Geoffray Labar

Crystal Structure of the Human Monoacylglycerol Lipase, a Key Actor in Endocannabinoid Signaling

2‐Arachidonoylglycerol plays a major role in endocannabinoid signaling, and is tightly regulated by the monoacylglycerol lipase (MAGL). Here we report the crystal structure of human MAGL. The protein crystallizes as a dimer, and despite structural homologies to haloperoxidases and esterases, it distinguishes itself by a wide and hydrophobic access to the catalytic site. An apolar helix covering the active site also gives structural insight into the amphitropic character of MAGL, and likely explains how MAGL interacts with membranes to recruit its substrate. Docking of 2‐arachidonoylglycerol highlights a hydrophobic and a hydrophilic cavity that accommodate the lipid into the catalytic site. Moreover, we identified Cys201 as the crucial residue in MAGL inhibition by N‐arachidonylmaleimide, a sulfhydryl‐reactive compound. Beside the advance in the knowledge of endocannabinoids degradation routes, the structure of MAGL paves the way for future medicinal chemistry works aimed at the design of new drugs exploiting 2‐arachidonoylglycerol transmission.

Lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis in vitro.

Two compounds, URB602 and URB754, have been reported in the literature to be selective inhibitors of monoacylglycerol lipase, although a recent study has questioned their ability to prevent 2‐arachidonoyl hydrolysis by brain homogenates and cerebellar membranes. In the present study, the ability of these compounds to inhibit monoacylglycerol lipase and fatty acid amide hydrolase has been reinvestigated.

Synthesis and in Vitro Evaluation of N-Substituted Maleimide Derivatives as Selective Monoglyceride Lipase Inhibitors

The endocannabinoid 2-arachidonoylglycerol (2-AG) plays a major role in many physiological processes, and its action is quickly terminated via enzymatic hydrolysis catalyzed by monoglyceride lipase (MGL). Regulating its endogenous level could offer therapeutic opportunities; however, few selective MGL inhibitors have been described so far. Here, we describe the synthesis of N-substituted maleimides and their pharmacological evaluation on the recombinant human fatty acid amide hydrolase (FAAH) and on the purified human MGL. A few N-arylmaleimides were previously described ( Saario , S. M. ; Salo , O. M. ; Nevalainen , T. ; Poso , A. ; Laitinen , J. T. ; Jarvinen , T. ; Niemi , R. Characterization of the Sulfhydryl-Sensitive Site in the Enzyme Responsible for Hydrolysis of 2-Arachidonoylglycerol in Rat Cerebellar Membranes . Chem. Biol. 2005 , 12 , 649 - 656 ) as MGL inhibitors, and along these lines, we present a new set of maleimide derivatives that showed low micromolar IC(50) and high selectivity toward MGL vs FAAH. Then, structure-activity relationships have been investigated and, for instance, 1-biphenyl-4-ylmethylmaleimide inhibits MGL with an IC(50) value of 790 nM. Furthermore, rapid dilution experiments reveal that these compounds act as irreversible inhibitors. In conclusion, N-substituted maleimides constitute a promising class of potent and selective MGL inhibitors.

A Review on the Monoacylglycerol Lipase: At the Interface Between Fat and Endocannabinoid Signalling

Together with anandamide, 2-arachidonoylglycerol (2-AG) constitutes one of the main representatives of a family of endogenous lipids known as endocannabinoids. These act by binding to CB(1) and CB(2) cannabinoid receptors, the molecular target of the psychoactive compound Delta(9)-THC, both in the periphery and in the central nervous system, where they behave as retrograde messengers to modulate synaptic transmission. These last years, evidence has accumulated to demonstrate the lead role played by the monoacylglycerol lipase (MAGL) in the regulation of 2-arachidonoylglycerol (2-AG) levels. Considering the numerous physiological functions played by this endocannabinoid, MAGL is now considered a promising target for therapeutics, as inhibitors of this enzyme could reveal useful for the treatment of pain and inflammatory disorders, as well as in cancer research, among others. Here we review the milestones that punctuated MAGL history, from its discovery to recent advances in the field of inhibitors development. An emphasis is given on the recent elucidation of the tridimensional structure of the enzyme, which could offer new opportunities for rational drug design.

CAY10499, a novel monoglyceride lipase inhibitor evidenced by an expeditious MGL assay

Monoglyceride lipase (MGL) plays a major role in the metabolism of the lipid transmitter 2‐arachidonoylglycerol (2‐AG). This endocannabinoid is known to mediate a large number of physiological processes, and its regulation is thought to be of great therapeutic potential. However, the number of available monoglyceride lipase inhibitors is limited, mostly due to the lack of rapid and accurate pharmacological assays for the enzyme. We have developed a 96‐well‐format assay for MGL using a nonradiolabeled substrate, 4‐nitrophenylacetate. The IC50 values that were obtained for known inhibitors of MGL using 4‐nitrophenylacetate were similar to those reported by using the radiolabeled form of an endogenous substrate, 2‐oleoylglycerol. In a first small‐scale screening, we identified CAY10499 as a novel monoglyceride lipase inhibitor. Thus, we report here the characterization of this submicromolar inhibitor, which acts on MGL through an unprecedented mechanism for inhibitors of this enzyme.

Disulfiram is an inhibitor of human purified monoacylglycerol lipase, the enzyme regulating 2-arachidonoylglycerol signaling.

