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.

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.

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.

3D-QSAR and molecular docking studies of peptide-hybrids as dengue virus NS2B/NS3 protease inhibitors.

Global warming and climate change have made dengue disease a global health issue. More than 50 % of the world's population is at danger of dengue virus (DENV) infection, according to the World Health Organization (WHO). Therefore, a clinically approved dengue fever vaccination and effective treatment are needed. Peptide medication development is new pharmaceutical research. Here we intend to recognize the structural features inhibiting the DENV NS2B/NS3 serine protease for a series of peptide-hybrid inhibitors (R1-R2-Lys-R3-NH2) by the 3D-QSAR technique. Comparative molecular field analysis (q2 = 0.613, r2 = 0.938, r2pred = 0.820) and comparative molecular similarity indices analysis (q2 = 0.640, r2 = 0.928, r2pred = 0.693) were established, revealing minor, electropositive, H-bond acceptor groups at the R1 position, minor, electropositive, H-bond donor groups at the R2 position, and bulky, hydrophobic groups at the R3 position for higher inhibitory activity. Docking studies revealed extensive H-bond and hydrophobic interactions in the binding of tripeptide analogues to the NS2B/NS3 protease. This study provides an insight into the key structural features for the design of peptide-based inhibitors of DENV NS2B/NS3 protease.

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.

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.

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

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

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.

Inhibition of NS2B-NS3 protease from all four serotypes of dengue virus by punicalagin, punicalin and ellagic acid identified from Punica granatum.

Despite a major threat to the public health in tropical and subtropical regions, dengue virus (DENV) infections are untreatable. Therefore, efforts are needed to investigate cost-effective therapeutic agents that could cure DENV infections in future. The NS2B-NS3 protease encoded by the genome of DENV is considered a critical target for the development of anti-dengue drugs. The objective of the current study was to find out a specific inhibitor of the NS2B-NS3 proteases from all four serotypes of DENV. To begin with, nine plant extracts with a medicinal history were evaluated for their role in inhibiting the NS2B-NS3 proteases by Fluorescence Resonance Energy Transfer (FRET) assay. Among the tested extracts, Punica granatum was found to be the most effective one. The metabolic profiling of this extract revealed the presence of several active compounds, including ellagic acid, punicalin and punicalagin, which are well-established antiviral agents. Further evaluation of IC50 values of these three antiviral molecules revealed punicalagin as the most potent anti-NS2B-NS3 protease drug with IC50 of 0.91 ± 0.10, 0.75 ± 0.05, 0.42 ± 0.03, 1.80 ± 0.16 µM against proteases from serotypes 1, 2, 3 and 4, respectively. The docking studies demonstrated that these compounds interacted at the active site of the enzyme, mainly with His and Ser residues. Molecular dynamics simulations analysis also showed the structural stability of the NS2B-NS3 proteases in the presence of punicalagin. In summary, this study concludes that the punicalagin can act as an effective inhibitor against NS2B-NS3 proteases from all four serotypes of DENV.Communicated by Ramaswamy H. Sarma.

Inhibition of dengue viruses by N-methylcytisine thio derivatives through targeting viral envelope protein and NS2B-NS3 protease.

Dengue virus (DENV) is a significant global health threat, causing millions of cases worldwide each year. Developing antiviral drugs for DENV has been a challenging endeavor. Our previous study identified anti-DENV properties of two (-)-cytisine derivatives contained substitutions within the 2-pyridone core from a pool of 19 (-)-cytisine derivatives. This study aimed to expand on the previous research by investigating the antiviral potential of N-methylcytisine thio (mCy thio) derivatives against DENV, understanding the molecular mechanisms of antiviral activity for the active thio derivatives. The inhibitory assays on DENV-2-induced cytopathic effect and infectivity revealed that mCy thio derivatives 3 ((1R,5S)-3-methyl-1,2,3,4,5,6-hexahydro-8H-1,5-methanopyrido[1,2-a][1,5]diazocine-8-thione) and 6 ((1S,5R)-3-methyl-2-thioxo-1,2,3,4,5,6-hexahydro-8H-1,5-methanopyrido[1,2-a][1,5]diazocin-8-one) were identified as the active compounds against both DENV-1 and DENV-2. Derivative 6 displayed robust antiviral activity against DENV-2, with EC50 values ranging from 0.002 to 0.005 µM in different cell lines. Derivative 3 also exhibited significant antiviral activity against DENV-2. The study found that these compounds are effective at inhibiting DENV-2 at both the entry stage (including virus attachment) and post-entry stages of the viral life cycle. The study also investigated the inhibition of the DENV-2 NS2B-NS3 protease activity by these compounds. Derivative 6 demonstrated notably stronger inhibition compared to mCy thio 3, revealing its dual antiviral action at both the entry and post-entry stages. Molecular docking simulations indicated that mCy thio derivatives 3 and 6 bind to the domain I and III of the DENV E protein, as well as the active of NS2B-NS3 protease, suggesting their molecular interactions with the virus. The study demonstrates the antiviral efficacy of N-methylcytisine thio derivatives against DENV. It provides valuable insights into the potential interactions between these compounds and viral target proteins, which could be useful in the development of antiviral drugs for DENV.

