Nematicidal activity of paucimannose-type glycoconjugates from acacia honey.

Natural honey contains glycoconjugates as minor components. We characterized acacia honey glycoconjugates with molecular masses in the range of 2-5 kDa. The glycoconjugates were separated by RP-HPLC into three peaks (termed RP-2-5 k-I, RP-2-5 k-II, and RP-2-5 k-III) which demonstrated paralyzing effects on the model nematode C. elegans (ED50 of 50 ng glycoconjugates/µL). To examine molecular mechanisms underlying the nematicidal effects of honey glycoconjugates, expressional analyses of genes that are essential for the growth, development, reproduction, and movement of C. elegans were carried out. Quantitative PCR-based assays showed that these molecules moderately regulate the expression of genes involved in the citric acid cycle (mdh-1 and idhg-1) and cytoskeleton (act-1 and act-2). MALDI-ToF-MS/MS analysis of RP-HPLC peaks revealed the presence of paucimannose-like N-glycans which are known to play important roles in invertebrates e.g., worms and flies. These findings provided novel information regarding the structure and nematicidal function of honey glycoconjugates.

Understanding the impact of structural modifications at the NNAT gene's post-translational acetylation site: in silico approach for predicting its drug-interaction role in anorexia nervosa.

PURPOSE: Anorexia nervosa (AN) is a neuropsychological public health concern with a socially disabling routine and affects a person's healthy relationship with food. The role of the NNAT (Neuronatin) gene in AN is well established. The impact of mutation at the protein's post-translational modification (PTM) site has been exclusively associated with the worsening of the protein's biochemical dynamics. METHODS: To understand the relationship between genotype and phenotype, it is essential to investigate the appropriate molecular stability of protein required for proper biological functioning. In this regard, we investigated the PTM-acetylation site of the NNAT gene in terms of 19 other specific amino acid probabilities in place of wild type (WT) through various in silico algorithms. Based on the highest pathogenic impact computed through the consensus classifier tool, we generated 3 residue-specific (K59D, P, W) structurally modified 3D models of NNAT. These models were further tested through the AutoDock Vina tool to compute the molecular drug binding affinities and inhibition constant (Ki) of structural variants and WT 3D models. RESULTS: With trained in silico machine learning algorithms and consensus classifier; the three structural modifications (K59D, P, W), which were also the most deleterious substitution at the acetylation site of the NNAT gene, showed the highest structural destabilization and decreased molecular flexibility. The validation and quality assessment of the 3D model of these structural modifications and WT were performed. They were further docked with drugs used to manage AN, it was found that the ΔGbind (kcal/mol) values and the inhibition constants (Ki) were relatively lower in structurally modified models as compared to WT. CONCLUSION: We concluded that any future structural variation(s) at the PTM-acetylation site of the NNAT gene due to possible mutational consequences, will serve as a basis to explore its relationship with the propensity of developing AN. LEVEL OF EVIDENCE: No level of evidence-open access bioinformatics research.

Characterization of the genome and serine protease of a novel Bacillus subtilis isolate.

Current study was undertaken to carry out the genome-wide analysis of a multipotent isolate from desert soil which was previously identified as Bacillus tequilensis based on 16S rDNA analysis. This study also aims to characterize the serine protease and its biocatalytic potentials implying a combination of empirical and in-silico approaches. Next generation sequencing and short read de novo assembly generated the 4,235,084 bp draft genome of Bacillus sp. ZMS-2. Genome sequence analysis by digital DNA:DNA hybridization (dDDH) and average nucleotide identity classified the isolate as Bacillus subtilis ZMS-2 (Bioproject ID: PRJNA691551). Genome annotation revealed 10 antibiotic resistance genes, 8 antibiotic/antifungal gene clusters and 25 genes encoding proteases including subtilisin E, an extracellular alkaline protease. This extracellular protease (ZMS-2 protease) was produced using a statistically optimized medium, purified partially and characterized as alkaline serine protease. The partially purified ZMS-2 protease (780 U/mL) showed a 21 mm zone of casein hydrolysis and dehaired goat skin by pulling out hair with roots. These catalytic potentials of ZMS-2 protease were further confirmed using scanning electron microscopy of casein beads and dehaired skin. The study concludes B. subtilis ZMS-2 as a potent producer of a protease with promising potentials of commercial importance.

