Dengue Virus NS2B-NS3 Protease Research Articles

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.

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.

Potential inhibitors isolated from Curcuma aeruginosa against dengue virus type 2 (DENV-2) NS2B-NS3 protease activity

Dengue virus (DENV) infection remains a formidable public health concern, causing significant fatalities worldwide. The unavailability of specific anti-DENV therapeutics and poor response towards dengue vaccine have impeded efforts to prevent and control this tropical disease, thereby, there is a pressing need for effective alternative therapeutics. Curcuma species including Curcuma aeruginosa have a long history of traditional use for the prevention and treatment of various ailments, including viral infections. While it is evident that several compounds isolated from these plants have antiviral activity against various viruses, their specific effects against DENV have not been thoroughly explored. The study aimed to investigate the potential of major compounds isolated from C. aeruginosa (demethoxycurcumin, curcumenol, furanodienone germacrone and zederone) in inhibiting DENV-2 NS2B-NS3 protease activity both in vitro and in silico. The results demonstrated that demethoxycurcumin (at 100 μM), furanodienone and germacrone (both at 25 μM) exhibited inhibitory effects on NS2B-NS3 protease activity comparable to aprotinin, a potent NS2B-NS3 protease inhibitor, albeit to a lesser extent. Importantly, these inhibitory concentrations did not adversely affect the viability of normal fibroblast cell line, L-929 cells, suggesting that the compounds’ effects were specific to the protease inhibition. Further in silico molecular docking analyses indicated that demethoxycurcumin, furanodienone and germacrone displayed significant binding affinities with both DENV E protein and DENV NS2B-NS3 protease, corroborating their inhibitory activity observed in the protease inhibition assay. These promising findings collectively suggest that demethoxycurcumin, furanodienone and germacrone from C. aeruginosa merit further development as distinctive inhibitors for treating DENV infection.

The effects of allosteric and competitive inhibitors on ZIKV protease conformational dynamics explored through smFRET, nanoDSF, DSF, and 19F NMR

ABSTRACT: Zika and dengue viruses cause mosquito-borne diseases of high epidemic relevance. The viral NS2B-NS3 proteases play crucial roles in the pathogen replication cycle and are validated drug targets. They can adopt at least two conformations depending on the position of the NS2B cofactor. Recently, we reported ligand-induced conformational changes of dengue virus NS2B-NS3 protease by single-molecule Förster resonance energy transfer (smFRET). Here, we investigated the conformational dynamics of the homologous Zika virus protease through an integrated methodological approach combining smFRET, thermal shift assays (DSF and nanoDSF) and 19F NMR spectroscopy. Our results show that allosteric inhibitors favor the open conformation and competitive inhibitors stabilize the closed conformation of the Zika virus protease.

Molecular docking and drug-likeness study of nirmatrelvir as promising drug candidates of dengue virus NS2B-NS3 protease

Molecular docking and drug-likeness study of nirmatrelvir as promising drug candidates of dengue virus NS2B-NS3 protease

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

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

A Critical Observation on the Design and Development of Reported Peptide Inhibitors of DENV NS2B-NS3 Protease in the Last Two Decades.

Dengue is one of the neglected tropical diseases, which remains a reason for concern as cases seem to rise every year. The failure of the only dengue vaccine, Dengvaxia® , has made the problem more severe and humanity has no immediate respite from this global burden. Dengue virus (DENV) NS2B-NS3 protease is an attractive target partly due to its role in polyprotein processing. Also, since it is among the most conserved domains in the viral genome, it could produce a broad scope of opportunities toward antiviral drug discovery in general. This review has made a detailed analysis of each case of the design and development of peptide inhibitors against DENV NS2B-NS3 protease in the last two decades. Also, we have discussed the reasons attributed to their inhibitory activity, and wherever possible, we have highlighted the concerns raised, challenges met, and suggestions to improve the inhibitory activity. Thus, we attempt to take the readers through the designing and development of reported peptide inhibitors and gain insight from these developments, which could further contribute toward strategizing the designing and development of peptide inhibitors of DENV protease with improved properties in the coming future.

