Design, Synthesis and Biological Characterization of Potential Antiatherogenic Nitric Oxide Releasing-Tocopherol Analogs.

PostprintSynthesis and biological characterization of a series of tocopherol analogs with NO-releasing capacity are reported. The selected NO-donor moieties were nitrooxy and furoxan. All products were tested for their in vitro NO-releasing capacities, vasodilating properties and antiplatelet activity. They were also capable to prevent LDL oxidation

Second generation of α-tocopherol analogs-nitric oxide donors: Synthesis, physicochemical and biological characterization.

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Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis

Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO2-OA) is an electrophilic fatty acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis upon activation of Nrf2 signaling. The effects of NO2-OA on intracellular GSH homeostasis were evaluated. In addition to upregulation of GSH biosynthesis, we observed that NO2-OA increased intracellular GSSG in an oxidative stress-independent manner. NO2-OA directly inhibited GR in vitro by covalent modification of the catalytic Cys61, with kon of (3.45±0.04)×103 M−1 s−1, koff of (4.4±0.4)×10−4 s−1, and Keq of (1.3±0.1)×10−7 M. Akin to NO2-OA, the electrophilic Nrf2 activators bardoxolone-imidazole (CDDO-Im), bardoxolone-methyl (CDDO-Me) and dimethyl fumarate (DMF) also upregulated GSH biosynthesis while promoting GSSG accumulation, but without directly inhibiting GR activity. In vitro assays in which GR was treated with increasing GSH concentrations and GSH depletion experiments in cells revealed that GR activity is finely regulated via product inhibition, an observation further supported by theoretical (kinetic modeling of cellular GSSG:GSH levels) approaches. Together, these results describe two independent mechanisms by which electrophiles modulate the GSH/GSSG couple, and provide a novel conceptual framework to interpret experimentally determined values of GSH and GSSG

Electrophilic nitroalkene-tocopherol derivatives: synthesis, physicochemical characterization and evaluation of anti-inflammatory signaling responses

Inflammation plays a major role in the onset and development of chronic non-communicable diseases like obesity, cardiovascular diseases and cancer. Combined, these diseases represent the most common causes of death worldwide, thus development of novel pharmacological approaches is crucial. Electrophilic nitroalkenes derived from fatty acids are formed endogenously and exert anti-inflammatory actions by the modification of proteins involved in inflammation signaling cascades. We have developed novel nitroalkenes derived from α-tocopherol aiming to increase its salutary actions by adding anti-inflammatory properties to a well-known nutraceutical. We synthesized and characterized an α-tocopherol-nitroalkene (NATOH) and two hydrosoluble analogues derived from Trolox (NATxME and NATx0). We analyzed the kinetics of the Michael addition reaction of these compounds with thiols in micellar systems aiming to understand the effect of hydrophobic partition on the reactivity of nitroalkenes. We studied NATxME in vitro showing it exerts non-conventional anti-inflammatory responses by inducing Nrf2-Keap1-dependent gene expression and inhibiting the secretion of NF-κB dependent pro-inflammatory cytokines. NATxME was also effective in vivo, inhibiting neutrophil recruitment in a zebrafish model of inflammation. This work lays the foundation for the rational design of a new therapeutic strategy for the prevention and treatment of metabolic and inflammation-related disease

Early events following phosphorus restriction involve changes in proteome and affects nitric oxide metabolism in soybean leaves

Phosphorus (P) is a macronutrient with structural and regulatory functions, essential for energy transfer. Under limited P availability, plant cells respond to internal signals, adjusting their metabolic pathways in order to reorganize physiological priorities. This work was conducted with the aim to explore the initial changes following P deprivation, before neither growth nor photosynthesis was strongly affected. The first unifoliate leaves of nine-day-old soybean plants (Glycine max, cv. Williams 82) were analyzed. Plants grown hydroponically under control conditions (+P, nutrient solution with 500 μM H2PO4 −) were compared with those restricted in phosphate (-P, without H2PO4 − in the nutrient solution). No visible symptoms of P-deficiency, such as changes in pigment intensity and leaf number or size, were observed up to 48 h of P deprivation. Neither fresh weight nor shoot/root FW ratio was affected in plants as a consequence of early P-restriction. Shotgun proteomic analyses of leaves from plants exposed to 24 h of P-deprivation, revealed that a total of 202 proteins were exclusively detected and 232 increased their relative abundance under –P conditions. The proteins affected belong mainly to the catalytic activity group according to Gene Ontology Consortium including proteins like 6-phosphogluconate dehydrogenase (from pentose phosphate pathway), pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit alpha, alpha-1,4 glucan phosphorylases (from carbohydrate metabolic processes), and other enzymes involved in metabolic processes such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and fructose-bisphosphate aldolase (from gycolisis), and nitrate reductase (from nitrogen assimilation). Early events in leaves also involve higher levels of the bioactive molecule nitric oxide (NO), detected employing confocal laser microscopy, accompanied with a parallel increase in nitrate reductase activity. Furthermore, the presence of nitrated proteins was detected using shotgun liquid chromatography–mass spectrometry (LCeMS/MS). This post-translational protein modification, affected proteins mainly related to photosynthesis (chlorophyll a–b binding protein, ribulose bisphosphate carboxylase), and some other metabolic processes (glutamine synthetase, methionine synthase, lipoxygenase). This methodology allowed the confirmation of nitration sites in proteins previously described as putatively nitrated. Taken together the data presented suggest important metabolic changes, including nitric oxide metabolism, during the first hours of P deprivation.Fil: Ramos Artuso, Facundo Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales; ArgentinaFil: Galatro, Andrea Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Lima, Analia Ethel. Instituto Pasteur de Montevideo; Uruguay. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Batthyány, Carlos. Instituto Pasteur de Montevideo; UruguayFil: Simontacchi, Marcela Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales; Argentin

