West Nile Virus Protease Research Articles

A New Class of Dengue and West Nile Virus Protease Inhibitors with Submicromolar Activity in Reporter Gene DENV-2 Protease and Viral Replication Assays.

Dengue and West Nile virus are rapidly spreading global pathogens for which no specific therapeutic treatments are available. One of the promising targets for drug discovery against dengue and other flaviviruses is the viral serine protease NS2B-NS3. We present the design, synthesis, and in vitro and cellular characterization of a novel chemotype of potent small-molecule non-peptidic dengue protease inhibitors derived from 4-benzyloxyphenylglycine. A newly developed luciferase-based DENV-2 protease reporter system in HeLa cells (DENV2proHeLa) was employed to determine the activity of the compounds in a cellular environment. Specificity and selectivity of the DENV2proHeLa system were confirmed by viral titer reduction assays. The compounds reach low micromolar to upper nanomolar inhibitory potency in cell-based assays, are selective against other serine proteases, and do not show relevant cytotoxicity. An extensive structure-activity relationship study provides a perspective for further drug development against flaviviral infections.

Catching a Moving Target: Comparative Modeling of Flaviviral NS2B-NS3 Reveals Small Molecule Zika Protease Inhibitors.

The pivotal role of viral proteases in virus replication has already been successfully exploited in several antiviral drug design campaigns. However, no efficient antivirals are currently available against flaviviral infections. In this study, we present lead-like small molecule inhibitors of the Zika Virus (ZIKV) NS2B-NS3 protease. Since only few nonpeptide competitive ligands are known, we take advantage of the high structural similarity with the West Nile Virus (WNV) NS2B-NS3 protease. A comparative modeling approach involving our in-house software PyRod was employed to systematically analyze the binding sites and develop molecular dynamics-based 3D pharmacophores for virtual screening. The identified compounds were biochemically characterized revealing low micromolar affinity for both ZIKV and WNV proteases. Their lead-like properties together with rationalized binding modes represent valuable starting points for future lead optimization. Since the NS2B-NS3 protease is highly conserved among flaviviruses, these compounds may also drive the development of pan-flaviviral antiviral drugs.

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.

Backbone modifications in peptidic inhibitors of flaviviral proteases.

The NS2B-NS3 protease is a promising target for the development of drugs against dengue virus (DENV), West Nile virus (WNV) and related flaviviruses. We report the systematic variation of the peptide backbone of the two lead compounds Bz-Arg-Lys-d-Phg-NH2 and Bz-Arg-Lys-d-Phg(OBn)-NH2. While inhibitory activity against WNV protease was generally decreased, the inhibitory potency against DENV protease could be conserved and increased in several peptidomimetics, particularly in those containing a (NMe)arginine fragment or an N-terminal α-keto amide. Methylation at the α-position of the C-terminal phenylglycine led to a 6-fold higher potency against DENV protease. Peptidomimetics with modified backbone showed increased resistance against hydrolytic attack by trypsin and α-chymotrypsin.

One Step Beyond: Design of Substrates Spanning Primed Positions of Zika Virus NS2B-NS3 Protease.

Although the mosquito-borne Zika virus was discovered in the late 1940s of the 20th century, for years it was neglected, as the disease in humans was rare and relatively mild. Viral NS2B-NS3 protease is essential for virus replication, and except for maturation of viral proteins, it also modulates the infection microenvironment to facilitate virus invasion. Here, we report the combinatorial chemistry approach for the synthesis of internally quenched substrates of the Zika virus NS2B-NS3 protease that were optimized in prime positions of the peptide chain. Final substrate ABZ-Val-Lys-Lys-Arg-Ala-Ala-Trp-Tyr(3-NO2)-NH2 displays an excellent kinetic parameter (k cat/K M reaching nearly 1.26 × 108 M-1 × s-1), which is over 10 times greater than previously reported (7.7 × 106 M-1 × s-1) substrate. Moreover, it was found to be selective over West Nile virus protease.

