Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi

The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite’s drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+ biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi. The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

Visfatin regulates insulin secretion, insulin receptor signalling and mRNA expression of diabetes-related genes in mouse pancreatic beta-cells

The role of the adipocyte-derived factor visfatin in metabolism remains controversial, although some pancreatic ß-cell-specific effects have been reported. This study investigated the effects of visfatin upon insulin secretion, insulin receptor activation and mRNA expression of key diabetes-related genes in clonal mouse pancreatic ß-cells. ß-TC6 cells were cultured in RPMI 1640 and were subsequently treated with recombinant visfatin. One-hour static insulin secretion was measured by ELISA. Phospho-specific ELISA and western blotting were used to detect insulin receptor activation. Real-time SYBR Green PCR array technology was used to measure the expression of 84 diabetes-related genes in both treatment and control cells. Incubation with visfatin caused significant changes in the mRNA expression of several key diabetes-related genes, including marked up-regulation of insulin (9-fold increase), hepatocyte nuclear factor (HNF)1ß (32-fold increase), HNF4a (16-fold increase) and nuclear factor ?B (40-fold increase). Significant down-regulation was seen in angiotensin-converting enzyme (-3.73-fold) and UCP2 (-1.3-fold). Visfatin also caused a significant 46% increase in insulin secretion compared to control (P<0.003) at low glucose, and this increase was blocked by co-incubation with the specific nicotinamide phosphoribosyltransferase inhibitor FK866. Both visfatin and nicotinamide mononucleotide induced activation of both insulin receptor and extracellular signal-regulated kinase (ERK)1/2, with visfatin-induced insulin receptor/ERK1/2 activation being inhibited by FK866. We conclude that visfatin can significantly regulate insulin secretion, insulin receptor phosphorylation and intracellular signalling and the expression of a number of ß-cell function-associated genes in mouse ß-cells.

Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors as Therapeutics: Rationales, Controversies, Clinical Experience.

Nicotinamide adenine dinucleotide (NAD+) biosynthesis from nicotinamide is used by mammalian cells to replenish their NAD+ stores and to avoid unwanted nicotinamide accumulation. Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in this biosynthetic pathway, almost invariably leads to intracellular NAD+ depletion and, when protracted, to ATP shortage and cell demise. Cancer cells and activated immune cells express high levels of NAMPT and are highly susceptible to NAMPT inhibitors, as shown by the activity of these agents in models of malignant and inflammatory disorders. As the spectrum of conditions which could benefit from pharmacological NAMPT inhibition becomes broader, the mechanisms accounting for their activity are also eventually becoming apparent, including the induction of autophagy and the impairment of Ca(2+) - and NF-κB-dependent signaling. Here, we discuss the rationales for exploiting NAMPT inhibitors in cancer and inflammatory diseases and provide an overview of the preclinical and clinical studies in which these agents have been evaluated.

A multicenter study to validate the reproducibility of MSI testing with a panel of 5 quasimonomorphic mononucleotide repeats.

Microsatellite instability (MSI) testing in clinics is becoming increasingly widespread; therefore, there is an urgent need for methodology standardization and the availability of quality control. This study is aimed to assess the interlaboratory reproducibility of MSI testing in archive samples by using a panel of 5 recently introduced, mononucleotide repeats (MNR). The quality control involved 8 European institutions. Participants were supplied with DNA extracted from 15 archive colon carcinoma samples and from the corresponding normal tissues. Every group was asked to assess the MSI status of the samples by using the BAT25, BAT26, NR21, NR24, and NR27 mononucleotide markers. Four institutions repeated the analysis using the NCI reference panel to confirm the results obtained with the MNR markers. The overall concordance among institutions for MSI analyses at single locus level was 97.7% when using the MNR panel and 95.0% with the NCI one. The laboratories obtained a full agreement in scoring the MSI status of each patient sample, both using the mononucleotide and the NCI marker sets. With the NCI marker set, however, concordance was lowered to 85.7% when considering the MSI-Low phenotype. Concordance between the 2 panels in scoring the MSI status of each sample was complete if no discrimination was made between MSI-Stable and MSI-L, whereas it dropped to 76.7% if MSI-L was considered. In conclusion, the use of the MNR panel seems to be a robust approach that yields a very high level of reproducibility. The results obtained with the 5 MNR are diagnostically consistent with those obtained by the use of the NCI markers, except for the MSI-Low phenotype.

The anti-lymphoma activity of APO866, an inhibitor of nicotinamide adenine dinucleotide biosynthesis, is potentialized when used in combination with anti-CD20 antibody.

Abstract APO866 is an inhibitor of nicotinamide adenine dinucleotide (NAD) biosynthesis that exhibits potent anti-lymphoma activity. Rituximab (RTX), an anti-CD20 antibody, kills lymphoma cells by direct apoptosis and antibody- and complement-dependent cell-mediated cytotoxicities, and has clinical efficacy in non-Hodgkin cell lymphomas. In the present study, we evaluated whether RTX could potentiate APO866-induced human B-lymphoma cell death and shed light on death-mediated mechanisms associated with this drug combination. We found that RTX significantly increases APO866-induced death in lymphoma cells from patients and lines. Mechanisms include enhancement of autophagy-mediated cell death, activation of caspase 3 and exacerbation of mitochondrial depolarization, but not increase of reactive oxygen species (ROS) production, when compared with those induced by each drug alone. In vivo, combined administration of APO866 with RTX in a laboratory model of human aggressive lymphoma significantly decreased tumor burden and prolonged survival over single-agent treatment. Our study demonstrates that the combination of RTX and APO866 optimizes B-cell lymphoma apoptosis and therapeutic efficacy over both compounds administered separately.

