Des études récentes ont montré que l'anandamide, le principal ligand endogène des récepteurs aux cannabinoïdes CB1 et CB2, possède des effets analgésiques, antidépresseurs et anti-inflammatoires. Dans la perspective de traitement des maladies inflammatoires chroniques de l'intestin (MICI), notre approche a été de développer de nouveaux ligands sélectifs du récepteur CB2 permettant de moduler l’inflammation sans provoquer d’effets secondaires centraux, et de nouveaux inhibiteurs de la principale enzyme du métabolisme de l’anandamide, la fatty acid amide hydrolase (FAAH). Ainsi, sur la base des travaux antérieurs de notre groupe, une nouvelle série d’agonistes sélectifs du récepteur CB2 et deux nouvelles séries d'inhibiteurs de FAAH s’articulant autour de plusieurs hétérocycles différents ont été conçues, synthétisées et évaluées pour leur activité biologique. Les résultats pharmacologiques ont révélé des affinités sélectives pour le récepteur CB2 et des activités inhibitrices de la FAAH pour certains composés. Ces travaux ont permis d'établir des relations structure-activité essentielles pour la conception d’agoniste CB2 mais aussi pour la conception de composés prometteurs à double activités: agonistes CB2 / inhibiteurs FAAH. Enfin, deux composés agonistes sélectifs CB2 ont été évalués pour leurs propriétés anti-inflammatoires au niveau intestinal sur modèle murin de colite induite au DSS. / Recent investigations showed that anandamide, the main endogenous ligand of CB1 and CB2 cannabinoid receptors, possesses analgesic, antidepressant and anti-inflammatory effects. In the perspective to treat inflammatory bowel disease (IBD), our approach was to develop new CB2-selective ligands which are able to modulate inflammation without triggering psychotropic effects, and new inhibitors of the main anandamide-degradation enzyme, the fatty acid amide hydrolase (FAAH). Therefore, based on previous works in our group, a new series of CB2-selective agonists and two new series of FAAH inhibitors based on different scaffolds was designed, synthesized and evaluated for their biological activity. The pharmacological results showed CB2-selective agonist activities and FAAH-inhibitory activities for some compounds. This work helped to establish essential structure-activity relationships for the design of CB2-selective agonist but also for the design of promising multitarget compounds: CB2 agonists / FAAH inhibitors. Finally, two CB2-selective agonist compounds were evaluated for their anti-inflammatory properties on DSS-induced colitis mouse model.

N-Acylethanolamines (NAEs) are a class of bioactive lipids, and FAAH is one of the enzymes responsible for degrading NAEs in both plants and animals. in plants, FAAH appears to be closely associated with ABA, a phytohormone which has long been associated with plant stress responses, since the overexpression of FAAH in Arabidopsis results in ABA hypersensitivity. Therefore, it is reasonable to speculate that alterations in FAAH transcript levels will result in altered stress responses in plants. to investigate this hypothesis experiments were carried out in which wild type (WT), FAAH-overexpressing (OE), and T-DNA insertional FAAH knockouts of Arabidopsis (faah) were grown in MS media under stress conditions. the stress conditions tested included chilling stress, heavy metal stress induced by cadmium or copper, nutrient limitations induced by low phosphorus or low nitrogen, salt stress induced with NaCl, and osmotic stress induced with mannitol. the OE plants were consistently hypersensitive to all stress conditions in relation to wild type plants. Inactive FAAH overexpressors did not have the hypersensitivity to the salt and osmotic stress of the active OE plants and were instead tolerant to these stresses. FAAH2 (faah2) knockouts and FAAH 1 and 2 double knockouts (faah 1+2) were based on some root development parameters somewhat more tolerant than WT plants, but more sensitive in terms of shoot growth. Collectively the data suggests that FAAH activity may interact with stress-responsive pathways in plants, perhaps including pathways involving ABA.