In silico identification of the rare-coding pathogenic mutations and structural modeling of human NNAT gene associated with anorexia nervosa.

PURPOSE: Increased susceptibility towards anorexia nervosa (AN) was reported with reduced levels of neuronatin (NNAT) gene. We sought to investigate the most pathogenic rare-coding missense mutations, non-synonymous single-nucleotide polymorphisms (nsSNPs) of NNAT and their potential damaging impact on protein function through transcript level sequence and structure based in silico approaches. METHODS: Gene sequence, single nucleotide polymorphisms (SNPs) of NNAT was retrieved from public databases and the putative post-translational modification (PTM) sites were analyzed. Distinctive in silico algorithms were recruited for transcript level SNPs analyses and to characterized high-risk rare-coding nsSNPs along with their impact on protein stability function. Ab initio 3D-modeling of wild-type, alternate model prediction for most deleterious nsSNP, validation and recognition of druggable binding pockets were also performed. AN 3D therapeutic compounds that followed rule of drug-likeness were docked with most pathogenic variant of NNAT to estimate the drugs' binding free energies. RESULTS: Conclusively, 10 transcript (201-205)-based nsSNPs from 3 rare-coding missense variants, i.e., rs539681368, rs542858994, rs560845323 out of 840 exonic SNPs were identified. Transcript-based functional impact analyses predicted rs539681368 (C30Y) from NNAT-204 as the high-risk rare-coding pathogenic nsSNP, deviating protein functions. The 3D-modeling analysis of AN drugs' binding energies indicated lowest binding free energy (ΔG) and significant inhibition constant (Ki) with mutant models C30Y. CONCLUSIONS: Mutant model (C30Y) exhibiting significant drug binding affinity and the commonest interaction observed at the acetylation site K59. Thus, based on these findings, we concluded that the identified nsSNP may serve as potential targets for various studies, diagnosis and therapeutic interventions. LEVEL OF EVIDENCE: No level of evidence-open access bioinformatics research.

3-substituted coumarin derivatives over-activate the HslV protease: A potential drug target for antibacterial activity.

The bacterial HslVU complex consists of two different proteins, i.e., the HslV protease and the HslU ATPase. The functional HslVU enzyme complex forms only when the HslU c-terminal helix is inserted into the cavity located between two adjacent HslV monomers in order to allosterically activate the HslV protease. Based on its essential role in maintaining microbial proteostasis as well its absence from human beings, it is considered a promising therapeutic target for designing antibacterial agents. The goal of the present study was to find out potential drug candidates that could over-activate the HslV protease and produce aberrant proteolysis in pathogenic bacteria. Derivatives of 3-substituted coumarin have been identified as potential HslV protease activators based on their highest docking scores, ideal interaction patterns, and significant in-vitro HslV activation potential. Their ED50 values were in the sub-micromolar range, i.e., 0.4-0.48µM. The conformational stability of the contacts between the HslV dimer and the active compounds was further confirmed by molecular dynamics studies. Correspondingly, the ADMET characteristics of these lead molecules considerably demonstrated their significant non-toxic drug-like abilities. This research not only identified small non-peptidic HslV protease activators but also improved the understanding of the mode of action of 3-substituted coumarin derivatives as antibacterials.

Analysis of Inflammatory Gene Expression Profile of Peripheral Blood Leukocytes in Type 2 Diabetes.

