NS2B NS3 Research Articles

Structural Insights into Plasticity and Discovery of Flavonoid Allosteric Inhibitors of Flavivirus NS2B–NS3 Protease

Flaviviruses are among the most critical pathogens in tropical regions; they cause various severe diseases in developing countries but are not restricted to these countries. The development of antiviral therapeutics is crucial for managing flavivirus outbreaks. Ten proteins are encoded in the flavivirus RNA. The N2B–NS3pro protein complex plays a fundamental role in flavivirus replication and is a promising drug target; however, no flavivirus protease inhibitors have progressed to the preclinical stage. This study analyzed the structural models and plasticity of the NS2B–NS3pro protein complex of five medically important non-dengue flaviviruses (West Nile, Rocio, Ilhéus, yellow fever, and Saint Louis encephalitis). The flavonoids amentoflavone, tetrahydrorobustaflavone, and quercetin were selected for their exceptional binding energies as potential inhibitors of the NS2B–NS3pro protein complex. AutoDock Vina results ranged from −7.0 kcal/mol to −11.5 kcal/mol and the compounds preferentially acted non-competitively. Additionally, the first structural model for the NS2B–NS3pro protein complex was proposed for Ilhéus and Rocio viruses. The NS2B–NS3pro protease is an attractive molecular target for drug development. The three identified natural flavonoids showed great inhibitory potential against the viral species. Nevertheless, further in silico and in vitro studies are required to obtain more information regarding NS2B–NS3pro inhibition by these flavonoids and their therapeutic potential.

Designing photoaffinity tool compounds for the investigation of the DENV NS2B-NS3 protease allosteric binding pocket.

Dengue virus (DENV) infection still lacks specific antiviral therapy, making the NS2B-NS3 protease an attractive target for drug development. However, allosteric inhibitors that bind to a site other than the active site still need to be better understood. In this study, we designed and synthesised tool compounds for photoaffinity labelling (PAL) to investigate the binding site of allosteric inhibitors on the DENV protease. These tool compounds contained an affinity moiety, a photoreactive group, and a reporter tag for detection. Upon irradiation, the photoreactive group formed a covalent bond with the protease, allowing for binding site identification. SDS-PAGE-based assays confirmed the qualitative binding of the designed inhibitors to the allosteric pocket, and pull-down experiments validated the interaction. Tryptic protein digestion following liquid chromatography/mass spectrometry analysis further supported the binding of the inhibitor to the proposed pocket revealing photo-attachment to an NS3 loop close to the C-terminus. These results enhance our understanding of allosteric inhibitors and their mechanism of action against the DENV protease. The developed tool compounds and PAL are potent tools for future drug discovery efforts and investigations targeting the DENV protease.

Dynamic Interactions of Post Cleaved NS2B Cofactor and NS3 Protease Identified by Integrative Structural Approaches.

Diseases caused by flaviviruses such as dengue virus (DENV) and West Nile Virus (WNV), are a serious threat to public health. The flavivirus single-stranded RNA genome is translated into a polyprotein which is cleaved into three structural proteins and seven non-structural proteins by the viral and cellular proteases. Non-structural (NS) protein 3 is a multifunctional protein that has N-terminal protease and C-terminal helicase domains. The NS3 protease requires co-factor NS2B for enzymatic activity and folding. Due to its essential role in viral replication, NS2B-NS3 protease is an attractive target for antiviral drugs. Despite the availability of crystal structures, dynamic interactions of the N- and C-termini of NS2B co-factor have been elusive due to their flexible fold. In this study, we employ integrative structural approaches combined with biochemical assays to elucidate the dynamic interactions of the flexible DENV4 NS2B and NS3 N- and C-termini. We captured the crystal structure of self-cleaved DENV4 NS2B47NS3 protease in post cleavage state. The intermediate conformation adopted in the reported structure can be targeted by allosteric inhibitors. Comparison of our new findings from DENV4 against previously studied ZIKV NS2B-NS3 proteins reveals differences in NS2B-NS3 function between the two viruses. No inhibition of protease activity was observed for unlinked DENV NS2B-NS3 in presence of the cleavage site while ZIKV NS2B-NS3 cleavage inhibits protease activity. Another difference is that binding of the NS2B C-terminus to DENV4 eNS2B47NS3Pro active site is mediated via interactions with P4-P6 residues while for ZIKV, the binding of NS2B C-terminus to active site is mediated by P1-P3 residues. The mapping of NS2B N- and C-termini with NS3 indicates that these intermolecular interactions occur mainly on the beta-barrel 2 of the NS3 protease domain. Our integrative approach enables a comprehensive understanding of the folding and dynamic interactions of DENV NS3 protease and its cofactor NS2B.

