Benzothiophene carboxamide derivatives as inhibitors of Plasmodium falciparum enoyl-ACP reductase

Benzothiophene derivatives like benzothiophene sulphonamides, biphenyls, or carboxyls have been synthesized and have found wide pharmacological usage. Here we report, bromo-benzothiophene carboxamide derivatives as potent, slow tight binding inhibitors of Plasmodium enoyl-acyl carrier protein (ACP) reductase (PfENR). 3-Bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) is the most potent inhibitor with an IC50 of 115 nM for purified PfENR. The inhibition constant (Ki) of compound 6 was 18 nM with respect to the cofactor and 91 nM with respect to crotonoyl-CoA. These inhibitors showed competitive kinetics with cofactor and uncompetitive kinetics with the substrate. Thus, these compounds hold promise for the development of potent antimalarials.     

Selection of Optimal Host Strain for Molecular Pathogenesis Studies on <Emphasis Type="Italic">Cryptococcus gattii</Emphasis>

Encapsulated yeast, Cryptococcus gattii (Cg) is a primary and emerging fungal pathogen in North America. It has a predilection for invading the central nervous system of both healthy and immunocompromised humans and animals. Recently, we initiated molecular pathogenesis studies in Cg strain NIH444 (ATCC 32609). In this report, we compared the biology and pathogenic potential of NIH444 to those of WM276, an Australian environmental isolate that is being used for the whole genome-sequencing project. Our data indicated that NIH444 is comparatively more virulent in a mouse model of cryptococcosis than is WM 276. We found robust mating of NIH444, and no mating of WM276, when tested against Cg MATa strain, NIH198. WM276 but not NIH444 was defective in filamentation and sporulation (haploid fruiting). Interestingly, NIH444 has a VGII/AFLP6 genotype similar to that of the genotype of the recent outbreak strains from Vancouver Island, British Columbia, Canada. Additionally, comparisons of nucleotide sequences of various genes also showed differences between NIH444 and WM276. Based on these observations, we conclude that NIH444 should remain the strain of choice for understanding Cg pathogenesis, especially on the North American continent.     

Conformation and activity of delta-lysin and its analogs

Delta-Lysin is a 26-residue hemolytic peptide secreted by Staphylococcus aureus. Unlike the bee venom peptide melittin, delta-lysin does not exhibit antibacterial activity. We have synthesized delta-lysin and several analogs wherein the N-terminal residues of the toxin were sequentially deleted. The toxin has three aspartic acids, four lysines and no prolines. Analogs were also generated in which all the aspartic acids were replaced with lysines. A proline residue was introduced in the native sequences as well as in the analogs where aspartic acids were replaced with lysines. We observed that 20- and 22-residue peptides corresponding to residues 7-26 and 5-26 of delta-lysin, respectively, had greater hemolytic activity than the parent peptide. These shorter peptides, unlike delta-lysin, did not self-associate to adopt alpha-helical conformation in water, at lytic concentrations. Introduction of proline or substitution of aspartic acids by lysines resulted in loss in propensity to adopt helical conformation in water. When proline was introduced in the peptides corresponding to the native toxin sequence, loss of hemolytic activity was observed. Substitution of all the aspartic acids with lysines resulted in enhanced hemolytic activity in all the analogs. However, when both proline and aspartic acid to lysine changes were made, only antibacterial activity was observed in the shorter peptides. Our investigations on delta-lysin and its analogs provide insights into the positioning of anionic, cationic residues and proline in determining hemolytic and antibacterial activities.     

Synthesis of (1,4)-naphthoquinono-[3,2-c]-1H-pyrazoles and their (1,4)-naphthohydroquinone derivatives as antifungal, antibacterial, and anticancer agents

A series of (1,4)-naphthoquinono [3,2-c]-1H-pyrazoles and their (1,4)-naphthohydroquinone derivatives 2-7 were synthesized and evaluated for antifungal, antibacterial, and anticancer activities. The structure-activity relationship of these compounds was studied and the results show that the compound 2b exhibited in vitro antifungal activity against Candida albicans and Cryptococcus neoformans, and also possessed antibacterial profile against Klebsiella pneumoniae and Escherichia coli whereas 1c showed anticancer activity against Walker 256 Carcinosarcoma in rats.     