The pathophysiology of type 2 diabetes (T2DM) is associated with perturbation of innate immune response. Several studies indicated alteration of pro-inflammatory and anti-inflammatory cytokines, chemokines and other mediators of innate immune response in T2DM. This study was designed to perform quantitative PCR-based expression profiling of genes involved in inflammation (i.e. CASP1, CASP5, CCL5, CXC11, CCR5, NF-Κb, IL-4, PPARG and PGC1α) in peripheral blood leukocytes of T2DM patients. The T2DM patients are often prescribed with metformin and insulin while metformin has also been reported to possess anti-inflammatory activity. To address the question whether metformin exerts any effect on inflammatory mediators in bloodstream, human subjects in this study were divided into four groups on the basis of medication they were taking during last 6 month. These groups included NT-T2DM (T2DM patients not taking medication, n = 34), Met-T2DM (T2DM patients taking metformin, n = 33), INS-T2DM (T2DM patients taking insulin, n = 15) and NGT (normoglycemic subjects, n = 34) groups. Differential expression of gene transcripts at a cutoff of fourfold was considered significant. In the NT-T2DM group, transcripts of inflammation-related genes (i.e. CASP1, CASP5, CCL5, CCR5 and NF-kB) were up-regulated while transcripts of PPARG and PGC1α genes were down-regulated compared to NGT group. On the other hand, down-regulation of CASP1, CASP5, CCL5, CCR5 and NF-kB transcripts was evident in Met-T2DM and INS-T2DM groups when compared to the NT-T2DM group. The Met-T2DM group and INS-T2DM group showed a significant difference in the transcript level of CASP1 and CCL5 which are more down-regulated in the Met-T2DM group compared to INS-T2DM group. These findings indicated that (a) in T2DM, expression of inflammation-related genes is up-regulated and (b) anti-inflammatory activity of metformin appears to be independent of its anti-hyperglycemic activity.

Mutational landscape of head and neck squamous cell carcinomas in a South Asian population.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type globally and contributes significantly to burden of disease in South Asia. In Pakistan, HNSCC is among the most commonly diagnosed cancer in males and females. The increasing regional burden of HNSCC along with a unique set of risk factors merited a deeper investigation of the disease at the genomic level. Whole exome sequencing of HNSCC samples and matched normal genomic DNA analysis (n=7) was performed. Significant somatic single nucleotide variants (SNVs) were identified and pathway analysis performed to determine frequently affected signaling pathways. We identified significant, novel recurrent mutations in ASNS (asparagine synthetase) that may affect substrate binding, and variants in driver genes including TP53, PIK3CA, FGFR2, ARID2, MLL3, MYC and ALK. Using the IntOGen platform, we identified MAP kinase, cell cycle, actin cytoskeleton regulation, PI3K-Akt signaling and other pathways in cancer as affected in the samples. This data is the first of its kind from the Pakistani population. The results of this study can guide a better mechanistic understanding of HNSCC in the population, ultimately contributing new, rational therapeutic targets for the treatment of the disease.

Nematicidal activity of 'major royal jelly protein'-containing glycoproteins from Acacia honey.

Parasitic nematodes infect more than two billion people worldwide particularly in developing countries. We previously reported nematicidal activity of natural honey using model nematode Caenorhabditis elegans. In this study, characterization of nematicidal effects of natural honey and its glycoproteins has been carried out. Chromatographically separated honey glycoproteins showed potent anti-C. elegans activity (LD50 = 100 ng proteins/µL). Honey glycoproteins with molecular masses of ∼260 kD and ∼160 kD comprised of 'major royal jelly protein-1'-containing complexes. In these complexes, MRJP1 was present in different glycosylation forms. Quantitative PCR based gene expression assays described molecular functions of C. elegans affected by honey and honey glycoproteins. Expression of 14 gene transcripts associated with key cellular and molecular functions including energy metabolism, cytoskeleton, cell division, transcription and translation was analyzed. Acacia honey exerted a concentration-dependent alteration of gene transcripts involved in the citric acid cycle (mdh-1 and idhg-1) and cytoskeleton (act-1, act-2, and arp6). Likewise, MRJP1-containing glycoproteins caused down-regulation of arp-6 and idhg-1; and up-regulation of act-1 and mdh-1 gene transcripts. Consistent down-regulation of isocitrate dehydrogenase encoding idhg-1 gene which is among the rate-controlling enzymes of the citric acid cycle was considered as main biochemical factor involved in the nematicidal activity of honey and MRJP-containing glycoproteins. Acacia honey suppressed the expression of gene transcripts encoding actin-2, while honey glycoproteins did not. Hence, honey partly exerted anti-C. elegans activity by decreasing the transcription of actin-2 gene transcripts, demonstrated by a defect in the movement and egg laying. Moreover, arp-6 gene transcripts encoding actin-related protein 6 was significantly and constantly down-regulated by honey and honey proteins.