A short survey of dengue protease inhibitor development in the past 6 years (2015–2020) with an emphasis on similarities between DENV and SARS-CoV-2 proteases

Dengue remains a disease of significant concern, responsible for nearly half of all arthropod-borne disease cases across the globe. Due to the lack of potent and targeted therapeutics, palliative treatment and the adoption of preventive measures remain the only available options. Compounding the problem further, the failure of the only dengue vaccine, Dengvaxia®, also delivered a significant blow to any hopes for the treatment of dengue fever. However, the success of Human Immuno-deficiency Virus (HIV) and Hepatitis C Virus (HCV) protease inhibitors in the past have continued to encourage researchers to investigate other viral protease targets. Dengue virus (DENV) NS2B-NS3 protease is an attractive target partly due to its role in polyprotein processing and also for being the most conserved domain in the viral genome. During the early days of the COVID-19 pandemic, a few cases of Dengue-COVID 19 co-infection were reported. In this review, we compared the substrate-peptide residue preferences and the residues lining the sub-pockets of the proteases of these two viruses and analyzed the significance of this similarity. Also, we attempted to abridge the developments in anti-dengue drug discovery in the last six years (2015–2020), focusing on critical discoveries that influenced the research.

Peptide-β-lactam Inhibitors of Dengue and West Nile Virus NS2B-NS3 Protease Display Two Distinct Binding Modes.

The β-lactam ring represents a valuable moiety that can induce covalent binding of an inhibitor to its target. In this study, we explored di- and tripeptides with β-lactam electrophilic warheads as inhibitors of dengue and West Nile virus NS2B-NS3 protease. Tripeptides with a (3S)-β-lactam moiety displayed the highest activity, with IC50 and EC50 values in the lower micromolar range in biochemical and cellular assays. The activity against dengue protease was in general higher than against West Nile virus protease. The compounds were inactive against the off-targets thrombin and trypsin. Liquid chromatography-mass spectrometry experiments revealed that tripeptide-β-lactam inhibitors bind to the protease in two distinct binding modes. Only one binding mode leads to a covalent, but reversible, interaction of the β-lactam ring with the catalytic serine, followed by release of the inhibitor with opened β-lactam ring. The other binding mode leads to the cleavage of the peptide backbone. This observation provides the first experimental evidence that benzyloxyphenylglycine in flaviviral protease inhibitors is positioned in the prime site of the enzyme.

Discovery of Ganoderma lucidum triterpenoids as potential inhibitors against Dengue virus NS2B-NS3 protease

Dengue virus (DENV) infection causes serious health problems in humans for which no drug is currently available. Recently, DENV NS2B-NS3 protease has been proposed as a primary target for anti-dengue drug discovery due to its important role in new virus particle formation by conducting DENV polyprotein cleavage. Triterpenoids from the medicinal fungus Ganoderma lucidum have been suggested as pharmacologically bioactive compounds and tested as anti-viral agents against various viral pathogens including human immunodeficiency virus. However, no reports are available concerning the anti-viral activity of triterpenoids from Ganoderma lucidum against DENV. Therefore, we employed a virtual screening approach to predict the functional triterpenoids from Ganoderma lucidum as potential inhibitors of DENV NS2B-NS3 protease, followed by an in vitro assay. From in silico analysis of twenty-two triterpenoids of Ganoderma lucidum, four triterpenoids, viz. Ganodermanontriol (−6.291 kcal/mol), Lucidumol A (−5.993 kcal/mol), Ganoderic acid C2 (−5.948 kcal/mol) and Ganosporeric acid A (−5.983 kcal/mol) were predicted to be viral protease inhibitors by comparison to reference inhibitor 1,8-Dihydroxy-4,5-dinitroanthraquinone (−5.377 kcal/mol). These results were further studied for binding affinity and stability using the molecular mechanics/generalized Born surface area method and Molecular Dynamics simulations, respectively. Also, in vitro viral infection inhibition suggested that Ganodermanontriol is a potent bioactive triterpenoid.

