RESUMEN
5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the only product of the proinflammatory 5-lipoxygenase pathway with potent chemoattractant effects for human eosinophils, suggesting an important role in eosinophilic diseases such as asthma. 5-Oxo-ETE, acting through its selective OXE receptor, induces dermal eosinophilia in both humans and monkeys. To block its effects, we designed selective indole-based OXE antagonists containing hexyl (S-230) or phenylhexyl (S-C025 and S-Y048) side chains, which inhibit allergen-induced dermal and pulmonary inflammation in monkeys, suggesting that they may be useful therapeutic agents in humans. In this study we identified two metabolic pathways for the phenylhexyl-containing antagonists in liver microsomes: benzylic and N-methyl hydroxylation, resulting in ω-hydroxy, ω-oxo, and NH-containing products with reduced potencies that were identified by mass spectrometry and comparison with synthetic standards. Products of both pathways were also identified in monkey plasma following oral administration of S-C025 and S-Y025, but were less abundant than the α-hydroxy metabolites that we previously identified. Interestingly, the α-hydroxy compounds were not detected in microsomal incubations, suggesting a different origin. The relative rates of metabolism of these antagonists were S-230 >> S-C025 > S-Y048, which may help to explain the differences in their plasma half-lives (S-230 < S-C025 < S-Y048). In conclusion, S-C025 and S-Y048 are metabolized by liver microsomes by benzylic and N-methyl hydroxylation but not by α-hydroxylation, whereas all three pathways exist in vivo. Addition of a phenyl group to the hexyl side chain of these antagonists dramatically reduced their rates of metabolism, which would explain their prolonged in vivo half-lives.
Asunto(s)
Eosinófilos , Receptores Eicosanoides , Animales , Antiinflamatorios/farmacología , Factores Quimiotácticos/farmacología , Haplorrinos/metabolismoRESUMEN
BACKGROUND AND PURPOSE: The 5-lipoxygenase product, 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid), is a potent chemoattractant for eosinophils and neutrophils. However, little is known about its pathophysiological role because of the lack of a rodent ortholog of the oxoeicosanoid (OXE) receptor. The present study aimed to determine whether the selective OXE receptor antagonist S-Y048 can inhibit allergen-induced pulmonary inflammation in a monkey model of asthma. EXPERIMENTAL APPROACH: Monkeys sensitized to house dust mite antigen (HDM) were treated with either vehicle or S-Y048 prior to challenge with aerosolized HDM, and bronchoalveolar (BAL) fluid was collected 24 h later. After 6 weeks, animals that had initially been treated with vehicle received S-Y048 and vice versa for animals initially treated with S-Y048. Eosinophils and neutrophils in BAL and lung tissue samples were evaluated, as well as mucus-containing cells in bronchi. KEY RESULTS: HDM significantly increased the numbers of eosinophils, neutrophils, and macrophages in BAL fluid 24 h after challenge. These responses were all significantly inhibited by S-Y048, which also reduced the numbers of eosinophils and neutrophils in lung tissue 24 h after challenge with HDM. S-Y048 also significantly reduced the numbers of bronchial epithelial cells staining for mucin and MUC5AC after antigen challenge. CONCLUSION AND IMPLICATIONS: This study provides the first evidence that 5-oxo-ETE may play an important role in inducing allergen-induced pulmonary inflammation and could also be involved in regulating MUC5AC in goblet cells. OXE receptor antagonists such as S-Y048 may useful therapeutic agents in asthma and other eosinophilic as well as neutrophilic diseases.