Zirconium catalyzed synthesis of 2-arylidene Indan-1,3-diones and evaluation of their inhibitory activity against NS2B-NS3 WNV protease

A simple and efficient Knoevenagel procedure for the synthesis of 2-arylidene indan-1,3-diones is herein reported. These compounds were prepared via ZrOCl2·8H2O catalyzed reactions of indan-1,3-dione with several aromatic aldehydes and using water as the solvent. The 2-arylidene indan-1,3-diones were obtained with 53%-95% yield within 10–45 min. The synthesized compounds were evaluated as inhibitors of the NS2B-NS3 protease of West Nile Virus (WNV). It was found that hydroxylated derivatives impaired enzyme activity with varying degrees of effectiveness. The most active hydroxylated derivatives, namely 2-(4-hydroxybenzylidene)-1H-indene-1,3(2H)-dione (14) and 2-(3,4-dihydroxybenzylidene)-1H-indene-1,3(2H)-dione (17), were characterized as noncompetitive enzymes inhibitors, with IC50 values of 11 μmol L−1 and 3 μmol L−1, respectively. Docking and electrostatic potential surfaces investigations provided insight on the possible binding mode of the most active compounds within an allosteric site.

Effects of NS2B-NS3 protease and furin inhibition on West Nile and Dengue virus replication

West Nile virus (WNV) and Dengue virus (DENV) replication depends on the viral NS2B-NS3 protease and the host enzyme furin, which emerged as potential drug targets. Modification of our previously described WNV protease inhibitors by basic phenylalanine analogs provided compounds with reduced potency against the WNV and DENV protease. In a second series, their decarboxylated P1-trans-(4-guanidino)cyclohexylamide was replaced by an arginyl-amide moiety. Compound 4-(guanidinomethyl)-phenylacetyl-Lys-Lys-Arg-NH2 inhibits the NS2B-NS3 protease of WNV with an inhibition constant of 0.11 µM. Due to the similarity in substrate specificity, we have also tested the potency of our previously described multibasic furin inhibitors. Their further modification provided chimeric inhibitors with additional potency against the WNV and DENV proteases. A strong inhibition of WNV and DENV replication in cell culture was observed for the specific furin inhibitors, which reduced virus titers up to 10,000-fold. These studies reveal that potent inhibitors of furin can block the replication of DENV and WNV.

Peptide-Boronic Acid Inhibitors of Flaviviral Proteases: Medicinal Chemistry and Structural Biology.

A thousand-fold affinity gain is achieved by introduction of a C-terminal boronic acid moiety into dipeptidic inhibitors of the Zika, West Nile, and dengue virus proteases. The resulting compounds have Ki values in the two-digit nanomolar range, are not cytotoxic, and inhibit virus replication. Structure-activity relationships and a high resolution X-ray cocrystal structure with West Nile virus protease provide a basis for the design of optimized covalent-reversible inhibitors aimed at emerging flaviviral pathogens.

High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease

The mosquito-borne dengue virus serotypes 1–4 (DENV1-4) and West Nile virus (WNV) cause serious illnesses worldwide associated with considerable morbidity and mortality. According to the World Health Organization (WHO) estimates, there are about 390 million infections every year leading to ∼500,000 dengue haemorrhagic fever (DHF) cases and ∼25,000 deaths, mostly among children. Antiviral therapies could reduce the morbidity and mortality associated with flaviviral infections, but currently there are no drugs available for treatment. In this study, a high-throughput screening assay for the Dengue protease was employed to screen ∼120,000 small molecule compounds for identification of inhibitors. Eight of these inhibitors have been extensively analyzed for inhibition of the viral protease in vitro and cell-based viral replication using Renilla luciferase reporter replicon, infectivity (plaque) and cytotoxicity assays. Three of these compounds were identified as potent inhibitors of DENV and WNV proteases, and viral replication of DENV2 replicon and infectious RNA. Fluorescence quenching, kinetic analysis and molecular modeling of these inhibitors into the structure of NS2B-NS3 protease suggest a mode of inhibition for three compounds that they bind to the substrate binding pocket.

Discovery of Nanomolar Dengue and West Nile Virus Protease Inhibitors Containing a 4-Benzyloxyphenylglycine Residue.

