Functional characterization by genetic complementation of aroB-Encoded dehydroquinate synthase from Mycobactetium tuberculosis H37Rv and its heterologous expression and purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.

Thiosemicarbazones, semicarbazones, dithiocarbazates and hydrazide/hydrazones: Anti-Mycobacterium tuberculosis activity and cytotoxicity

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Vanadium complexes with thiosemicarbazones: Synthesis, characterization, crystal structures and anti-Mycobacterium tuberculosis activity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Photocatalytic decomposition of diclofenac potassium using silver-modified TiO(2) thin films

The effects of silver insertion on the TiO(2) photocatalytic activity for the degradation of diclofenac potassium were reported here. Techniques such as X-ray diffraction, scanning electron microscopy and UV-Vis spectroscopy were used to comprehend the relation between structure and properties of the silver-modified TiO(2), thin films obtained by the sol-gel method. The lattice parameters and the crystallinity of TiO(2) anatase phase were affected by inserted silver, and the film thickness increased about 4 nm for each 1 wt.% of silver inserted. The degradation of diclofenac potassium and by-products reached an efficiency of 4.6 mg(C) W(-1) when the material was modified with silver. Although the first step of degradation involves only the photochemical process related to the loss of the chlorine and hydrogen atoms. This cyclization reaction leads to the formation of intermediate, which degradation is facilitated by the modified material. (C) 2007 Elsevier B.V. All rights reserved.

Palladium(II) Complexes with Thiosemicarbazones. Syntheses, Characterization and Cytotoxicity against Breast Cancer Cells and Anti-Mycobacterium tuberculosis Activity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Design, synthesis and biological evaluation of new aryl thiosemicarbazone as antichagasic candidates

The present work reports on the synthesis, biological assaying and docking studies of a series of 12 aryl thiosemicarbazones, which were planned to act over two main enzymes, cruzain and trypanothione reductase. These enzymes are used as targets of trypanocidal activity in Chagas disease control with a minimal mutagenic profile. Three p-nitroaromatic thiosemicarbazones showed high activity against Trypanosoma cruzi in in vitro assays (IC50 < 57 μM), and no mutagenic profile was observed in micronucleous tests. Although the in vitro inhibition test showed that 10-μM doses of eight compounds inhibited cruzain activity, no correlation was found between cruzain inhibition and trypanocidal activity. © 2013 Elsevier Masson SAS. All rights reserved.

Manganese(II) complexes with thiosemicarbazones as potential anti-Mycobacterium tuberculosis agents

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cobalt(III) complexes with thiosemicarbazones as potential anti-Mycobacterium tuberculosis agents

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural characterization of diastereoisomeric ethano adducts derived from the reaction of 2'-deoxyguanosine with trans,trans-2,4-decadienal

Background levels of exocyclic DNA adducts have been detected in rodent and human tissues. Several studies have focused on bifunctional electrophiles generated from lipid peroxidation as one of the endogenous sources of these lesions. We have previously shown that the reaction of 2'-deoxyguanosine (dGuo) with trans,trans-2,4-decadienal (DDE), a highly cytotoxic aldehyde generated as a product of lipid peroxidation in cell membranes, results in the formation of a number of different base derivatives. Three of these derivatives have been fully characterized as 1,N-2-etheno-2'-deoxyguanosine adducts. In the present work, four additional adducts, designated A3-A6, were isolated from in vitro reactions by reversed-phase HPLC and fully characterized on the basis of spectroscopic measurements. Adducts A3-A6 are four diastereoisomeric 1,N-2-hydroxyethano-2'-deoxyguanosine derivatives possessing a carbon side chain with a double bond and a hydroxyl group. The systematic name of these adducts is 6-hydroxy3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-7-((E)-1-hydroxy-oct-2-enyl)-3,5,6,7-tetrahydro-imidazo- [1,2-a]purin-9-one. The proposed reaction mechanism yielding adducts A3-A6 involves DDE epoxidation at C2, followed by nucleophilic addition of the exocyclic amino group of dGuo to the C1 of the aldehyde and cyclization, via nucleophilic attack, on the C2 epoxy group by N-1. The formation of adducts A1-A6 has been investigated in acidic, neutral, and basic pH in the presence of H2O2 or tent-butyl hydroperoxide. Neutral conditions, in the presence of H2O2, have favored the formation of adducts A1 and A2, with minor amounts of A3-A6, which were prevalent under basic conditions. These data indicate that DDE can modify DNA bases through different oxidative pathways involving its two double bonds. It is important to structurally characterize DNA base derivatives induced by alpha,beta-unsaturated aldehydes so that the genotoxic risks associated with the lipid peroxidation process can be assessed.

Formation of 1,N-6-etheno-2 '-deoxyadenosine adducts by trans,trans-2,4-decadienal

trans,trans-2,4-Decadienal (DDE) is an important breakdown product of lipid peroxidation. This aldehyde is cytotoxic to mammalian cells and is known to be implicated in DNA damage. Therefore, attempts were made in this work to assess the reactivity of DDE with 2'-deoxyadenosine (dAdo). It was shown that DDE is able to bind to 2'-deoxyadenosine, yielding highly fluorescent products. Besides 1,N-6-etheno-2'-deoxyadenosine (epsilon dAdo), two other related adducts, 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)3H-imidazo[2,1-i]purin-7-yl]-1,2,3-octanetriol and 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imidazo[2,1-i]purin-7-yl]-1,2-heptanediol, were isolated by reverse phase high-performance liquid chromatography and characterized on the basis of their UV, fluorescence, nuclear magnetic resonance, and mass spectrometry features. The reaction mechanism for the formation of the DDE-2'-deoxyadenosine adducts involves 2,4-decadienal epoxidation and subsequent addition to the N-2 amino group of 2'-deoxyadenosine, followed by cyclization at the N-1 site. Adducts differ by the length of carbon side chain and the number of hydroxyl groups. The present data indicate that DDE can be epoxidized by peroxides, and the resulting products are able to form several adducts with 2'-deoxyadenosine and/or DNA. Endogenous DNA adduct formation can contribute to the already reported high cytotoxicity of DDE to mammalian cells.