West Nile Virus NS2B-NS3 Protease Research Articles

Synthesis and Molecular Docking Studies of New Dispiropyrrolidines on West Nile Virus NS2B-NS3 Protease

West Nile virus (WNV) is among the other four flavivirus genus, rapidly spreading worldwide. The number of cases increases globally as there are no clinically available approved drugs and vaccines against this disease. Based on our previous finding related to a flavivirus, a series of spiropyrrolidine derivatives were regioselectively synthesized via [3+2]-cycloaddition reaction of three components between isatins, sarcosine, and (E)-3,5-bis (arylidene)-4-piperidones. The yield of synthesized compounds was in a range between 81–95%. The structures of all the synthesized compounds were characterized using FT-IR, 1D- and 2D-NMR, and HRMS. Molecular docking studies of spiropyrrolidines on NS2B-NS3 protease were done to understand and explore the ligand-receptor interactions and hypothesize the drug's refinements. The inhibition of NS2B-NS3 protease has been considered a promising strategy because this enzyme is responsible for the viral replication process. Among them, compound 5c shows an excellent binding affinity with ‒7.71 kcal/mol free binding energy and an inhibition constant of 1.73 μM. It also showed the binding orientation into the active site of WNV NS2B-NS3 protease on Asn84, Tyr1161, Gly1151, and Gly1153.

Exploiting Water Dynamics for Pharmacophore Screening.

Three-dimensional pharmacophore models have been proven extremely valuable in exploring novel chemical space through virtual screening. However, traditional pharmacophore-based approaches need ligand information and rely on static snapshots of highly dynamic systems. In this chapter, we describe PyRod, a novel tool to generate three-dimensional pharmacophore models based on water traces of a molecular dynamics simulation of an apo-protein.The protocol described herein was successfully applied for the discovery of novel drug-like inhibitors of West Nile virus NS2B-NS3 protease. By using this recent example, we highlight the key steps of the generation and validation of PyRod-derived pharmacophore models and their application for virtual screening.

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 novel West Nile Virus protease inhibitor based on isobenzonafuranone and triazolic derivatives of eugenol and indan-1,3-dione scaffolds.

The West Nile Virus (WNV) NS2B-NS3 protease is an attractive target for the development of therapeutics against this arboviral pathogen. In the present investigation, the screening of a small library of fifty-eight synthetic compounds against the NS2-NB3 protease of WNV is described. The following groups of compounds were evaluated: 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones; eugenol derivatives bearing 1,2,3-triazolic functionalities; and indan-1,3-diones with 1,2,3-triazolic functionalities. The most promising of these was a eugenol derivative, namely 4-(3-(4-allyl-2-methoxyphenoxy)-propyl)-1-(2-bromobenzyl)-1H-1,2,3-triazole (35), which inhibited the protease with IC50 of 6.86 μmol L-1. Enzyme kinetic assays showed that this derivative of eugenol presents competitive inhibition behaviour. Molecular docking calculations predicted a recognition pattern involving the residues His51 and Ser135, which are members of the catalytic triad of the WNV NS2B-NS3 protease.

Breathing new life into West Nile virus therapeutics; discovery and study of zafirlukast as an NS2B-NS3 protease inhibitor

The West Nile virus (WNV) has spread throughout the world causing neuroinvasive diseases with no treatments available. The viral NS2B-NS3 protease is essential for WNV survival and replication in host cells and is a promising drug target. Through an enzymatic screen of the National Institute of Health clinical compound library, we report the discovery of zafirlukast, an FDA approved treatment for asthma, as an inhibitor for the WNV NS2B-NS3 protease. Zafirlukast was determined to inhibit the protease through a mixed mode mechanism with an IC50 value of 32 μM. A structure activity relationship study of zafirlukast revealed the cyclopentyl carbamate and N-aryl sulfonamide as structural elements crucial for NS2B-NS3 protease inhibition. Replacing the cyclopentyl with a phenyl improved inhibition, resulting in an IC50 of 22 μM. Experimental and computational docking analysis support the inhibition model of zafirlukast and analogs binding at an allosteric site on the NS3 protein, thereby disrupting the NS2B cofactor from binding, resulting in protease inhibition.

