral residues, all of these inhibitors possess Ki values >1 µM. However, it is possible to replace the P2 lysine with the less basic 3-aminomethylphenylalanine, which provides a similarly potent inhibitor of the ZIKV protease (Ki = 2.69 nM). Crystal structure investigations showed that the P2 benzylamine structure forms comparable interactions with the protease as lysine. Twelve additional structures of these inhibitors in complex with the protease were determined, which explain many, but not all, SAR data obtained in this study. All individual modifications in the P2 or P3 position resulted in inhibitors with low antiviral efficacy in cell culture. Therefore, a third inhibitor series with combined modifications was synthesized; all of them contain a more hydrophobic d-cyclohexylalanine in the linker segment. At a concentration of 40 µM, two of these compounds possess similar antiviral potency as ribavirin at 100 µM. Due to their reliable crystallization in complex with the ZIKV protease, these cyclic compounds are very well suited for a rational structure-based development of improved inhibitors.