Carbohydrazones as new class of carbonic anhydrase inhibitors: Synthesis, kinetics, and ligand docking studies.

Discovery and development of carbonic anhydrase inhibitors is crucial for their clinical use as antiepileptic, diurectic and antiglaucoma agents. Keeping this in mind, we have synthesized carbohydrazones 1-27 and evaluated them for their in vitro carbonic anhydrase inhibitory potential. Out of twenty-seven compounds, compounds 1 (IC50=1.33±0.01µM), 2 (IC50=1.85±0.24µM), 3 (IC50=1.37±0.06µM), and 9 (IC50=1.46±0.12µM) have showed carbonic anhydrase inhibition better than the standard drug zonisamide (IC50=1.86±0.03µM). Moreover, compounds 4 (IC50=2.32±0.04µM), 5 (IC50=3.96±0.35µM), 7 (IC50=2.33±0.02µM), and 8 (IC50=2.67±0.01µM) showed good inhibitory activity. Cheminformatic analysis has shown that compounds 1 and 2 possess lead-like properties. In addition, kinetic and molecular docking studies were also performed to investigate the binding interaction between carbohydrazones and carbonic anhydrase enzyme. This study has identified a novel and potent class of carbonic anhydrase inhibitors with the potential to be investigated further.

Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard.

We analyzed the genome sequence of an endophytic bacterial strain Pseudomonas putida TJI51 isolated from mango bark tissues. Next generation DNA sequencing and short read de novo assembly generated the 5,805,096 bp draft genome of P. putida TJI51. Out of 6,036 protein coding genes in P. putida TJI51 sequences, 4,367 (72%) were annotated with functional specifications, while the remaining encoded hypothetical proteins. Comparative genome sequence analysis revealed that the P. putida TJI51genome contains several regions, not identified in so far sequenced P. putida genomes. Some of these regions were predicted to encode enzymes, including acetylornithine deacetylase, betaine aldehyde dehydrogenase, aldehyde dehydrogenase, benzoylformate decarboxylase, hydroxyacylglutathione hydrolase, and uroporphyrinogen decarboxylase. The genome of P. putida TJI51 contained three nonribosomal peptide synthetase gene clusters. Genome sequence analysis of P. putidaTJI51 identified this bacterium as an endophytic resident. The endophytic fitness might be linked with alginate, which facilitates bacterial colonization in plant tissues. Genome sequence analysis shed light on the presence of a diverse spectrum of metabolic activities and adaptation of this isolate to various niches.

Characterization of mango (Mangifera indica L.) transcriptome and chloroplast genome.