Synthesis and biological activity of imidazole phenazine derivatives as potential inhibitors for NS2B-NS3 dengue protease.

A series of new imidazole-phenazine derivatives were synthesized via a two-step process. The condensation of 2,3-diaminophenazine and benzaldehyde derivatives proceeds with intermediate formation of an aniline Schiff base, which undergoes subsequent cyclodehydrogenation in situ. The structures of the synthesized compounds were characterized by 1D and 2D NMR, FTIR and HRMS. A total of thirteen imidazole phenazine derivatives were synthesized and validated for their inhibitory activity as anti-dengue agents by an in vitro DENV2 NS2B-NS3 protease assay using a fluorogenic Boc-Gly-Arg-Arg-AMC substrate. Two para-substituted imidazole phenazines, 3e and 3k, were found to be promising lead molecules for novel NS2B-NS3 protease inhibitors with IC50 of 54.8 µM and 71.9 µM, respectively, compared to quercetin as a control (IC50 104.8 µM). The in silico study was performed using AutoDock Vina to identify the binding energy and conformation of 3e and 3k with the active site of the DENV2 NS2B-NS3 protease Wichapong model. The results indicate better binding properties of 3e and 3k with calculated binding energies of -8.5 and -8.4 kcal mol-1, respectively, compared to the binding energy of quercetin of -7.2 kcal mol-1, which corroborates well with the experimental observations.

Evaluation of the inhibitory potency of anti-dengue phytocompounds against DENV-2 NS2B-NS3 protease: virtual screening, ADMET profiling and molecular dynamics simulation investigations.

Dengue fever has been a worldwide concern, with 50-100 million new infections each year mainly due to five different serotypes of the Dengue virus (DENV). Designing a perfect anti-dengue agent that can inhibit all the serotypes by distinguishing antigenic differences is quite difficult. Previous anti-dengue researches have included chemical compounds screening against DENV enzymes. The ongoing analysis is meant for investigation of the plant-based compounds as antagonistic to DENV-2 focusing on the specific NS2B-NS3Pro target, a trypsin like serine protease that cuts the DENV polyprotein into separate proteins crucial for viral reproduction. Initially, a virtual library of more than 130 phytocompounds was prepared from previously published reports of plants with anti-dengue properties, which were then virtually screened and shortlisted against the WT, H51N and S135A mutant of DENV-2 NS2B-NS3Pro. The three top-most compounds were viewed as Gallocatechin (GAL), Flavokawain-C (FLV), and Isorhamnetin (ISO) showing docking scores of -5.8, -5.7, -5.7 kcal/mol for WT, -7.5, -6.8, -7.6 kcal/mol for the H51N, and -6.9, -6.5, -6.1 kcal/mol for the S135A mutant protease, respectively. 100 ns long MD simulations and MM-GBSA based free energy calculations were performed on the NS2B-NS3Pro complexes to witness the relative binding affinity of the compounds and favourable molecular interactions network. A comprehensive analysis of the study reveals some promising outcomes with ISO as the topmost compound with favourable pharmacokinetic properties for the WT and mutants (H51N and S135A) as well, suggesting as a novel anti-NS2B-NS3Pro agent with better adapting characters in both the mutants.Communicated by Ramaswamy H. Sarma.

Investigating Lycotoxin-An1a (An1a), a defense antiviral peptide from Alopecosa nagpag venom as prospective anti-dengue agent against DENV-2 NS2B-NS3 protease.

