Tetracyclic 1,4-Naphthoquinone Thioglucoside Conjugate U-556 Blocks the Purinergic P2X7 Receptor in Macrophages and Exhibits Anti-Inflammatory Activity In Vivo.

P2X7 receptors (P2X7Rs) are ligand-gated ion channels that play a significant role in inflammation and are considered a potential therapeutic target for some inflammatory diseases. We have previously shown that a number of synthetic 1,4-naphthoquinones are capable of blocking P2X7Rs in neuronal and macrophage cells. In the present investigation, we have demonstrated the ability of the tetracyclic quinone-thioglucoside conjugate U-556, derived from 1,4-naphthoquinone thioglucoside, to inhibit ATP-induced Ca2+ influx and YO-PRO-1 dye uptake, which indicates blocking P2X7R in RAW 264.7 macrophages. This process was accompanied by the inhibition of ATP-induced reactive oxygen species production in macrophages, as well as the macrophage survival strengthening under ATP toxic effects. Nevertheless, U-556 had no noticeable antioxidant capacity. Naphthoquinone-thioglucoside conjugate U-556 binding to the extracellular part of the P2X7R was confirmed by SPR analysis, and the kinetic characteristics of this complex formation were established. Computer modeling predicted that U-556 binds the P2X7R allosteric binding site, topographically similar to that of the specific A438079 blocker. The study of biological activity in in vivo experiments shows that tetracylic conjugate significantly reduces inflammation provoked by carrageenan. The data obtained points out that the observed physiological effects of U-556 may be due to its ability to block the functioning of the P2X7R.

Structural Insights into the Substrate Transport Mechanisms in GTR Transporters through Ensemble Docking.

Glucosinolate transporters (GTRs) are part of the nitrate/peptide transporter (NPF) family, members of which also transport specialized secondary metabolites as substrates. Glucosinolates are defense compounds derived from amino acids. We selected 4-methylthiobutyl (4MTB) and indol-3-ylmethyl (I3M) glucosinolates to study how GTR1 from Arabidopsis thaliana transports these substrates in computational simulation approaches. The designed pipeline reported here includes massive docking of 4MTB and I3M in an ensemble of GTR1 conformations (in both inward and outward conformations) extracted from molecular dynamics simulations, followed by clustered and substrate-protein interactions profiling. The identified key residues were mutated, and their role in substrate transport was tested. We were able to identify key residues that integrate a major binding site of these substrates, which is critical for transport activity. In silico approaches employed here represent a breakthrough in the plant transportomics field, as the identification of key residues usually takes a long time if performed from a purely wet-lab experimental perspective. The inclusion of structural bioinformatics in the analyses of plant transporters significantly speeds up the knowledge-gaining process and optimizes valuable time and resources.

Diverse Excretion Pathways of Benzyl Glucosinolate in Humans after Consumption of Nasturtium (Tropaeolum majus L.)-A Pilot Study.

SCOPE: Different metabolic and excretion pathways of the benzyl glucosinolate breakdown products benzyl isothiocyanate and benzyl cyanide are investigated to obtain information about their multiple fate after ingestion. Detailed focus is on the so far underestimated transformation/excretion pathways-protein conjugation and exhalation. METHODS AND RESULTS: Metabolites, protein conjugates, and non-conjugated isothiocyanates are determined in plasma, urine, and breath of seven volunteers after consuming freeze-dried nasturtium or bread enriched with nasturtium. Samples are collected up to 48 h at selected time points. The metabolites of the mercapturic acid pathway are detectable in plasma up to 24 h after consumption. Additionally, mercapturic acid is the main metabolite in urine, but non-conjugated benzyl isothiocyanate is detectable as well. Protein conjugates show high amounts in plasma even 48 h after consumption. In breath, benzyl isothiocyanate and benzyl cyanide are detectable up to 48 h after consumption. CONCLUSION: Isothiocyanates are not only metabolized via the mercapturic acid pathway, but also form protein conjugates in blood and are exhaled. To balance intake and excretion, it is necessary to investigate all potential metabolites and excretion routes. This has important implications for the understanding of physiological and pharmacological effects of isothiocyanate-containing products.

Bioavailability and metabolism of benzyl glucosinolate in humans consuming Indian cress (Tropaeolum majus L.).