We characterized mango leaf transcriptome and chloroplast genome using next generation DNA sequencing. The RNA-seq output of mango transcriptome generated >12 million reads (total nucleotides sequenced >1 Gb). De novo transcriptome assembly generated 30,509 unigenes with lengths in the range of 300 to ≥3,000 nt and 67× depth of coverage. Blast searching against nonredundant nucleotide databases and several Viridiplantae genomic datasets annotated 24,593 mango unigenes (80% of total) and identified Citrus sinensis as closest neighbor of mango with 9,141 (37%) matched sequences. The annotation with gene ontology and Clusters of Orthologous Group terms categorized unigene sequences into 57 and 25 classes, respectively. More than 13,500 unigenes were assigned to 293 KEGG pathways. Besides major plant biology related pathways, KEGG based gene annotation pointed out active presence of an array of biochemical pathways involved in (a) biosynthesis of bioactive flavonoids, flavones and flavonols, (b) biosynthesis of terpenoids and lignins and (c) plant hormone signal transduction. The mango transcriptome sequences revealed 235 proteases belonging to five catalytic classes of proteolytic enzymes. The draft genome of mango chloroplast (cp) was obtained by a combination of Sanger and next generation sequencing. The draft mango cp genome size is 151,173 bp with a pair of inverted repeats of 27,093 bp separated by small and large single copy regions, respectively. Out of 139 genes in mango cp genome, 91 found to be protein coding. Sequence analysis revealed cp genome of C. sinensis as closest neighbor of mango. We found 51 short repeats in mango cp genome supposed to be associated with extensive rearrangements. This is the first report of transcriptome and chloroplast genome analysis of any Anacardiaceae family member.

Genome characterization of a novel Burkholderia cepacia complex genomovar isolated from dieback affected mango orchards.

We characterized the genome of the antibiotic resistant, caseinolytic and non-hemolytic Burkholderia sp. strain TJI49, isolated from mango trees (Mangifera indica L.) with dieback disease. This isolate produced severe disease symptoms on the indicator plants. Next generation DNA sequencing and short-read assembly generated the 60X deep 7,631,934 nucleotide draft genome of Burkholderia sp. TJI49 which comprised three chromosomes and at least one mega plasmid. Genome annotation studies revealed a total 8,992 genes, out of which 8,940 were protein coding genes. Comparative genomics and phylogenetics identified Burkholderia sp. TJI49 as a distinct species of Burkholderia cepacia complex (BCC), closely related to B. multivorans ATCC17616. Genome-wide sequence alignment of this isolate with replicons of BCC members showed conservation of core function genes but considerable variations in accessory genes. Subsystem-based gene annotation identified the active presence of wide spread colonization island and type VI secretion system in Burkholderia sp. TJI49. Sequence comparisons revealed (a) 28 novel ORFs that have no database matches and (b) 23 ORFs with orthologues in species other than Burkholderia, indicating horizontal gene transfer events. Fold recognition of novel ORFs identified genes encoding pertactin autotransporter-like proteins (a constituent of type V secretion system) and Hap adhesion-like proteins (involved in cell-cell adhesion) in the genome of Burkholderia sp. TJI49. The genomic characterization of this isolate provided additional information related to the 'pan-genome' of Burkholderia species.

Identification of potential therapeutic intervening targets by in-silico analysis of nsSNPs in preterm birth-related genes.

Prematurity is the foremost cause of death in children under 5 years of age. Genetics contributes to 25-40% of all preterm births (PTB) yet we still need to identify specific targets for intervention based on genetic pathways. This study involved the effect of region-specific non-synonymous variations and their transcript level mutational impact on protein functioning and stability by various in-silico tools. This investigation identifies potential therapeutic targets to manage the challenge of PTB, corresponding protein cavities and explores their binding interactions with intervening compounds. We searched 20 genes coding 55 PTB proteins from NCBI. Single Nucleotide Polymorphisms (SNPs) of concerned genes were extracted from ENSEMBL, and filtration of exonic variants (non-synonymous) was performed. Several in-silico downstream protein functional effect prediction tools were used to identify damaging variants. Rare coding variants were selected with an allele frequency of ≤1% in 1KGD, further supported by South Asian ALFA frequencies and GTEx gene/tissue expression database. CNN1, COL24A1, IQGAP2 and SLIT2 were identified with 7 rare pathogenic variants found in 17 transcript sequences. The functional impact analyses of rs532147352 (R>H) of CNN1 computed through PhD-SNP, PROVEAN, SNP&GO, PMut and MutPred2 algorithms showed impending deleterious effects, and the presence of this pathogenic mutation in CNN1 resulted in large decrease in protein structural stability (ΔΔG (kcal/mol). After structural protein identification, homology modelling of CNN1, which has been previously reported as a biomarker for the prediction of PTB, was performed, followed by the stereochemical quality checks of the 3D model. Blind docking approach were used to search the binding cavities and molecular interactions with progesterone, ranked with energetic estimations. Molecular interactions of CNN1 with progesterone were investigated through LigPlot 2D. Further, molecular docking experimentation of CNN1 showed the significant interactions at S102, L105, A106, K123, Y124 with five selected PTB-drugs, Allylestrenol (-7.56 kcal/mol), Hydroxyprogesterone caproate (-8.19 kcal/mol), Retosiban (-9.43 kcal/mol), Ritodrine (-7.39 kcal/mol) and Terbutaline (-6.87 kcal/mol). Calponin-1 gene and its molecular interaction analysis could serve as an intervention target for the prevention of PTB.

