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 K_i values of 4.9 and 10.5 μM. 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.     

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 K_m of 3.35 ± 0.31 μM, a k_cat of 0.0717 ± 0.0016 s^?1 and a k_cat/K_m of 21,400 ± 2000 M^?1 s^?1.     

Identification of novel thiadiazoloacrylamide analogues as inhibitors of dengue-2 virus NS2B/NS3 protease

Dengue virus is endemic throughout tropical and subtropical regions, and cause severe epidemic diseases. The NS2B/NS3 protease is a promising drug target for dengue virus. Herein, we report the discovery and modification of a novel class of thiadiazoloacrylamide derivatives with potent inhibitory activity against the NS2B/NS3 protease. Thiadiazolopyrimidinone 1 was firstly determined as a new chemical structure against NS2B/NS3 from a commercial compound library. Then, we sought to identify similar compounds with the thiadiazoloacrylamide core that would exhibit better activity. A series of analogues were synthesized and fourteen of them were identified with strong inhibitory activities, in which the nitrile group in the linker part was discovered as an essential group for the inhibitory activity. The best of these (8b) demonstrated an IC₅₀ at 2.24 μM based on in vitro DENV2 NS2B-NS3pro assays.     

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.     

Synthesis and disulfide bond connectivity–activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B–NS3 protease

Kalata B1 is a plant protein with remarkable thermal, chemical and enzymatic stability. Its potential applications could be centered on the possibility of using its cyclic structure and cystine knot motif as a scaffold for the design of stable pharmaceuticals. To discover potent dengue NS2B–NS3 protease inhibitors, we have prepared various kalata B1 analogues by varying the amino acid sequence. Mass spectrometric and biochemical investigations of these analogues revealed a cyclopeptide whose two fully oxidized forms are substrate-competitive inhibitors of the dengue viral NS2B–NS3 protease. Both oxidized forms showed potent inhibition with K_i of 1.39 ± 0.35 and 3.03 ± 0.75 μM, respectively.     

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.     

Fusion of Protegrin-1 and Plectasin to MAP30 Shows Significant Inhibition Activity against Dengue Virus Replication

Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC₅₀) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.     

Design,synthesis and characterization of novel 1,2-benzisothiazol-3(2H)-one and 1,3,4-oxadiazole hybrid derivatives: Potent inhibitors of Dengue and West Nile virus NS2B/NS3 proteases

1,2-Benzisothiazol-3(2H)-ones and 1,3,4-oxadiazoles individually have recently attracted considerable interest in drug discovery, including as antibacterial and antifungal agents. In this study, a series of functionalized 1,2-benzisothiazol-3(2H)-one—1,3,4-oxadiazole hybrid derivatives were synthesized and subsequently screened against Dengue and West Nile virus proteases. Ten out of twenty-four compounds showed greater than 50% inhibition against DENV2 and WNV proteases ([I] = 10 μM). The IC₅₀ values of compound 7n against DENV2 and WNV NS2B/NS3 were found to be 3.75 ± 0.06 and 4.22 ± 0.07 μM, respectively. The kinetics data support a competitive mode of inhibition by compound 7n. Molecular modeling studies were performed to delineate the putative binding mode of this series of compounds. This study reveals that the hybrid series arising from the linking of the two scaffolds provides a suitable platform for conducting a hit-to-lead optimization campaign via iterative structure–activity relationship studies, in vitro screening and X-ray crystallography.     

Structure–activity relationship and improved hydrolytic stability of pyrazole derivatives that are allosteric inhibitors of West Nile Virus NS2B-NS3 proteinase

West Nile Virus (WNV) is a potentially deadly mosquito-borne flavivirus which has spread rapidly throughout the world. Currently there is no effective vaccine against flaviviral infections. We previously reported the identification of pyrazole ester derivatives as allosteric inhibitors of WNV NS2B-NS3 proteinase. These compounds degrade rapidly in pH 8 buffer with a half life of 1–2 h. We now report the design, synthesis and in vitro evaluation of pyrazole derivatives that are inhibitors of WNV NS2B-NS3 proteinase with greatly improved stability in the assay medium.     