SCOPE: Benzyl isothiocyanate (BITC), which occurs in Brassicales, has demonstrated chemopreventive potency and cancer treatment properties in cell and animal studies. However, fate of BITC in human body is not comprehensively studied. Therefore, the present human intervention study investigates the metabolism of the glucosinolate (GSL) glucotropaeolin and its corresponding BITC metabolites. Analyzing BITC metabolites in plasma and urine should reveal insights about resorption, metabolism, and excretion. METHODS AND RESULTS: Fifteen healthy men were randomly recruited for a cross-over study and consumed 10 g freeze-dried Indian cress as a liquid preparation containing 1000 µmol glucotropaeolin. Blood and urine samples were taken at several time points and investigated by LC-ESI-MS/MS after sample preparation using SPE. Plasma contained high levels of BITC-glutathione (BITC-GSH), BITC-cysteinylglycine (BITC-CysGly), and BITC-N-acetyl-L-cysteine (BITC-NAC) 1-5 h after ingestion, with BITC-CysGly appearing as the main metabolite. Compared to human plasma, the main urinary metabolites were BITC-NAC and BITC-Cys, determined 4-6 h after ingestion. CONCLUSION: This study confirms that consumption of Indian cress increases the concentration of BITC metabolites in human plasma and urine. The outcome of this human intervention study supports clinical research dealing with GSL-containing innovative food products or pharmaceutical preparations.

Quantification of plant surface metabolites by matrix-assisted laser desorption-ionization mass spectrometry imaging: glucosinolates on Arabidopsis thaliana leaves.

The localization of metabolites on plant surfaces has been problematic because of the limitations of current methodologies. Attempts to localize glucosinolates, the sulfur-rich defense compounds of the order Brassicales, on leaf surfaces have given many contradictory results depending on the method employed. Here we developed a matrix-assisted laser desorption-ionization (MALDI) mass spectrometry protocol to detect surface glucosinolates on Arabidopsis thaliana leaves by applying the MALDI matrix through sublimation. Quantification was accomplished by spotting glucosinolate standards directly on the leaf surface. The A. thaliana leaf surface was found to contain approximately 15 nmol of total glucosinolate per leaf with about 50 pmol mm(-2) on abaxial (bottom) surfaces and 15-30 times less on adaxial (top) surfaces. Of the major compounds detected, 4-methylsulfinylbutylglucosinolate, indol-3-ylmethylglucosinolate, and 8-methylsulfinyloctylglucosinolate were also major components of the leaf interior, but the second most abundant glucosinolate on the surface, 4-methylthiobutylglucosinolate, was only a trace component of the interior. Distribution on the surface was relatively uniform in contrast to the interior, where glucosinolates were distributed more abundantly in the midrib and periphery than the rest of the leaf. These results were confirmed by two other mass spectrometry-based techniques, laser ablation electrospray ionization and liquid extraction surface analysis. The concentrations of glucosinolates on A. thaliana leaf surfaces were found to be sufficient to attract the specialist feeding lepidopterans Plutella xylostella and Pieris rapae for oviposition. The methods employed here should be easily applied to other plant species and metabolites.

Turning the 'mustard oil bomb' into a 'cyanide bomb': aromatic glucosinolate metabolism in a specialist insect herbivore.

Plants have evolved a variety of mechanisms for dealing with insect herbivory among which chemical defense through secondary metabolites plays a prominent role. Physiological, behavioural and sensorical adaptations to these chemicals provide herbivores with selective advantages allowing them to diversify within the newly occupied ecological niche. In turn, this may influence the evolution of plant metabolism giving rise to e.g. new chemical defenses. The association of Pierid butterflies and plants of the Brassicales has been cited as an illustrative example of this adaptive process known as 'coevolutionary armsrace'. All plants of the Brassicales are defended by the glucosinolate-myrosinase system to which larvae of cabbage white butterflies and related species are biochemically adapted through a gut nitrile-specifier protein. Here, we provide evidence by metabolite profiling and enzyme assays that metabolism of benzylglucosinolate in Pieris rapae results in release of equimolar amounts of cyanide, a potent inhibitor of cellular respiration. We further demonstrate that P. rapae larvae develop on transgenic Arabidopsis plants with ectopic production of the cyanogenic glucoside dhurrin without ill effects. Metabolite analyses and fumigation experiments indicate that cyanide is detoxified by ß-cyanoalanine synthase and rhodanese in the larvae. Based on these results as well as on the facts that benzylglucosinolate was one of the predominant glucosinolates in ancient Brassicales and that ancient Brassicales lack nitrilases involved in alternative pathways, we propose that the ability of Pierid species to safely handle cyanide contributed to the primary host shift from Fabales to Brassicales that occured about 75 million years ago and was followed by Pierid species diversification.

