Dengue Virus NS2B-NS3 Protease Research Articles

Discovery and SAR studies of methionine–proline anilides as dengue virus NS2B-NS3 protease inhibitors

A series of methionine–proline dipeptide derivatives and their analogues were designed, synthesized and assayed against the serotype 2 dengue virus NS2B-NS3 protease, and methionine–proline anilides 1 and 2 were found to be the most active DENV 2 NS2B-NS3 competitive inhibitors with Ki values of 4.9 and 10.5μM. The structure and activity relationship and the molecular docking revealed that l-proline, l-methionine and p-nitroaniline in 1 and 2 are the important characters in blocking the active site of NS2B-NS3 protease. Our current results suggest that the title dipeptidic scaffold represents a promising structural core to discover a new class of active NS2B-NS3 competitive inhibitors.

NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex

The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker ("linked protease"), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a (1)H-(15)N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV.

Synthesis of (±)‐Panduratin A and Related Natural Products Using the High Pressure Diels–Alder Reaction

Under pressure: Panduratin A (1) has been synthesized in six steps including a high pressure Diels–Alder reaction. This divergent sequence also allowed the synthesis of 4-hydroxypanduratin A, panduratin H, panduratin I, nicolaioidesin B, and 2-hydroxyisopanduratin A. The binding of panduratin A to the dengue virus NS2B-NS3 protease was investigated by NMR spectroscopy.

Repurposing of FDA-Approved Drugs for the Discovery of Inhibitors of Dengue Virus NS2B-NS3 Protease by Docking, Consensus Scoring, and Molecular Dynamics Simulations

Dengue viruses (DENV) are transmitted by mosquitoes and infect ∼50 million people annually with an additional 2.5 billion people at risk living in tropical areas. However, there are no approved vaccines or antiviral therapies to combat the disease. DENV genome is translates into a single polyprotein comprising 3 structural and 7 non-structural (NS) proteins. The polyprotein precursor is cleaved by both host proteases and the two-component virus protease NS2B-NS3. Thus, this protease is considered as a promising target for antiviral design. In order to identify novel inhibitors of the DENV NS2B-NS3 protease we focused our strategy on the allosteric inhibitors capable of targeting the NS2B-NS3 interaction rather than the NS3 active site. The computational protocol was performed for 7,240- FDA-approved drugs retrieved from the ZINC database. We firstly implement a structure-based virtual screening to identify a preliminary set of inhibitors against the catalytic domain in active form of DENV NS2B-NS3 by docking analysis. The preliminary set inhibitors was then used to perform a consensus scoring of docking poses, based on scoring functions from the DrugScore and Xscore packages. Six docked poses were ranked among the top 50 compounds according to consensus scoring and were used to molecular dynamics (MD) simulation and free-energy calculation. Three compounds belonging to the piperazine derivatives family were finally proposed as potential inhibitors for DENV NS2B-NS3. These compounds target the allosteric-binding site of protease so that compound binding produces a conformational change able to affect the interaction among the protease and peptide substrates. The computational drug discovery strategy employed in our study could be applied for the identification of inhibitors of other flaviviral proteases.

Critical Effect of Peptide Cyclization on the Potency of Peptide Inhibitors against Dengue Virus NS2B-NS3 Protease

Dengue virus (DENV) infection is a serious public health threat worldwide that demands effective treatment. In the search for potent virus protease inhibitors, several cone snail venoms were screened against serotype 2 DENV NS2B-NS3 protease, and one conotoxin, MrIA, was identified to have inhibitory activity. The inhibitory activity was attributed to a disulfide bond-mediated loop, from which rational optimization was made to improve the potency and stability. An eight-residue cyclic peptide inhibitor was finally obtained with high potency (inhibitory constant 2.2 μM), stability, and cell permeability. This inhibitor can thus serve as a good lead for DENV drug development. In addition, this work highlights the critical effect of peptide cyclization on the potency of oligopeptide inhibitors against DENV protease, which may advance the design of peptide inhibitors for homologous virus proteases.

Retro peptide-hybrids as selective inhibitors of the Dengue virus NS2B-NS3 protease

New chemotherapeutics against Dengue virus and related flaviviruses are of growing interest in antiviral drug discovery. The viral serine protease NS2B-NS3 is a promising target for the development of such agents. Drug-like inhibitors of this protease with high affinity to the target are not available at the moment. The present work describes the discovery of new retro di- and tripeptide hybrids that do not necessarily require an electrophilic “warhead” to achieve affinities in the low micromolar range. The most active sequence in this series is the tripeptide R-Arg-Lys-Nle-NH2. By variation of the N-terminal groups (R) it could be shown that the previously described arylcyanoacrylamide moiety is a preferable group in this position. Retro tripeptide hybrids were found to be more active and more selective than retro dipeptide hybrids. A significant selectivity towards the Dengue virus protease could be shown in a counterscreen with thrombin and the West Nile virus protease. Alternative sequences to R-Arg-Lys-Nle-NH2 did not have higher affinities towards the Dengue virus protease, similar to retro-inverse sequences with d-lysine and d-arginine residues. The results of a competition assay with the known inhibitor aprotinin indicate that the N-terminal arylcyanoacrylamide residue of this compound class binds near the catalytic center of the enzyme.