Flavonoid from Carica papaya inhibits NS2B-NS3 protease and prevents Dengue 2 viral assembly

Dengue virus belongs to the virus family Flaviviridae. Dengue hemorrhagic disease caused by dengue virus is a public health problem worldwide. The viral non structural 2B and 3 (NS2B-NS3) protease complex is crucial for virus replication and hence, it is considered to be a good anti-viral target. Leaf extracts from Carica papaya is generally prescribed for patients with dengue fever, but there are no scientific evidences for its anti-dengue activity; hence we intended to investigate the anti-viral activity of compounds present in the leaves of Carica papaya against dengue 2 virus (DENV-2). We analysed the anti-dengue activities of the extracts from Carica papaya by using bioinformatics tools. Interestingly, we find the flavonoid quercetin with highest binding energy against NS2B-NS3 protease which is evident by the formation of six hydrogen bonds with the amino acid residues at the binding site of the receptor. Our results suggest that the flavonoids from Carica papaya have significant anti-dengue activities.

Abbreviations

ADME - Absorption, distribution, metabolism and excretion, BBB - Blood brain barrier, CYP - Cytochrome P450, DENV - – Dengue virus, DHF - Dengue hemorrhagic fever, DSS - Dengue shock syndrome, GCMS - – Gas chromatography- Mass spectrometry, MOLCAD - Molecular Computer Aided Design, NS - Non structural, PDB - Protein data bank, PMF - Potential Mean Force.     

Psychrophilic α-amylase from Aeromonas veronii NS07 isolated from farm soils

Among 120 isolates examined in this study, three isolates were selected for amylase production on starch agar plates following incubation at 10 °C. Identification by 16SrRNA on selected bacterium disclosed the highest similarity for protean regions of this gene as Aeromonas veronii NS07. A 63 kDa psychrophilic amylase enzyme from NS07 strain was purified by two-steps chromatography. The enzyme had the highest specific activity at pH 4 and was active at the range of temperatures from 0 to 50 °C, although the optimum temperature for enzyme activity was found at 10 °C. Analysis of the N-terminal amino acid sequencing disclosed 20 amino acids from purified amylase which had no similarity with other known α-amylases, indicating that the presented enzyme was novel. Amylase activity was enhanced in relation to optimum activity with the presence of sodium sulphate (161%), MnCl₂ (298%), CaCl₂ (175%), FeCl₂ (182%), MgCl₂ (237%), ZnCl₂ (169%), NiCl₂ (139%), NaCl (158%), each at 5 mM, while EDTA, phenylmethane sulphonylfluoride (PMSF) (3 mM), urea (8 M) and SDS (1%) inhibited the enzyme up to 5%, 2%, 80% and 18%, respectively. NS07 strain seems to be suitable as biocatalyst for practical use in liquefaction of starch at low temperatures, detergent and textile industries.     

Novel Peptides Inhibit Zika NS2B-NS3 Serine Protease and Virus Replication in Human Hepatic Cell Line

Zika virus (ZIKV) is a Flavivirus associated with several neurological complications. Currently, there are no vaccines or cures available and an efficient antiviral treatment is urgently needed to combat ZIKV infection. Herein, we targeted ZIKV NS2B-NS3 serine protease with short peptides to inhibit ZIKV replication in human hepatic cell line (WRL-68). The short peptide inhibitors were designed using Hyperchem 8.0.10 software. Docking energy and binding configuration were calculated using HADDOCK webserver. ZIKV NS2B-NS3 protease was produced as a recombinant single peptide in Escherichia coli and the protease activity was examined by measuring the cleavage of a fluorescent substrate in the presence of the peptides or aprotinin as a standard protease inhibitor. Computational analysis revealed that the short peptides, AYA2 and AYA9, exhibited lower docking energy to ZIKV protease than aprotinin. Both peptides also possessed lower half maximal inhibitory concentration (IC₅₀), 30.9 and 22.1 µM respectively, against ZIKV protease activity when compared to aprotinin (35.4 µM). Interestingly, AYA2 and AYA9 exhibited minimal cytotoxic effects in WRL-68 cells and showed considerable inhibition against ZIKV replication in vitro at half maximal effective concentration (EC₅₀) of 40.73?±?2.3 µM and 34.65?±?1.8 µM respectively. Fusion of these two peptides to MAP30 peptide substantially reduced the IC₅₀ of ZIKV protease inhibition to 1.1 µM and inhibited ZIKV replication at EC₅₀ of 0.5157?±?0.03 µM. In sum, we reported novel peptides that effectively inhibited ZIKV replication in vitro. This study represents a cost-effective strategy of developing peptide inhibitors by shortening the peptides and producing them in recombinant form.