Engineering of benzylglucosinolate in tobacco provides proof-of-concept for dead-end trap crops genetically modified to attract Plutella xylostella (diamondback moth).

Glucosinolates are biologically active natural products characteristic of crucifers, including oilseed rape, cabbage vegetables and the model plant Arabidopsis thaliana. Crucifer-specialist insect herbivores, like the economically important pest Plutella xylostella (diamondback moth), frequently use glucosinolates as oviposition stimuli. This suggests that the transfer of a glucosinolate biosynthetic pathway to a non-crucifer would stimulate oviposition on an otherwise non-attractive plant. Here, we demonstrate that stable genetic transfer of the six-step benzylglucosinolate pathway from A. thaliana to Nicotiana tabacum (tobacco) results in the production of benzylglucosinolate without causing morphological alterations. Benzylglucosinolate-producing tobacco plants were more attractive for oviposition by female P. xylostella moths than wild-type tobacco plants. As newly hatched P. xylostella larvae were unable to survive on tobacco, these results represent a proof-of-concept strategy for rendering non-host plants attractive for oviposition by specialist herbivores with the long-term goal of generating efficient dead-end trap crops for agriculturally important pests.

N-phenylglucosylamine hydrolysis: a mechanistic probe of ß-glucosidase.

The spontaneous hydrolysis of glycosylamines, where the aglycone is either a primary amine or ammonia, is over a hundred million-times faster than that of O- or S-glycosides. The reason for this (as pointed out by Capon and Connett in 1965) is that, in contrast to the mechanism for O- or S-glycoside hydrolysis, hydrolysis of these N-glycosides (e.g., glc-NHR) involves an endocyclic C-O bond cleavage resulting in formation of an imine (iminium ion) which then reacts with water. Since ring-opening is kinetically favored with glycosylamines, compounds such as phenylglucosylamine can be a useful probes of enzymes that have been suggested to possibly follow this mechanism. With ß-glucosidase from sweet almonds, the enzyme is highly efficient in catalyzing the hydrolysis of phenyl glucoside (k(cat)/k(non)∼10(14)) and phenyl thioglucoside (k(cat)/k(non)∼10(10)) while with either the almond or the Aspergillus niger enzyme or with yeast α-glucosidase, there is no detectable catalysis of phenylglucosylamine hydrolysis (k(cat)/k(non)<20). These results are consistent with the generally accepted mechanism involving exocyclic bond cleavage by these enzymes.

Modulation of sulfur metabolism enables efficient glucosinolate engineering.

BACKGROUND: Metabolic engineering in heterologous organisms is an attractive approach to achieve efficient production of valuable natural products. Glucosinolates represent a good example of such compounds as they are thought to be the cancer-preventive agents in cruciferous plants. We have recently demonstrated that it is feasible to engineer benzylglucosinolate (BGLS) in the non-cruciferous plant Nicotiana benthamiana by transient expression of five genes from Arabidopsis thaliana. In the same study, we showed that co-expression of a sixth Arabidopsis gene, γ-glutamyl peptidase 1 (GGP1), resolved a metabolic bottleneck, thereby increasing BGLS accumulation. However, the accumulation did not reach the expected levels, leaving room for further optimization. RESULTS: To optimize heterologous glucosinolate production, we have in this study performed a comparative metabolite analysis of BGLS-producing N. benthamiana leaves in the presence or absence of GGP1. The analysis revealed that the increased BGLS levels in the presence of GGP1 were accompanied by a high accumulation of the last intermediate, desulfoBGLS, and a derivative thereof. This evidenced a bottleneck in the last step of the pathway, the transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to desulfoBGLS by the sulfotransferase AtSOT16. While substitution of AtSOT16 with alternative sulfotransferases did not alleviate the bottleneck, experiments with the three genes involved in the formation and recycling of PAPS showed that co-expression of adenosine 5'-phosphosulfate kinase 2 (APK2) alone reduced the accumulation of desulfoBGLS and its derivative by more than 98% and increased BGLS accumulation 16-fold. CONCLUSION: Adjusting sulfur metabolism by directing sulfur from primary to secondary metabolism leads to a remarkable improvement in BGLS accumulation and thereby represents an important step towards a clean and efficient production of glucosinolates in heterologous hosts. Our study emphasizes the importance of considering co-substrates and their biological nature in metabolic engineering projects.