Asunto(s)
Asma , Neumonía , Alérgenos , Animales , Asma/tratamiento farmacológico , Eosinófilos , Neumonía/tratamiento farmacológico , Neumonía/prevención & control , Primates , Receptores EicosanoidesRESUMEN
Oxylipins constitute a huge class of compounds produced by oxidation of long-chain unsaturated fatty acids either chemically (by radicals such as reactive oxygen species, ROS) or enzymatically (by lipoxygenases, LOX; cyclooxygenases, COX; or cytochrome P450 pathways). This process generates fatty acids peroxides, which can then be further modified in a broad range to epoxy, hydroxy, keto, ether fatty acids, and also hydrolyzed to generate small aldehydes and alcohols. In general, oxylipins are present in almost all living organisms and have a wide range of signaling, metabolic, physiological, and ecological roles depending on the particular organism and on their structure. In plants, oxylipins have been extensively studied over the past 35 years. However, these studies have focused mainly on the jasmonates and so-called green leaves volatiles. The function of early LOX products (like keto and hydroxy fatty acids) is yet not well understood in plants, where they are mainly analyzed by indirect methods or by GC-MS what requires a laborious sample preparation. Here, we developed and validated a straightforward, precise, accurate, and sensitive method for quantifying oxylipins in plant tissues using HPLC-MS/MS, with a one-step extraction procedure using low amount of plant tissues. We successfully applied this method to quantify the oxylipins in different plant species and Arabidopsis thaliana plants treated with various biotic and abiotic stress conditions.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Oxilipinas/química , Extractos Vegetales/química , Plantas/química , Espectrometría de Masas en Tándem/métodos , Estructura Molecular , Oxidación-ReducciónRESUMEN
5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is an arachidonic acid metabolite formed by oxidation of the 5-lipoxygenase (5-LO) product 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5S-HETE) by the NADP+-dependent enzyme 5-hydroxyeicosanoid dehydrogenase. It is the only 5-LO product with appreciable chemoattractant activity for human eosinophils. Its actions are mediated by the selective OXE receptor, which is highly expressed on eosinophils, basophils, neutrophils and monocytes. Orthologs of the OXER1 gene, which encodes this receptor, are found in many species except for rodents. Intradermal injection of 5-oxo-ETE into humans and monkeys elicits eosinophil infiltration into the skin, raising the possibility that it may play a pathophysiological role in eosinophilic diseases. To investigate this and possibly identify a novel therapy we sought to prepare synthetic antagonists that could selectively block the OXE receptor. We synthesized a series of indole-based compounds bearing substituents that mimic the regions of 5-oxo-ETE that are required for biological activity, which we modified to reduce metabolism. The most potent of these OXE receptor antagonists is S-Y048, which is a potent inhibitor of 5-oxo-ETE-induced calcium mobilization (IC50, 20 pM) and has a long half-life following oral administration. S-Y048 inhibited allergen-induced eosinophil infiltration into the skin of rhesus monkeys that had been experimentally sensitized to house dust mite and inhibited pulmonary inflammation resulting from challenge with aerosolized allergen. These data provide the first evidence for a pathophysiological role for 5-oxo-ETE in mammals and suggest that potent and selective OXE receptor antagonists such as S-Y048 may be useful therapeutic agents in asthma and other eosinophilic diseases.
Asunto(s)
Antiasmáticos/farmacología , Ácidos Araquidónicos/metabolismo , Asma/tratamiento farmacológico , Asma/metabolismo , Receptores Eicosanoides/metabolismo , Animales , Antiasmáticos/síntesis química , Antiasmáticos/química , Ácidos Araquidónicos/farmacología , Membrana Basal/efectos de los fármacos , Membrana Basal/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Peroxidación de Lípido , Terapia Molecular Dirigida/métodos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Receptores Eicosanoides/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
Leaf senescence is a catabolic process that emits volatile organic compounds (VOCs). In densely planted monocultures these VOC emissions occur in outbursts that might be relevant for the local air quality since these VOCs are typically oxygenated. The VOC emissions of a high-density poplar (Populus) bioenergy plantation were monitored along with meteorological parameters, CO2 and H2O exchanges, canopy greenness, and leaf area index during the second half of the year 2015. The emissions of 25 VOCs peaked at the beginning of September, coinciding with the onset of senescence. Together these VOC emissions amounted to a total of 2.85 mmol m-2, translated into 98.3 mg C m-2. The emission peak was mainly composed of oxygenated VOCs as methanol, acetic acid, and lipoxygenase products that are all typical for catabolic processes. So, the senescence process of the poplar plantation was very well reflected in the peak of VOC emissions.