     

Design,synthesis, X-ray studies,and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1′-P2′ ligands

Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1′-P2′ tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (K_i = 13.2 nM, IC₅₀ = 22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (K_i = 62 pM and 14 pM, respectively) and antiviral activity (IC₅₀ = 5.3 nM and 2.0 nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.     

Analogs design,synthesis and biological evaluation of peptidomimetics with potential anti-HCV activity

Two series of peptidomimetics were designed, prepared and evaluated for their anti-HCV activity. One series possesses a C-terminal carboxylate functionality. In the other series, the electrophilic vinyl sulfonate moiety was introduced as a novel class of HCV NS3/4A protease inhibitors. In vitro based studies were then performed to evaluate the efficacies of the inhibitors using Human hepatoma cells, with the vinyl sulfonate ester (10) in particular, found to have highly potent anti-HCV activity with an EC₅₀ = 0.296 μM. Finally, molecular modeling studies were performed through docking of the synthesized compounds in the HCV NS3/4A protease active site to assess their binding modes with the enzyme and gain further insight into their structure–activity relationships.     

Antibacterial and anticancer activity of a series of novel peptides incorporating cyclic tetra-substituted Cα amino acids

Eleven antimicrobial peptides (AMP) based on the incorporation of cyclic tetra substituted C^α amino acids, as well as other unnatural amino acids were designed, synthesized and screened for in vitro activity against 18 strains of bacteria as well as 12 cancer cell lines. The AMPs discussed herein are derived from the following peptide sequence: Ac-GF(X)G(X)B(X)G(X)F(X)G(X)GB(X)BBBB-amide, X = any one of the following residues, A5c, A6c, Tic or Oic and B = any one of the following residues, Arg, Lys, Orn, Dpr or Dab. A diversity of in vitro inhibitory activity was observed for these AMPs. Several analogs exhibited single digit μM activity against drug resistant bacteria including; multiple drug resistant Mycobacterium tuberculosis, extremely drug resistant Mycobacterium tuberculosis and MRSA. The physicochemical properties of the basic amino acid residues incorporated into these AMPs seem to play a major role in defining antibacterial activity. Overall hydrophobicity seems to play a limited role in defining antibacterial activity. The ESKAPE pathogens were used to compare the activity of these AMPs to another family of synthetic AMPs incorporating the unnatural amino acids Tic and Oic. In most cases similarly substituted members of both families exhibited similar inhibitory activity against the ESKAPE pathogens. In specific cases differences in activity as high as 15 fold were observed between analogs. In addition four of these AMPs exhibited promising IC₅₀ (<7.5 μM) values against 12 different and diverse cancer cell lines. Five other AMPs exhibited promising IC₅₀ (<7.5 μM) values against selected cancer cell lines.     

Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue

NS5 is the largest and most conserved protein among the four dengue virus (DENV) serotypes. It has been the target of interest for antiviral drug development due to its major role in replication. NS5 consists of two domains, the N-terminal methyltransferase domain and C-terminal catalytic RNA-dependent RNA polymerase (RdRp) domain. It is an unstable protein and is prone to inactivation upon prolonged incubation at room temperature, thus affecting the inhibitor screening assays. In the current study, we expressed and purified DENV RdRp alone in Esherichia coli (E. coli) cells. The N-terminally His-tagged construct of DENV RdRp was transformed into E. coli expression strain BL-21 (DE3) pLysS cells. Protein expression was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mM. The induced cultures were then grown for 20 h at 18 °C and cells were harvested by centrifugation at 6000 x g for 15 min at 4 °C. The recombinant protein was purified using HisTrap affinity column (Ni-NTA) and then the sample was subjected to size exclusion chromatography, which successfully removed the degradation product obtained during the previous purification step. The in vitro polymerase activity of RdRp was successfully demonstrated using homopolymeric polycytidylic acid (poly(rC)) RNA template. This study describes the high level production of enzymatically active DENV RdRp protein which can be used to develop assays for testing large number of compounds in a high-throughput manner. RdRp has the de novo initiation activity and the in vitro polymerase assays for the protein provide a platform for highly robust and efficient antiviral compound screening systems.     