Attenuation of West Nile Virus NS2B/NS3 Protease by Amino Terminal Copper and Nickel Binding (ATCUN) Peptides.

West Nile virus NS2B/NS3 protease (WNVP) is a viable target for the development of antiviral compounds. To that end, catalytic metallopeptides that incorporate the copper-binding ATCUN motif into either the N- or C-terminus of known WNVP targeting peptides have been developed as new families of peptide-based inhibitors. Each metallopeptide was evaluated based on its inhibitory constant (KI), time-dependent inactivation of the protein, Michaelis-Menten parameters, and the ability to oxidatively modify WNVP. Following catalytic inactivation of WNVP, sequencing by LC-MS/MS demonstrated active site residues Ser135, Thr134, and Thr132, as well as residues in the S2 binding pocket, to be modified by oxidative chemistry. Results from a DNPH-based assay to detect oxidative damage showed the formation of carbonyls in WNVP treated with metallopeptides. These results suggest that the metallopeptides are attenuating WNVP activity by irreversible oxidation of amino acids essential to substrate binding and catalysis.

STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease

West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3pro, we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3pro. Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC50 ∼ 500 μM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3pro. To identify the matching protein binding site, chemical shift perturbation studies employing 1H,15N-TROSY-HSQC experiments with uniformly 2H,15N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3pro inhibitors.

Miniature bovine pancreatic trypsin inhibitors (m-BPTIs) of the West Nile virus NS2B-NS3 protease

The mosquito-borne West Nile virus (WNV) causes a wide range of symptoms ranging from fever to the often fatal viral encephalitis. To date, no vaccine or drug therapy is available. The trypsin-like WNV NS2B-NS3 protease is deemed a plausible drug target and was shown to be inhibited by bovine pancreatic trypsin inhibitor (BPTI), a 58-residue protein isolated from bovine lung. Herein, we report a protein truncation study that resulted in a novel 14-residue cyclic peptide with equipotent inhibitory activity to native BPTI. We believe our truncation strategy can be further applied in the development of peptide-based inhibitors targeting trypsin-like proteases.

Synthesis and in vitro Evaluation of West Nile Virus Protease Inhibitors Based on the 1,3,4,5‐Tetrasubstituted 1H‐Pyrrol‐2(5H)‐one Scaffold

West Nile virus (WNV), a member of the Flaviviridae family, is a mosquito-borne pathogen that causes a large number of human infections each year. There are currently no vaccines or antiviral therapies available for human use against WNV. Therefore, efforts to develop new chemotherapeutics against this virus are highly desired. In this study, a WNV NS2B-NS3 protease inhibitor with a 1,3,4,5-tetrasubstituted 1H-pyrrol-2(5H)-one scaffold was identified by screening a small library of nonpeptidic compounds. Optimization of this initial hit by the synthesis and screening of a focused library of compounds with this scaffold led to the identification of a novel uncompetitive inhibitor ((-)-1a16, IC₅₀ =2.2±0.7 μM) of the WNV NS2B-NS3 protease. Molecular docking of the chiral compound onto the WNV protease indicates that the R enantiomer of 1a16 interferes with the productive interactions between the NS2B cofactor and the NS3 protease domain and is thus the preferred isomer for inhibition of the WNV NS2B-NS3 protease.

A FRET-based assay for the discovery of West Nile Virus NS2B-NS3 protease inhibitors

The West Nile Virus (WNV) has been a worldwide epidemic since the early 1990s. Currently there are no therapeutic treatments for WNV infections. One particular avenue of treatment is inhibition of the NS2B-NS3 protease, an enzyme that is crucial for WNV replication. In our effort to increase the number of NS2B-NS3 protease inhibitors, we report a novel FRET-based high throughput assay for the discovery of WNV NS2B-NS3 protease inhibitors. For this assay, a FRET-based peptide substrate was synthesized and kinetically characterized with the NS2B-NS3 protease. The new substrate exhibits a Km of 3.35±0.31μM, a kcat of 0.0717±0.0016s−1 and a kcat/Km of 21,400±2000M−1s−1.

