Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite.

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107-108 M-1⋅s-1 range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107 M-1⋅s-1 at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2 insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions.

Evaluation of cytogenotoxicity, antioxidant and hypoglycemiant activities of isolate compounds from Mansoa hirsuta D.C. (Bignoniaceae).

Mansoa hirsuta (Bignoniaceae) is a native plant from caatinga in Brazilian semiarid. This plant has been locally used as antimicrobial and hypoglycemiant agents, but their action mechanisms and toxicity remain largely unknown. Therefore, we evaluated the composition and antioxidant, cytoprotective and hypoglycemiant effects of raw extract, fractions and compounds from leaves of M. hirsuta. The cytogenotoxic effects of ursolic and oleanolic acids, the main phytotherapic components of this plant, were assessed. The raw extract and fractions presented steroids, saponins, flavonols, flavanonols, flavanones, xanthones, phenols, tannins, anthocyanins, anthocyanidins and flavonoids. The ethyl acetate fraction inhibited efficiently the cascade of lipid peroxidation while the hydroalcoholic fraction was richer in total phenols and more efficient in capturing 2,2-diphenyl-1-picrylhydrazyl (·DPPH) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS·+) radicals. The isolated fraction of M. hirsuta also inhibited the α-amylase activity. Cytotoxic effects were absent in both raw extract and fractions while ursolic+oleanolic acids were efficient in protecting cells after exposure to hydrogen peroxide. Moreover, this mixture of acid shad no significant interference on the mitotic index and frequency of nuclear and/or chromosomal abnormalities in Allium cepa test. Therefore, M. hirsuta represents a potential source of phytochemicals against inflammatory and oxidative pathologies, including diabetes.

Melipona mondury produces a geopropolis with antioxidant, antibacterial and antiproliferative activities.

Geopropolis is a special type of propolis produced by stingless bees. Several pharmacological properties have been described for different types of geopropolis, but there have been no previous studies of the geopropolis from Melipona mondury. In this study, we investigated the antioxidant, antibacterial, and antiproliferative activities of M. mondury geopropolis, and determined its chemical profile. The antioxidant activity was determined using in vitro ABTS·+, ·DPPH, and ß-carotene/linoleic acid co-oxidation methods. The antibacterial activity was determined using a microdilution method with Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant S. aureus. The antiproliferative effect was determined in tumor cell lines using the Alamar Blue assay. The chemical profile was obtained using UHPLC-MS and UHPLC-MS/MS. The butanolic fraction had the highest concentration of phenolic compounds and more potent antioxidant properties in all assays. This fraction also had bacteriostatic and bactericidal effects against all bacterial strains at low concentrations, especially S. aureus. The hexane fraction had the highest antiproliferative potential, with IC50 values ranging from 24.2 to 46.6 µg/mL in HL-60 (human promyelocytic leukemia cell) and K562 (human chronic myelocytic leukemia cell), respectively. Preliminary chemical analysis indicates the presence of terpenes and gallic acid in the geopropolis. Our results indicate the therapeutic potential of geopropolis from M. mondury against inflammatory, oxidative, infectious, and neoplastic diseases.

Cytochrome c reacts with cholesterol hydroperoxides to produce lipid- and protein-derived radicals.

Lipid peroxidation is a well-known process that has been implicated in many diseases. Recent evidence has shown that mitochondrial cholesterol levels are increased under specific conditions, making it an important target for peroxidation inside the mitochondria. Cholesterol peroxidation generates, as primary products, several hydroperoxides (ChOOH), which can react with transition metals and metalloproteins. In this sense, cytochrome c (CYTC), a heme protein largely found in the mitochondria, becomes a candidate to react with ChOOH. Using CYTC associated with SDS micelles to mimic mitochondrial conditions, we show that ChOOH induces dose-dependent CYTC Soret band bleaching, indicating that it is using ChOOH as a substrate. This reaction leads to protein oligomerization, suggesting the formation of a protein radical that, subsequently, recombines, giving dimers, trimers, and tetramers. EPR experiments confirmed the production of carbon-centered radicals from both protein and lipid in the presence of ChOOH. Similar results were obtained with linoleic acid hydroperoxides (LAOOH). In addition, replacing SDS micelles by cardiolipin-containing liposomes as the mitochondrial mimetic led to similar results with either ChOOH or LAOOH. Importantly, kinetic experiments show that CYTC bleaching is faster with ChOOH than with H2O2, suggesting that these hydroperoxides could be relevant substrates for CYTC peroxidase-like activity in biological media. Altogether, these results show that CYTC induces homolytic cleavage of lipid-derived hydroperoxides, producing lipid and protein radicals.