Synthesis, Structure-Activity Relationships, and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B-NS3 Protease.

Flaviviruses, including Zika, dengue, and West Nile viruses, are important human pathogens. The highly conserved NS2B-NS3 protease of Flavivirus is essential for viral replication and therefore a promising drug target. Through compound screening, followed by medicinal chemistry studies, a novel series of 2,5,6-trisubstituted pyrazine compounds are found to be potent, allosteric inhibitors of Zika virus protease (ZVpro) with IC50 values as low as 130 nM. Their structure-activity relationships are discussed. The ZVpro inhibitors also inhibit homologous proteases of dengue and West Nile viruses, and their inhibitory activities are correlated. The most potent compounds 47 and 103 potently inhibited Zika virus replication in cells with EC68 values of 300-600 nM and in a mouse model of Zika infection. These compounds represent novel pharmacological leads for drug development against Flavivirus infections.

Crystal structure of the NS3-like helicase from Alongshan virus.

Alongshan virus (ALSV) is an emerging human pathogen that was identified in China and rapidly spread to the European continent in 2019, raising concerns about public health. ALSV belongs to the distinct Jingmenvirus group within the Flaviviridae family with segmented RNA genomes. While segments 2 and 4 of the ALSV genome encode the VP1-VP3 proteins of unknown origin, segments 1 and 3 encode the NS2b-NS3 and NS5 proteins, which are related to Flavivirus nonstructural proteins, suggesting an evolutionary link between segmented and unsegmented viruses within the Flaviviridae family. Here, the enzymatic activity of the ALSV NS3-like helicase (NS3-Hel) was characterized and its crystal structure was determined to 2.9 Å resolution. ALSV NS3-Hel exhibits an ATPase activity that is comparable to those measured for Flavivirus NS3 helicases. The structure of ALSV NS3-Hel exhibits an overall fold similar to those of Flavivirus NS3 helicases. Despite the limited amino-acid sequence identity between ALSV NS3-Hel and Flavivirus NS3 helicases, structural features at the ATPase active site and the RNA-binding groove remain conserved in ALSV NS3-Hel. These findings provide a structural framework for drug design and suggest the possibility of developing a broad-spectrum antiviral drug against both Flavivirus and Jingmenvirus.

A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus-host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus-host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.

Intermolecular interactions of cn-716 and acyl-KR-aldehyde dipeptide inhibitors against Zika virus

The emergent Zika virus (ZIKV) infection has become a threat to global health due to its association with severe neurological abnormalities, namely Guillain-Barré Syndrome (GBS) in adults and Congenital Zika virus Syndrome (CZS) in neonates. Many studies are nowadays being conducted to find an effective antiviral drug against ZIKV. In particular, NS2B-NS3 protease is an attractive drug target due to its essential function in viral replication, although a drug is not yet commercially available. In this context, we present here a comparative structural study, based on quantum chemistry calculations, to analyze the intermolecular binding energies between the crystallographic structure of NS2B-NS3 protease and dipeptide boronic acid (cn-716) and aldehyde (acyl-KR-aldehyde) peptidomimetic inhibitors, by using the molecular fractionation with conjugate caps (MFCC) scheme within the density functional theory (DFT) formalism. Most intermolecular interactions in cn-716/NS2B-NS3 (acyl-KR-aldehyde/NS2B-NS3) are due to the amino acid residues Asp83*, His51, Asp129, Ser81*, Gly133, Ala132, Tyr161, Asn152 and Asp75 (Asp83*, Asp129, His51, Asn152, Tyr161, Tyr130, Gly153, Gly151, Asp75, Pro131, and Gly82). Additionally, we have considered missense mutation analysis of these residues to evaluate the destabilization and the increase of the flexibility of the protease, showing that mutation of the residues Tyr161 and Tyr130 causes more impact. Our simulations are a valuable tool for a better understanding of the binding mechanism of recognized inhibitors of NS2B-NS3 protease, and can lead to the rational design and development of novel anti-Zika drugs with improved efficiency.

An Antiviral Peptide from Alopecosa nagpag Spider Targets NS2B-NS3 Protease of Flaviviruses.

