Pedro F. Pinheiro

Distribution of Cepacian Biosynthesis Genes among Environmental and Clinical Burkholderia Strains and Role of Cepacian Exopolysaccharide in Resistance to Stress Conditions

ABSTRACT The genus Burkholderia includes strains pathogenic to animals and plants, bioremediators, or plant growth promoters. Genome sequence analyses of representative Burkholderia cepacia complex (Bcc) and non-Bcc strains for the presence of the bce-I gene cluster, directing the biosynthesis of the exopolysaccharide (EPS) cepacian, further extended this previously described cluster by another 9 genes. The genes in the bce-II cluster were named bceM to bceU and encode products putatively involved in nucleotide sugar precursor biosynthesis and repeat unit assembly, modification, and translocation across the cytoplasmic membrane. Disruption of the B. cepacia IST408 bceQ and bceR genes, encoding a putative repeat unit flippase and a glycosyltransferase, respectively, resulted in the abolishment of cepacian biosynthesis. A mutation in the bceS gene, encoding a putative acyltransferase, did not affect EPS production yield significantly but decreased its acetylation content by approximately 20%. Quantitative real-time reverse transcription-PCR experiments confirmed the induction of genes in the bce-I and bce-II clusters in a Burkholderia multivorans EPS producer clinical isolate in comparison to the level for its isogenic EPS-defective strain. Fourier Transform infrared spectroscopy analysis confirmed that the exopolysaccharide produced by 10 Burkholderia isolates tested was cepacian. The ability of Burkholderia strains to withstand desiccation and metal ion stress was higher when bacteria were incubated in the presence of 2.5 g/liter of cepacian, suggesting that this EPS plays a role in the survival of these bacteria by contributing to their ability to thrive in different environments.

Structural Analysis of Flavonoids and Related Compounds - A Review of Spectroscopic Applications

In 1936 St. Rusznyak and A. Szent-Gyorgyi described, in a paper in Nature (Rusznyak & Szent-Gyorgy, 1936), the relief of certain pathological conditions, characterized by an increased permeability or fragility of the capillary wall, by extracts of Hungarian red pepper containing flavonols, a type of flavonoids, which were then named “vitamin P”. The following five decades saw a slow but steady rise in the interest in the group of flavonoids, and their benefits in the treatment of a vast number of diseases and conditions, including pregnancy toxaemia, rheumatic fever, diabetes and cancer. In the late 1980s and throughout the 1990s flavonoids were intensely studied concerning their actions as mutagenic agents and as antioxidants and pro-oxidants as their likely roles in biological systems (for example, Aviram & Fuhrman, 1998; Lambert & Elias, 2010). In the early 90s the antioxidant activity of flavonoids was extensively studied in vitro, and it was assumed that such activity would be at the basis of the health promoting benefits of these compounds. However, in the late 90s and early 00s the metabolism of flavonoids was deeply scrutinized, and the results indicated that their antioxidant activity in vivo could not account for the overall actions attributed to them (Fraga et al., 2010). The paradigm for flavonoid action changed towards the establishment of flavonoids as inflammation modulators, and more recently their role in neuroprotection, memory and cognition has been under scrutiny (Gomes et al., 2008; Spencer et al., 2009; Spencer, 2010). However, exact mechanisms for many of the actions attributed to flavonoids have not yet been established, but the relationship between their activity and the presence of specific functional groups in the molecules is undeniable. Moreover, each role attributed to flavonoids has been linked to different structural features – for example, while antioxidant activity depends essentially on the number and location of OH groups in the molecules, their antagonist effect towards adenosine receptors depends more on the overall planarity than on the hydroxyl groups; in fact, the latter even appear to be counter-productive (Gonzalez et al., 2007).

Biochemical and Functional Studies on the Burkholderia cepacia Complex bceN Gene, Encoding a GDP-D-Mannose 4,6-Dehydratase

This work reports the biochemical and functional analysis of the Burkholderia cenocepacia J2315 bceN gene, encoding a protein with GDP-D-mannose 4,6-dehydratase enzyme activity (E.C.4.2.1.47). Data presented indicate that the protein is active when in the tetrameric form, catalyzing the conversion of GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. This sugar nucleotide is the intermediary necessary for the biosynthesis of GDP-D-rhamnose, one of the sugar residues of cepacian, the major exopolysaccharide produced by environmental and human, animal and plant pathogenic isolates of the Burkholderia cepacia complex species. Vmax and Km values of 1.5±0.2 µmol.min−1.mg−1 and 1024±123 µM, respectively, were obtained from the kinetic characterization of the B. cenocepacia J2315 BceN protein by NMR spectroscopy, at 25°C and in the presence of 1 mol MgCl2 per mol of protein. The enzyme activity was strongly inhibited by the substrate, with an estimated Ki of 2913±350 µM. The lack of a functional bceN gene in a mutant derived from B. cepacia IST408 slightly reduced cepacian production. However, in the B. multivorans ATCC17616 with bceN as the single gene in its genome with predicted GMD activity, a bceN mutant did not produce cepacian, indicating that this gene product is required for cepacian biosynthesis.