Les MICI (maladies inflammatoires chroniques de l'intestin) invalident 200 000 personnes en France. La région Nord-Pas-de-Calais est particulièrement touchée par ces affections et les traitements disponibles pour ces pathologies demeurent coûteux et peu nombreux. Des études récentes ont suggéré que le système endocannabinoïde, exprimé au seing du tractus gastro-intestinal, est une cible thérapeutique prometteuse pour le traitement des MICI. Ce système se compose des récepteurs aux cannabinoïdes CB1 et CB2, des ligands endogènes de ces récepteurs, notamment l'anandamide et le 2-arachidonoylglycérol et des protéines impliquées dans l'anabolisme et le catabolisme des ligands. L'anandamide a présenté des capacités à prévenir la colite induite par le TNBS à des rongeurs. Toutefois, in vivo ce composé possède un temps de demi-vie court et est rapidement dégradé par une amidase à sérine, la FAAH (Fatty Acid Amide Hydrolase). Nous avons ainsi envisagé la conception, la synthèse et l'évaluation pharmacologique de nouveau inhibiteurs de la FAAH. L'une de nos molécules, le composé 95, présentant une CI50 sur l'enzyme de 88 nM a ensuite été injectée par voie intrapéritonéale à des souris dont la colite a été induite trois jours plus tard par l'injection intrarectale de TNBS. L'évaluation des scores macroscopiques et microscopiques des dommages causés sur le côlon par l'agent irritant a ensuite été effectuée. L'inflammation du côlon a été significativement réduite chez le groupe de souris ayant été traité par le composé 95, montrant que l'inhibition de la FAAH est une stratégie thérapeutique efficace dans le traitement des MICI. / IBD (Inflammatory Bowel Diseases) invalidate 200 000 persons in France. Nord-Pas-de-Calais region is particularly touched by these diseases and the available treatments for these pathologies are few and expensive. Recent studies have suggested that endocannabinoid system expressed in the gastrointestinal tract, was a promising therapeutic target for the IBD treatment. This system is made up of cannabinoids receptors CB1 and CB2, endogenous ligands of these receptors, notably anandamide and 2-arachidonoylglycerol, and proteins involved in ligands metabolism. Anandamide has shown properties to prevent TNBS-induced colitis in mice. However, in vivo, anandamide possesses a short life time and is rapidly hydrolyzed by a serine amidase, the FAAH (Fatty Acid Amide Hydrolase). In this context, we have considered the design, the synthesis and the pharmacological evaluation of new FAAH inhibitors. One of our molecules, compound 95, inhibiting the enzyme with an IC50 value of 88 nM has been injected intraperitonally to mice which the colitis was induced three days later by intrarectal TNBS-administration. The assessment of macroscopic and microscopic scores of colonic damages was undergone. Colonic inflammation was significatively reduced in the group of mice which has been treated by 95, showing evidence that FAAH inhibition was an effective therapeutic target for the treatment of IBD.

N-Acylethanolamines (NAEs) are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE). In animal systems this reaction is part of the “endocannabinoid” signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH), which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440) lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA), enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD) and inductive long day (LD) conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT) gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3), were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways.

No description available.

In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 18C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, its metabolism and functional implications in plants however, remains incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. We identified FAAH in Physcomitrella patens and expressed heterologously in E. coli using Gateway cloning system. Radiolabeled NAE 16:0 and 20:4 were used as substrates to test amide hydrolase activity in vitro. In order to understand the role of PpFAAH in vivo, knock out (KO) and overexpressors (OE) were generated by homologous recombination. PpFAAH KO construct was generated by inserting 5‟- and 3‟-flanking regions into pMP1159 plasmid. Full length PpFAAH with stop codon was cloned into pTHUBlGATE vector in order to make OE construct. KO and OE constructs were then transformed into protoplasts of P. patens by using PEG-mediated transformation to generate mutant lines. To identify potential interacting proteins of PpFAAH, it was cloned into pDEST15 plasmid with N-terminus GST tag. Interaction between GST-tagged PpFAAH and proteins from 14-day old protonema will be visualized by SDS-PAGE and then subjected to LC-MS/MS analysis for identification. Our long-term goal is to conduct comprehensive analyses of NAE metabolite mutants to determine their role in growth and development, and mediating stress responses in P. patens.

N-acylethanolamines (NAEs) are present in wide range of organisms and belong to family of functionally diverse signaling lipids. They consist of a fatty acid with varying chain lengths and ethanolamine linked via an amide bond. The level of NAEs is modulated by their hydrolysis to ethanolamine and fatty acid by an enzyme fatty acid amide hydrolase (FAAH). FAAH is an integral membrane protein that belongs to “amidase signature” superfamily of proteins, which is characterized by highly conserved region rich in serine, glycine and alanine. FAAH directly or indirectly plays a role in modulation of various physiological processes by regulating NAE levels. Although the role of NAEs and its key modulator FAAH has been studied in other plants, their role in tomato model is limited and unknown. More recently, SlFAAH1, an ortholog of AtFAAH1, was identified in tomato and cloned into bacterial expression system. However, putative SlFAAH1 function and distinct features are yet to be determined. It is hypothesized that the putative SlFAAH1 catalyzes the hydrolysis of NAEs and modulates the level of NAEs during the seedling development in tomato. To this extent, a putative SlFAAH1 (previously identified and cloned in pET-23a vector) will be biochemically characterized and also effect of NAEs on seedling development will be studied. Thus far, SlFAAH1 cloned in pET-23a vector was expressed in RIL cell line (prokaryotic expression system) followed by conformation of positive transformant by colony PCR. Currently, protein expression and confirmation of SlFAAH in the positive transformant is being done. The expressed protein will be characterized for its hydrolytic activity using radiolabelled substrate. The effect of exogenous NAEs during seedling development will be studied with regards to expression level of SlFAAH1 by qPCR and composition of NAE during the seedling development to determine the role of NAE during seedling development. Thus, this study is expected to not only characterize a protein in tomato but also determine its role in mediating NAE metabolism and seedling development. Long-term studies will identify the significance of highly conserved NAE pathway in eukaryotes.

