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.

Fatty Acid Amide Hydrolase (FAAH), a serine hydrolase family protein, hydrolyzes N-acylethanolamines (NAEs) by cleaving the amide bond linking the acyl group with ethanolamine to produce free fatty acids. Highly conserved ‘Amidase Signature (AS)’ sequence rich in serine, glycine and alanine residues characterize the protein. FAAH plays role in various physiological processes by regulating NAE levels, such as seedling growth, defense response. Understanding of the role of NAEs and FAAH has been however, limited to model plant Arabidopsis. Here, with interest to understand the role of FAAH in modulating NAE composition, tomato was chosen as a model system. Recently, SlFAAH1, an ortholog of AtFAAH1 was identified in tomato and was successfully expressed in prokaryotic expression system. Protein assay with lysate of cells expressing recombinant putative SlFAAH1 showed the ability to hydrolyze a polyunsaturated NAE (NAE20:4). Currently, additional assays are being carried out to determine optimal pH, temp, substrate specificity and associated enzyme kinetics. In parallel, the effect of exogenous NAEs on SlFAAH1 expression levels and during seedling development is being evaluated. Together, this study is expected to not only characterize a protein in tomato but also determine its role in mediating NAE metabolism and seedling development, and further allows for comparison with Arabidopsis and mammalian FAAH to determine its functional conservation.

Endocannabinoid signaling is well studied in mammals and known to be involved in numerous pathological and physiological processes. Fatty acid amide hydrolase (FAAH) terminates endocannabinoid signaling in mammals. In Physcomitrella patens, we identified nine orthologs of FAAH (PpFAAH1 to PpFAAH9) with the characteristic catalytic triad and amidase signature sequence. Kinetics of PpFAAH1 showed specificity towards anandamide (AEA) at 37°C and pH 8.0. Further biophysical and bioinformatic analyses revealed that, structurally, PpFAAH1 to PpFAAH4 were closely associated to the plant FAAH whereas PpFAAH6 to PpFAAH9 were more closely associated to the animal FAAH. A substrate entry gate or ‘dynamic paddle’ in FAAH is fully formed in vertebrates but absent or not fully developed in non-vertebrates and plants. In planta analysis revealed that PpFAAH responded differently with saturated and unsaturated N-acylethanolamines (NAEs). In vivo amidohydrolase activity showed specificity associated with developmental stages. Additionally, overexpression of PpFAAH1 indicated the need for NAEs in developmental transition. To understand and identify key molecules related to endocannabinoid signaling in P. patens, we used high-throughput RNA sequencing. We analyzed temporal expression of mRNA and long non-coding RNA (lncRNA) in response not only to exogenous anandamide but also its precursor arachidonic acid and abscisic acid (ABA, a stress hormone). From the 40 RNA-seq libraries generated, we identified 4244 novel lncRNAs. The highest number of differentially expressed genes (DEGs) for both mRNA and lncRNA were detected on short-term exposure (1 h) to AEA. Furthermore, gene ontology enrichment analysis showed that 17 genes related to activation of the G protein-coupled receptor signaling pathway were highly expressed along with a number of genes associated with organelle relocation and localization. We identified key signaling components of AEA that showed significant difference when compared with ABA. This study provides a fundamental understanding of novel endocannabinoid signaling in early land plants and a future direction to elucidate its functional role.

Endocannabinoids were known to exist only among Animalia but recent report of their occurrence in early land plants prompted us to study its function and metabolism. In mammals, anandamide, as an endocannabinoid ligand, mediates several neurological and physiological processes, which are terminated by fatty acid amide hydrolase (FAAH). We identified nine orthologs of FAAH in the moss Physcomitrella patens (PpFAAH1 to PpFAAH9) with amidase signature and catalytic triad. The optimal amidase activity for PpFAAH1 was at 37 °C and pH 8.0, with higher specificity to anandamide. Further, the phylogeny and predicted structural analyses of the nine paralogs revealed that PpFAAH1 to PpFAAH4 were closely related to plant FAAH while PpFAAH6 to PpFAAH9 were to the rat FAAH, categorized based on the membrane binding cap, membrane access channel and substrate binding pocket. We also identified that a true ‘dynamic paddle’ that is responsible for tighter regulation of FAAH is recent in vertebrates and absent or not fully emerged in plants and non-vertebrates. These data reveal evolutionary and functional relationship among eukaryotic FAAH orthologs and features that contribute to versatility and tighter regulation of FAAH. Future studies will utilize FAAH mutants of moss to elucidate the role of anandamide in early land plants.