Pharmacological inhibition of nicotinamide phosphoribosyltransferase/visfatin enzymatic activity identifies a new inflammatory pathway linked to NAD.

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFalpha levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders.

Nicotinamide phosphoribosyltransferase as a target in inflammation- related disorders.

NAD(+) biosynthesis through nicotinamide phosphoribosyltransferase (NAMPT) holds potential as a target for the treatment of inflammatory disorders due to NAD(+)'s role in immune cell signaling and metabolism. In addition to its activity as an enzyme, NAMPT is also secreted in the extracellular space where it acts as a pro-inflammatory and proangiogenic cytokine. NAMPT inhibition with FK866 has anti-inflammatory activity in different models of immune disorders and it prevents ischemia-reperfusion-induced heart damage by dampening the production of neutrophil chemoattractants. NAMPT blockade with a neutralizing antibody has beneficial effects in an acute lung injury model. Last, but not least, the anticancer activity of NAMPT inhibitors may also reflect, at least in part, their ability to modify the cancer microenvironment through their anti-inflammatory properties. Overall, NAMPT inhibition holds potential for the treatment of inflammation-related disorders and the development of effective and safe NAMPT inhibitors remains an area of strong interest in pharmaceutical research.

Electronic transport properties of ascorbic acid and nicotinamide adsorbed on single-walled carbon nanotubes

Ab initio simulations of carbon nanotubes interacting with ascorbic acid and nicotinamide are reported. The electronic transport properties of these systems are studied using a combination of density functional theory and non-equilibrium Green`s functions methods. The adsorptions of both molecules are observed to depend strongly on their functionalization. The interaction through the appropriate functionalized species modifies the structural and electronic properties of the original system, resulting in a chemisorption regime. Changes in the electronic transport properties are also observed, with reductions on the total electronic transmission probabilities. Nevertheless, when the molecules interact through the pristine form, a physisorption interaction is observed with insignificant structural and electronic transport changes. (c) 2011 Elsevier B.V. All rights reserved.

Protection of spruce seedlings against pine weevil attacks by treatment of seeds or seedlings with nicotinamide, nicotinic acid and jasmonic acid

Sustainable methods are required to protect newly planted tree seedlings from insect herbivore attack. To this end, here Norway spruce (Picea abies (L.) Karst.) seeds were treated with 2.5 mM nicotinamide (NIC), 2.5 mM nicotinic acid (NIA), 3 mM jasmonic acid (JA) or 0.2 mM 5-azacytidine (5-Aza), and 6-month-old seedlings grown from these seeds were planted at a reforestation area in central Sweden. Attack by pine weevils (Hylobius abietis) was reduced by 50 per cent by NIC treatment, 62.5 per cent by JA treatment and 25 per cent by 5-Aza treatment, when compared with seedlings grown from untreated seeds. Watering 18-month-old spruce seedlings with 2 mM NIC or 2 mM NIA did reduce attack during the first season in the field by 40 and 53 per cent, respectively, compared with untreated plants. Girdling was also reduced by the different treatments. Analysis of conifer seedlings treated with 5-Aza points at a possible involvement of epigenetic mechanisms in this defensive capacity. This is supported by a reduced level of DNA methylation in the needles of young spruce seedlings grown in a greenhouse from NIC-treated seeds. Seed treatment for seedling defense potentiation is simple, inexpensive and also a new approach for forestry with many potential applications.

Chorismate synthase: An attractive target for drug development against orphan diseases

The increase in incidence of infectious diseases worldwide, particularly in developing countries, is worrying. Each year, 14 million people are killed by infectious diseases, mainly HIV/AIDS, respiratory infections, malaria and tuberculosis. Despite the great burden in the poor countries, drug discovery to treat tropical diseases has come to a standstill. There is no interest by the pharmaceutical industry in drug development against the major diseases of the poor countries, since the financial return cannot be guaranteed. This has created an urgent need for new therapeutics to neglected diseases. A possible approach has been the exploitation of the inhibition of unique targets, vital to the pathogen such as the shikimate pathway enzymes, which are present in bacteria, fungi and apicomplexan parasites but are absent in mammals. The chorismate synthase (CS) catalyses the seventh step in this pathway, the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. The strict requirement for a reduced flavin mononucleotide and the anti 1,4 elimination are both unusual aspects which make CS reaction unique among flavin-dependent enzymes, representing an important target for the chemotherapeutic agents development. In this review we present the main biochemical features of CS from bacterial and fungal sources and their difference from the apicomplexan CS. The CS mechanisms proposed are discussed and compared with structural data. The CS structures of some organisms are compared and their distinct features analyzed. Some known CS inhibitors are presented and the main characteristics are discussed. The structural and kinetics data reviewed here can be useful for the design of inhibitors. © 2007 Bentham Science Publishers Ltd.

The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

Background. The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results. In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMN ox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion. This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents. © 2008 Ely et al; licensee BioMed Central Ltd.

Expression profiles of fetal membrane nicotinamide adenine dinucleotide phosphate oxidases (NOX) 2 and 3 differentiates spontaneous preterm birth and pPROM pathophysiologies

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using the nicotinamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) for Bartonella species in domiciled and stray cats in Brazil

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