NS2B-NS3 Protease Research Articles

Evaluation of methoxyflavones as dengue NS2B-NS3 protease inhibitors: an in silico and in vitro studies.

Dengue is one of the most prevalent viruses transmitted by the Aedes aegypti mosquitoes. Currently, no specific medication is available to treat dengue diseases. The NS2B-NS3 protease is vital during post-translational processing, which is a key target in this study. Due to methoxy group substitution, methoxyflavones are more bioavailable and metabolically stable than hydroxylated flavones. To date, research on the anti-dengue activity of methoxyflavones is limited. Hence, this work aims to determine the inhibitory activity of methoxyflavones against the dengue NS2B-NS3. Methoxyflavones derivatives were screened using molecular docking. The result showed a strong binding interaction of compound 1 and compound 2 with NS2B-NS3 protease. Both compounds exhibited comparable binding energy as the reference compound, quercetin, with values lower than - 8.1kcal/mol. Molecular dynamics simulation using GROMACS revealed the stability and stiffness of the complexes over a 100ns time scale. In addition, an in vitro assay for NS2B-NS3 protease inhibition revealed inhibitory effects of compounds 1 and 2 with IC50 values of 316.80µM and 463.30µM, respectively. The ADMET analyses showed favorable pharmacokinetics profiles that comply with Lipinski's Rule of Five. Collectively, our findings suggest that compounds 1 and 2 inhibit dengue NS2B-NS3 activity. These findings hold promise of methoxyflavones as starting compounds for potential dengue treatment, highlighting the need for further investigation.

Identification and Evaluation of Natural Compounds as Potential Inhibitors of NS2B-NS3 Zika Virus Protease: A Computational Approach.

The Zika virus (ZIKV), an arbovirus within the Flavivirus genus, is associated with severe neurological complications, including Guillain-Barré syndrome in affected individuals and microcephaly in infants born to infected mothers. With no approved vaccines or antiviral treatments available, there is an urgent need for effective therapeutic options. This study aimed to identify new natural compounds with inhibitory potential against the NS2B-NS3 protease (PDB ID: 5LC0), an essential enzyme in viral replication. An e-pharmacophore model was generated using a five-point (ADDRR) feature approach in the PHASE module of Schrodinger and used for the virtual screening of 26,689 natural compounds from the PubChem database. The screening yielded 14,277 prioritized compounds based on fitness scores, further refined through extra precision (XP) docking in GLIDE, resulting in 24 compounds. Eight top hits were selected following ADME analysis with SwissADME, and toxicity screening with ProTox-II identified four non-toxic lead candidates. Molecular dynamic simulations confirmed the stability of the three most promising leads, CID 44418637, CID 163078083, and CID 68734190, with binding affinities of - 7.721, - 8.226, and - 8.307kcal/mol, respectively. MM/GBSA analysis revealed that Compounds 68734190 (- 50.192kcal/mol) and 163078083 (- 49.947kcal/mol) possess superior binding affinities to the ZIKV NS2B-NS3 protease compared to the reference compound (- 38.347kcal/mol). Given their natural origin, these compounds may offer safer options to mitigate severe ZIKV-related symptoms while providing a favourable safety and pharmacokinetic profile. This study lays the groundwork for developing targeted ZIKV therapies, potentially addressing a significant unmet need in public health by reducing the incidence of ZIKV-related complications. Further experimental validation is required to confirm efficacy and address potential development challenges.

Identifying Allosteric Small-Molecule Binding Sites of Inactive NS2B-NS3 Proteases of Pathogenic Flaviviridae.