Flaviviruses are single-stranded RNA viruses predominantly transmitted by the widely distributed Aedes mosquitoes in nature. As important human pathogens, the geographic reach of Flaviviruses and their threats to public health are increasing, but there is currently no approved specific drug for treatment. In recent years, the development of peptide antivirals has gained much attention. Natural host defense peptides which uniquely evolved to protect the hosts have been shown to have antiviral properties. In this study, we firstly collected the venom of the Alopecosa nagpag spider from Shangri-La County, Yunnan Province. A defense peptide named Av-LCTX-An1a (Antiviral-Lycotoxin-An1a) was identified from the spider venom, and its anti-dengue serotype-2 virus (DENV2) activity was verified in vitro. Moreover, a real-time fluorescence-based protease inhibition assay showed that An1a functions as a DENV2 NS2B–NS3 protease inhibitor. Furthermore, we also found that An1a restricts zika virus (ZIKV) infection by inhibiting the ZIKV NS2B–NS3 protease. Together, our findings not only demonstrate that An1a might be a candidate for anti-flavivirus drug but also indicate that spider venom is a potential resource library rich in antiviral precursor molecules.

Mutational landscape of Zika virus strains worldwide and its structural impact on proteins

Zika virus (ZIKV) has spread globally and has been linked to the onset of microcephaly and other brain abnormalities. The ZIKV genome consists of an ~10.7 kb positive-stranded RNA molecule that encodes three structural (C, prM and E) and seven nonstructural (5′-NS1–NS2A–NS2B–NS3– NS4A/2K–NS4B–NS5-3′) proteins. In this work, we looked for genetic variants in 485 ZIKV complete genomes from GenBank (NCBI) and performed a computational systematic approach using MAESTROweb server to assess the impact of nonsynonymous mutations in ZIKV proteins (C, M, E, NS1, NS2A, NS2B-NS3 protease, NS3 helicase and NS5). Then, we merged the data and correlated it with the phenotypic reports of ZIKV circulating strains. The sensitivity profile of the proteins showed 96 mutational hotspots. We found 22 relevant mutations in proteins C (I80T), NS2A (I34M/T/V, I45V, I80T/V, L113F, A117V, I118V, L128P, V143A, T151A, M199I/V, R207K and L208I) and NS3 helicase (D436G, Y498H, R525K, Q528R and R583K) of the circulating strains. Our analysis exploited the impact of nonsynonymous mutations on ZIKV proteins, their structural and functional insights. The results presented here could advance our current understanding on ZIKV proteins functions and pathogenesis.

Docking of Phytoconstituents of Cynodon dactylon on NS2B NS3 protease domain of Dengue virus

Docking of Phytoconstituents of <i>Cynodon dactylon</i> on NS2B NS3 protease domain of Dengue virus

Trimethylsilyl tag for probing protein-ligand interactions by NMR.

Protein-ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined from straightforward 1D 1H-NMR spectra not only in the fast but also in the slow exchange limit. The tag is easily attached to cysteine residues and a sensitive reporter of ligand binding also at sites where it does not interfere with ligand binding or catalytic efficiency of the target protein. Its utility is demonstrated for the Zika virus NS2B-NS3 protease and the human prolyl isomerase FK506 binding protein.

Molecular Recognition Features in Zika Virus Proteome

Viruses have compact genomes that encode limited number of proteins in comparison to other biological entities. Interestingly, viral proteins have shown natural abundance of either completely disordered proteins that are recognized as intrinsically disorder proteins (IDPs) or partially disordered segments known as intrinsically disordered protein regions (IDPRs). IDPRs are involved in interactions with multiple binding partners to accomplish signaling, regulation, and control functions in cells. Tuning of IDPs and IDPRs are mediated through post-translational modification and alternative splicing. Often, the interactions of IDPRs with their binding protein partner(s) lead to transition from the state of disorder to ordered form. Such interaction-prone protein IDPRs are identified as molecular recognition features (MoRFs). Molecular recognition is an important initial step for the biomolecular interactions and their functional proceedings. Although previous studies have established occurrence of the IDPRs in Zika virus proteome, which provide the functional diversity and structural plasticity to viral proteins, the MoRF analysis has not been performed as of yet. Many computational methods have been developed for the identification of the MoRFs in protein sequences including ANCHOR, MoRFpred, DISOPRED3, and MoRFchibi_web server. In the current study, we have investigated the presence of MoRF regions in structural and non-structural proteins of Zika virus using an aforementioned set of computational techniques. Furthermore, we have experimentally validated the intrinsic disorderness of NS2B cofactor region of NS2B–NS3 protease. NS2B has one of the longest MoRF regions in Zika virus proteome. In future, this study may provide valuable information while investigating the virus host protein interaction networks.