Synthetic peptide optimization improves the inhibition of dengue NS2B-NS3 protease and dengue replication in vitro.

Dengue virus (DENV) infection is one of the most widely-spread flavivirus infections with no effective antiviral drugs available. Peptide inhibitors have been considered as one of the best drug candidates due to their high specificity, selectivity in their interactions and minimum side effects. In this study, we employed computational studies using YASARA, HADDOCK server and PyMOL software to generate short and linear peptides based on a reference peptide, CP5-46A, to block DENV NS2B-NS3 protease. The inhibition potencies of the peptides were evaluated using in-house DENV2 serine protease and fluorogenic peptide substrates. In vitro analyses were performed to determine the peptides cytotoxicity and the inhibitory effects against DENV2 replication in WRL-68 cells. Our computational analyses revealed that the docking energy of AYA3, a 16 amino acid (aa) (-81.2 ± 10.6 kcal/mol) and AYA9, a 15 aa peptide (-83.8 ± 6.8 kcal/mol) to DENV NS2B-NS3 protease were much lower than the reference peptide (46 aa; -70.9 ± 7.8 kcal/mol) and the standard protease inhibitor, aprotinin (58 aa; -48.2 ± 10.6 kcal/mol). Both peptides showed significant inhibition against DENV2 NS2B-NS3 protease activity with IC50 values of 24 µM and 23 µM, respectively. AYA3 and AYA9 peptides also demonstrated approximately 68% and 83% of viral plaque reduction without significantly affecting cell viability at 50 µM concentration. In short, we generated short linear peptides with lower cytotoxic effect and substantial antiviral activities against DENV2. Further studies are required to investigate the inhibitory effects of these peptides in vivo. Keywords: peptide inhibitors; dengue virus; NS2B-NS3 protease; plaque reduction.

Cysteine Disulfide Traps Reveal Distinct Conformational Ensembles in Dengue Virus NS2B-NS3 Protease

The dengue virus protease (NS2B-NS3pro) plays a critical role in the dengue viral life cycle, making it an attractive drug target for dengue-related pathologies, including dengue hemorrhagic fever. A number of studies indicate that NS2B-NS3pro undergoes a transition between two widely different conformational states: an "open" (inactive) conformation and a "closed" (active) conformation. For the past several years, the equilibrium between these states and the resting conformation of NS2B-NS3pro have been debated, although a strong consensus is emerging. To investigate the importance of such conformational states, we developed versions of NS2B-NS3pro that allow us to trap the enzyme in various distinct conformations. Our data from these variants suggest that the enzymatic activity appears to be dependent on the movement of NS2B and may rely on the flexibility of the protease core. Locking the enzyme into the "closed" conformation dramatically increased activity, strongly suggesting that the "closed" conformation is the active conformation. The observed resting state of the enzyme depends largely on the construct used to express the NS2B-NS3pro complex. In an "unlinked" construct, in which the NS2B and NS3 regions exist as independent, co-expressed polypeptides, the enzyme rests predominantly in a "closed", active conformation. In contrast, in a "linked" construct, in which NS2B and NS3 are attached by a nine-amino acid linker, NS2B-NS3pro adopts a more relaxed, alternative conformation. Nevertheless, even the unlinked construct samples both the "closed" and other alternative conformations. Given our findings, and the more realistic resemblance of NS2B-NS3pro to the native enzyme, these data strongly suggest that studies should focus on the "unlinked" constructs moving forward. Additionally, the results from these studies provide a more detailed understanding of the various poses of the dengue virus NS2B-NS3 protease and should help guide future drug discovery efforts aimed at this enzyme.

