Influenza virus non-structural protein 1 (NS1) counteracts host antiviral innate immune responses by inhibiting Retinoic acid inducible gene-I (RIG-I) activation. However, whether NS1 also specifically regulates RIG-I transcription is unknown. Here, we identify a CCAAT/Enhancer Binding Protein beta (C/EBPβ) binding site in the RIG-I promoter as a repressor element, and show that NS1 promotes C/EBPβ phosphorylation and its recruitment to the RIG-I promoter as a C/EBPβ/NS1 complex. C/EBPβ overexpression and siRNA knockdown in human lung epithelial cells resulted in suppression and activation of RIG-I expression respectively, implying a negative regulatory role of C/EBPβ. Further, C/EBPβ phosphorylation, its interaction with NS1 and occupancy at the RIG-I promoter was associated with RIG-I transcriptional inhibition. These findings provide an important insight into the molecular mechanism by which influenza NS1 commandeers RIG-I transcriptional regulation and suppresses host antiviral responses.
A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus. The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein. A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein. The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region. Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein. The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests.
The risk of liver cancer in patients infected with the hepatitis B virus (HBV) and their clinical response to interferon alpha therapy vary based on the HBV genotype. The mechanisms underlying these differences in HBV pathogenesis remain unclear. In HepG2 cells transfected with a mutant HBV(G2335A) expression plasmid that does not transcribe the 2.2-kb doubly spliced RNA (2.2DS-RNA) expressed by wild-type HBV genotype A, the level of HBV pregenomic RNA (pgRNA) was higher than that in cells transfected with an HBV genotype A expression plasmid. By using cotransfection with HBV genotype D and 2.2DS-RNA expression plasmids, we found that a reduction of pgRNA was observed in the cells even in the presence of small amounts of the 2.2DS-RNA plasmid. Moreover, ectopic expression of 2.2DS-RNA in the HBV-producing cell line 1.3ES2 reduced the expression of pgRNA. Further analysis showed that exogenously transcribed 2.2DS-RNA inhibited a reconstituted transcription in vitro. In Huh7 cells ectopically expressing 2.2DS-RNA, RNA immunoprecipitation revealed that 2.2DS-RNA interacted with the TATA-binding protein (TBP) and that nucleotides 432 to 832 of 2.2DS-RNA were required for efficient TBP binding. Immunofluorescence experiments showed that 2.2DS-RNA colocalized with cytoplasmic TBP and the stress granule components, G3BP and poly(A)-binding protein 1 (PABP1), in Huh7 cells. In conclusion, our study reveals that 2.2DS-RNA acts as a repressor of HBV transcription through an interaction with TBP that induces stress granule formation. The expression of 2.2DS-RNA may be one of the viral factors involved in viral replication, which may underlie differences in clinical outcomes of liver disease and responses to interferon alpha therapy between patients infected with different HBV genotypes.
Arachidonic acid and its metabolites have generated high level of interest among researchers due to their vital role in inflammation. The inhibition of enzymes involved in arachidonic acid metabolism has been considered as synergistic anti-inflammatory effect. A series of novel α,β-unsaturated carbonyl based compounds were synthesized and evaluated for their inhibitory activity on secretory phospholipase A₂ (sPLA₂), cyclooxygenases (COX), soybean lipoxygenase (LOX) in addition to proinflammatory cytokines comprising IL-6 and TNF-α. Six α,β-unsaturated carbonyl based compounds (2, 3, 4, 12, 13 and 14) exhibited strong inhibition of sPLA₂ activity, with IC₅₀ values in the range of 2.19-8.76 μM. Nine compounds 1-4 and 10-14 displayed inhibition of COX-1 with IC₅₀ values ranging from 0.37 to 1.77 μM (lower than that of reference compound), whereas compounds 2, 10, 13 and 14 strongly inhibited the COX-2. The compounds 10-14 exhibited strong inhibitory activity against LOX enzyme. All compounds were evaluated for the inhibitory activities against LPS-induced TNF-α and IL-6 release in the macrophages. On the basis of screening results, five active compounds 3, 4, 12, 13 and 14 were found strong inhibitors of TNF-α and IL-6 release in a dose-dependent manner. Molecular docking experiments were performed to clarify the molecular aspects of the observed COX and LOX inhibitory activities of the investigated compounds. Present findings increases the possibility that these α,β-unsaturated carbonyl based compounds might serve as beneficial starting point for the design and development of improved anti-inflammatory agents.