Camphor-based CCR5 blocker lead compounds – a computational and experimental approach

The C–C chemokine receptor type 5 (CCR5) is a transmembrane receptor that plays a pivotal role as a HIV anchor to human cell membranes, mediating viral entry. CCR5 antagonists, acting by blocking the receptor and preventing its interaction with the HIV proteins, are key agents towards effective anti-viral therapy. This work describes the computational study, synthesis and viral inhibition assay of a number of camphor derivatives as a first step towards new drug leads to block this specific entry pathway. Viral inhibition assays have identified three molecules, camphor carboxylic acid, its tri(hydroxymethyl)aminomethane amide derivative, and an hydroxyl-imide camphor derivative as promising agents to develop new drugs, with IC50 values (0.16, 0.22 and 1.02 μM, respectively) one order below that of maraviroc (0.02 μM), a clinically used CCR5 antagonist.

Structure-based virtual screening toward the discovery of novel inhibitors of the DNA repair activity of the human apurinic/apyrimidinic endonuclease 1.

The DNA repair activity of human apurinic/apyrimidinic endonuclease 1 (APE1) has been recognized as a promising target for the development of small‐molecule inhibitors to be used in combination with anticancer agents. In an attempt to identify novel inhibitors of APE1, we present a structure‐based virtual screening (SBVS) study based on molecular docking analysis of the compounds of NCI database using the GOLD 5.1.0 (Genetic Optimization for Ligand Docking) suite of programs. Compounds selected in this screening were tested with a fluorescence‐based APE1 endonuclease activity assay. Two compounds (37 and 41) were able to inhibit the multifunctional enzyme APE1 in the micromolar range, while compound 22 showed inhibitory effects at nanomolar concentrations. These results were confirmed by a plasmid DNA nicking assay. In addition, the potential APE1 inhibitors did not affect the cell viability of non‐tumor MCF10A cells. Overall, compounds 22, 37, and 41 appear to be important scaffolds for the design of novel APE1 inhibitors and this study highlights the relevance of in silico‐based approaches as valuable tools in drug discovery.

Sex differences in hepatic and intestinal contributions to nevirapine biotransformation in rats

The understanding of the intestine contribution to drug biotransformation improved significantly in recent years. However, the sources of inter-individual variability in intestinal drug biotransformation, namely sex-differences, are still elusive. Nevirapine (NVP) is an orally taken anti-HIV drug associated with severe idiosyncratic reactions elicited by toxic metabolites, with women at increased risk. As such, NVP is a good model to assess sex-dimorphic metabolism. The aim of this study was to perform a comparative profiling of NVP biotransformation in rat intestine and liver and evaluate whether or not it is organ- and sex-dependent. Therefore, nevirapine-containing solutions were perfused through the intestine, in a specially designed chamber, or incubated with liver slices, from male and female Wistar rats. The levels of NVP and its Phase I metabolites were quantified by HPLC-UV. Liver incubation experiments yielded the metabolites 2-, 3-, 8-, and 12-OH-NVP, being 12-OH-NVP and 2-OH-NVP the major metabolites in males and females, respectively. Inter-sex differences in the metabolic profile were also detected in the intestine perfusion experiments. Herein, the metabolites 3- and 12-OH-NVP were only found in male rats, whereas 2-OH-NVP levels were higher in females, both in extraluminal (p<0.01) and intraluminal media. The metabolite 8-OH-NVP was not detected in the intraluminal media from either males or females. In this study, important inter-sex differences were detected in both organs, providing further clues to the sex-dimorphic profile of NVP toxicity. Moreover, an extra-hepatic contribution to NVP biotransformation was observed, strengthening the relevance of the intestinal contribution in the biotransformation of orally taken-drugs.

Nevirapine modulation of paraoxonase-1 in the liver: An in vitro three-model approach.