Computational insight into dengue virus NS2B-NS3 protease inhibition: A combined ligand- and structure-based approach

The NS2B-NS3 protease is essential for the replication process of Dengue Virus, which make it an attractive target for anti-virus drugs. Since a considerable number of NS2B-NS3 protease inhibitors have been reported so far, it is significant for the discovery of more effective antivirus compounds with the essential structure-activity relationship extracted from known inhibitors. In this perspective, the relationship between the chemical features of inhibitors and their biological activities was investigated with a combined ligand- and structure-based approach. Furthermore, 3D pharmacophore models were generated with the best selected, which consisted of five chemical features: one ring aromatic group, one hydrophobic group, one hydrogen bond donor and two hydrogen bond acceptors (RHDAA). Subsequently, molecular docking was employed to explore the specific allosteric site for non-peptidic inhibitors to bind, which was proved to be located behind the catalytic triad. Taken the results of both molecular docking and pharmacophore modeling into consideration, a model of receptor-ligand interaction was obtained with four essential chemical features including aromatic rings and hydrogen bonds. This research provided an accurate binding model for the discovery and optimization of NS2B-NS3 protease inhibitors.

Discovery of novel cyclic peptide inhibitors of dengue virus NS2B-NS3 protease with antiviral activity

NS2B-NS3 protease is an essential enzyme for the replication of dengue virus (DENV), which continues to be a serious threat to worldwide public health. We designed and synthesized a series of cyclic peptides mimicking the substrates of this enzyme, and assayed their activity against the DENV-2 NS2B-NS3 protease. The introduction of aromatic residues at the appropriate positions and conformational restriction generated the most promising cyclic peptide with an IC50 of 0.95μM against NS2B-NS3 protease. Cyclic peptides with proper positioning of additional arginines and aromatic residues exhibited antiviral activity against DENV. Furthermore, replacing the C-terminal amide bond of the polybasic amino acid sequence with an amino methylene moiety stabilized the cyclic peptides against hydrolysis by NS2B-NS3 protease, while maintaining their enzyme inhibitory activity and antiviral activity.

Sensitive NMR Approach for Determining the Binding Mode of Tightly Binding Ligand Molecules to Protein Targets.

Structure-guided drug design relies on detailed structural knowledge of protein-ligand complexes, but crystallization of cocomplexes is not always possible. Here we present a sensitive nuclear magnetic resonance (NMR) approach to determine the binding mode of tightly binding lead compounds in complex with difficult target proteins. In contrast to established NMR methods, it does not depend on rapid exchange between bound and free ligand or on stable isotope labeling, relying instead on a tert-butyl group as a chemical label. tert-Butyl groups are found in numerous protein ligands and deliver an exceptionally narrow and tall (1)H NMR signal. We show that a tert-butyl group also produces outstandingly intense intra- and intermolecular NOESY cross-peaks. These enable measurements of pseudocontact shifts generated by lanthanide tags attached to the protein, which in turn allows positioning of the ligand on the protein. Once the ligand has been located, assignments of intermolecular NOEs become possible even without prior resonance assignments of protein side chains. The approach is demonstrated with the dengue virus NS2B-NS3 protease in complex with a high-affinity ligand containing a tert-butyl group.

Conformational change study of dengue virus NS2B-NS3 protease using 19F NMR spectroscopy

The dengue virus NS2B-NS3 protease (NS2B-NS3p), an important antiviral target for drug development, has been reported to adopt an open or closed conformation in crystal structures with different NS2B C-terminus (NS2Bc) positioning. In solution, nevertheless, NS2B-NS3p forms a mixture of open, closed and maybe other intermediate conformations, which is difficult to characterize using conventional biophysical and biochemical techniques. In this study, we developed a new strategy to analyze these conformational changes using 19F NMR spectroscopy. Low pH or bovine pancreatic trypsin inhibitor (BPTI) binding promote the conformation change from open to closed, showing the importance of charge forces in the interaction between NS2Bc and NS3p. The mutation H51A impairs the charge interaction and the pH dependence of the conformational changes. It stabilizes the open conformation, while the addition of BPTI still converts NS2B-NS3p from open to closed conformation.