The dengue virus (DENV) and West Nile Virus (WNV) NS2B-NS3 proteases are attractive targets for the development of dual-acting therapeutics against these arboviral pathogens. We present the synthesis and extensive biological evaluation of inhibitors that contain benzyl ethers of 4-hydroxyphenylglycine as non-natural peptidic building blocks synthesized via a copper-complex intermediate. A three-step optimization strategy, beginning with fragment growth of the C-terminal 4-hydroxyphenylglycine to the benzyloxy ether, followed by C- and N-terminal optimization, and finally fragment merging generated compounds with in vitro affinities in the low nanomolar range. The most promising derivative reached Ki values of 12 nM at the DENV-2 and 39 nM at the WNV proteases. Several of the newly discovered protease inhibitors yielded a significant reduction of dengue and West Nile virus titers in cell-based assays of virus replication, with an EC50 value of 3.4 μM at DENV-2 and 15.5 μM at WNV for the most active analogue.

Identification of covalent active site inhibitors of dengue virus protease.

Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.

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.

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly.

The molecular mechanisms that define the specificity of flavivirus RNA encapsulation are poorly understood. Virions composed of the structural proteins of one flavivirus and the genomic RNA of a heterologous strain can be assembled and have been developed as live attenuated vaccine candidates for several flaviviruses. In this study, we discovered that not all combinations of flavivirus components are possible. While a West Nile virus (WNV) subgenomic RNA could readily be packaged by structural proteins of the DENV2 strain 16681, production of infectious virions with DENV2 strain New Guinea C (NGC) structural proteins was not possible, despite the very high amino acid identity between these viruses. Mutagenesis studies identified a single residue (position 101) of the DENV capsid (C) protein as the determinant for heterologous virus production. C101 is located at the P1' position of the NS2B/3 protease cleavage site at the carboxy terminus of the C protein. WNV NS2B/3 cleavage of the DENV structural polyprotein was possible when a threonine (Thr101 in strain 16681) but not a serine (Ser101 in strain NGC) occupied the P1' position, a finding not predicted by in vitro protease specificity studies. Critically, both serine and threonine were tolerated at the P1' position of WNV capsid. More extensive mutagenesis revealed the importance of flanking residues within the polyprotein in defining the cleavage specificity of the WNV protease. A more detailed understanding of the context dependence of viral protease specificity may aid the development of new protease inhibitors and provide insight into associated patterns of drug resistance. West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause considerable morbidity and mortality in humans. No specific antiflavivirus therapeutics are available for treatment of infection. Proteolytic processing of the flavivirus polyprotein is an essential step in the replication cycle and is an attractive target for antiviral development. The design of protease inhibitors has been informed by insights into the molecular details of the interactions of proteases and their substrates. In this article, studies of the processing of WNV and DENV capsid proteins by the WNV protease identified an unexpected contribution of the sequence surrounding critical residues within the cleavage site on protease specificity. This demonstration of context-dependent protease cleavage has implications for the design of chimeric flaviviruses, new therapeutics, and the interpretation of flavivirus protease substrate specificity studies.

NMR Structural Characterization for Proteases of Dengue and West Nile Viruses and its Insight into Drug Discovery

Flaviviruses are a major cause of infectious disease in human, which include the Dengue Virus (DENV), West Nile virus (WNV). The genomic RNA encodes a polyprotein precursor which is processed proteolytically upon translation to 10 proteins, including three structural proteins (capsid [C], premembrane [prM] and envelope [Env]), and seven nonstructural (NS) proteins (NS1, NS2A/B, NS3, NS4A/B, and NS5). The NS3 is of great interest in drug discovery because of its N-terminal domain has the protease activity required for viral replication. The activity of NS3 is regulated by membrane protein NS2B. Previous studies were using a construct that contains 40 amino acids from NS2B fused with NS3 protease domain though an artificial G4SG4 linker. We have developed a series of peptidic inhibitors targeting WNV and DENV proteases. We analyzed their interactions with the protease using both chemical shift perturbation and docking studies. For the DENV protease, there is still no potent inhibitor available so far. Using NMR spectroscopy, we discovered that the conventional DENV protease construct in which NS2B cofactor region linked with NS3 protease through a flexible liner is not suitable for drug discovery due to the protein dynamics. Using a co-expression system, we obtained a protease complex that contains the NS3 protease domain and 50-residue segment of the NS2B. Our results show that this protease complex exists as a close conformation and active under physiological conditions, which might be a better construct for drug discovery targeting DENV. We also expressed and purified a natural form of DENV protease which was shown to be active in detergent micelles. Our studies will be useful for development of DENV protease inhibitors.