New benzimidazole derivatives as antiplasmodial agents and plasmepsin inhibitors: synthesis and analysis of structure-activity relationships.

The newly synthesized benzimidazole compounds were suggested to be inhibitors of Plasmodium falciparum plasmepsin II and human cathepsin D by virtual screening of an internal library of synthetic compounds. This was confirmed by enzyme inhibition studies that gave IC(50) values in the low micromolar range (2-48µM). Ligand docking studies with plasmepsin II predicted binding of benzimidazole compounds at the center of the extended substrate-binding cleft. According to the plausible mode of binding, the pyridine ring of benzimidazole compounds interacted with S1' subsite residues whereas the acetophenone moiety was in contact with S1-S3 subsites of plasmepsin II active center. The benzimidazole derivatives were evaluated for capacity to inhibit the growth of intraerythrocytic P. falciparum in culture. Four benzimidazole compounds inhibited parasite growth at ⩽3µM. The most active compound 10, 1-(4-phenylphenyl)-2[2-(pyridinyl-2-yl)-1,3-benzdiazol-1-yl]ethanone showed an IC(50) of 160nM. The substitution of a phenyl group and a chlorine atom at the para position of the acetophenone moiety were shown to be crucial for antiplasmodial activity.

Effect of natural honey on human platelets and blood coagulation proteins.

Present study was conducted to determine the effects of honey on blood hemostasis, in-vitro effect of honey was observed on platelet aggregation and blood coagulation employing, activated partial prothrombin time (aPTT), prothrombin time (PT), thrombin time (TT) and fibrinogen levels in blood. Honey samples showed moderate inhibition of platelet aggregation with IC(50) 5-7.5%. The coagulation assays showed that at higher concentrations (>15%) honey samples increased whole blood clotting time. When assayed in platelet poor plasma (PPP), honey samples significantly (P>0.005) prolonged aPTT, PT, and TT. The honey samples (at 3.75% and 7.5% concentrations) cause mean increment of aPTT = 19±10% and 62±10%; PT 6±5% and 40±5%; TT 35±15% and 112±30% respectively. Moreover, PPP isolated from whole blood pre-incubated with honey samples (9.0% for 10 minutes) showed mean prolongation of aPTT, PT and TT of 45±21%, 26±9% and 105±24% respectively. Interestingly, incubation of honey at 6.25% and 11.75% concentrations in PPP considerably (P≥0.005) reduced fibrinogen levels i.e. 13±4% and 86±30% respectively. The present study outlines the inhibitory effect of natural honey on platelet aggregation and blood coagulation. These observations provide first line data for modulatory role(s) of honey on process of hemostasis.

Characterisation of hydrazides and hydrazine derivatives as novel aspartic protease inhibitors.

Virtual screening of an in-house virtual library of synthetic compounds using FlexX, followed by enzyme inhibition, identified hydrazide and hydrazine derivatives as novel aspartic protease inhibitors. These compounds inhibited human cathepsin D and Plasmodium falciparum plasmepsin-II with low micromolar concentrations (IC(50) = 1-2.5 microM). Modelling studies with plasmepsin-II predicted binding of ligands at the centre of the extended substrate-binding cleft, where hydrazide/hydrazine parts of the inhibitors acted as the transition state mimic by forming electrostatic interactions with catalytic aspartates.