Proteomic analysis of Saccharomyces cerevisiae to study the effects of red wine polyphenols on oxidative stress

Understanding the molecular mechanisms underlying the “French paradox” has contributed to a growing interest in the investigation of the biological activity of red wine polyphenols (RWP). The main goal of this research is to provide valuable information on how RWP could exert their biological action at the cellular level. So, we report a proteomic analysis of S. cerevisiae exposed to both pro-oxidant (H2O2) and antioxidant (wine) agents. Cellular proteome analysis shows that RWP modify the level of certain proteins. Under both normal conditions (Wine treatment) and oxidative stress situations (Wine + H2O2 treatment), the proteins involved in the metabolism and biosynthesis of biomolecules were down-regulated, while one ribosomal protein was up-regulated, probably performing its ribosome-independent functions, and so contributing to the stress defense system. Considering this action mechanism, we suggest that RWP may be acting as mild pro-oxidants and, therefore, exerting a hormetic effect that leads to the strengthening of cells’ antioxidant capacity.Fil: Lingua, Mariana Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Neme Tauil, Ricardo Martin. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Batthyány, Carlos. Universidad de la Republica, Facultad de Medicina, Departamento de Bioquímica, Unidad de Bioquímica y Proteómica Analítica, IPMON; UruguayFil: Wunderlin, Daniel Alberto. Universidad Nacional de Córdoba. Secretaría de Ciencia y Tecnología. Instituto Superior de Investigación, Desarrollo y Servicio de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Baroni, María Verónica. Universidad Nacional de Córdoba. Secretaría de Ciencia y Tecnología. Instituto Superior de Investigación, Desarrollo y Servicio de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentin

Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe–S center

International audiencePknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition

Decreased proteasomal cleavage at nitrotyrosine sites in proteins and peptides

Removal of moderately oxidized proteins is mainly carried out by the proteasome, while highly modified proteins are no longer degradable. However, in the case of proteins modified by nitration of tyrosine residues to 3-nitrotyrosine (NO2Y), the role of the proteasome remains to be established. For this purpose, degradation assays and mass spectrometry analyses were performed using isolated proteasome and purified fractions of native cytochrome c (Cyt c) and tyrosine nitrated proteoforms (NO2Y74-Cyt c and NO2Y97-Cyt c). While Cyt c treated under mild conditions with hydrogen peroxide was preferentially degraded by the proteasome, NO2Y74- and NO2Y97-Cyt c species did not show an increased degradation rate with respect to native Cyt c. Peptide mapping analysis confirmed a decreased chymotrypsin-like cleavage at C-terminal of NO2Y sites within the protein, with respect to unmodified Y residues. Additionally, studies with the proteasome substrate suc-LLVY-AMC (Y-AMC) and its NO2Y-containing analog, suc-LLVNO2Y-AMC (NO2Y-AMC) were performed, both using isolated 20S-proteasome and astrocytoma cell lysates as the proteasomal source. Comparisons of both substrates showed a significantly decreased proteasome activity towards NO2Y-AMC. Moreover, NO2Y-AMC, but not Y-AMC degradation rates, were largely diminished by increasing the reaction pH, suggesting an inhibitory influence of the additional negative charge contained in NO2Y-AMC secondary to nitration. The mechanism of slowing of proteasome activity in NO2Y-contaning peptides was further substantiated in studies using the phenylalanine and nitro-phenylalanine peptide analog substrates. Finally, degradation rates of Y-AMC and NO2Y-AMC with proteinase K were the same, demonstrating the selective inability of the proteasome to readily cleave at nitrotyrosine sites. Altogether, data indicate that the proteasome has a decreased capability to cleave at C-terminal of NO2Y residues in proteins with respect to the unmodified residues, making this a possible factor that decreases the turnover of oxidized proteins, if they are not unfolded, and facilitating the accumulation of nitrated proteins

A Focused Library of NO-Donor Compounds with Potent Antiproliferative Activity Based on Green Multicomponent Reactions

Cancer is the second leading cause of death worldwide. Herein, a strategy to quickly and efficiently identify novel lead compounds to develop anticancer agents, using green multicomponent reactions followed by antiproliferative activity and structure–activity relationship studies, is described. A second-generation focused library of nitric oxide-releasing compounds was prepared by microwave-assisted Passerini and Ugi reactions. Nearly all compounds displayed potent antiproliferative activities against a panel of human solid tumor cell lines, with 1-phenyl-1-[(tert-butylamino)carbonyl]methyl 3-[(3-phenylsulfonyl-[1,2,5]oxadiazol-4-yl N-oxide)oxy]benzoate (4 k) and N-[1-(tert-butylaminocarbonyl)-1-phenylmethyl]-N-(4-methylphenyl)-3-(3-phenylsulfonyl-[1,2,5]oxadiazol-4-yl N-oxide)oxyphenyl carboxamide (6 d) exhibiting the strongest activity on SW1573 lung cell line (GI=110 and 21 nm) with selectivity indices of 70 and 470, respectively. Preliminary mechanistic studies suggest a relationship between NO release and antiproliferative activity. Our strategy allowed the rapid identification of at least two molecules as future candidates for the development of potent antitumor drugs.This work was supported by Agencia Nacional de Investigación e Innovación (ANII‐Fondo Clemente Estable, FCE‐2‐2011‐1‐5717), PEDECIBA‐Química, Uruguay.Peer Reviewe