Dengue, West Nile, Zika, Yellow fever, and Japanese encephalitis viruses persist as significant global health threats. The development of new therapeutic strategies based on inhibiting essential viral enzymes or viral-host protein interactions is problematic due to the fast mutation rate and rapid emergence of drug resistance. This study focuses on the NS2B-NS3 protease as a promising target for antiviral drug development. Promising allosteric binding sites were identified in two conformationally distinct inactive states and characterized for five flaviviruses and four Dengue virus subtypes. Their shapes, druggability, inter-viral similarity, sequence variation, and susceptibility to drug-resistant mutations have been studied. Two identified allosteric inactive state pockets appear to be feasible alternatives to a larger closed pocket near the active site, and they can be targeted with specific drug-like small-molecule inhibitors. Virus-specific sequence and structure implications and the feasibility of multi-viral inhibitors are discussed.

Discovery of Potent Dengue Virus NS2B-NS3 Protease Inhibitors Among Glycyrrhizic Acid Conjugates with Amino Acids and Dipeptides Esters.

This study investigated a library of known and novel glycyrrhizic acid (GL) conjugates with amino acids and dipeptide esters, as inhibitors of the DENV NS2B-NS3 protease. We utilized docking algorithms to evaluate the interactions of these GL derivatives with key residues (His51, Asp75, Ser135, and Gly153) within 10 Å of the DENV-2 NS2B-NS3 protease binding pocket (PDB ID: 2FOM). It was found that compounds 11 and 17 exhibited unique binding patterns, forming hydrogen bonds with Asp75, Tyr150, and Gly153. Based on the molecular docking data, conjugates 11 with L-glutamic acid dimethyl ester, 17 with β-alanine ethyl ester, and 19 with aminoethantic acid methyl ester were further demonstrated as potent inhibitors of DENV-2 NS3 protease, with IC50 values below 1 μM, using NS3-mediated cleavage assay. Compound 11 was the most potent, with EC50 values of 0.034 μM for infectivity, 0.042 μM for virus yield, and a selective index over 2000, aligning with its strong NS3 protease inhibition. Compound 17 exhibited better NS3 protease inhibition than compound 19 but showed weaker effects on infectivity and virus yield. While all compounds strongly inhibited viral infectivity post-entry, compound 19 also blocked viral entry. This study provided valuable insights into the interactions between active GL derivatives and DENV-2 NS2B-NS3 protease, offering a comprehensive framework for identifying lead compounds for further drug optimization and design as NS2B-NS3 protease inhibitors against DENV.

Antiviral Drug Discovery with an Optimized Biochemical Dengue Protease Assay: Improved Predictive Power for Antiviral Efficacy

The viral NS2B-NS3 protease is a promising drug target to combat dengue virus (DENV) and other emerging flaviviruses. The discovery of novel DENV protease inhibitors with antiviral efficacy is hampered by the low predictive power of biochemical assays. We herein present a comparative evaluation of biochemical DENV protease assay conditions and their benchmarking against antiviral efficacy and a protease-specific reporter gene assay. Variations were performed with respect to pH, type of detergent, buffer, and substrate. The revised assay conditions were applied in a medicinal chemistry effort aimed at phenylglycine protease inhibitors. This validation study demonstrated a considerably improved predictive power for antiviral efficacy in comparison to previous approaches. An extensive evaluation of phenylglycine-based DENV protease inhibitors with highly diverse N-terminal caps indicates further development potential in this structural region. Furthermore, the phenylglycine moiety may be less essential than previously assumed, providing a development option towards reduced lipophilicity and thereby an improved pharmacokinetic and toxicity profile.

NS2B-D55E and NS2B-E65D Variations Are Responsible for Differences in NS2B-NS3 Protease Activities Between Japanese Encephalitis Virus Genotype I and III in Fluorogenic Peptide Model.