3D-QSAR pharmacophore-based virtual screening, molecular docking and molecular dynamics simulation toward identifying lead compounds for NS2B–NS3 protease inhibitors

NS2B–NS3 protease has been identified to serve as lead drug design target due to its significant role in West Nile viral (WNV) and dengue virus (DENV) reproduction and replication. There are currently no approved chemotherapeutic drugs and effective vaccines to inhibit DENV and WNV infections. In this work, 3D-QSAR pharmacophore model has been developed to discover potential inhibitory candidates. Validation through Fischer’s model and decoy test indicate that the developed 3D pharmacophore model is highly predictive for DENV inhibitors, which was then employed to screen ZINC chemical library to obtain reasonable hits. Following ADMET filtering, 15 hits were subjected to further filter through molecular docking and CoMFA modeling. Finally, top three hits were identified as lead compounds or potential inhibitory candidates with IC50 values of ∼0.4637 µM and fitness of ∼57.73. It is implied from CoMFA modeling that substituents at the side site of benzotriazole such as a p-nitro group (e.g. biphenyl head) and a carbonyl (e.g. carboxylate function) at the side site of furan or amino group may improve bioactivity of ZINC85645245, respectively. Molecular dynamics simulations (MDS) were performed to discover new interactions and reinforce the binding modes from docking for the hits also. The QSAR and MDS results obtained from this work should be useful in determining structural requirements for inhibitor development as well as in designing more potential inhibitors for NS2B–NS3 protease.

Progress and prospects on DENV protease inhibitors

New treatments are desperately required to combat increasing rate of dengue fever cases reported in tropical and sub-tropical parts of the world. Among the ten proteins (structural and non-structural) encoded by dengue viral genome, NS2B–NS3 protease is an ideal target for drug discovery. It is responsible for the processing of poly protein that is required for genome replication of the virus. Moreover, inhibitors designed against proteases were found successful in Human Immuno-deficiency Virus (HIV) and Hepatitis C Virus (HCV). Complete molecular mechanism and a survey of inhibitors reported against dengue protease will be helpful in designing effective and potent inhibitors. This review provides an insight on molecular mechanism of dengue virus protease and covers up-to-date information on different inhibitors reported against dengue proteases with medicinal chemistry perspective.

Simultaneous uncoupled expression and purification of the Dengue virus NS3 protease and NS2B co-factor domain

Dengue Virus (DENV) infection is responsible for the world's most significant insect-borne viral disease. Despite an increasing global impact, there are neither prophylactic nor therapeutic options available for the effective treatment of DENV infection. An attractive target for antiviral drugs is the virally encoded trypsin-like serine protease (NS3pro) and its associated cofactor (NS2B). The NS2B–NS3pro complex is responsible for cleaving the viral polyprotein into separate functional viral proteins, and is therefore essential for replication. Recombinant expression of an active NS2B–NS3 protease has primarily been based on constructs linking the C-terminus of the approximately 40 amino acid hydrophilic cofactor domain of NS2B to the N-terminus of NS3pro via a flexible glycine linker. The resulting complex can be expressed in high yield, is soluble and catalytically active and has been used for most in vitro screening, inhibitor, and X-ray crystallographic studies over the last 15 years. Despite extensive analysis, no inhibitor drug candidates have been identified yet. Moreover, the effect of the artificial linker introduced between the protease and its cofactor is unknown. Two alternate methods for bacterial expression of non-covalently linked, catalytically active, NS2B–NS3pro complex are described here along with a comparison of the kinetics of substrate proteolysis and binding affinities of substrate-based aldehyde inhibitors. Both expression methods produced high yields of soluble protein with improved substrate proteolysis kinetics and inhibitor binding compared to their glycine-linked equivalent. The non-covalent association between NS2B and NS3pro is predicted to be more relevant for examining inhibitors that target cofactor-protease interactions rather than the protease active site. Furthermore, these approaches offer alternative strategies for the high yield co-expression of other protein assemblies.

Synthesis and molecular modelling studies of novel sulphonamide derivatives as dengue virus 2 protease inhibitors

Development of antivirals for dengue is now based on rational approach targeting the enzymes involved in its life cycle. Among the targets available for inhibition of dengue virus, non-structural protein NS2B–NS3 protease is considered as a promising target for the development of anti-dengue agents. In the current study we have synthesized a series of 4-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)benzene-1-sulphonamide derivatives and screened for DENV2 protease activity. Compounds 16 and 19 showed IC50 of DENV2 Protease activity with 48.2 and 121.9μM respectively. Molecular docking and molecular dynamic simulation studies were carried out to know the binding mode responsible for the activity. MD simulations revealed that, NS2B/NS3 protease was more stable when it binds with the active compound. Structure optimization of the lead compounds 16 and 19 and their co-crystallization studies are underway.