ProBiS is a new method to identify the binding site of protein through local structural alignment against the nonredundant Protein Data Bank (PDB), which may result in unique findings compared to the energy-based, geometry-based, and sequence-based predictors. In this work, binding sites of Hemagglutinin (HA), which is an important target for drugs and vaccines in influenza treatment, have been revisited by ProBiS. For the first time, the identification of conserved binding sites by local structural alignment across all subtypes and strains of HA available in PDB is presented. ProBiS finds three distinctive conserved sites on HA's structure (named Site 1, Site 2, and Site 3). Compared to other predictors, ProBiS is the only one that accurately defines the receptor binding site (Site 1). Apart from that, Site 2, which is located slightly above the TBHQ binding site, is proposed as a potential novel conserved target for membrane fusion inhibitor. Lastly, Site 3, located around Helix A at the stem domain and recently targeted by cross-reactive antibodies, is predicted to be conserved in the latest H7N9 China 2013 strain as well. The further exploration of these three sites provides valuable insight in optimizing the influenza drug and vaccine development.
We previously showed that 2,6-bis-(4-hydroxyl-3-methoxybenzylidine)cyclohexanone (BHMC), suppressed the synthesis of various proinflammatory mediators. In this study we explain the mechanism of action of BHMC in lipopolysaccharide (LPS)-induced U937 monocytes and further show that BHMC prevents lethality of CLP-induced sepsis. BHMC showed dose-dependent inhibitory effects on p38, JNK and ERK 1/2 activity as determined by inhibition of phosphorylation of downstream transcription factors ATF-2, c-Jun and Elk-1 respectively. Inhibition of these transcription factors subsequently caused total abolishment of AP-1-DNA binding. BHMC inhibited p65 NF-κB nuclear translocation and DNA binding of p65 NF-κB only at the highest concentration used (12.5μM) but failed to alter phosphorylation of JNK, ERK1/2 and STAT-1. Since the inhibition of p38 activity was more pronounced we evaluated the possibility that BHMC may bind to p38. Molecular docking experiments confirmed that BHMC fits well in the highly conserved hydrophobic pocket of p38 MAP kinase. We also show that BHMC was able to improve survival from lethal sepsis in a murine caecal-ligation and puncture (CLP) model.
Crystal structure analysis of the zinc complex establishes it as a distorted octahedral complex, bis(3-methylpicolinato-kappa(2) N,O)(2)(1,10-phenanthroline-kappa(2) N,N)-zinc(II) pentahydrate, [Zn(3-Me-pic)(2)(phen)]x5H(2)O. The trans-configuration of carbonyl oxygen atoms of the carboxylate moieties and orientation of the two planar picolinate ligands above and before the phen ligand plane seems to confer DNA sequence recognition to the complex. It cannot cleave DNA under hydrolytic condition but can slightly be activated by hydrogen peroxide or sodium ascorbate. Circular Dichroism and Fluorescence spectroscopic analysis of its interaction with various duplex polynucleotides reveals its binding mode as mainly intercalation. It shows distinct DNA sequence binding selectivity and the order of decreasing selectivity is ATAT > AATT > CGCG. Docking studies lead to the same conclusion on this sequence selectivity. It binds strongly with G-quadruplex with human tolemeric sequence 5'-AG(3)(T(2)AG(3))(3)-3', can inhibit topoisomerase I efficiently and is cytotoxic against MCF-7 cell line.