Asunto(s)
Envejecimiento , Populus , Compuestos Orgánicos Volátiles , Envejecimiento/metabolismo , Contaminantes Atmosféricos/análisis , Contaminantes Atmosféricos/metabolismo , Hojas de la Planta/metabolismo , Populus/metabolismo , Compuestos Orgánicos Volátiles/análisis , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
Opium poppy (Papaver somniferum L.) is an ancient medicinal plant producing pharmaceutically important benzylisoquinoline alkaloids. In the present work we focused on the study of enzyme lipoxygenase (LOX, EC 1.13.11.12) from opium poppy cultures. LOX is involved in lipid peroxidation and lipoxygenase oxidation products of polyunsaturated fatty acids have a significant role in regulation of growth, development and plant defense responses to biotic or abiotic stress. The purpose of this study was to isolate and characterize LOX enzyme from opium poppy callus cultures. LOX was purified by ammonium sulfate precipitation and then followed by hydrophobic chromatography using Phenyl-Sepharose CL-4B and hydroxyapatite chromatography using HA Ultrogel sorbent. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting revealed that LOX from opium poppy cultures was a single monomeric protein showing the relative molecular weight of 83 kDa. To investigate the positional specificity of the LOX reaction, purified LOX was incubated with linoleic acid and the products were analyzed by high-performance liquid chromatography in two steps, firstly with reverse phase (120-5 Nucleosil C18 column) and secondly with normal phase (Zorbax Rx-SIL column). LOX converted linoleic acid primarily to 13-hydroperoxy-(9Z,11E)-octadecadienoic acids (78%) and to a lesser extent 9-hydroperoxy-(10E,12Z)-octadecadienoic acids (22%). Characterization of LOX from opium poppy cultures provided valuable information in understanding LOX involvement in regulation of signaling pathways leading to biosynthesis of secondary metabolites with significant biological activity.
Asunto(s)
Ácido Linoleico/metabolismo , Lipooxigenasa/aislamiento & purificación , Lipooxigenasa/metabolismo , Papaver/crecimiento & desarrollo , Precipitación Química , Cromatografía Líquida de Alta Presión , Durapatita/química , Peroxidación de Lípido , Peso Molecular , Papaver/enzimología , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Metabolismo Secundario , Sefarosa/análogos & derivados , Sefarosa/químicaRESUMEN
We previously identified the indole 264 as a potent in vitro antagonist of the human OXE receptor that mediates the actions of the powerful eosinophil chemoattractant 5-oxo-ETE. No antagonists of this receptor are currently commercially available or are being tested in clinical studies. The lack of a rodent ortholog of the OXE receptor has hampered progress in this area because of the unavailability of commonly used mouse or rat animal models. In the present study, we examined the feasibility of using the cynomolgus monkey as an animal model to investigate the efficacy of orally administered 264 in future in vivo studies. We first confirmed that 264 is active in monkeys by showing that it is a potent inhibitor of 5-oxo-ETE-induced actin polymerization and chemotaxis in granulocytes. The major microsomal metabolites of 264 were identified by cochromatography with authentic chemically synthesized standards and LC-MS/MS as its ω2-hydroxy and ω2-oxo derivatives, formed by ω2-oxidation of its hexyl side chain. Small amounts of ω1-oxidation products were also identified. None of these metabolites have substantial antagonist potency. High levels of 264 appeared rapidly in the blood following oral administration to both rats and monkeys, and declined to low levels by 24â¯h. As with microsomes, its major plasma metabolites in monkeys were ω2-oxidation products. We conclude that the monkey is a suitable animal model to investigate potential therapeutic effects of 264. This, or a related compound with diminished susceptibility to ω2-oxidation, could be a useful therapeutic agent in eosinophilic disorders such as asthma.
Asunto(s)
Ácidos Araquidónicos/farmacología , Factores Quimiotácticos/farmacología , Eosinófilos/efectos de los fármacos , Indoles/farmacocinética , Receptores Eicosanoides/antagonistas & inhibidores , Administración Oral , Animales , Quimiotaxis/efectos de los fármacos , Eosinófilos/metabolismo , Femenino , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Haplorrinos , Masculino , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Oxidación-Reducción/efectos de los fármacos , RatasRESUMEN
We have developed a selective indole antagonist (230) targeting the OXE receptor for the potent eosinophil chemoattractant 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid), that may be useful for the treatment of eosinophilic diseases such as asthma. In previous studies we identified ω2-oxidation of the hexyl side chain of racemic 230 as a major metabolic route in monkeys, but also obtained evidence for another pathway that appeared to involve hydroxylation of the hexyl side chain close to the indole. The present study was designed to investigate the metabolism of the active S-enantiomer of 230 (S230) and to identify the novel hydroxy metabolite and its chirality. Following oral administration, S230 rapidly appeared in the blood along with metabolites formed by a novel and highly stereospecific α-hydroxylation pathway, resulting in the formation of αS-hydroxy-S230. The chirality of α-hydroxy-S230 was determined by the total synthesis of the relevant diastereomers. Of the four possible diastereomers of α-hydroxy-230 only αS-hydroxy-S230 has significant OXE receptor antagonist activity and only this diastereomer was found in significant amounts in blood following oral administration of S230. Other novel metabolites of S230 identified in plasma by LC-MS/MS were αS,ω2-dihydroxy-S230 and glucuronides of S230 and ω2-hydroxy-S230. Thus the alkyl side chain of S230, which is essential for its antagonist activity, is also the major target of the metabolic enzymes that terminate its antagonist activity. Modification of this side chain might result in the development of related antagonists with improved metabolic stability and efficacy.