Japanese encephalitis virus (JEV) NS2B-NS3 is a protein complex composed of NS3 proteases and an NS2B co-factor. The N-terminal protease domain (180 residues) of NS3 (NS3(pro)) interacts directly with a central 40-amino acid hydrophilic domain of NS2B (NS2B(H)) to form an active serine protease. In this study, the recombinant NS2B(H)-NS3(pro) proteases were prepared in E. coli and used to compare the enzymatic activity between genotype I (GI) and III (GIII) NS2B-NS3 proteases. The GI NS2B(H)-NS3(pro) was able to cleave the sites at the internal C, NS2A/NS2B, NS2B/NS3, and NS3/NS4A junctions that were identical to the sites proteolytically processed by GIII NS2B(H)-NS3(pro). Analysis of the enzymatic activity of recombinant NS2B(H)-NS3(pro) proteases using a model of fluorogenic peptide substrate revealed that the proteolytical processing activity of GIII NS2B(H)-NS3(pro) was significantly higher than that of GI NS2B(H)-NS3(pro). There were eight amino acid variations between GI and GIII NS2B(H)-NS3(pro), which may be responsible for the difference in enzymatic activities between GI and GIII proteases. Therefore, recombinant mutants were generated by exchanging the NS2B(H) and NS3(pro) domains between GI and GIII NS2B(H)-NS3(pro) and subjected to protease activity analysis. Substitution of NS2B(H) significantly altered the protease activities, as compared to the parental NS2B(H)-NS3(pro), suggesting that NS2B(H) played an essential role in the regulation of NS3(pro) protease activity. To further identify the amino acids responsible for the difference in protease activities, multiple substitution mutants including the individual and combined mutations at the variant residues 55 and 65 of NS2B(H) were generated and subjected to protease activity analysis. Replacement of NS2B-55 and NS2B-65 of GI to GIII significantly increased the enzymatic activity of GI NS2B(H)-NS3(pro) protease, whereas mutation of NS2B-55 and NS2B-65 of GIII to GI remarkably reduced the enzymatic activity of GIII NS2B(H)-NS3(pro) protease. Overall, these data demonstrated that NS2B-55 and NS2B-65 variations in the hydrophilic domain of NS2B co-contributed to the difference in NS2B(H)-NS3(pro) protease activities between GI and GIII. However, it will be crucial to explore these mutations in other in vivo and/or in vitro models. Collectively, these observations will be useful for understanding the replication of JEV GI and GIII viruses.

Identifying Novel Inhibitors for Dengue NS2B-NS3 Protease by Combining Topological similarity, Molecular Dynamics, MMGBSA and SiteMap Analysis.

DENV NS2B-NS3 protease inhibitors were designed based upon the reference molecule, 4-(1,3-dioxoisoindolin-2-yl)-N-(4-ethylphenyl) benzenesulfonamide, reported by our team with the aim to optimize lead compound via rational approach. Top five best scoring molecules with zinc ids ZINC23504872, ZINC48412318, ZINC00413269, ZINC13998032 and ZINC75249613 bearing 'pyrimidin-4(3H)-one' basic scaffold have been identified as a promising candidate against DENV protease enzyme. The shape and electrostatic complementary between identified HITs and reference molecules were found to be Tanimotoshape 0.453, 0.690, 0.680, 0.685 & 0.672 respectively and Tanimotoelectrostatic 0.211, 0.211, 0.441, 0.442, 0.442 and 0.442 respectively. The molecular docking studies suggested that the identified HITs displayed the good interactions with active site residues and lower binding energies. The stability of docked complexes was assessed by MD simulations studies. The RMSD values of protein backbone (1.6779, 3.1563, 3.3634, 3.3893 & 3.0960 Å) and protein backbone RMSF values (1.0126, 1.0834, 1.0890, 0.9974 & 1.0080 Å respectively) for all top five HITs were stable and molecules did not fluctuate from the active pocket during entire 100ns MD run. The druggability Dscore below 1 indicate the tightly binding of ligand at the active site. Dscore for ZINC23504872 was found to be 1.084 while for the second class of compounds ZINC48412318, ZINC00413269, ZINC13998032 and ZINC75249613, 0.503, 0.484, 0.487 and 0.501 Dscores were observed. In-silico ADMET calculations suggested that all five HITs were possessed the drug likeliness properties and did not violate the Lipinski's rule of five. Summing up, these in-silico generated data suggested that the identified molecules bearing pyrimidin-4(3H)-one would be promising scaffold for DENV protease inhibitors. However, experimental results are needed to prove the obtained results.