Structural bioinformatics of Vibrio cholerae aminopeptidase A (PepA) monomer.

Aminopeptidase A (PepA) is a metalloexopeptidase found in Vibrio cholerae .It functions as a transcriptional repressor in regulatory cascade that controls virulence gene expression in V. cholerae. It is involved in protein degradation and in the metabolism of biologically active peptides. We proposed a 3D model of PepA based upon the crystal structure of PepA from Escherichia coli (E. coli) with an intention to evaluate the active site of the enzyme and to predict the properties of this enzyme, study of its 3D structure will help in understanding its role in DNA binding.

Exome sequencing identifies a novel frameshift variant causing hypomagnesemia with secondary hypocalcemia.

Hypomagnesemia with secondary hypocalcemia is a rare autosomal-recessive disorder characterized by intense hypomagnesemia associated with hypocalcemia (HSH). Mutations in the TRPM6 gene, encoding the epithelial Mg2+ channel TRPM6, have been proven to be the molecular cause of this disease. This study identified causal mutations in a 2-month-old male patient of hypomagnesemia from a consanguineous marriage. Biochemical analyses indicated the diagnosis of HSH due to primary gastrointestinal loss of magnesium. Whole exome sequencing of the trio (i.e. proband and both parents) was carried out with mean coverage of > 150×. ANNOVAR was used to annotate functional consequences of genetic variation from exome sequencing data. After variant filtering and annotation, a number of single nucleotide variants (SNVs) and 2 bp deletion at exon26:c.4402_4403delCT in TRPM6 gene were identified. This deletion which resulted in a novel frameshift mutation in exon 26 of this gene was confirmed by Sanger sequencing. With these investigations in hand, the patient was managed with magnesium sulphate. The patient remained asymptomatic and was developmentally and neurologically normal till his last follow up.

Identification of acridinyl hydrazides as potent aspartic protease inhibitors.

We have identified acridinyl derivatives as potent aspartic protease inhibitors by virtual screening of in-house library of synthetic compounds. Enzyme inhibition experiments showed that both compounds inhibit human cathepsin D and Plasmodium falciparum plasmepsin-II in nanomolar ranges. The IC(50) values against cathepsin D and plasmepsin-II of compound-Nar103 were found to be 9.0+/-2.0 and 4.0+/-1.0nM and of compound-Nar110 were 0.5+/-0.05 and 0.13+/-0.03nM, respectively. Ligand docking predicted the binding of acridinyl derivatives at the substrate-binding cleft, where hydrazide part of the inhibitors interact with the S1-S1' subsite residues including catalytic aspartates. The phenyl ring and acridinyl moiety of the inhibitors were predicted to interact with S2/S3 and S2'/S3' subsite residues.

Homology modeling of hemagglutinin/protease [HA/P (vibriolysin)] from Vibrio cholerae: sequence comparision, residue interactions and molecular mechanism.

Vibrio cholerae produces a zinc-containing and calcium-stabilized soluble hemagglutinin/protease, which has been earlier shown to have the ability to cleave several physiologically important substrates including mucin, fibronectin and lactoferin. This study presents homology modeling of hemagglutinin/protease (vibriolysin) from Vibrio cholerae in the presence of inhibitor HPI [N-(1-carboxy-3-phenylpropyl)-phenylalanyl-alpha-aspargine]. The 3D structure was predicted based on its sequence homology with Pseudomonas aeruginosa elastase (PAE). Comparison of the 3D structures of PAE and HA/P reveals a remarkable similarity having a conserved alpha + beta domain. The inhibitor shows similar binding features as seen in other metalloproteases of M4 peptidase family. The study also highlights the key catalytic residues as well as the residues at the S1 and S1' binding sub-sites. The similarities between the two proteins provide support for the hypothesis that the two enzymes have similar three-dimensional structures and a common mechanism of action. The fact that both enzymes are secreted as zinc-containing proteases, led us to further hypothesize that they may play similar role in pathogenesis.