5-Nitro-2-pyridyl-1-thioglucosides: application in synthesis of analogues of glycosyltransferases natural substrates.

5-Nitro-2-pyridyl-1-thioglucosides were used in synthesis of complex uridine derivatives (13-16) in two different sequences of reactions. In one route, the first step was glycosylation of selectively protected 5-nitro-2-pyridyl-1-thioglucoside 1 with two different glycosyl donors (5 or 6), next, the nitro group in aglycone of obtained disaccharides 7 or 8 was reduced and then obtained products 9 or 10 were condensed with uridine derivatives 3 or 4 using DMT-MM as condensing agent under microwave irradiation. In the second route, condensation and glycosylation reactions were applied in reverse order. As it turned up, a sequence of reactions affected the yield of final glycoconjugates 13-16 and depended on the type of uridine derivatives used.

Herbicidal activity of glucosinolate degradation products in fermented meadowfoam ( Limnanthes alba ) seed meal.

Meadowfoam ( Limnanthes alba ) is an oilseed crop grown in western Oregon. After extraction of the oil from the seeds, the remaining seed meal contains 2-4% of the glucosinolate glucolimnanthin. This study investigated the effect of fermentation of seed meal on its chemical composition and the effect of the altered composition on downy brome ( Bromus tectorum ) coleoptile emergence. Incubation of enzyme-inactive seed meal with enzyme-active seeds (1% by weight) resulted in complete degradation of glucolimnanthin and formation of 3-methoxybenzyl isothiocyanate in 28% yield. Fermentation in the presence of an aqueous solution of FeSO(4) (10 mM) resulted in the formation of 3-methoxyphenylacetonitrile and 2-(3-methoxyphenyl)ethanethioamide, a novel natural product. The formation of the isothiocyanate, the nitrile, and the thioamide, as a total, correlated with an increase of herbicidal potency of the seed meal (r(2) = 0.96). The results of this study open new possibilities for the refinement of glucosinolate-containing seed meals for use as bioherbicides.

Towards engineering glucosinolates into non-cruciferous plants.

Glucosinolates are amino acid-derived secondary metabolites present in cruciferous plants. Glucosinolates and their hydrolysis products are involved in defence against insects and pathogens, but are also known for their characteristic flavor and their cancer-preventive and antibacterial properties. This wide range of bioactivities has prompted a desire to engineer glucosinolates into non-cruciferous plants. We report the one-step transfer of the last three steps of the benzylglucosinolate pathway (comprising the C-S lyase, glycosyltransferase and sulfotransferase) from Arabidopsis to tobacco. This was achieved using an expression construct consisting of a single 2A polycistronic open reading frame, which allowed the expression of the three coding-sequences from a single promoter. When compared to wildtype plants, transgenic tobacco lines showed increased ability to convert the intermediate phenylacetothiohydroxamate to benzylglucosinolate upon in vivo feeding. Enzymatic assays using plant extracts demonstrated that the individual activities required for this conversion were enhanced in the transgenic plants. The relatively high conversion by wildtype plants in feeding assays supports the hypothesis that the last part of the glucosinolate pathway was recruited from existing detoxification reactions. Immunoblots confirmed that individual proteins were being successfully produced from the 2A polycistronic open reading frame, albeit fusion proteins could also be detected. In summary, we transferred the last three steps of the benzylglucosinolate pathway to tobacco as a first step towards engineering glucosinolates into non-cruciferous plants.