The carbonylation and covalent dimerization of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity is inhibited by the radical scavenger tempol.

Tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of various diseases via mechanisms that are not completely understood. Recently, we reported that high doses of tempol moderately increased survival in a rat model of ALS (amyotrophic lateral sclerosis) while decreasing the levels of oxidized hSOD1 (human Cu,Zn-superoxide dismutase) in spinal cord tissues. To better understand such a protective effect in vivo, we studied the effects of tempol on hSOD1 oxidation in vitro. The chosen oxidizing system was the bicarbonate-dependent peroxidase activity of hSOD1 that consumes H2O2 to produce carbonate radical, which oxidizes the enzyme. Most of the experiments were performed with 30 µM hSOD1, 25 mM bicarbonate, 1 mM H2O2, 0.1 mM DTPA (diethylenetriaminepenta-acetic acid) and 50 mM phosphate buffer at a final pH of 7.4. The results showed that tempol (5-75 µM) does not inhibit hSOD1 turnover, but decreases its resulting oxidation to carbonylated and covalently dimerized forms. Tempol acted by scavenging the carbonate radical produced and by recombining with hSOD1-derived radicals. As a result, tempol was consumed nearly stoichiometrically with hSOD1 monomers. MS analyses of turned-over hSOD1 and of a related peptide oxidized by the carbonate radical indicated the formation of a relatively unstable adduct between tempol and hSOD1-Trp32•. Tempol consumption by the bicarbonate-dependent peroxidase activity of hSOD1 may be one of the reasons why high doses of tempol were required to afford protection in an ALS rat model. Overall, the results of the present study confirm that tempol can protect against protein oxidation and the ensuing consequences.

Inhibition of the chlorinating activity of myeloperoxidase by tempol: revisiting the kinetics and mechanisms.

The nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of inflammation by mechanisms that are not completely understood. MPO (myeloperoxidase), which plays a fundamental role in oxidant production by neutrophils, is an important target for anti-inflammatory action. By amplifying the oxidative potential of H2O2, MPO produces hypochlorous acid and radicals through the oxidizing intermediates MPO-I [MPO-porphyrin•+-Fe(IV)=O] and MPO-II [MPO-porphyrin-Fe(IV)=O]. Previously, we reported that tempol reacts with MPO-I and MPO-II with second-order rate constants similar to those of tyrosine. However, we noticed that tempol inhibits the chlorinating activity of MPO, in contrast with tyrosine. Thus we studied the inhibition of MPO-mediated taurine chlorination by tempol at pH 7.4 and re-determined the kinetic constants of the reactions of tempol with MPO-I (k=3.5×105 M-1·s-1) and MPO-II, the kinetics of which indicated a binding interaction (K=2.0×10-5 M; k=3.6×10-2 s-1). Also, we showed that tempol reacts extremely slowly with hypochlorous acid (k=0.29 and 0.054 M-1·s-1 at pH 5.4 and 7.4 respectively). The results demonstrated that tempol acts mostly as a reversible inhibitor of MPO by trapping it as MPO-II and the MPO-II-tempol complex, which are not within the chlorinating cycle. After turnover, a minor fraction of MPO is irreversibly inactivated, probably due to its reaction with the oxammonium cation resulting from tempol oxidation. Kinetic modelling indicated that taurine reacts with enzyme-bound hypochlorous acid. Our investigation complements a comprehensive study reported while the present study was underway

Methoxylated flavonols from Vellozia dasypus Seub ethyl acetate active myeloperoxidase extract: in vitro and in silico assays.