Isosorbide-based peptidomimetics as inhibitors of hepatitis C virus serine protease

Hepatitis C infection is a cause of chronic liver diseases such as cirrhosis and carcinoma. The current therapy for hepatitis C has limited efficacy and low tolerance. The HCV encodes a serine protease which is critical for viral replication, and few protease inhibitors are currently on the market. In this paper, we describe the synthesis and screening of novel isosorbide-based peptidomimetic inhibitors, in which the compounds 1d, 1e, and 1i showed significant inhibition of the protease activity in vitro at 100 µM. The compound 1e also showed dose-response (IC₅₀ = 36 ± 3 µM) and inhibited the protease mutants D168A and V170A at 100 µM, indicating it as a promising inhibitor of the HCV NS3/4A protease. Our molecular modeling studies suggest that the activity of 1e is associated with a change in the interactions of S2 and S4 subsites, since that the increased flexibility favors a decrease in activity against D168A, whereas the appearance of a hydrophobic cavity in the S4 subsite increase the inhibition against V170A strain.     

A Crystal Structure of the Dengue Virus Non-structural Protein 5 (NS5) Polymerase Delineates Interdomain Amino Acid Residues That Enhance Its Thermostability and de Novo Initiation Activities

The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263–271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1–4 proteins and improves their de novo polymerase initiation activities by enhancing the turnover of the RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication. We report the structure at 2.6-Å resolution of an RdRp fragment from DENV3 spanning residues 265–900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272–900) reported previously. This new orthorhombic crystal form (space group P2₁2₁2) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed “preinitiation” conformation similar to the one that was captured previously in space group C222₁ with one molecule per asymmetric unit. The structure reveals that residues 269–271 interact with the RdRp domain and suggests that residues 263–268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp.     

Comparison of the susceptibility of porcine and human dipeptidyl-peptidase IV to inhibition by protein-derived peptides

The enzyme dipeptidyl-peptidase IV (DPP-IV) is recognized to be a promising target for the management of type 2 diabetes. Over the last decade, numerous synthetic molecules and more recently, peptides from dietary proteins, have been reported to be able to inhibit DPP-IV activity. Most studies that have investigated the in vitro effect of these inhibitors have used porcine or human DPP-IV. Although structurally alike, it is unclear whether these two species display similar inhibition patterns. Therefore, the objective of this study was to compare the effects of protein-derived peptides on the activity of porcine and recombinant human DPP-IV. The two species showed different inhibition susceptibility to 43 of the 62 peptide sequences investigated. While 37 protein-derived peptides were more effective at inhibiting the porcine DPP-IV, only six caused a stronger inhibition of the activity of the human enzyme. Although the peptides WR, IPIQY and WCKDDQNPHS were found to be among the most potent inhibitors of both species, the inhibitory effect was greater on the porcine enzyme than on human DPP-IV (αK_i or K_i = 11.5, 13.4, 13.3 μM and 31.4, 28.2, 75.0 μM for porcine and human DPP-IV, respectively). Investigation into the mode of action of the most effective inhibitory peptides revealed that both species were inhibited in a similar manner by short fragments (≤5 amino acid residues), but that some of the longer peptides acted differently on the enzymes. This study shows that porcine DPP-IV is generally inhibited with greater potency by protein-derived peptides than is the human enzyme.     

Antiviral activity of benzimidazole derivatives. II. Antiviral activity of 2-phenylbenzimidazole derivatives

Seventy-six 2-phenylbenzimidazole derivatives were synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. The most commonly affected viruses were, in decreasing order, CVB-2, BVDV, Sb-1, HSV-1, and YFV, while HIV-1 and VSV were not affected, and RSV, VV and Reo-1 were only susceptible to a few compounds. Thirty-nine compounds exhibited high activity (EC₅₀ = 0.1–10 μM) against at least one virus, and four of them were outstanding for their high and selective activity against VV (24, EC₅₀ = 0.1 μM) and BVDV (50, 51, and 53 with EC₅₀ = 1.5, 0.8, and 1.0 μM, respectively). The last compounds inhibited at low micromolar concentrations the NS5B RdRp of BVDV and also of HCV, the latter sharing structural similarity with the former. The considered compounds represent attractive leads for the development of antiviral agents against poxviruses, pestiviruses and even HCV, which are important human and veterinary pathogens.