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.

Employing Machine Learning-Based QSAR for Targeting Zika Virus NS3 Protease: Molecular Insights and Inhibitor Discovery.

Zika virus infection is a mosquito-borne viral disease that has become a global health concern recently. Zika virus belongs to the Flavivirus genus and is primarily transmitted by Aedes mosquitoes. Prevention of Zika virus infection involves avoiding mosquito bites by using repellent, wearing protective clothing, and staying in screened areas, especially for pregnant women. Treatment focuses on managing symptoms with rest, fluids, and acetaminophen, with close monitoring for pregnant women. Currently, there is no specific antiviral treatment or vaccine for the Zika virus, highlighting the importance of prevention strategies to control its spread. Therefore, in this study, the Zika virus non-structural protein NS3 was targeted to inhibit Zika infection by identifying the novel inhibitor through an in silico approach. Here, 2864 natural compounds were screened using a machine learning-based QSAR model, and later docking was performed to select the potential target. Subsequently, Tanimoto similarity and clustering were performed to obtain the potential target. The three most potential compounds were obtained: (a) 5297, (b) 432449, and (c) 85137543. The protein-ligand complex's stability and flexibility were then investigated by dynamic modelling. The 300 ns simulation showed that 5297 exhibited the steadiest deviation and constant creation of hydrogen bonds. Compared to the other compounds, 5297 demonstrated a superior binding free energy (ΔG = -20.81 kcal/mol) with the protein when the MM/GBSA technique was used. The study determined that 5297 showed significant therapeutic potential and justifies further experimental investigation as a possible inhibitor of the NS2B-NS3 protease target implicated in Zika virus infection.

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.

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

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. 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. 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. 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. The results highlight the antiviral potential of the GA-Hecate bioconjugate and open the door for the development of new antivirals.

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

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.

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 hydrophobicd-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.

A high-throughput cell-based screening method for Zika virus protease inhibitor discovery

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors.

Genetic Encoding of Fluoro-l-tryptophans for Site-Specific Detection of Conformational Heterogeneity in Proteins by NMR Spectroscopy.

The substitution of a single hydrogen atom in a protein by fluorine yields a site-specific probe for sensitive detection by 19F nuclear magnetic resonance (NMR) spectroscopy, where the absence of background signal from the protein facilitates the detection of minor conformational species. We developed genetic encoding systems for the site-selective incorporation of 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, and 7-fluorotryptophan in response to an amber stop codon and used them to investigate conformational heterogeneity in a designed amino acid binding protein and in flaviviral NS2B-NS3 proteases. These proteases have been shown to present variable conformations in X-ray crystal structures, including flips of the indole side chains of tryptophan residues. The 19F NMR spectra of different fluorotryptophan isomers installed at the conserved site of Trp83 indicate that the indole ring flip is common in flaviviral NS2B-NS3 proteases in the apo state and suppressed by an active-site inhibitor.

Antiviral Activity of Flavonoids from Bauhinia holophylla Leaves against Zika virus

Zika virus (ZIKV) is involved in the etiology of serious nervous system pathologies. Currently, there are no specific and effective vaccines or antiviral drugs to prevent the diseases caused by ZIKV. This study aimed to assess the activity of flavonoids present in crude hydroethanolic extract (CHE) and fractions obtained from B. holophylla leaves against ZIKV. O-glycosylated flavonoids were characterized by high-performance liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS). The cytotoxic concentration and the effective concentration for 50% of the cells (CC50 and EC50, respectively) were determined, and the selectivity index (SI) was calculated. Molecular networks were constructed based on the chemical composition of the samples and global antiviral activity data using the Global Natural Products Social Molecular Networking (GNPS) platform. Protein–ligand docking was performed in the NS2B-NS3 protease, NS3 helicase, and NS5 methyltransferase of the ZIKV. CHE showed greater antiviral activity at a multiplicity of infection (MOI) of 1.0, with an EC50 of 11.93 µg/mL, SI = 13.38, and reduced cytopathic effects. Molecular networks indicated that O-glycosylated flavonoids are responsible for the activity against ZIKV, being quercetin-O-deoxyhexoside more selective and effective. Molecular docking confirmed the inhibitory activity of quercetin-O-deoxyhexoside, which showed an affinity for the tested targets, especially for NS2B-NS3 protease. The results showed that B. holophylla has flavonoids with potential for future therapeutic applications against ZIKV.