This study aimed to evaluate the effect of a methoxylated fraction from Vellozia dasypus Seub on myeloperoxidase (MPO)-chlorinating activity and subsequent in silico assays for binding profile prediction. Therefore, the ethyl acetate extract of aerial parts from Vellozia dasypus Seub was fractionated on open-column chromatography containing SiO2 and eluted with solvent in crescent polarity to yield a fraction with a mixture of flavonols quercetin 3-O-methyl ether (1) and 6-C-methyl quercetin 3-O-methyl ether (2). Their chemical structures were proposed by HPLC coupled to photodiode array (HPLC-DAD) and mass spectrometer using electrospray ionization multistage analysis (HPLC-MS/MS). The fraction enriched with compounds 1 and 2 inhibited more efficiently the in vitro MPO-chlorinating activity (IC50 = 40 µg/mL) than the ethyl acetate extract (IC50 = 64.0 µg/mL). Molecular docking studies revealed that these compounds interact with MPO active pocket similarly to trifluoromethyl-substituted aromatic hydroxamate, a well-known MPO inhibitor, co-crystallized at the MPO binding site (PDB ID: 4C1M). Molecular dynamics trajectories confirmed that these two molecules interact with the MPO binding site with a similar energetic pattern when compared to the crystallographic ligand. Taken together, these data expand the sources of phenolic natural compounds that may be further investigated against inflammation-related diseases. Communicated by Ramaswamy H. Sarma.

Preparation, validation and use of a vasoactive tryptophan-derived hydroperoxide and relevant control compounds.

The L-tryptophan-derived tricyclic hydroperoxide cis-WOOH was recently identified as a novel and biologically important factor for regulating vascular tone and blood pressure under inflammatory conditions and potentially other cellular redox signaling events. cis-WOOH is highly labile and currently not available commercially. In this protocol, we provide procedures for the synthesis, purification, quantification and characterization of cis-WOOH, its epimer trans-WOOH and their respective alcohols (cis-WOH and trans-WOH). Photo-oxidation of L-tryptophan (L-Trp) results in a mixture containing cis-WOOH and trans-WOOH, which are separated and purified by semi-preparative HPLC. cis-WOH and trans-WOH are then produced by sodium borohydride reduction and purified by semi-preparative HPLC. Characterization of cis-WOOH and trans-WOOH and the reduced alcohol variants is achieved using HPLC, fluorescence, NMR and liquid chromatography-tandem mass spectrometry. The protocol provides instructions for storage and quantification, as well as ways to test the stability of these hydroperoxides in commonly used buffers and media. Finally, we describe examples of how to monitor the formation of cis-WOOH in biological samples. The protocol ensures reasonable yield (11%) and purity (>99%) of cis-WOOH and control compounds in 5-6 d and outlines conditions under which cis-WOOH is stable for several months.

Hydrogen peroxide signaling via its transformation to a stereospecific alkyl hydroperoxide that escapes reductive inactivation.

During systemic inflammation, indoleamine 2,3-dioxygenase 1 (IDO1) becomes expressed in endothelial cells where it uses hydrogen peroxide (H2O2) to oxidize L-tryptophan to the tricyclic hydroperoxide, cis-WOOH, that then relaxes arteries via oxidation of protein kinase G 1α. Here we show that arterial glutathione peroxidases and peroxiredoxins that rapidly eliminate H2O2, have little impact on relaxation of IDO1-expressing arteries, and that purified IDO1 forms cis-WOOH in the presence of peroxiredoxin 2. cis-WOOH oxidizes protein thiols in a selective and stereospecific manner. Compared with its epimer trans-WOOH and H2O2, cis-WOOH reacts slower with the major arterial forms of glutathione peroxidases and peroxiredoxins while it reacts more readily with its target, protein kinase G 1α. Our results indicate a paradigm of redox signaling by H2O2 via its enzymatic conversion to an amino acid-derived hydroperoxide that 'escapes' effective reductive inactivation to engage in selective oxidative activation of key target proteins.

