Peptide-Bismuth Bicycles: In Situ Access to Stable Constrained Peptides with Superior Bioactivity.

Constrained peptides are promising next-generation therapeutics. We report here a fundamentally new strategy for the facile generation of bicyclic peptides using linear precursor peptides with three cysteine residues and a non-toxic trivalent bismuth(III) salt. Peptide-bismuth bicycles form instantaneously at physiological pH, are stable in aqueous solution for many weeks, and much more resistant to proteolysis than their linear precursors. The strategy allows the in situ generation of bicyclic ligands for biochemical screening assays. We demonstrate this for two screening campaigns targeting the proteases from Zika and West Nile viruses, revealing a new lead compound that displayed inhibition constants of 23 and 150 nM, respectively. Bicyclic peptides are up to 130 times more active and 19 times more proteolytically stable than their linear analogs without bismuth.

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.

Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication.

West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.

A Hyperactive Kunjin Virus NS3 Helicase Mutant Demonstrates Increased Dissemination and Mortality in Mosquitoes.

The unwinding of double-stranded RNA intermediates is critical for the replication and packaging of flavivirus RNA genomes. This unwinding activity is achieved by the ATP-dependent nonstructural protein 3 (NS3) helicase. In previous studies, we investigated the mechanism of energy transduction between the ATP and RNA binding pockets using molecular dynamics simulations and enzymatic characterization. Our data corroborated the hypothesis that motif V is a communication hub for this energy transduction. More specifically, mutations T407A and S411A in motif V exhibit a hyperactive helicase phenotype, leading to the regulation of translocation and unwinding during replication. However, the effect of these mutations on viral infection in cell culture and in vivo is not well understood. Here, we investigated the role of motif V in viral replication using West Nile virus (Kunjin subtype) T407A and S411A mutants (T407A and S411A Kunjin, respectively) in cell culture and in vivo We were able to recover S411A Kunjin but unable to recover T407A Kunjin. Our results indicated that S411A Kunjin decreased viral infection and increased cytopathogenicity in cell culture compared to wild-type (WT) Kunjin. Similarly, decreased infection rates in surviving S411A Kunjin-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin infection. Additionally, S411A Kunjin infection increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin infection. These data suggest that S411A Kunjin increases viral pathogenesis in mosquitoes. Overall, these data indicate that NS3 motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus.IMPORTANCE Kunjin and West Nile viruses belong to the arthropod-borne flaviviruses, which can result in severe symptoms, including encephalitis, meningitis, and death. Flaviviruses have expanded into new populations and emerged as novel pathogens repeatedly in recent years, demonstrating that they remain a global threat. Currently, there are no approved antiviral therapeutics against either Kunjin or West Nile viruses. Thus, there is a pressing need for understanding the pathogenesis of these viruses in humans. In this study, we investigated the role of the Kunjin virus helicase on infection in cell culture and in vivo This work provides new insight into how flaviviruses control pathogenesis and mosquito transmission through the nonstructural protein 3 helicase.

Structure-Based Macrocyclization of Substrate Analogue NS2B-NS3 Protease Inhibitors of Zika, West Nile and Dengue viruses.

A series of cyclic active-site-directed inhibitors of the NS2B-NS3 proteases from Zika (ZIKV), West Nile (WNV), and dengue-4 (DENV4) viruses has been designed. The most potent compounds contain a reversely incorporated d-lysine residue in the P1 position. Its side chain is connected to the P2 backbone, its α-amino group is converted into a guanidine to interact with the conserved Asp129 side chain in the S1 pocket, and its C terminus is connected to the P3 residue via different linker segments. The most potent compounds inhibit the ZIKV protease with Ki values <5 nM. Crystal structures of seven ZIKV protease inhibitor complexes were determined to support the inhibitor design. All the cyclic compounds possess high selectivity against trypsin-like serine proteases and furin-like proprotein convertases. Both WNV and DENV4 proteases are inhibited less efficiently. Nonetheless, similar structure-activity relationships were observed for these enzymes, thus suggesting their potential application as pan-flaviviral protease inhibitors.

Profiling of flaviviral NS2B-NS3 protease specificity provides a structural basis for the development of selective chemical tools that differentiate Dengue from Zika and West Nile viruses.

West Nile virus (WNV) and Dengue virus (DENV) are mosquito-borne pathogenic flaviviruses. The NS2B-NS3 proteases found in these viruses are responsible for polyprotein processing and are therefore considered promising medical targets. Another ortholog of these proteases is found in Zika virus (ZIKV). In this work, we applied a combinatorial chemistry approach - Hybrid Combinatorial Substrate Library (HyCoSuL), to compare the substrate specificity profile at the P4-P1 positions of the NS2B-NS3 proteases found in all three viruses. The obtained data demonstrate that Zika and West Nile virus NS2B-NS3 proteases display highly overlapping substrate specificity in all binding pockets, while the Dengue ortholog has slightly different preferences toward natural and unnatural amino acids at the P2 and P4 positions. We used this information to extract specific peptide sequences recognized by the Dengue NS2B-NS3 protease. Next, we applied this knowledge to design a selective substrate and activity-based probe for the Dengue NS2B-NS3 protease. Our work provides a structural framework for the design of inhibitors, which could be used as a lead structure for drug development efforts.