Glycine conjugates in a lepidopteran insect herbivore--the metabolism of benzylglucosinolate in the cabbage white butterfly, Pieris rapae.

Herbivores have developed a wide array of countermeasures to overcome plants' chemical defences. Larvae of the cabbage white butterfly, Pieris rapae, feed exclusively on plants of the Brassicales order, which are defended by the glucosinolate-myrosinase system. The defensive function of this system comes from toxic isothiocyanates that are formed when glucosinolates are hydrolysed by myrosinases upon tissue damage. Here we show that P. rapae larvae convert benzylglucosinolate to phenylacetylglycine, which is released in their faeces. Feeding experiments with isotopic tracers suggest that phenylacetonitrile and phenylacetic acid are intermediates in this conversion. We also identified additional glycine and isoserine (2-hydroxy-3-aminopropanoic acid) conjugates with benzoate and indole-3-carboxylate from P. rapae faeces extracts. This is the first description of such conjugates from lepidopteran insects.

Thiosugar nucleosides. Synthesis and biological activity of 1,3,4-thiadiazole, thiazoline and thiourea derivatives of 5-thio-D-glucose.

New acylated 5-thio-beta-D-glucopyranosylimino-disusbstituted 1,3,4-thiadiazols 8, and 11 were prepared, via spontaneous rearrangements, by cycloaddition of the glycosyl isothiocyanate 2 with the reactive intermediates 1-aza-2-azoniaallene hexachloroantimonates 4 and 6, respectively. Reaction of 2 with aminoacetone or chloroethylamine afforded the acylated 5-thio-beta-D-glucopyranosyl-4-imidazoline-2-thione nucleoside 16 and glucopyranosylamino-2-thiazoline derivative 18, respectively. Deblocking of 8, 11, 17 and 19 furnished the free nucleoside analogues 9, 12, 18 and 20, respectively. Analogously, treatment of 2 with chloroethylamine in the 1:2 ratio afforded the thioureylendisaccharide 21. No in vitro antiviral activity against HIV-1, HIV-2, human cytomegallovirus (HMCV), has been found for the new synthesized compounds.

Selective transport systems mediate sequestration of plant glucosides in leaf beetles: a molecular basis for adaptation and evolution.

Chrysomeline larvae respond to disturbance and attack by everting dorsal glandular reservoirs, which release defensive secretions. The ancestral defense is based on the de novo synthesis of monoterpene iridoids. The catabolization of the host-plant O-glucoside salicin into salicylaldehyde is a character state that evolved later in two distinct lineages, which specialized on Salicaceae. By using two species producing monoterpenes (Hydrothassa marginella and Phratora laticollis) and two sequestering species (Chrysomela populi and Phratora vitellinae), we studied the molecular basis of sequestration by feeding the larvae structurally different thioglucosides resembling natural O-glucosides. Their accumulation in the defensive systems demonstrated that the larvae possess transport systems, which are evolutionarily adapted to the glycosides of their host plants. Minor structural modifications in the aglycon result in drastically reduced transport rates of the test compounds. Moreover, the ancestral iridoid-producing leaf beetles already possess a fully functional import system for an early precursor of the iridoid defenses. Our data confirm an evolutionary scenario in which, after a host-plant change, the transport system of the leaf beetles may play a pivotal role in the adaptation on new hosts by selecting plant-derived glucosides that can be channeled to the defensive system.

In vitro digestion of sinigrin and glucotropaeolin by single strains of Bifidobacterium and identification of the digestive products.

Three strains of Bifidobacterium sp., B. pseudocatenulatum, B. adolescentis, and B. longum were studied for their ability to digest glucosinolates, sinigrin (SNG) and glucotropaeolin (GTL), in vitro. All strains digested both glucosinolates during 24-48 h cultivation, accompanied by a decline in the medium pH from 7.1 to 5.2. The digestion of glucosinolates by a cell-free extract prepared from sonicated cells of B. adolescentis, but not cultivated broth, increased in the presence of 0.5 mM l-ascorbic acid. Also, a time-dependent formation of allyl isothiocyanate (AITC) was observed when the cell-free extract was incubated with 0.25 mM SNG for 120 min at pH 7.0. These reaction features suggest that the digestive activity may have been due to an enzyme similar to myrosinase, an enzyme of plant origin. GC-MS analysis of the Bifidobacterial cultured broth showed that the major products were 3-butenenitrile (BCN) and phenylacetonitrile (PhACN), from SNG and GTL, respectively and nitriles, probably due to a decrease in the pH of the media. AITC and benzyl isothiocyanate (BzITC) were barely detectable in the broth. It was concluded that the three species of Bifidobacteria could be involved in digestive degradation of glucosinolates in the human intestinal tract.