Flavonoids as noncompetitive inhibitors of Dengue virus NS2B-NS3 protease: Inhibition kinetics and docking studies

NS2B-NS3 is a serine protease of the Dengue virus considered a key target in the search for new antiviral drugs. In this study flavonoids were found to be inhibitors of NS2B-NS3 proteases of the Dengue virus serotypes 2 and 3 with IC50 values ranging from 15 to 44μM. Agathisflavone (1) and myricetin (4) turned out to be noncompetitive inhibitors of dengue virus serotype 2 NS2B-NS3 protease with Ki values of 11 and 4.7μM, respectively. Docking studies propose a binding mode of the flavonoids in a specific allosteric binding site of the enzyme. Analysis of biomolecular interactions of quercetin (5) with NT647-NHS-labeled Dengue virus serotype 3 NS2B-NS3 protease by microscale thermophoresis experiments, yielded a dissociation constant KD of 20μM. Our results help to understand the mechanism of inhibition of the Dengue virus serine protease by flavonoids, which is essential for the development of improved inhibitors.

An inhibition model of BPTI to unlinked dengue virus NS2B-NS3 protease.

One approach to treating the dengue virus infection is to inhibit its NS2B-NS3 protease that plays a vital role in virus maturation. However, the lack of structural information on the active conformation of the protease hindered related drug design. With a co-expression system, we obtained the active two-component protease in its unlinked form. BPTI shows strong competitive inhibitory activity (Ki = 6.5 nM) against this unlinked protease, which adopts a closed conformation. Based on the biochemical and NMR perturbation information, an inhibition model of BPTI to NS2B-NS3 protease is proposed.

The dengue virus NS2B-NS3 protease retains the closed conformation in the complex with BPTI.

The C-terminal β-hairpin of NS2B (NS2Bc) in the dengue virus NS2B-NS3 protease is required for full enzymatic activity. In crystal structures without inhibitor and in the complex with bovine pancreatic trypsin inhibitor (BPTI), NS2Bc is displaced from the active site. In contrast, nuclear magnetic resonance (NMR) studies in solution only ever showed NS2Bc in the enzymatically active closed conformation. Here we demonstrate by pseudocontact shifts from a lanthanide tag that NS2Bc remains in the closed conformation also in the complex with BPTI. Therefore, the closed conformation is the best template for drug discovery.

Binding mode of the activity‐modulating C‐terminal segment of NS2B to NS3 in the dengue virus NS2B–NS3 protease

The two-component dengue virus NS2B-NS3 protease (NS2B-NS3pro) is an established drug target but inhibitor design is hampered by uncertainties about its 3D structure in solution. Crystal structures reported very different conformations for the functionally important C-terminal segment of the NS2B cofactor (NS2Bc), indicating open and closed conformations in the absence and presence of inhibitors, respectively. An earlier NMR study in solution indicated that a closed state is the preferred conformation in the absence of an artificial linker engineered between NS2B and NS3pro. To obtain direct structural information on the fold of unlinked NS2B-NS3pro in solution, we tagged NS3pro with paramagnetic tags and measured pseudocontact shifts by NMR to position NS2Bc relative to NS3pro. NS2Bc was found to bind to NS3pro in the same way as reported in a previously published model and crystal structure of the closed state. The structure is destabilized, however, by high ionic strength and basic pH, showing the importance of electrostatic forces to tie NS2Bc to NS3pro. Narrow NMR signals previously thought to represent the open state are associated with protein degradation. In conclusion, the closed conformation of the NS2B-NS3 protease is the best model for structure-guided drug design.