A method to map the specific site on dengue virus envelope protein (E) that interacts with cells and a neutralizing antibody is developed using serially truncated dengue virus type 2 (DENV-2) E displayed on M13 phages as recombinant E-g3p fusion proteins. Recombinant phages displaying the truncated E consisting of amino acids 297-423 (EB2) and amino acids 379-423 (EB4) were neutralized by DENV-2 patient sera and the DENV-2 E-specific 3H5-1 monoclonal antibodies suggesting that the phages retained the DENV-2 E antigenic properties. The EB4 followed by EB2 recombinant phages bound the most to human monocytes (THP-1), African green monkey kidney (Vero) cells, mosquito (C6/36) cells, ScFv specific against E and C6/36 cell proteins. Two potential cell attachment sites were mapped to loop I (amino acids 297 to 312) and loop II (amino acids 379-385) of the DENV-2 E using the phage-displayed truncated DENV-2 E fragments and by the analysis of the E structure. Loop II was present only in EB4 recombinant phages. There was no competition for binding to C6/36 cell proteins between EB2 and EB4 phages. Loop I and loop II are similar to the sub-complex specific and type-specific neutralizing monoclonal antibody binding sites, respectively. Hence, it is proposed that binding and entry of DENV involves the interaction of loop I to cell surface glycosaminoglycan-motif and a subsequent highly specific interaction involving loop II with other cell proteins. The phage displayed truncated DENV-2 E is a powerful and useful method for the direct determination of DENV-2 E cell binding sites.
Peroxisome proliferator activated receptor alpha has been implicated as a regulator of acute phase response genes in hepatocytes. Interleukin-6 is widely known as a major cytokine responsible in the regulation of acute phase proteins and, therefore, acute phase response. Unfortunately, to date, very little is understood about the molecular mechanisms by which interleukin-6 regulates the gene expression of peroxisome proliferator activated receptor alpha. Here, we report the molecular mechanisms by which peroxisome proliferator activated receptor alpha was regulated by interleukin-6 in human HepG2 cells. Interleukin-6 was shown to down-regulate the peroxisome proliferator activated receptor alpha gene expression at the level of gene transcription. Functional dissection of human peroxisome proliferator activated receptor alpha promoter B revealed the role of predicted CCAAT/enhancer-binding protein binding site (-164/+34) in mediating the interleukin-6 inhibitory effects on peroxisome proliferator activated receptor alpha mRNA expression and electrophoretic mobility shift assay showed the binding of CCAAT/enhancer-binding protein isoforms to this cis-acting elements was increased in interleukin-6-treated HepG2 cells. Co-transfection experiments, then, demonstrated that CCAAT/enhancer-binding protein beta either in homodimer or heterodimer with CCAAT/enhancer-binding protein alpha and CCAAT/enhancer-binding protein delta plays a predominant role in inhibiting the transcriptional activity of peroxisome proliferator activated receptor alpha promoter B, thus, reducing the peroxisome proliferator activated receptor alpha mRNA expression. These studies, therefore, suggest a novel mechanism for interleukin-6-mediated inhibition of peroxisome proliferator activated receptor alpha gene expression that involves the activation of CCAAT/enhancer-binding protein isoforms with CCAAT/enhancer-binding protein beta may play a major role.
Various works have been carried out in developing therapeutics against dengue. However, to date, no effective vaccine or anti-dengue agent has yet been discovered. The development of protease inhibitors is considered as a promising option, but most previous works have involved competitive inhibition. In this study, we focused on rational discovery of potential anti-dengue agents based on non-competitive inhibition of DEN-2 NS2B/NS3 protease. A homology model of the DEN-2 NS2B/NS3 protease (using West Nile Virus NS2B/NS3 protease complex, 2FP7, as the template) was used as the target, and pinostrobin, a flavanone, was used as the standard ligand. Virtual screening was performed involving a total of 13 341 small compounds, with the backbone structures of chalcone, flavanone, and flavone, available in the ZINC database. Ranking of the resulting compounds yielded compounds with higher binding affinities compared with the standard ligand. Inhibition assay of the selected top-ranking compounds against DEN-2 NS2B/NS3 proteolytic activity resulted in significantly better inhibition compared with the standard and correlated well with in silico results. In conclusion, via this rational discovery technique, better inhibitors were identified. This method can be used in further work to discover lead compounds for anti-dengue agents.