Synthesis and in vitro Evaluation of West Nile Virus Protease Inhibitors Based on the 2‐{6‐[2‐(5‐Phenyl‐4H‐{1,2,4]triazol‐3‐ylsulfanyl)acetylamino]benzothiazol‐2‐ylsulfanyl}acetamide Scaffold

In recent years, clinical symptoms resulting from West Nile virus (WNV) infection have worsened in severity, with an increased frequency in neuroinvasive diseases among the elderly. As there are presently no successful therapies against WNV for use in humans, continual efforts to develop new chemotherapeutics against this virus are highly desired. The viral NS2B-NS3 protease is a promising target for viral inhibition due to its importance in viral replication and its unique substrate preference. In this study, a WNV NS2B-NS3 protease inhibitor with a 2-{6-[2-(5-phenyl-4H-[1,2,4]triazol-3-ylsulfanyl)acetylamino]benzothiazol-2-ylsulfanyl}acetamide scaffold was identified during screening. Optimization of this initial hit by synthesis and screening of a focused compound library with this scaffold led to the identification of a novel uncompetitive inhibitor (1 a24, IC50 =3.4±0.2 μM) of the WNV NS2B-NS3 protease. Molecular docking of 1 a24 into the WNV protease showed that the compound interferes with productive interactions of the NS2B cofactor with the NS3 protease and is an allosteric inhibitor of the WNV NS3 protease.

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

A series of new substrate analogue inhibitors of the WNV NS2B-NS3 protease containing decarboxylated arginine mimetics at the P1 position was developed. Among the various analogues, trans-(4-guanidino)cyclohexylmethylamide (GCMA) was identified as the most suitable P1 residue. In combination with dichloro-substituted phenylacetyl groups at the P4 position, three inhibitors with inhibition constants of <0.2 μM were obtained. These GCMA inhibitors have a better selectivity profile than the previously described agmatine analogues, and possess negligible affinity for the trypsin-like serine proteases thrombin, factor Xa, and matriptase. A crystal structure in complex with the WNV protease was determined for one of the most potent inhibitors, 3,4-dichlorophenylacetyl-Lys-Lys-GCMA (K(i)=0.13 μM). The inhibitor adopts a horseshoe-like conformation, most likely due to a hydrophobic contact between the P4 phenyl ring and the P1 cyclohexyl group, which is further stabilized by an intramolecular hydrogen bond between the P1 guanidino group and the P4 carbonyl oxygen atom. These inhibitors are stable, readily accessible, and have a noncovalent binding mode. Therefore, they may serve as suitable lead structures for further development.

Exploring the binding of peptidic West Nile virus NS2B–NS3 protease inhibitors by NMR

West Nile virus (WNV) NS2B–NS3 protease is an important drug target since it is an essential protein for the replication of the virus. In order to determine the minimum pharmacophore for protease inhibition, a series of dipeptide aldehydes were synthesized. The 50% inhibitory concentration (IC50) measurements revealed that a simple acetyl-KR-aldehyde was only threefold less active than 4-phenyl-phenylacetyl-KKR-aldehyde (1) (Stoermer et al., 2008) that was used as the reference compound. The ligand efficiency of 0.40kcal/mol/HA (HA=heavy atom) for acetyl-KR-aldehyde is much improved compared to the reference compound 1 (0.23kcal/mol/HA). The binding of the inhibitors was examined using 1H-15N-HSQC experiments and differential chemical shifts were used to map the ligand binding sites. The biophysical studies show that the conformational mobility of WNV protease has a major impact on the design of novel inhibitors, since the protein conformation changes profoundly depending on the structure of the bound ligand.