In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 18C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, its metabolism and functional implications in plants however, remains incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. We identified FAAH in Physcomitrella patens and expressed heterologously in E. coli using Gateway cloning system. Radiolabeled NAE 16:0 and 20:4 were used as substrates to test amide hydrolase activity in vitro. In order to understand the role of PpFAAH in vivo, knock out (KO) and overexpressors (OE) were generated by homologous recombination. PpFAAH KO construct was generated by inserting 5'- and 3'-flanking regions into pMP1159 plasmid. Full length PpFAAH with stop codon was cloned into pTHUBlGATE vector in order to make OE construct. KO and OE constructs were then transformed into protoplasts of P. patens by using PEG-mediated transformation to generate mutant lines. To identify potential interacting proteins of PpFAAH, it was cloned into pDEST15 plasmid with Nterminus GST tag. Interaction between GST-tagged PpFAAH and proteins from 14-day old protonema will be visualized by SDS-PAGE and then subjected to LC-MS/MS analysis for identification. Our long-term goal is to conduct comprehensive analyses of NAE metabolite mutants to determine their role in growth and development, and mediating stress responses in P. patens.

N-acylethanolamines (NAEs) are present in wide range of organisms and belong to family of functionally diverse signaling lipids. They consist of a fatty acid with varying chain lengths and ethanolamine linked via an amide bond. The level of NAEs is modulated by their hydrolysis to ethanolamine and fatty acid by an enzyme fatty acid amide hydrolase (FAAH). FAAH is an integral membrane protein that belongs to “amidase signature” superfamily of proteins, which is characterized by highly conserved region rich in serine, glycine and alanine. FAAH directly or indirectly plays a role in modulation of various physiological processes by regulating NAE levels. Although the role of NAEs and its key modulator FAAH has been studied in other plants, their role in tomato model is limited and unknown. More recently, SlFAAH1, an ortholog of AtFAAH1, was identified in tomato and cloned into bacterial expression system. However, putative SlFAAH1 function and distinct features are yet to be determined. It is hypothesized that the putative SlFAAH1 catalyzes the hydrolysis of NAEs and modulates the level of NAEs during the seedling development in tomato. To this extent, a putative SlFAAH1 (previously identified and cloned in pET-23a vector) will be biochemically characterized and also effect of NAEs on seedling development will be studied. Thus far, SlFAAH1 cloned in pET-23a vector was expressed in RIL cell line (prokaryotic expression system) followed by conformation of positive transformant by colony PCR. Currently, protein expression and confirmation of SlFAAH in the positive transformant is being done. The expressed protein will be characterized for its hydrolytic activity using radiolabelled substrate. The effect of exogenous NAEs during seedling development will be studied with regards to expression level of SlFAAH1 by qPCR and composition of NAE during the seedling development to determine the role of NAE during seedling development. Thus, this study is expected to not only characterize a protein in tomato but also determine its role in mediating NAE metabolism and seedling development. Longterm studies will identify the significance of highly conserved NAE pathway in eukaryotes.

N-acylethanolamines (NAEs) are fatty acid ethanolamides that mediate stress responses in plants and animals. NAEs such as NAE 20:4 (anandamide) have only been reported in mammals and they regulate processes like neuroprotection and pain perception. Interestingly, we discovered the unique occurrence of anandamide in moss, Physcomitrella patens, a stress tolerant early land plant. Since NAEs including anandamide are degraded by fatty acid amide hydrolase (FAAH), it is hypothesized that a functional homolog of FAAH occurs in P. patens. I specifically propose to biochemically characterize FAAH enzyme that degrades anandamide. For this, Arabidopsis FAAH (AtFAAH) homolog was identified in moss database using BLASTP. The predicted protein structure of putative moss FAAH (PpFAAH) closely resembled to that of AtFAAH with conserved amidase signature sequence and catalytic triad residues: Lys205, Ser281, Ser305. Transcript levels of PpFAAH increased five-fold when moss was grown on excess NAE containing media. PpFAAH cDNA was PCR amplified and cloned into pET23a expression vector and transformed into RIL E. coli cells and confirmed by colony PCR. Heterologously expressed protein will be purified by Ni+2 affinity column chromatography and confirmed by western blot using anti-His-tag antibody. For biochemical characterization, enzyme will be presented with 14C NAE 20:4 substrate and rate of product free fatty acid formed will be quantified by extracting lipids from reaction mixture and separating by thin layer chromatography followed by radiometric scanning. E. coli cells expressing AtFAAH enzyme will be used as control. A complete characterization of the PpFAAH enzyme will be carried out to determine the kinetics, optimal temperature and pH conditions. Characterization of the enzyme that hydrolyzes anandamide in moss is expected to lead us to develop NAE metabolite mutants that will subsequently allow us to study the physiological role of anandamide in early land plants.