Monoacylglycerol lipase (MAGL) is a key enzyme responsible for the termination of endocannabinoid signaling. Its crucial role in 2‐arachidonoylglycerol (2‐AG) metabolism, together with the numerous pharmacological properties mediated by this endocannabinoid, emphasize the interest in MAGL as therapeutic target, along with the need to design potent and selective inhibitors. Meanwhile, the complexity of 2‐AG degradation pathways underscores the need to use a purified source of enzyme in evaluation studies of new inhibitors. We report here the first heterologous expression and purification of human MAGL. A highly pure protein was obtained and allowed us to measure the affinity of several MAGL inhibitors for the human enzyme. Importantly, disulfiram (tetraethylthiuram disulfide), a compound used to treat alcoholism, and other disulfide‐containing compounds were shown to inhibit MAGL with good potency, likely through an interaction with cysteine residues.

Bis(dialkylaminethiocarbonyl)disulfides as Potent and Selective Monoglyceride Lipase Inhibitors.

Monoglyceride lipase (MGL) inhibition may offer an approach in treating diseases in which higher 2-arachidonoyglycerol activity would be beneficial. We report here the synthesis and pharmacological evaluation of bis(dialkylaminethiocarbonyl)disulfide derivatives as irreversible MGL inhibitors. Inhibition occurs through interactions with MGL C208 and C242 residues, and these derivatives exhibit high inhibition selectivity over fatty acid amide hydrolase, another endocannabinoid-hydrolyzing enzyme.

Synthesis and Pharmacological Evaluation of 2,4-Dinitroaryldithiocarbamate Derivatives as Novel Monoacylglycerol Lipase Inhibitors

Monoacylglycerol lipase (MAGL) is responsible for signal termination of 2-arachidonoylglycerol (2-AG), an endocannabinoid neurotransmitter endowed with several physiological effects. Previously, we showed that the arylthioamide scaffold represents a privileged template for designing MAGL inhibitors. A series of 37 compounds resulting from pharmacomodulations around the arylthioamide template were synthesized and tested to evaluate their inhibitory potential on MAGL activity as well as their selectivity over fatty acid amide hydrolase (FAAH), another endocannabinoid-hydrolyzing enzyme. We have identified 2,4-dinitroaryldithiocarbamate derivatives as a novel class of MAGL inhibitors. Among the synthesized compounds, we identified [2,4-dinitrophenyl-4-(4-tert-butylbenzyl)piperazine-1-carbodithioate] (CK37), as the most potent MAGL inhibitor within this series (IC(50) = 154 nM). We have also identified [2,4-dinitrophenyl-4-benzhydrylpiperazine-1-carbodithioate] (CK16) as a selective MAGL inhibitor. These compounds are irreversible MAGL inhibitors that probably act by interacting with Cys208 or Cys242 and Ser122 residues of the enzyme. Moreover, CK37 is able to raise 2-arachidonoylglycerol (2-AG) levels in intact cells.

Development and characterization of endocannabinoid hydrolases FAAH and MAGL inhibitors bearing a benzotriazol-1-yl carboxamide scaffold

A series of (1H-benzo[d][1,2,3]triazol-1-yl)(4-benzylpiperazin-1-yl)methanones and of (1H-benzo[d][1,2,3]triazol-1-yl)(4-phenylpiperazin-1-yl)methanones has been prepared and tested on human fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). In the benzylpiperazinyl series, compound 29 (ML30) exhibited an IC(50) value of 0.54 nM on MAGL, combined with a 1000-fold selectivity versus FAAH, while compounds 11 and 16 acted as potent dual FAAH-MAGL inhibitors (IC(50)<10 nM). In the phenylpiperazinyl series, compounds 37, 38, 42, and 43 displayed IC(50) values against MAGL in the nanomolar range, whilst being between one and two orders of magnitude less potent on the FAAH, while compounds 31 and 32 were potent FAAH inhibitors (IC(50)<20 nM) and over 12-fold selective versus MAGL. The key structural determinants driving the structure-activity relationships were explored by the minimization of the inhibitors inside the active site of both enzymes.

PET imaging of fatty acid amide hydrolase in the brain: synthesis and biological evaluation of an 11C-labelled URB597 analogue

INTRODUCTION Fatty acid amide hydrolase (FAAH) is part of the endocannabinoid system (ECS) and has been linked to the aetiology of several neurological and neuropsychiatric disorders. So far no useful PET or SPECT tracer for in vivo visualisation of FAAH has been reported. We synthesized and evaluated a carbon-11-labeled URB597 analogue, biphenyl-3-yl [(11)C]-4-methoxyphenylcarbamate or [(11)C]-1, as potential FAAH imaging agent. METHODS The inhibitory activity of 1 was determined in vitro using recombinant FAAH. Radiosynthesis of [(11)C]-1 was performed by methylation using [(11)C]-CH(3)I, followed by HPLC purification. Biological evaluation was done by biodistribution studies in wild-type and FAAH knock-out mice, and by ex vivo and in vivo metabolite analysis. The influence of URB597 pretreatment on the metabolisation profile was assessed. RESULTS [(11)C]-1 was obtained in good yields and high radiochemical purity. Biodistribution studies revealed high brain uptake in wild-type and FAAH knock-out mice, but no retention of radioactivity could be demonstrated. Metabolite analysis and URB597 pretreatment confirmed the non-FAAH-mediated metabolisation of [(11)C]-1. The inhibition mechanism was determined to be reversible. In addition, the inhibition of URB597 appeared slowly reversible. CONCLUSIONS Although [(11)C]-1 inhibits FAAH in vitro and displays high brain uptake, the inhibition mechanism seems to deviate from the proposed carbamylation mechanism. Consequently, it does not covalently bind to FAAH and will not be useful for mapping the enzyme in vivo. However, it represents a potential starting point for the development of in vivo FAAH imaging tools.