Characterisation of ten NS2B-NS3 proteases: Paving the way for pan-flavivirus drugs

Flaviviruses can cause severe illness in humans. Effective and safe vaccines are available for some species; however, for many flaviviruses disease prevention or specific treatments remain unavailable. The viral replication cycle depends on the proteolytic activity of the NS2B-NS3 protease, which releases functional viral proteins from a non-functional polyprotein precursor, rendering the protease a promising drug target. In this study, we characterised recombinant NS2B-NS3 proteases from ten flaviviruses including three unreported proteases from the Usutu, Kyasanur forest disease and Powassan viruses. All protease constructs comprise a covalent Gly4-Ser-Gly4 linker connecting the NS3 serine protease domain with its cofactor NS2B. We conducted a comprehensive cleavage site analysis revealing areas of high conversion. While all proteases were active in enzymatic assays, we noted a 1000-fold difference in catalytic efficiency across proteases from different flaviviruses. Two bicyclic peptide inhibitors displayed anti-pan-flaviviral protease activity with inhibition constants ranging from 10 to 1000 nM.

Exploring the therapeutic potential of DV-B-120 as an inhibitor of dengue virus infection.

Dengue fever, an infectious disease prevalent in subtropical and tropical regions, currently lacks effective small-molecule drugs as treatment. In this study, we used a fluorescence peptide cleavage assay to screen seven compounds to assess their inhibition of the dengue virus (DENV) NS2B-NS3 protease. DV-B-120 demonstrated superior inhibition of NS2B-NS3 protease activity and lower toxicity compared to ARDP0006. The selectivity index of DV-B-120 was higher than that of ARDP0006. In vivo assessments of the antiviral efficacy of DV-B-120 against DENV replication demonstrated delayed mortality of suckling mice treated with the compound, with 60-80% protection against life-threatening effects, compared to the outcomes of DENV-infected mice treated with saline. The lower clinical scores of DENV-infected mice treated with DV-B-120 indicated a reduction in acute-progressive illness symptoms, underscoring the potential therapeutic impact of DV-B-120. Investigations of DV-B-120's ability to restore the antiviral type I IFN response in the brain tissue of DENV-infected ICR suckling mice demonstrated its capacity to stimulate IFN and antiviral IFN-stimulated gene expression. DV-B-120 not only significantly delayed DENV-2-induced mortality and illness symptoms but also reduced viral numbers in the brain, ultimately restoring the innate antiviral response. These findings strongly suggest that DV-B-120 holds promise as a therapeutic agent against DENV infection and highlight its potential contribution in addressing the current lack of effective treatments for this infectious disease.IMPORTANCEThe prevalence of dengue virus (DENV) infection in tropical and subtropical regions is escalating due to factors like climate change and mosquito vector expansion. With over 300 million annual infections and potentially fatal outcomes, the urgent need for effective treatments is evident. While the approved Dengvaxia vaccine has variable efficacy, there are currently no antiviral drugs for DENV. This study explores seven compounds targeting the NS2B-NS3 protease, a crucial protein in DENV replication. These compounds exhibit inhibitory effects on DENV-2 NS2B-NS3, holding promise for disrupting viral replication and preventing severe manifestations. However, further research, including animal testing, is imperative to assess therapeutic efficacy and potential toxicity. Developing safe and potent treatments for DENV infection is critical in addressing the rising global health threat posed by this virus.

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.