Neural cell adhesion molecule-associated polysialic acid potentiates alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor currents

The highly negatively charged polysialic acid (PSA) is a carbohydrate predominantly carried by the neural cell adhesion molecule (NCAM) in mammals. NCAM and, in particular, PSA play important roles in cellular and synaptic plasticity. Here we investigated whether PSA modulates the activity of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors (AMPA-Rs). Single channel recordings of affinity-purified AMPA-Rs reconstituted in lipid bilayers revealed that bacterially derived PSA, called colominic acid, prolonged the open channel time of AMPA-R-mediated currents by severalfold and altered the bursting pattern of the receptor channels but did not modify AMPA-R single channel conductance. This effect was reversible, concentration-dependent, and specific, since monomers of sialic acid and another negatively charged carbohydrate, chondroitin sulfate, did not potentiate single channel AMPA-R currents. Recombinant PSA-NCAM also potentiated currents mediated by reconstituted AMPA-Rs. In pyramidal neurons acutely isolated from the CA1 region of the early postnatal hippocampus, l-glutamate or AMPA (applied in the presence of antagonists blocking voltage-gated Na(+) and K(+) currents and N-methyl-d-aspartate and metabotropic glutamate receptors) induced inward currents, which were significantly increased by co-application of colominic acid. Chondroitin sulfate did not affect AMPA-R-mediated currents in CA1 neurons. The effect of colominic acid was age-dependent, since in pyramidal neurons from adult hippocampus, colominic acid failed to potentiate glutamate responses. Thus, our study demonstrates age-dependent potentiation of AMPA receptors by PSA via a mechanism probably involving direct PSA-AMPA-R interactions. This mechanism might amplify AMPA-R-mediated signaling in immature cells, thereby affecting their development.     

Identification of aspirin and diclofenac binding proteins in the red complex pathogens

Red complex organisms are a group of organisms (Porphyromonas gingivalis ATCC 33277, Treponema denticola ATCC 35405, Tannerella forsythia ATCC 43037) that have been identified for the causation of periodontal diseases. Aspirin and diclofenac have been used as regular analgesics. Therefore, it is of interest to document the identification of aspirin and diclofenac binding proteins in the red complex pathogens using the STITCH v.5 pipeline. The virulence properties of these proteins were analyzed using VICMPred and VirulentPred software. Thus, we document 000 number of proteins having optimal binding features with the known analgesics.     

Application of fluorescent probes to study structural changes in Aspergillus fumigatus exposed to amphotericin B, itraconazole, and voriconazole

The broad objective of this study was to document patterns of structural changes following antifungal treatment, and to determine any relationship with minimum inhibitory concentration (MIC) of an antifungal. Three clinical isolates of Aspergillus fumigatus, with high, intermediate, and low amphotericin B (AB), itraconazole (IZ), and voriconazole (VZ) MICs were studied in 24-well plates with cover slips. The fluorescent probes used were Calcofluor White (cell wall), propidium iodide (nucleus), and MitoTracker Green FM (mitochondria). Fluorescent microscopy as early as 3-h after exposure revealed that AB treated hyphae had intact cell wall with deformed mitochondria and nuclei while IZ and VZ treated hyphae revealed no intact cell wall, and deformation of mitochondria and nuclei. At 48 h, AB treated cells revealed rupture of hyphae and disintegration of mitochondria, and nuclei, IZ treated hyphae were swollen with disintegration of mitochondria, and nuclei while VZ treated hyphae showed rupture and disintegration of mitochondria and nuclei. The structural changes for the three strains studied were similar in fluorescent microscopy as long as the incubation time and their respective MICs were used. Thus, AB, IZ, and VZ induced gross organelle defects in A. fumigatus nuclei, mitochondria, and cell wall, which were consistent with respective MICs of antifungals used.