Dengue fever is a global health concern with no effective therapy. Screening synthetic chemicals, animal-originated compounds, and phytocompounds against Dengue virus (DENV) targets has failed to find dengue antivirals. The current study examines animal drugs as antagonists against NS2B-NS3Pro, one of DENV's most promising therapeutic targets for dengue fever. Antiviral-Lycotoxin-An1a (An1a), a defence antiviral peptide isolated from the venom of Alopecosa nagpag, a toxic spider. Based on prior in vitro research, it was discovered that the venom peptide suppresses the action of DENV-2 NS2B-NS3Pro. An1a peptide with NS2B-NS3Pro wild type (WT) and two mutants (H51N and S135A) was tested for anti-dengue characteristics using in silico analysis. The WT NS2B-NS3Pro has a catalytic triad of His51, Asp75, and Ser135 in the active site, but the mutants have N51 instead of His51 and Ala135 instead of Ser135. The dynamic sites of the three proteases (WT, H51N, S135A) and the peptide toxin (An1a) were taken into account to achieve molecular docking of An1a with WT NS2B-NS3Pro in conjunction with H51N and S135A. Cluspro-2 performs rigid-flexible docking to predict peptide binding affinity, effectiveness, and inhibitory consistency. Since the ligand had a higher binding affinity, docking score, and molecular interaction network, MD simulations and MM-GBSA free energy calculations were used to investigate the stability of the three protein-peptide complexes. The computer-aided screening and manufacture of spider venom-based anti-dengue medicines yielded intriguing results in the preliminary studies. This study is significant in defining the ideal therapeutic candidate against dengue infections.

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 a NS2B cofactor. 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 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 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 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 residue 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 hydrophilic domain of NS2B co-contributed to the difference in NS2B(H)-NS3(pro) protease activities between GI and GIII. These observations gain an insight into the role of NS2B in regulation of NS3 protease activities, which is useful for understanding the replication of JEV GI and GIII viruses.

Lambda-free light chain: A serum marker of dengue disease via NS3 protease-mediated antibody cleavage.

Dengue poses a significant global public health threat, with diverse clinical manifestations due to complex interactions between the host and the pathogen. Recent reports have highlighted elevated serum-free light chain (FLC) levels in viral infectious diseases. Hence, our study aimed to investigate serum FLC levels in dengue patients. The findings revealed elevated serum λ FLCs, which were associated with the severity of dengue. Receiver operating characteristic curve (ROC) analysis demonstrated that λ FLCs may serve as a serum marker for identifying dengue disease (AUC: 0.7825, sensitivity: 80, specificity: 71.43) and classifying severe dengue (AUC: 0.8102, sensitivity: 75, specificity: 79.52). The viral protease, Dengue virus (DENV) nonstructural protein 3 (NS3), acts as a protease that cleaves viral polyproteins as well as host substrates. Therefore, we proposed that antibodies might be potential targets of NS3 protease, leading to an increase in FLCs. LC/MS-MS analysis confirmed that λ FLCs were the predominant products after antibody degradation by NS3 protease. Additionally, purified NS3 protease cleaved both human IgG and DENV2-neutralizing antibodies, resulting in the presence of λ FLCs. Moreover, NS3 protease administration in vitro led to a reduction in the neutralizing efficacy of DENV2-neutralizing antibodies. In summary, the elevated serum λ FLC levels effectively differentiate dengue patients from healthy individuals and identify severe dengue. Furthermore, the elevation of serum λ FLCs is, at least in part, mediated through NS3 protease-mediated antibody cleavage. These findings provide new insights for developing diagnostic tools and understanding the pathogenesis of DENV infection.

Discovery and structural optimization of a new series of N-acyl-2-aminobenzothiazole as inhibitors of Zika virus.

Zika virus infection is associated to severe diseases such as congenital microcephaly and Zika fever causing serious harm to humans and special concern to health systems in low-income countries. Currently, there are no approved drugs against the virus, and the development of anti-Zika virus drugs is thus urgent. The present investigation describes the discovery and hit expansion of a N-acyl-2-aminobenzothiazole series of compounds against Zika virus replication. A structure-activity relationship study was obtained with the synthesis and evaluation of anti-Zika virus activity and cytotoxicity on Vero cells of nineteen derivatives. The three optimized compounds were 2.2-fold more potent than the initial hit and 20.9, 7.7 and 6.4-fold more selective. Subsequent phenotypic and biochemical assays were performed to evidence whether non-structural proteins, such as the complex NS2B-NS3pro, are related to the mechanism of action of the most active compounds.