INTRODUCTION Nevirapine is associated with severe hepatotoxicity, through the formation of reactive metabolites. Paraoxonase-1 (PON-1) is a promiscuous enzyme involved in the metabolism of xeno- and endobiotics and proposed as a biomarker of hepatotoxicity. The aim of this work was to explore the effects of nevirapine and its phase I metabolites, 2-hydroxy-nevirapine and 12-hydroxy-nevirapine, on PON-1 activities. MATERIAL AND METHODS 2D and 3D primary cultures of rat hepatocytes, and also HepG2 2D cell cultures, were exposed to nevirapine, 2-hydroxy-nevirapine, and 12-hydroxy-nevirapine. The paraoxonase (POase), arylesterase (AREase) and lactonase (LACase) activities of PON-1 were quantified. RESULTS Effects of nevirapine and its metabolites were only observed in the 3D cell model. Both nevirapine and 12-hydroxy-nevirapine increased POase (p<0.05, p<0.01) and LACase activities (p<0.05, p<0.001). The AREase activity was increased only upon 12-hydroxy-nevirapine exposure (p<0.01). These modulatory effects were observed at 300μM concentrations of nevirapine and 12-hydroxy-nevirapine. CONCLUSIONS The formation of 12-hydroxy-nevirapine seems to be the main factor responsible for the increase of PON-1 activities induced by nevirapine exposure. This effect was only observed in the 3D model, suggesting that an in vivo-like system is necessary for this modulation to occur. The present data suggest that the 3D model is a more suitable in vitro model than the conventional ones to explore drug effects on PON-1.

Synthesis, antimicrobial activity and toxicity to nematodes of cyclam derivatives

The antimicrobial activity and toxicity to nematodes of the cyclam salt [H2{H2(4-CF3PhCH2)2Cyclam}](CH3COO)2⋅(CH3COOH)2 were evaluated. Estimated minimum inhibitory concentrations (MICs) of 9, 261 and 15 µg/mL were obtained for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, respectively. For selected Candida spp., the estimated MICs obtained ranged from 32 µg/mL to 63 µg/mL. Bactericidal activity was demonstrated but the compound was not reliably fungicidal. Concentrations of the cyclam salt up to 32 µg/mL did not significantly affect survival of the nematode Caenorhabditis elegans; however, concentrations equal or above this value significantly affected nematode survival in a dose-dependent manner.

Quinoid derivatives of the nevirapine metabolites 2-hydroxy- and 3-hydroxy-nevirapine: activation pathway to amino acid adducts

Nevirapine (NVP) is the non-nucleoside HIV-1 reverse transcriptase inhibitor most commonly used in developing countries, both as a component of combined antiretroviral therapy and to prevent mother-to-child transmission of the virus; however, severe hepatotoxicity and serious adverse cutaneous effects raise concerns about its safety. NVP metabolism yields several phenolic derivatives conceivably capable of undergoing further metabolic oxidation to electrophilic quinoid derivatives prone to react with bionucleophiles and initiate toxic responses. We investigated the ability of two phenolic NVP metabolites, 2-hydroxy-NVP and 3-hydroxy-NVP, to undergo oxidation and subsequent reaction with bionucleophiles. Both metabolites yielded the same ring-contraction product upon oxidation with Fremys salt in aqueous medium. This is consistent with the formation of a 2,3-NVP-quinone intermediate, which upon stabilization by reduction was fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Additionally, we established that the oxidative activation of 2-hydroxy-NVP involved the transient formation of both the quinone and a quinone-imine, whereas 3-hydroxy-NVP was selectively converted into 2,3-NVP-quinone. The oxidations of 2-hydroxy-NVP and 3-hydroxy-NVP in the presence of the model amino acids ethyl valinate (to mimic the highly reactive N-terminal valine of hemoglobin) and N-acetylcysteine were also investigated. Ethyl valinate reacted with both 2,3-NVP-quinone and NVP-quinone-imine, yielding covalent adducts. By contrast, neither 2,3-NVP-quinone nor NVP-derived quinone-imine reacted with N-acetylcysteine. The product profile observed upon Fremys salt oxidation of 2-hydroxy-NVP in the presence of ethyl valinate was replicated with myeloperoxidase-mediated oxidation. Additionally, tyrosinase-mediated oxidations selectively yielded 2,3-NVP-quinone-derived products, while quinone-imine-derived products were obtained upon lactoperoxidase catalysis. These observations suggest that the metabolic conversion of phenolic NVP metabolites into quinoid electrophiles is biologically plausible. Moreover, the lack of reaction with sulfhydryl groups might hamper the in vivo detoxification of NVP-derived quinone and quinone-imine metabolites via glutathione conjugation. As a result, these metabolites could be available for reaction with nitrogen-based bionucleophiles (e.g., lysine residues of proteins) ultimately eliciting toxic events.