Production of lysozyme and lysozyme-superoxide dismutase dimers bound by a ditryptophan cross-link in carbonate radical-treated lysozyme.

Despite extensive investigation of the irreversible oxidations undergone by proteins in vitro and in vivo, the products formed from the oxidation of Trp residues remain incompletely understood. Recently, we characterized a ditryptophan cross-link produced by the recombination of hSOD1-tryptophanyl radicals generated from attack of the carbonate radical produced during the bicarbonate-dependent peroxidase activity of the enzyme. Here, we examine whether the ditryptophan cross-link is produced by the attack of the carbonate radical on proteins other than hSOD1. To this end, we treated hen egg white lysozyme with photolytically and enzymatically generated carbonate radical. The radical yields were estimated and the lysozyme modifications were analyzed by SDS-PAGE, western blot, enzymatic activity and MS/MS analysis. Lysozyme oxidation by both systems resulted in its inactivation and dimerization. Lysozyme treated with the photolytic system presented monomers oxidized to hydroxy-tryptophan at Trp(28) and Trp(123) and N-formylkynurenine at Trp(28), Trp(62) and Trp(123). Lysozyme treated with the enzymatic system rendered monomers oxidized to N-formylkynurenine at Trp(28). The dimers were characterized as lysozyme-Trp(28)-Trp(28)-lysozyme and lysozyme-Trp(28)-Trp(32)-hSOD1. The results further demonstrate that the carbonate radical is prone to causing biomolecule cross-linking and hence, may be a relevant player in pathological mechanisms. The possibility of exploring the formation of ditryptophan cross-links as a carbonate radical biomarker is discussed.

Nitroxides attenuate carrageenan-induced inflammation in rat paws by reducing neutrophil infiltration and the resulting myeloperoxidase-mediated damage.

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and other cyclic nitroxides have been shown to inhibit the chlorinating activity of myeloperoxidase (MPO) in vitro and in cells. To examine whether nitroxides inhibit MPO activity in vivo we selected acute carrageenan-induced inflammation on the rat paw as a model. Tempol and three more hydrophobic 4-substituted derivatives (4-azido, 4-benzenesulfonyl, and 4-(4-phenyl-1H-1,2,3-triazol-1-yl)) were synthesized, and their ability to inhibit the in vitro chlorinating activity of MPO and carrageenan-induced inflammation in rat paws was evaluated. All of the tested nitroxides inhibited the chlorinating activity of MPO in vitro with similar IC(50) values (between 1.5 and 1.8 µM). In vivo, the attenuation of carrageenan-induced inflammation showed some correlation with the lipophilicity of the nitroxide at early time points but the differences in the effects were small (<2-fold) compared with the differences in lipophilicity (>200-fold). No inhibition of MPO activity in vivo was evident because the levels of MPO activity in rat paws correlated with the levels of MPO protein. Likewise, paw edema, levels of nitrated and oxidized proteins, and levels of plasma exudation correlated with the levels of MPO protein in the paws of the animals that were untreated or treated with the nitroxides. The effects of the nitroxides in vivo were compared with those of 4-aminobenzoic hydrazide and of colchicine. Taken together, the results indicate that nitroxides attenuate carrageenan-induced inflammation mainly by reducing neutrophil migration and the resulting MPO-mediated damage. Accordingly, tempol was shown to inhibit rat neutrophil migration in vitro.