The Antiviral Activity of Varenicline against Dengue Virus Replication during the Post-Entry Stage.

Dengue virus (DENV) poses a significant global health challenge, with millions of cases each year. Developing effective antiviral drugs against DENV remains a major hurdle. Varenicline is a medication used to aid smoking cessation, with anti-inflammatory and antioxidant effects. In this study, varenicline was investigated for its antiviral potential against DENV. This study provides evidence of the antiviral activity of varenicline against DENV, regardless of the virus serotype or cell type used. Varenicline demonstrated dose-dependent effects in reducing viral protein expression, infectivity, and virus yield in Vero and A549 cells infected with DENV-1 and DENV-2, with EC50 values ranging from 0.44 to 1.66 µM. Time-of-addition and removal experiments demonstrated that varenicline had a stronger inhibitory effect on the post-entry stage of DENV-2 replication than on the entry stage, as well as the preinfection and virus attachment stages. Furthermore, cell-based trans-cleavage assays indicated that varenicline dose-dependently inhibited the proteolytic activity of DENV-2 NS2B-NS3 protease. Docking models revealed the formation of hydrogen bonds and van der Waals forces between varenicline and specific residues in the DENV-1 and DENV-2 NS2B-NS3 proteases. These results highlight the antiviral activity and potential mechanism of varenicline against DENV, offering valuable insights for further research and development in the treatment of DENV infection.

Repurposing of antiparasitic drugs against the NS2B-NS3 protease of the Zika virus.

To date, no approved drugs are available to treat the Zika virus (ZIKV) infection. Therefore, it is necessary to urgently identify potential drugs against the ZIKV infection. Here, the repurposing of 30 antiparasitic drugs against the NS2B-NS3 protease of the ZIKV has been carried out by using combined docking and molecular dynamics- (MD) simulations. Based on the docking results, 5 drugs, such as Amodiaquine, Primaquine, Paromomycin, Dichlorophene, and Ivermectin were screened for further analysis by MD simulations and free energy calculations. Among these drugs, Amodiaquine and Dichlorophen are found to produce the most stable complexes and possess relative binding free energies of about -44.3 ± 3.7 kcal/mol and -41.1 ± 5.3 kcal/mol respectively. Therefore, they would act as potent small-molecule inhibitors of the ZIKV protease.However, evaluations of biological and safety activities of these drugs against the ZIKV protease are required before their clinical use.Communicated by Ramaswamy H. Sarma.

Computer-aided identification of dengue virus NS2B/NS3 protease inhibitors: an integrated molecular modelling approach for screening of phytochemicals.

Globally, dengue (DENV) fever has appeared as the most widespread vector-borne disease, affecting more than 100 million individuals annually. No approved anti-DENV therapy or preventive vaccine is available yet. DENV NS3 protein is associated with protease activity and is essential for viral replication process within the host cell. NS2B is linked with NS3 protein as a cofactor. Hence, NS3/NS2B is a potential druggable target for developing inhibitors against dengue virus. In the present study, a dataset of Beta vulgaris L.-based natural compounds was developed. Virtual ligand screening of 30 phytochemicals was carried out to find novel inhibitors against the NS2B/NS3 protein. Spatial affinity, drug-likeness, and binding behaviors of selected phytochemicals were analyzed. Post-simulation analysis, including Principal Component Analysis (PCA), MMGBSA, and Co-relation analysis, was also performed to provide deep insight for elucidating protein-ligand complexes. This computer-aided screening scrutinized four potent phytochemicals, including betavulgaroside II, vitexin xyloside, epicatechin, and isovitexin2-O-xyloside inhibitors exhibiting optimal binding with viral NS3/NS2B protein. Our study brings novel scaffolds against DENV NS2B/NS3 of serotype-2 to act as lead molecules for further biological optimization. In future, this study will prompt the exploration and development of adjuvant anti-DENV therapy based on natural compounds.Communicated by Ramaswamy H. Sarma.