In the designed research work, a series of 2-furoyl piperazine based sulfonamide derivatives were synthesized as therapeutic agents to target the Alzheimer's disease. The structures of the newly synthesized compounds were characterized through spectral analysis and their inhibitory potential was evaluated against butyrylcholinesterase (BChE). The cytotoxicity of these sulfonamides was also ascertained through hemolysis of bovine red blood cells. Furthermore, compounds were inspected by Lipinki Rule and their binding profiles against BChE were discerned by molecular docking. The protein fluctuations in docking complexes were recognized by dynamic simulation. From our in vitro and in silico results 5c, 5j and 5k were identified as promising lead compounds for the treatment of targeted disease.
The alginate lyase AlyQ from Persicobacter sp. CCB-QB2 is a three-domained enzyme with a carbohydrate-binding module (CBM) from family 32. The CBM32 domain, AlyQB, binds enzymatically cleaved but not intact alginate. Co-crystallisation of AlyQB with the cleaved alginate reveals that it binds to the 4,5-unsaturated mannuronic acid of the non-reducing end. The binding pocket contains a conserved R248 that interacts with the sugar's carboxyl group, as well as an invariant W303 that stacks against the unsaturated pyranose ring. Targeting specifically the non-reducing end is more efficient than the reducing end since the latter consists of a mixture of mannuronic acid and guluronic acid. AlyQB also seems unable to bind these two saturated sugars as they contain OH groups that will clash with the pocket. Docking analysis of YeCBM32, which binds oligogalacturonic acid, shows that the stacking of the pyranose ring is shifted in order to accommodate the sugar's axial C1-OH, and its R69 is accordingly elevated to bind the sugar's carboxyl group. Unlike AlyQB, YeCBM32's binding pocket is able to accommodate both saturated and unsaturated galacturonic acid.
Development of multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis (TB) has been considered as major health burden, globally. In order to develop novel, potential molecules against drug resistant TB, twenty two (22) new 3-substituted-7-benzyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6a-k) and 3-substituted-7-benzyl-2-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7a-k) derivatives were designed and synthesized by using appropriate synthetic protocols. Pantothenate synthetase (PS) was considered as the target for the molecular docking studies and evaluated the binding pattern at active site, as PS plays a significant role in the biosynthesis of pantothenate in Mycobacterium tuberculosis (MTB). The preliminary in vitro antibacterial screening of test compounds was carried out against two strains of Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria. The antimycobacterial screening was performed against MTB H37Rv and an isoniazid-resistant clinical isolate of MTB. The compounds 6b, 6c, 6d, 6k, 7b, 7c, 7d and 7k exhibited promising antibacterial activity MIC in the range of 15-73 μM against all bacterial strains used and compounds 6d and 7b showed antimycobacterial activity (IC50 <340 μM in LRP assay) and (MIC <9 μM in broth microdilution method).
Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.
A high incidence of sensitization to Blomia tropicalis, the predominant house dust mite species in tropical regions, is strongly associated with allergic diseases in Singapore, Malaysia, and Brazil. IgE binding to the group 5 allergen, Blo t 5, is found to be the most prevalent among all B. tropicalis allergens. The NMR structure of Blo t 5 determined represents a novel helical bundle structure consisting of three antiparallel alpha-helices. Based on the structure and sequence alignment with other known group 5 dust mite allergens, surface-exposed charged residues have been identified for site-directed mutagenesis and IgE binding assays. Four charged residues, Glu76, Asp81, Glu86, and Glu91 at around the turn region connecting helices alpha2 and alpha3 have been identified to be involved in the IgE binding. Using overlapping peptides, we have confirmed that these charged residues are located on a major putative linear IgE epitope of Blo t 5 from residues 76-91 comprising the sequence ELKRTDLNILERFNYE. Triple and quadruple mutants have been generated and found to exhibit significantly lower IgE binding and reduced responses in skin prick tests. The mutants induced similar PBMC proliferation as the wild-type protein but with reduced Th2:Th1 cytokines ratio. Mass screening on a quadruple mutant showed a 40% reduction in IgE binding in 35 of 42 sera of atopic individuals. Findings in this study further stressed the importance of surface-charged residues on IgE binding and have implications in the cross-reactivity and use of Blo t 5 mutants as a hypoallergen for immunotherapy.
Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.
MTH1 is an enzyme that hydrolyzes 8-oxo-dGTP, which is an oxidatively damaged nucleobase, into 8-oxo-dGMP in nucleotide pools to prevent its mis-incorporation into genomic DNA. Selective and potent MTH1-binding molecules have potential as biological tools and drug candidates. We recently developed 8-halogenated 7-deaza-dGTP as an 8-oxo-dGTP mimic and found that it was not hydrolyzed, but inhibited enzyme activity. To further increase MTH1 binding, we herein designed and synthesized 7,8-dihalogenated 7-deaza-dG derivatives. We successfully synthesized multiple derivatives, including substituted nucleosides and nucleotides, using 7-deaza-dG as a starting material. Evaluations of the inhibition of MTH1 activity revealed the strong inhibitory effects on enzyme activity of the 7,8-dihalogenated 7-deaza-dG derivatives, particularly 7,8-dibromo 7-daza-dGTP. Based on the results obtained on kinetic parameters and from computational docking simulating studies, these nucleotide analogs interacted with the active site of MTH1 and competitively inhibited the substrate 8-oxodGTP. Therefore, novel properties of repair enzymes in cells may be elucidated using new compounds.
The CDKN2A (p16) gene plays a key role in pancreatic cancer etiology. It is one of the most commonly somatically mutated genes in pancreatic cancer, rare germline mutations have been found to be associated with increased risk of developing familiar pancreatic cancer and CDKN2A promoter hyper-methylation has been suggested to play a critical role both in pancreatic cancer onset and prognosis. In addition several unrelated SNPs in the 9p21.3 region, that includes the CDNK2A, CDNK2B and the CDNK2B-AS1 genes, are associated with the development of cancer in various organs. However, association between the common genetic variability in this region and pancreatic cancer risk is not clearly understood. We sought to fill this gap in a case-control study genotyping 13 single nucleotide polymorphisms (SNPs) in 2,857 pancreatic ductal adenocarcinoma (PDAC) patients and 6,111 controls in the context of the Pancreatic Disease Research (PANDoRA) consortium. We found that the A allele of the rs3217992 SNP was associated with an increased pancreatic cancer risk (ORhet=1.14, 95% CI 1.01-1.27, p=0.026, ORhom=1.30, 95% CI 1.12-1.51, p=0.00049). This pleiotropic variant is reported to be a mir-SNP that, by changing the binding site of one or more miRNAs, could influence the normal cell cycle progression and in turn increase PDAC risk. In conclusion, we observed a novel association in a pleiotropic region that has been found to be of key relevance in the susceptibility to various types of cancer and diabetes suggesting that the CDKN2A/B locus could represent a genetic link between diabetes and pancreatic cancer risk.
We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.
Compounds with activity at serotonin (5-hydroxytryptamine) 5-HT2 and α1 adrenergic receptors have potential for the treatment of central nervous system disorders, drug addiction or overdose. Isolaureline, dicentrine and glaucine enantiomers were synthesized, and their in vitro functional activities at human 5-HT2 and adrenergic α1 receptor subtypes were evaluated. The enantiomers of isolaureline and dicentrine acted as antagonists at 5-HT2 and α1 receptors with (R)-isolaureline showing the greatest potency (pKb = 8.14 at the 5-HT2C receptor). Both (R)- and (S)-glaucine also antagonized α1 receptors, but they behaved very differently to the other compounds at 5-HT2 receptors: (S)-glaucine acted as a partial agonist at all three 5-HT2 receptor subtypes, whereas (R)-glaucine appeared to act as a positive allosteric modulator at the 5-HT2A receptor.