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.

Synthesis and structure-activity relationships of small-molecular di-basic esters, amides and carbamates as flaviviral protease inhibitors.

Inhibitors of the flaviviral serine proteases, which are crucial for the replication of dengue and West-Nile virus, have attracted much attention over the last years. A dibasic 4-guanidinobenzoate was previously reported as inhibitor of the dengue protease with potency in the low-micromolar range. In the present study, this lead structure was modified with the intent to explore structure-activity relationships and obtain compounds with increased drug-likeness. Substitutions of the guanidine moieties, the aromatic rings, and the ester with other functionalities were evaluated. All changes were accompanied by a loss of inhibition, indicating that the 4-guanidinobenzoate scaffold is an essential element of this compound class. Further experiments indicate that the target recognition of the compounds involves the reversible formation of a covalent adduct.

Discovery, X-ray Crystallography and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B-NS3 Protease.

Flaviviruses, including dengue, West Nile and recently emerged Zika virus, are important human pathogens, but there are no drugs to prevent or treat these viral infections. The highly conserved Flavivirus NS2B-NS3 protease is essential for viral replication and therefore a drug target. Compound screening followed by medicinal chemistry yielded a series of drug-like, broadly active inhibitors of Flavivirus proteases with IC50 as low as 120 nM. The inhibitor exhibited significant antiviral activities in cells (EC68: 300-600 nM) and in a mouse model of Zika virus infection. X-ray studies reveal that the inhibitors bind to an allosteric, mostly hydrophobic pocket of dengue NS3 and hold the protease in an open, catalytically inactive conformation. The inhibitors and their binding structures would be useful for rational drug development targeting Zika, dengue and other Flaviviruses.

DDX3X Helicase Inhibitors as a New Strategy To Fight the West Nile Virus Infection.

Increased frequency of arbovirus outbreaks in the last 10 years represents an important emergence for global health. Climate warming, extensive urbanization of tropical regions, and human migration flows facilitate the expansion of anthropophilic mosquitos and the emerging or re-emerging of new viral infections. Only recently the human adenosinetriphosphatase/RNA helicase X-linked DEAD-box polypeptide 3 (DDX3X) emerged as a novel therapeutic target in the fight against infectious diseases. Herein, starting from our previous studies, a new family of DDX3X inhibitors was designed, synthesized, validated on the target enzyme, and evaluated against the West Nile virus (WNV) infection. Time of addition experiments after virus infection indicated that the compounds exerted their antiviral activities after the entry process, likely at the protein translation step of WNV replication. Finally, the most interesting compounds were then analyzed for their in vitro pharmacokinetic parameters, revealing favorable absorption, distribution, metabolism, and excretion values. The good safety profile together with a good activity against WNV for which no treatments are currently available, make this new class of molecules a good starting point for further in vivo studies.

Phosphonate inhibitors of West Nile virus NS2B/NS3 protease.

West Nile virus (WNV) is a member of the flavivirus genus belonging to the Flaviviridae family. The viral serine protease NS2B/NS3 has been considered an attractive target for the development of anti-WNV agents. Although several NS2B/NS3 protease inhibitors have been described so far, most of them are reversible inhibitors. Herein, we present a series of α-aminoalkylphosphonate diphenyl esters and their peptidyl derivatives as potent inhibitors of the NS2B/NS3 protease. The most potent inhibitor identified was Cbz-Lys-Arg-(4-GuPhe)P(OPh)2 displaying Ki and k2/Ki values of 0.4 µM and 28 265 M-1s-1, respectively, with no significant inhibition of trypsin, cathepsin G, and HAT 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.

Impaired heterologous protein-protein interaction is an essential cause for non-viability of WNV/DENV recombinants.

Flavivirus RNA replication starts at 3'-end, where it folds into a highly conserved stem-loop structure. We attempted to identify the viral non-structural proteins (NSPs) that might specifically interact with the 3'-stemloop (3'SL) through a genetic approach. WNV/DENV2 chimeric recombinants that contain Dengue2 (DENV2) gene(s) in West Nile virus (WNV) backbone were tested for replication competence. Three of seven recombinant viruses, containing the DENV2 NS1, NS2A, or NS4B gene and terminated with a mutated 3'SL (MutC 3'SL), were viable. Of these three, only those bearing the DENV2 NS1 and NS2A substitutions remained infectious when the MutC 3'SL was replaced by the wildtype WNV 3'SL. However, none of the seven chimeric recombinants bearing the DENV2 3'SL were viable. We then investigated the causes for failed replication of WNV/DENV2 chimeric recombinants. Proteolytic cleavage of NS polyproteins was defective by heterologous protease NS2B/3, but was efficient by homologous DENV2 NS2B/3 protease. Whereas, the heterologous polyproteins that contained DENV2 homologous protease were found to produce abnormal vesicles. WNV/DENV2 recombinants expressing the DENV2 homologous protease did not produce infectious virus either. We examined NS protein-protein interaction (PPI) and found that heterologous PPI (hPPI) between WNV and DENV2 NSPs were impaired to various degrees. Insufficient PPIs occurred mainly between heterologous NS2B and NS3; NS2B and NS4A; NS3 and NS5, correlating to those non-viability of substitution mutants. Our results indicate that impaired PPI may decrease protease activity and affect vesicle formation, and is the essential cause for non-viability of the WNV/DENV2 recombinants.