Analysis of urinary N-acetyl-beta-D-glucosaminidase using 2,4-dinitrophenyl-1-thio N-acetyl-beta-D-glucosaminide as the substrate.

2,4-Dinitrophenyl-1-thio N-acetyl-beta-D-glucosaminide was examined as a new substrate for analyzing the level of N-acetyl-beta-D-glucosaminidase in the urine of patients suffering from renal diseases. The analysis is based on the fact that the substrate, when hydrolyzed in the presence of N-acetyl-beta-D-glucosaminidase, liberates 2,4-dinitrothiophenol as the chromogen. The optimum pH for the enzyme reaction is 4.6, which is covered by the optimal range for the UV absorbance of the chromogen. The first-order rate of increase of the absorbance at this pH was linear to the enzyme concentration up to 600 U/L, with a high sensitivity. Analytical reagents with glucosaminides of 2,4-dinitrophenol and 2-chloro-4-nitrophenol are less stable than the reagent with glucosaminide of 2,4-dinitrothiophenol. The optimum pH for the absorbance of p-nitrophenol and 2-chloro-4-nitrophenol does not match that for the enzyme reaction.

Induction of myrosinase gene expression and myrosinase activity in radish hypocotyls by phototropic stimulation.

The role of myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) in the phototropic response in radish hypocotyls was investigated. Unilateral illumination with blue light abruptly up-regulated the activity of myrosinase, which releases bioactive 4-methylthio-3-butenyl isothiocyanate (MTBI) from inactive 4-methylthio-3-butenyl glucosinolate (MTBG), in the illuminated halves of radish hypocotyls 10 min after onset of phototropic stimulation, peaking after 30 min and decreasing thereafter. The myrosinase activity in the shaded halves also increased, but was significantly lower than that in the illuminated halves. Furthermore, whether blue light illumination induces myrosinase gene expression was studied. Northern blotting analysis indicated that myrosinase mRNA levels were increased markedly in unilaterally illuminated hypocotyls, reaching maximum signal intensity within 10 min after onset of blue illumination, declining nearly to the control level thereafter. These results suggested that phototropic stimulation promotes myrosinase gene expression and myrosinase activity in the illuminated side, resulting in the conversion of inactive MTBG to active MTBI and simultaneously producing more active raphanusanins, causing a phototropic response.

Identification of new derivatives of sinigrin and glucotropaeolin produced by the human digestive microflora using 1H NMR spectroscopy analysis of in vitro incubations.

One- and two-dimensional (1)H NMR spectroscopy were used to study the biotransformation of two dietary glucosinolates, sinigrin (SIN), and glucotropaeolin (GTL) by the human digestive microflora in vitro. The molecular structures of the new metabolites issued from the aglycone moiety of the glucosinolate were identified, and the modulation of carbon metabolism was studied by quantifying bacterial metabolites issued from the xenobiotic incubation in the presence or absence of a source of free glucose. Unambiguously and for the first time, it was shown that SIN and GTL were transformed quantitatively into allylamine and benzylamine, respectively. The comparison of the kinetics of transformation of SIN and GTL with and without glucose clearly showed that the presence of glucose did not modify either the nature of the metabolites or the rate of transformation of the glucosinolates (complete degradation within 30 h). The main end products of the glucose moiety of glucosinolates were characteristic of anaerobic carbon metabolism in the digestive tract (acetate, lactate, ethanol, propionate, formate, and butyrate) and similar to those released from free glucose. This work represents the first application of (1)H NMR spectroscopy to the study of xenobiotic metabolism by the human digestive microflora, demonstrating allyl- and benzylamine production from glucosinolates. Whether these amines are produced in vivo from dietary glucosinolates remains to be established. This would reduce the availability of other glucosinolate metabolites, notably cancer-protective isothiocyanates.