Fatty acid amide hydrolase (FAAH) belongs to a diverse class of enzymes in amidase signature family. In mammals, FAAH is targeted to affect neurological functions because it terminates endocannabinoid signaling by degrading anandamide, a 20C N-acylethanolamine (NAE 20:4). In higher plants, FAAH is known to modulate growth, development and stress tolerance by degrading 12-18C NAEs. Since anandamide was reported to exclusively occur in early land plants, we investigated its metabolic pathway in the moss Physcomitrella patens. Based on the highest identity with ratFAAH, we identified nine orthologs in moss, PpFAAH1 to PpFAAH9. Several bioinformatic tools were used to understand the structural basis of how catalytic residues fold for amidohydrolase activity. Based on these in silico analyses of PpFAAH homologs and their gene expression in response to saturated (NAE16:0) and unsaturated NAE (NAE 20:4) treatment, PpFAAH1 was selected for biochemical characterization. Heterologously expressed PpFAAH1 showed highest amidohydrolase activity at 37°C and pH 8.0. Both in vivo and in vitro studies showed that unsaturated NAE substrate is hydrolyzed faster than the saturated NAE (> 10-fold in vivo and 50-fold in vitro). Additionally, transgenic moss lines over expressing FAAH1 showed slower growth and disrupted gametophyte formation when compared to wild type. These data suggest that PpFAAH1-mediated NAE metabolism is likely involved in developmental transition in moss.

In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 20C 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, their metabolism and functional implications in plants however, remain 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. FAAH orthologs, putative PpFAAHs (Physcomitrella patens FAAH) were identified based on the sequence blast of ratFAAH, and named as PpFAAH1 to PpFAAH10. Based on the highest mRNA expression of the PpFAAH homologs upon NAE treatment, PpFAAH1 was selected for further in vitro characterization, which shares 31% sequence identity with ratFAAH. PpFAAH1 was heterologously expressed in E. coli and purified for characterization. Highest amidohydrolysis activity of PpFAAH1 was observed in vitro at pH 8.0 and temperature 37°C. Methoxy arachidonyl fluorophosphonate (MAFP), an inhibitor showed highest inhibition with 10mM concentration, however, one of the principal classes of FAAH inhibitor O-aryl carbamates (URB597) exhibited only 22% inhibition with the same concentration. Both in vivo and in vitro studies showed that unsaturated NAE substrate (NAE 20:4) is hydrolyzed faster than the saturated NAE (NAE16:0); more than 50- and 10-fold higher in vitro and in vivo assays, respectively. Amidohydrolase activity in vivo was mostly associated with microsomes compared with cytoplasmic fractions. Additionally, microsomal fraction of mature gametophytes showed higher amidohydrolase activity than of the protonemal or early gametophyte stages; however, PpFAAH expression was not significantly different between the developmental stages. Further functional characterization of NAE metabolic pathway is ongoing by generation of PpFAAH knock out (KO) and overexpressor (OE) to understand the biological implications of FAAH in growth and development of early land plants.

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.

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.