Inhibition of Dengue virus and West Nile virus proteases by click chemistry-derived benz[d]isothiazol-3(2H)-one derivatives

Two click chemistry-derived focused libraries based on the benz[d]isothiazol-3(2H)-one scaffold were synthesized and screened against Dengue virus and West Nile virus NS2B-NS3 proteases. Several compounds (4l, 7j–n) displayed noteworthy inhibitory activity toward Dengue virus NS2B-NS3 protease in the absence and presence of added detergent. These compounds could potentially serve as a launching pad for a hit-to-lead optimization campaign.

Binding of Low Molecular Weight Inhibitors Promotes Large Conformational Changes in the Dengue Virus NS2B-NS3 Protease: Fold Analysis by Pseudocontact Shifts

The two-component dengue virus NS2B-NS3 protease (DEN NS2B-NS3pro) is an established drug target, but inhibitor design is hampered by the lack of a crystal structure of the protease in its fully active form. In solution and without inhibitors, the functionally important C-terminal segment of the NS2B cofactor is dissociated from DEN NS3pro ("open state"), necessitating a large structural change to produce the "closed state" thought to underpin activity. We analyzed the fold of DEN NS2B-NS3pro in solution with and without bound inhibitor by nuclear magnetic resonance (NMR) spectroscopy. Multiple paramagnetic lanthanide tags were attached to different sites to generate pseudocontact shifts (PCS). In the face of severe spectral overlap and broadening of many signals by conformational exchange, methods for assignment of (15)N-HSQC cross-peaks included selective mutation, combinatorial isotope labeling, and comparison of experimental PCSs and PCSs back-calculated for a structural model of the closed conformation built by using the structure of the related West Nile virus (WNV) protease as a template. The PCSs show that, in the presence of a positively charged low-molecular weight inhibitor, the enzyme assumes a closed state that is very similar to the closed state previously observed for the WNV protease. Therefore, a model of the protease built on the closed conformation of the WNV protease is a better template for rational drug design than available crystal structures, at least for positively charged inhibitors. To assess the open state, we created a binding site for a Gd(3+) complex and measured paramagnetic relaxation enhancements. The results show that the specific open conformation displayed in the crystal of DEN NS2B-NS3pro is barely populated in solution. The techniques used open an avenue to the fold analysis of proteins that yield poor NMR spectra, as PCSs from multiple sites in combination with model building generate powerful information even from incompletely assigned (15)N-HSQC spectra.

Structure-guided fragment-based in silico drug design of dengue protease inhibitors

An in silico fragment-based drug design approach was devised and applied towards the identification of small molecule inhibitors of the dengue virus (DENV) NS2B-NS3 protease. Currently, no DENV protease co-crystal structure with bound inhibitor and fully formed substrate binding site is available. Therefore a homology model of DENV NS2B-NS3 protease was generated employing a multiple template spatial restraints method and used for structure-based design. A library of molecular fragments was derived from the ZINC screening database with help of the retrosynthetic combinatorial analysis procedure (RECAP). 150,000 molecular fragments were docked to the DENV protease homology model and the docking poses were rescored using a target-specific scoring function. High scoring fragments were assembled to small molecule candidates by an implicit linking cascade. The cascade included substructure searching and structural filters focusing on interactions with the S1 and S2 pockets of the protease. The chemical space adjacent to the promising candidates was further explored by neighborhood searching. A total of 23 compounds were tested experimentally and two compounds were discovered to inhibit dengue protease (IC(50)=7.7μM and 37.9μM, respectively) and the related West Nile virus protease (IC(50)=6.3μM and 39.0μM, respectively). This study demonstrates the successful application of a structure-guided fragment-based in silico drug design approach for dengue protease inhibitors providing straightforward hit generation using a combination of homology modeling, fragment docking, chemical similarity and structural filters.

Anthracene-based inhibitors of dengue virus NS2B–NS3 protease

Dengue virus (DENV) is a mosquito-borne flavivirus that has strained global healthcare systems throughout tropical and subtropical regions of the world. In addition to plaguing developing nations, it has re-emerged in several developed countries with recent outbreaks in the USA ( CDC, 2010), Australia ( Hanna et al., 2009), Taiwan ( Kuan et al., 2010) and France ( La Ruche et al., 2010). DENV infection can cause significant disease, including dengue fever, dengue hemorrhagic fever, dengue shock syndrome, and death. There are no approved vaccines or antiviral therapies to prevent or treat dengue-related illnesses. However, the viral NS2B–NS3 protease complex provides a strategic target for antiviral drug development since NS3 protease activity is required for virus replication. Recently, we reported two compounds with inhibitory activity against the DENV protease in vitro and antiviral activity against dengue 2 (DEN2V) in cell culture ( Tomlinson et al., 2009a). Analogs of one of the lead compounds were purchased, tested in protease inhibition assays, and the data evaluated with detailed kinetic analyses. A structure activity relationship (SAR) identified key atomic determinants (i.e. functional groups) important for inhibitory activity. Four “second series” analogs were selected and tested to validate our SAR and structural models. Here, we report improvements to inhibitory activity ranging between ∼2- and 60-fold, resulting in selective low micromolar dengue protease inhibitors.