Asunto(s)
Antiasmáticos/farmacocinética , Antiinflamatorios no Esteroideos/farmacocinética , Ácidos Araquidónicos/antagonistas & inhibidores , Factores Quimiotácticos/antagonistas & inhibidores , Indoles/farmacocinética , Cetoácidos/farmacocinética , Receptores Eicosanoides/antagonistas & inhibidores , Administración Oral , Alquilación , Animales , Antiasmáticos/administración & dosificación , Antiasmáticos/sangre , Antiasmáticos/farmacología , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/sangre , Antiinflamatorios no Esteroideos/farmacología , Ácidos Araquidónicos/metabolismo , Factores Quimiotácticos/metabolismo , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Eosinófilos/metabolismo , Femenino , Glucurónidos/sangre , Glucurónidos/química , Glucurónidos/farmacología , Humanos , Hidroxilación , Inactivación Metabólica , Indoles/administración & dosificación , Indoles/sangre , Indoles/química , Indoles/farmacología , Cetoácidos/administración & dosificación , Cetoácidos/sangre , Cetoácidos/química , Cetoácidos/farmacología , Macaca fascicularis , Estructura Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Receptores Eicosanoides/agonistas , Receptores Eicosanoides/metabolismo , EstereoisomerismoRESUMEN
The release of stress-driven volatiles throughout leaf development has been little studied. Therefore, we subjected poplar leaves during their developmental stage (from 2 days to 2 weeks old) to wounding by a single punch hole, and measured online the wound-induced volatile organic compound emissions. Our study shows that the emission of certain volatile compounds fades with increasing leaf age. Among these compounds we found lipoxygenase products (LOX products), acetaldehyde, methyl benzoate, methyl salicylate, and mono- and sesquiterpenes. In parallel, we studied the fading of constitutive emissions of methanol during leaf maturation, as well as the rise in isoprene constitutive emission during leaf maturation and its relationship to leaf photosynthetic capacity. We found highly significant relationships between leaf chlorophyll content, photosynthetic capacity, and leaf size during leaf ageing. As the level of constitutive defences increases with increasing leaf age, the strength of the volatile signal is expected to be gradually reduced. The higher elicitation of volatile organic compound emissions (especially LOX products) in younger leaves could be an evolutionary defence against herbivory, given that younger leaves are usually more subjected to infestation and herbivory.
Asunto(s)
Butadienos/metabolismo , Hemiterpenos/metabolismo , Pentanos/metabolismo , Populus/fisiología , Compuestos Orgánicos Volátiles/metabolismo , Clorofila/metabolismo , Herbivoria , Lipooxigenasa/metabolismo , Metanol/metabolismo , Fotosíntesis/fisiología , Hojas de la Planta/química , Hojas de la Planta/fisiología , Populus/química , Factores de TiempoRESUMEN
Foraging success generally depends on various environmental and physiological factors. Particularly for organisms with limited motility such as gastropods, food searching is a very cost-intensive process. As energy gain through foraging is dependent on both resource quality and quantity, consumers have to be able to differentiate between varying resource items. The effectiveness of food searching could be increased through the perception of diet-derived chemical signals that convey information about a food resource's quality over a certain distance. This strategy would clearly help to optimize movement decisions. In this study, we investigated the foraging behaviour of a freshwater gastropod towards volatile signal substances released from benthic algae grown under high and low nutrient availability, representing high and low food quality, using behavioural assays in the laboratory. Our results demonstrate that volatile organic compounds (VOCs) serve as foraging kairomones for these aquatic, benthic herbivores. Further, we were able to show for the first time that snails are able to differentiate between high- and low-quality food sources, only by the perception of food odours alone (volatile infochemicals). Gas chromatography coupled with mass spectrometry demonstrated quantitative as well as qualitative differences in the chemical composition of the VOCs bouquet, dependent on algal nutrient content. Our results suggest that the recognition of resource quality via the reception of signal substances is likely to be adaptive for consumers with low mobility to maximize ingestion of high-quality resources.