Delta9-tetrahydrocannbinol (THC), the primary active constituent of Cannabis sativa, has long been known to reduce opioid withdrawal symptoms. Although THC produces most of its pharmacological actions through the activation of CB1 and CB2 cannabinoid receptors, the role these receptors play in reducing opioid withdrawal symptoms remains unknown. The endogenous cannabinoids, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonylglycerol (2-AG), activate both cannabinoid receptors, but are rapidly metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The objective of this dissertation was to test whether increasing AEA or 2-AG, via inhibition of their respective hydrolytic enzymes, reduces morphine withdrawal symptoms in in vivo and in vitro models of opiate dependence. Morphine-dependent ICR mice subjected to acute naloxone challenge or abrupt withdrawal (via pellet removal) reliably displayed a profound withdrawal syndrome, consisting of jumping, paw tremors, head shakes, diarrhea, and weight loss. THC and the MAGL inhibitor, JZL184 dose-dependently reduced the intensity of precipitated withdrawal measures through the activation of CB1 receptors. The FAAH inhibitor, PF-3845, reduced the intensity of a subset of precipitated signs through the activation of CB1 receptors, but did not ameliorate the incidence of diarrhea or weight loss. In the next set of experiments, MAGL inhibition dose-dependently reduced the intensity of all spontaneous withdrawal signs (i.e jumps, paw flutters, head shakes, weight loss and diarrhea) in a CB1 receptor dependent manner. However, FAAH inhibition reduced the intensity of head shakes and paw flutters, but did not affect other signs. Strikingly, a combination of low-dose JZL184 and high-dose PF-3845 reduced abrupt withdrawal signs in a manner similar to complete MAGL inhibition, which suggests potential therapeutic advantages of dual enzyme inhibition. This combination elevated appropriate eCB levels and caused moderate CB1 receptor desensitization, but did not affect receptor number in whole brain. Since MAGL, but not FAAH inhibition, blocked diarrhea during opioid withdrawal in vivo, we investigated whether inhibitors of each enzyme would differentially attenuate naloxone-precipitated contractions and secretion in morphine-dependent ilea in vitro. Both enzyme inhibitors attenuated the intensity of naloxone-induced contractions, and blocked naloxone-precipitated hypersecretion. Thus, these models offer useful tools for investigating in vitro end-ponts of withdrawal, but not for elucidating anti-diarrheal mechanism of these inhibitors.If targeting endocannabinoid catabolic enzymes is indeed a viable approach to treat other abuse disorders, it is important to know whether these inhibitors would themselves have abuse or dependence liability. In the final series of experiments we tested whether prolonged elevation of endocannabinoid leads to the development of cannabinoid dependence, based on the occurrence of somatic withdrawal signs upon challenge with rimonabant, a CB1 receptor antagonist. Repeated treatment with high doses, but not low doses, of JZL184 led to cannabinoid dependnece. These results indicate that the strategy of increasing endogenous cannabinoids through the inhibition of their catabolic enzymes represents a promising approach to ameliorate opioid withdrawal symptoms.

N-acylethanolamines (NAEs) are fatty acid derivatives that occur naturally in plant and animal systems. In mammals, they regulate physiological functions, including neurotransmission, immune responses, vasodilation, embryo development and implantation, feeding behavior, and cell proliferation. NAEs are metabolized by fatty acid amide hydrolase (FAAH), which belongs to the amidase signature family. It is hypothesized that putative FAAH functions as the catalyst in the metabolism of N-acylethanolamine in tomato plants. To test the hypothesis, FAAH protein homologs were identified in tomato via in silico analysis. Among the six homologs identified, FAAH1 and FAAH2 were selected for further validation. This study is focused on 1) in silico analyses of SlFAAH2, 2) quantification of transcript levels for SlFAAH2, 3) determination of FAAH activity at various developmental stages of tomato, and 4) isolation of and synthesis of SlFAAH2 cDNA for cloning. Putative SlFAAH2 showed high homology to Arabidopsis FAAH1. Transcript levels, as measured by qPCR using RNA extracted from various developmental stages, were highest at 0 days and lowest at 4 days. Enzyme activity at certain developmental stages coincided with SlFAAH2 transcript levels. In order to confirm that putative SlFAAH2 encodes for an enzyme that hydrolyzes NAEs, SlFAAH2 gene was isolated from total RNA of tomato, cDNA was synthesized by reverse transcription and the gene was amplified by PCR for further cloning in a heterologous expression system for biochemical characterization. To gain better molecular and biochemical understanding of FAAH and determine its broader functions, it is pertinent to characterize FAAH in other plant species.