Identification of a novel inhibitor of dengue virus protease through use of a virtual screening drug discovery Web portal.

We report the discovery of a novel small-molecule inhibitor of the dengue virus (DENV) protease (NS2B-NS3pro) using a newly constructed Web-based portal (DrugDiscovery@TACC) for structure-based virtual screening. Our drug discovery portal, an extension of virtual screening studies performed using IBM's World Community Grid, facilitated access to supercomputer resources managed by the Texas Advanced Computing Center (TACC) and enabled druglike commercially available small-molecule libraries to be rapidly screened against several high-resolution DENV NS2B-NS3pro crystallographic structures. Detailed analysis of virtual screening docking scores and hydrogen-bonding interactions between each docked ligand and the NS2B-NS3pro Ser135 side chain were used to select molecules for experimental validation. Compounds were ordered from established chemical companies, and compounds with established aqueous solubility were tested for their ability to inhibit DENV NS2B-NS3pro cleavage of a model substrate in kinetic studies. As a proof-of-concept, we validated a small-molecule dihydronaphthalenone hit as a single-digit-micromolar mixed noncompetitive inhibitor of the DENV protease. Since the dihydronaphthalenone was predicted to interact with NS2B-NS3pro residues that are largely conserved between DENV and the related West Nile virus (WNV), we tested this inhibitor against WNV NS2B-NS3pro and observed a similar mixed noncompetitive inhibition mechanism. However, the inhibition constants were ∼10-fold larger against the WNV protease relative to the DENV protease. This novel validated lead had no chemical features or pharmacophores associated with adverse toxicity, carcinogenicity, or mutagenicity risks and thus is attractive for additional characterization and optimization.

Actinomycetes from Red Sea sponges: sources for chemical and phylogenetic diversity.

The diversity of actinomycetes associated with marine sponges collected off Fsar Reef (Saudi Arabia) was investigated in the present study. Forty-seven actinomycetes were cultivated and phylogenetically identified based on 16S rRNA gene sequencing and were assigned to 10 different actinomycete genera. Eight putatively novel species belonging to genera Kocuria, Mycobacterium, Nocardia, and Rhodococcus were identified based on sequence similarity values below 98.2% to other 16S rRNA gene sequences available in the NCBI database. PCR-based screening for biosynthetic genes including type I and type II polyketide synthases (PKS-I, PKS-II) as well as nonribosomal peptide synthetases (NRPS) showed that 20 actinomycete isolates encoded each at least one type of biosynthetic gene. The organic extracts of nine isolates displayed bioactivity against at least one of the test pathogens, which were Gram-positive and Gram-negative bacteria, fungi, human parasites, as well as in a West Nile Virus protease enzymatic assay. These results emphasize that marine sponges are a prolific resource for novel bioactive actinomycetes with potential for drug discovery.

Intrinsic Flexibility of West Nile Virus Protease in Solution Characterized Using Small-Angle X-ray Scattering

West Nile virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection can cause severe neurological disease and fatality in humans. Efforts are ongoing to develop antiviral drugs that inhibit the WNV protease, a viral enzyme required for polyprotein processing. Unfortunately, little is known about the solution structure of recombinant WNV protease (NS2B-NS3pro) used for antiviral drug discovery and development, although X-ray crystal structures and nuclear magnetic resonance (NMR) studies have provided valuable insights into the interactions between NS2B-NS3pro and peptide-based inhibitors. We completed small-angle X-ray scattering and Fourier transform infrared spectroscopy experiments to determine the solution structure and dynamics of WNV NS2B-NS3pro in the absence of a bound substrate or inhibitor. Importantly, these solution studies suggested that all or most of the NS2B cofactor was highly flexible and formed an ensemble of structures, in contrast to the NS2B tertiary structures observed in crystallographic and NMR studies. The secondary structure of NS2B-NS3pro in solution had high β-content, similar to the secondary structure observed in crystallographic studies. This work provided evidence of the intrinsic flexibility and conformational heterogeneity of the NS2B chain of the WNV protease in the absence of substratelike ligands, which should be considered during antiviral drug discovery and development efforts.

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.

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.