Identification of a 3-Alkylpyridinium Compound from the Red Sea Sponge Amphimedon chloros with In Vitro Inhibitory Activity against the West Nile Virus NS3 Protease.

Viruses are underrepresented as targets in pharmacological screening efforts, given the difficulties of devising suitable cell-based and biochemical assays. In this study we found that a pre-fractionated organic extract of the Red Sea sponge Amphimedon chloros was able to inhibit the West Nile Virus NS3 protease (WNV NS3). Using liquid chromatography⁻mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the identity of the bioactive compound was determined as a 3-alkylpyridinium with m/z = 190.16. Diffusion Ordered Spectroscopy (DOSY) NMR and NMR relaxation rate analysis suggest that the bioactive compound forms oligomers of up to 35 kDa. We observed that at 9.4 µg/mL there was up to 40⁻70% inhibitory activity on WNV NS3 protease in orthogonal biochemical assays for solid phase extracts (SPE) of A. chloros. However, the LC-MS purified fragment was effective at inhibiting the protease up to 95% at an approximate amount of 2 µg/mL with negligible cytotoxicity to HeLa cells based on a High-Content Screening (HCS) cytological profiling strategy. To date, 3-alkylpyridinium type natural products have not been reported to show antiviral activity since the first characterization of halitoxin, or 3-alkylpyridinium, in 1978. This study provides the first account of a 3-alkylpyridinium complex that exhibits a proposed antiviral activity by inhibiting the NS3 protease. We suggest that the here-described compound can be further modified to increase its stability and tested in a cell-based assay to explore its full potential as a potential novel antiviral capable of inhibiting WNV replication.

Nucleocytoplasmic shuttling of the West Nile virus RNA-dependent RNA polymerase NS5 is critical to infection.

West Nile virus (WNV) is a single-stranded, positive sense RNA virus of the family Flaviviridae and is a significant pathogen of global medical importance. Flavivirus replication is known to be exclusively cytoplasmic, but we show here for the first time that access to the nucleus of the WNV strain Kunjin (WNVKUN ) RNA-dependent RNA polymerase (protein NS5) is central to WNVKUN virus production. We show that treatment of cells with the specific nuclear export inhibitor leptomycin B (LMB) results in increased NS5 nuclear accumulation in WNVKUN -infected cells and NS5-transfected cells, indicative of nucleocytoplasmic shuttling under normal conditions. We used site-directed mutagenesis to identify the nuclear localisation sequence (NLS) responsible for WNVKUN NS5 nuclear targeting, observing that mutation of this NLS resulted in exclusively cytoplasmic accumulation of NS5 even in the presence of leptomycin B. Introduction of NS5 NLS mutations into FLSDX, an infectious clone of WNVKUN , resulted in lethality, suggesting that the ability of NS5 to traffic into the nucleus in integral to WNVKUN replication. This study thus shows for the first time that NLS-dependent trafficking into the nucleus during infection of WNVKUN NS5 is critical for viral replication. Excitingly, specific inhibitors of NS5 nuclear import reduce WNVKUN virus production, proving the principle that inhibition of WNVKUN NS5 nuclear import is a viable therapeutic avenue for antiviral drug development in the future.

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.

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.

Helicase Domain of West Nile Virus NS3 Protein Plays a Role in Inhibition of Type I Interferon Signalling.

West Nile virus (WNV) is a neurotropic flavivirus that can cause encephalitis in mammalian and avian hosts. In America, the virulent WNV strain (NY99) is causing yearly outbreaks of encephalitis in humans and horses, while in Australia the less virulent Kunjin strain of WNV strain has not been associated with significant disease outbreaks until a recent 2011 large outbreak in horses (but not in humans) caused by NSW2011 strain. Using chimeric viruses between NY99 and NSW2011 strains we previously identified a role for the non-structural proteins of NY99 strain and especially the NS3 protein, in enhanced virus replication in type I interferon response-competent cells and increased virulence in mice. To further define the role of NY99 NS3 protein in inhibition of type I interferon response, we have generated and characterised additional chimeric viruses containing the protease or the helicase domains of NY99 NS3 on the background of the NSW2011 strain. The results identified the role for the helicase but not the protease domain of NS3 protein in the inhibition of type I interferon signalling and showed that helicase domain of the more virulent NY99 strain performs this function more efficiently than helicase domain of the less virulent NSW2011 strain. Further analysis with individual amino acid mutants identified two amino acid residues in the helicase domain primarily responsible for this difference. Using chimeric replicons, we also showed that the inhibition of type I interferon (IFN) signalling was independent of other known functions of NS3 in RNA replication and assembly of virus particles.

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.

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.