Design, structure-based focusing and in silico screening of combinatorial library of peptidomimetic inhibitors of Dengue virus NS2B-NS3 protease

Serine protease activity of the NS3 protein of Dengue virus is an important target of antiviral agents that interfere with the viral polyprotein precursor processing catalyzed by the NS3 protease (NS3pro), which is important for the viral replication and maturation. Recent studies showed that substrate-based peptidomimetics carrying an electrophilic warhead inhibit the NS2B-NS3pro cofactor-protease complex with inhibition constants in the low micromolar concentration range when basic amino acid residues occupy P(1) and P(2) positions of the inhibitor, and an aldehyde warhead is attached to the P(1). We have used computer-assisted combinatorial techniques to design, focus using the NS2B-NS3pro receptor 3D structure, and in silico screen a virtual library of more than 9,200 peptidomimetic analogs targeted around the template inhibitor Bz-Nle-Lys-Arg-Arg-H (Bz-benzoyl) that are composed mainly of unusual amino acid residues in all positions P(1)-P(4). The most promising virtual hits were analyzed in terms of computed enzyme-inhibitor interactions and Adsorption, Distribution, Metabolism and Excretion (ADME) related physico-chemical properties. Our study can direct the interest of medicinal chemists working on a next generation of antiviral chemotherapeutics against the Dengue Fever towards the explored subset of the chemical space that is predicted to contain peptide aldehydes with NS3pro inhibition potencies in nanomolar range which display ADME-related properties comparable to the training set inhibitors.

NMR Analysis of the Dynamic Exchange of the NS2B Cofactor between Open and Closed Conformations of the West Nile Virus NS2B-NS3 Protease

BackgroundThe two-component NS2B-NS3 proteases of West Nile and dengue viruses are essential for viral replication and established targets for drug development. In all crystal structures of the proteases to date, the NS2B cofactor is located far from the substrate binding site (open conformation) in the absence of inhibitor and lining the substrate binding site (closed conformation) in the presence of an inhibitor.MethodsIn this work, nuclear magnetic resonance (NMR) spectroscopy of isotope and spin-labeled samples of the West Nile virus protease was used to investigate the occurrence of equilibria between open and closed conformations in solution.FindingsIn solution, the closed form of the West Nile virus protease is the predominant conformation irrespective of the presence or absence of inhibitors. Nonetheless, dissociation of the C-terminal part of the NS2B cofactor from the NS3 protease (open conformation) occurs in both the presence and the absence of inhibitors. Low-molecular-weight inhibitors can shift the conformational exchange equilibria so that over 90% of the West Nile virus protease molecules assume the closed conformation. The West Nile virus protease differs from the dengue virus protease, where the open conformation is the predominant form in the absence of inhibitors.ConclusionPartial dissociation of NS2B from NS3 has implications for the way in which the NS3 protease can be positioned with respect to the host cell membrane when NS2B is membrane associated via N- and C-terminal segments present in the polyprotein. In the case of the West Nile virus protease, discovery of low-molecular-weight inhibitors that act by breaking the association of the NS2B cofactor with the NS3 protease is impeded by the natural affinity of the cofactor to the NS3 protease. The same strategy can be more successful in the case of the dengue virus NS2B-NS3 protease.

Optimization of Assay Conditions for Dengue Virus Protease: Effect of Various Polyols and Nonionic Detergents

The aim of this work was to perform a systematic study of the effect of nonionic detergents on the activity of the dengue virus NS2B-NS3 protease. To ensure a high activity of the protease, the assay procedures for the dengue virus and other flaviviral proteases published to date are performed in the presence of up to 35% glycerol, which does not represent the cellular physicochemical environment. In addition, the high viscosity of glycerol-containing solutions leads to various experimental problems in miniaturized assays. Using an internally quenched peptide substrate, the authors show that glycerol is not essential for enzymatic activity if certain nonionic detergents are added to the assay buffer. In addition, nonionic detergents may help to avoid false-positive screening results caused by "promiscuous" inhibitors. Other polyalcohols can substitute glycerol and have less effect on the viscosity of the assay buffer. The assay was used to screen a compound library and allowed the identification of small-molecular nonpeptidic inhibitors of dengue NS3 protease. Finally, the authors discuss the mode of action of nonionic detergents and the influence that they may have on the conformational properties of the NS2B-NS3 protease.