NS2B/NS3 protease: allosteric effect of mutations associated with the pathogenicity of tick-borne encephalitis virus

The sequences of the protease domain of the tick-borne encephalitis (TBE) virus NS3 protein have two amino acid substitutions, 16 R→K and 45 S→F, in the highly pathogenic and poorly pathogenic strains of the virus, respectively. Two models of the NS2B-NS3 protease complex for the highly pathogenic and poorly pathogenic strains of the virus were constructed by homology modeling using the crystal structure of West Nile virus NS2B-NS3 protease as a template; 20 ns molecular dynamic simulations were performed for both models, the trajectories of the dynamic simulations were compared, and the averaged distance between the two models was calculated for each residue. Conformational differences between two models were revealed in the identified pocket. The different conformations of the pocket resulted in different orientations of the NS2B segment located near the catalytic triad. In the model of the highly pathogenic TBE virus the identified pocket had a more open conformation compared to the poorly pathogenic model. We propose that conformational changes in the active protease center, caused by two amino acid substitutions, can influence enzyme functioning and the virulence of the virus.

Discovery, Synthesis, and in vitro Evaluation of West Nile Virus Protease Inhibitors Based on the 9,10‐Dihydro‐3H,4aH‐1,3,9,10a‐tetraazaphenanthren‐4‐one Scaffold

West Nile virus (WNV), a member of the Flaviviridae family, is a mosquito-borne pathogen that causes a great number of human infections each year. Neither vaccines nor antiviral therapies are currently available for human use. In this study, a WNV NS2B-NS3 protease inhibitor with a 9,10-dihydro-3H,4aH-1,3,9,10a-tetraazaphenanthren-4-one scaffold was identified by screening a small library of non-peptidic compounds. This initial hit was optimized by solution-phase synthesis and screening of a focused library of compounds bearing this scaffold. This led to the identification of a novel, uncompetitive inhibitor (1a40, IC(50) = 5.41±0.45 μM) of WNV NS2B-NS3 protease. Molecular docking of this chiral compound onto the WNV protease indicates that the S enantiomer of 1a40 appears to interfere with the productive interactions between the NS2B cofactor and the NS3 protease domain; (S)-1a40 is a preferred isomer for inhibition of WNV NS3 protease.

Tripeptide inhibitors of dengue and West Nile virus NS2B–NS3 protease

A series of tripeptide aldehyde inhibitors were synthesized and their inhibitory effect against dengue virus type 2 (DENV2) and West Nile virus (WNV) NS3 protease was evaluated side by side with the aim to discover potent flaviviral protease inhibitors and to examine differences in specificity of the two proteases. The synthesized inhibitors feature a varied N-terminal cap group and side chain modifications of a P2-lysine residue. In general a much stronger inhibitory effect of the tripeptide inhibitors was observed toward WNV protease. The inhibitory concentrations against DENV2 protease were in the micromolar range while they were submicromolar against WNV. The data suggest that a P2-arginine shifts the specificity toward DENV2 protease while WNV protease favors a lysine in the P2 position. Peptides with an extended P2-lysine failed to inhibit DENV2 protease suggesting a size-constrained S2 pocket. Our results generally encourage the investigation of di- and tripeptide aldehydes as inhibitors of DENV and WNV protease.

Identification of palmatine as an inhibitor of West Nile virus

In this study, we investigated the specific inhibition of West Nile virus (WNV) NS2B-NS3 protease and viral propagation by palmatine, a chemical compound from Coptis chinensis Franch. It was demonstrated that palmatine could inhibit WNV NS2B-NS3 protease activity in an uncompetitive manner, with a 50% inhibitory concentration (IC(50)) of 96 microM. Palmatine suppressed WNV without detectable cytotoxicity (a 50% effective concentration [EC(50)] of 3.6 microM and a 50% cytotoxicity concentration [CC(50)] of 1,031 microM). Furthermore, palmatine could also suppress dengue virus and yellow fever virus in a dose-dependent manner. This compound could potentially be developed for the treatment of flavivirus infections.

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.

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B–NS3 protease

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

Substrate Inhibition Kinetic Model for West Nile Virus NS2B-NS3 Protease

West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested that peptide substrates could bind to two binding sites on the protease. Reaction product analogues also showed inhibition of the protease, demonstrating product inhibition in addition to and distinct from substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1'-P3' interaction surface (i.e., the S1'-S3' sites). Optimization of substrate analogue inhibitors that target these two independent sites may lead to novel anti-WNV drugs.