Evaluation of cytogenotoxicity, antioxidant and hypoglycemiant activities of isolate compounds from Mansoa hirsuta D.C. (Bignoniaceae)

ABSTRACT Mansoa hirsuta (Bignoniaceae) is a native plant from caatinga in Brazilian semiarid. This plant has been locally used as antimicrobial and hypoglycemiant agents, but their action mechanisms and toxicity remain largely unknown. Therefore, we evaluated the composition and antioxidant, cytoprotective and hypoglycemiant effects of raw extract, fractions and compounds from leaves of M. hirsuta. The cytogenotoxic effects of ursolic and oleanolic acids, the main phytotherapic components of this plant, were assessed. The raw extract and fractions presented steroids, saponins, flavonols, flavanonols, flavanones, xanthones, phenols, tannins, anthocyanins, anthocyanidins and flavonoids. The ethyl acetate fraction inhibited efficiently the cascade of lipid peroxidation while the hydroalcoholic fraction was richer in total phenols and more efficient in capturing 2,2-diphenyl-1-picrylhydrazyl (·DPPH) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS·+) radicals. The isolated fraction of M. hirsuta also inhibited the α-amylase activity. Cytotoxic effects were absent in both raw extract and fractions while ursolic+oleanolic acids were efficient in protecting cells after exposure to hydrogen peroxide. Moreover, this mixture of acid shad no significant interference on the mitotic index and frequency of nuclear and/or chromosomal abnormalities in Allium cepa test. Therefore, M. hirsuta represents a potential source of phytochemicals against inflammatory and oxidative pathologies, including diabetes.

Melipona mondury produces a geopropolis with antioxidant, antibacterial and antiproliferative activities

ABSTRACT Geopropolis is a special type of propolis produced by stingless bees. Several pharmacological properties have been described for different types of geopropolis, but there have been no previous studies of the geopropolis from Melipona mondury. In this study, we investigated the antioxidant, antibacterial, and antiproliferative activities of M. mondury geopropolis, and determined its chemical profile. The antioxidant activity was determined using in vitro ABTS·+, ·DPPH, and β-carotene/linoleic acid co-oxidation methods. The antibacterial activity was determined using a microdilution method with Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant S. aureus. The antiproliferative effect was determined in tumor cell lines using the Alamar Blue assay. The chemical profile was obtained using UHPLC-MS and UHPLC-MS/MS. The butanolic fraction had the highest concentration of phenolic compounds and more potent antioxidant properties in all assays. This fraction also had bacteriostatic and bactericidal effects against all bacterial strains at low concentrations, especially S. aureus. The hexane fraction had the highest antiproliferative potential, with IC50 values ranging from 24.2 to 46.6 µg/mL in HL-60 (human promyelocytic leukemia cell) and K562 (human chronic myelocytic leukemia cell), respectively. Preliminary chemical analysis indicates the presence of terpenes and gallic acid in the geopropolis. Our results indicate the therapeutic potential of geopropolis from M. mondury against inflammatory, oxidative, infectious, and neoplastic diseases.

MPO Inhibitors Selected by Virtual Screening.

The hemeprotein myeloperoxidase (MPO) participates in innate immune defense through its ability to generate potent microbicidal oxidants. However, these oxidants are also key mediators of the tissue damage associated with many inflammatory diseases. Thus, there is considerable interest in developing therapeutically useful MPO inhibitors. Here, we used structure-based drug design (SBDD) and ligand-based drug design (LBDD) to select for potentially new and selective MPO inhibitors. A pharmacophore model was developed based on the crystal structure of human MPO in complex with salicylhydroxamic acid (SHA), a known inhibitor of the enzyme. The pharmacophore model was used to screen the ZINC database for potential ligands, which were further filtered on the basis of their physical-chemical properties and docking score. The filtered compounds were visually inspected, and nine were purchased for experimental studies. Surprisingly, almost all of the selected compounds belonged to the aromatic hydrazide class, which had been previously described as MPO inhibitors. The compounds selected by virtual screening were shown to inhibit the chlorinating activity of MPO; the top four compounds displayed IC50 values ranging from 1.0 to 2.8 µM. MPO inactivation by the most effective compound was shown to be irreversible. Overall, our results show that SBDD and LBDD may be useful for the rational development of new MPO inhibitors.