Dual inhibitors of the dengue and West Nile virus NS2B–NS3 proteases: Synthesis, biological evaluation and docking studies of novel peptide-hybrids

Dengue virus (DENV) and West Nile virus (WNV) are mosquito-borne arboviruses responsible for causing acute systemic diseases and severe health conditions in humans. The discovery of therapies capable to prevent infections or treat infected individuals remains an important challenge, since no vaccine or specific efficient treatment could be developed so far. In this context, we present herein the synthesis, characterization, biological evaluation and docking studies of novel peptide-hybrids based on 2,4-thiazolidinedione scaffolds containing non-polar groups. The most promising compound has an IC50 of 0.75μM against WNV protease, which represents a seventyfold improvement in activity compared to our previously reported compounds. Experimental results and docking studies are in agreement with the hypothesis that a non-polar group in the scaffold is important to obtain interactions between the inhibitors and a hydrophobic pocket in the substrate recognition region of the DENV and WNV NS2B–NS3 serine proteases.

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.

Amodiaquine, an antimalarial drug, inhibits dengue virus type 2 replication and infectivity

Dengue virus serotypes 1–4 (DENV1–4) are transmitted by mosquitoes which cause most frequent arboviral infections in the world resulting in ∼390 million cases with ∼25,000 deaths annually. There is no vaccine or antiviral drug currently available for human use. Compounds containing quinoline scaffold were shown to inhibit flavivirus NS2B–NS3 protease (NS2B–NS3pro) with good potencies. In this study, we screened quinoline derivatives, which are known antimalarial drugs for inhibition of DENV2 and West Nile virus (WNV) replication using the corresponding replicon expressing cell-based assays. Amodiaquine (AQ), one of the 4-aminoquinoline drugs, inhibited DENV2 infectivity measured by plaque assays, with EC50 and EC90 values of 1.08±0.09μM and 2.69±0.47 μM, respectively, and DENV2 RNA replication measured by Renilla luciferase reporter assay, with EC50 value of 7.41±1.09μM in the replicon expressing cells. Cytotoxic concentration (CC50) in BHK-21 cells was 52.09±4.25μM. The replication inhibition was confirmed by plaque assay of the extracellular virions as well as by qRT-PCR of the intracellular and extracellular viral RNA levels. AQ was stable for at least 96h and had minor inhibitory effect on entry, translation, and post-replication stages in the viral life cycle. DENV protease, 5′-methyltransferase, and RNA-dependent RNA polymerase do not seem to be targets of AQ. Both p-hydroxyanilino and diethylaminomethyl moieties are important for AQ to inhibit DENV2 replication and infectivity. Our results support AQ as a promising candidate for anti-flaviviral therapy.

Protein–protein interactions among West Nile non-structural proteins and transmembrane complex formation in mammalian cells

To study the membrane orientation of flavivirus non-structural proteins (NSPs) in the replication complex, the seven major West Nile (WN) NSPs were separately expressed in monkey cells, and their subcellular localization was investigated by imaging-based techniques. First, we observed by confocal microscopy that four small transmembrane proteins (TP) (NS2A, NS2B, NS4A, and NS4B) were located to the endoplasmic reticulum (ER), whereas the largest NSPs, NS1, NS3, and NS5 were not. We then analyzed the colocalization and the association of WN NSPs using the methods of confocal microscopy, fluorescence resonance energy transfer (FRET), and biologic fluorescence complementation (BiFC). Through these combined imaging techniques, protein–protein interactions (PPI) among WNNSPs were detected. Our data demonstrate that there are interactions between NS2A and NS4A, and interactions of NS2B with three other TPs (NS2A, NS4A, and NS4B) as well as the expected interaction with NS3. PPI between NS2A and NS4B or between NS4A and NS4B were not detected. By the criteria of these techniques, NS5 interacted only with NS3, and NS1 was not shown to be in close proximity with other NSPs. In addition, homo-oligomerization of some NSPs was observed and three-way interactions between NS2A, NS4A, and NA4B with NS2B–NS3 were also observed, respectively. Our results suggest that the four TPs are required for formation of transmembrane complex. NS2B protein seems to play a key role in bringing the TPs together on the ER membrane and in bridging the TPs with non-membrane-associated proteins (NS3 and NS5).