Inhibitors of diacylglycerol lipases in neurodegenerative and metabolic disorders.

2-Arachidonoylglycerol (2-AG) is an endocannabinoid that activates the cannabinoid receptors type 1 and 2. It also serves as an important lipid precursor for the eicosanoid signaling pathway. Consequently, 2-AG is involved in many physiological functions, including anxiety, food intake, inflammation, memory, pain sensation and neurotransmission. Diacylglycerol lipases (DAGLs) are the main biosynthetic enzymes for 2-AG and their role in several pathophysiological conditions is currently under investigation. In this Digest we review all DAGL inhibitors reported to date and their effects in preclinical models of neurodegeneration and metabolic disorders.

A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase α (DAGL-α) in the CNS. Selective inhibitors of DAGL-α will provide valuable insights in the role of 2-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-α, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-α activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-α activity, using 1-stearoyl-2-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-α. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-α inhibitors.

Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons.

Diacylglycerol lipase (DAGL)-α and -ß are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes. Key to the success of this approach is the use of comparative and competitive activity-based proteome profiling (ABPP), in which broad-spectrum and tailor-made activity-based probes are combined to report on the inhibition of a protein family in its native environment. Competitive ABPP with broad-spectrum fluorophosphonate-based probes and specific ß-lactone-based probes led to the discovery of α-ketoheterocycle LEI105 as a potent, highly selective, and reversible dual DAGL-α/DAGL-ß inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metabolism including abhydrolase domain-containing protein 6, abhydrolase domain-containing protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concentration-dependently reduced 2-AG levels, but not anandamide levels, in Neuro2A cells. We show that cannabinoid CB1-receptor-mediated short-term synaptic plasticity in a mouse hippocampal slice model can be reduced by LEI105. Thus, we have developed a highly selective DAGL inhibitor and provide new pharmacological evidence to support the hypothesis that "on demand biosynthesis" of 2-AG is responsible for retrograde signaling.

Chiral disubstituted piperidinyl ureas: a class of dual diacylglycerol lipase-α and ABHD6 inhibitors.

Inhibitors of diacylglycerol lipases and α,ß-hydrolase domain containing protein 6 (ABHD6) are potential leads for the development of therapeutic agents for metabolic and neurodegenerative disorders. Here, we report the enantioselective synthesis and structure activity relationships of triazole ureas featuring chiral, hydroxylated 2-benzylpiperidines as dual inhibitors of DAGLα and ABHD6. The chirality of the carbon bearing the C2 substituent, as well as the position of the hydroxyl (tolerated at C5, but not at C3) has profound influence on the inhibitory activity of both DAGLα and ABHD6, as established using biochemical assays and competitive activity-based protein profiling on mouse brain extracts.

Triazole Ureas Act as Diacylglycerol Lipase Inhibitors and Prevent Fasting-Induced Refeeding.

Triazole ureas constitute a versatile class of irreversible inhibitors that target serine hydrolases in both cells and animal models. We have previously reported that triazole ureas can act as selective and CNS-active inhibitors for diacylglycerol lipases (DAGLs), enzymes responsible for the biosynthesis of 2-arachidonoylglycerol (2-AG) that activates cannabinoid CB1 receptor. Here, we report the enantio- and diastereoselective synthesis and structure-activity relationship studies. We found that 2,4-substituted triazole ureas with a biphenylmethanol group provided the most optimal scaffold. Introduction of a chiral ether substituent on the 5-position of the piperidine ring provided ultrapotent inhibitor 38 (DH376) with picomolar activity. Compound 38 temporarily reduces fasting-induced refeeding of mice, thereby emulating the effect of cannabinoid CB1-receptor inverse agonists. This was mirrored by 39 (DO34) but also by the negative control compound 40 (DO53) (which does not inhibit DAGL), which indicates the triazole ureas may affect the energy balance in mice through multiple molecular targets.

Comprehensive Analysis of Structure-Activity Relationships of α-Ketoheterocycles as sn-1-Diacylglycerol Lipase α Inhibitors.

Diacylglycerol lipase α (DAGLα) is responsible for the formation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα inhibitors are required to study the physiological role of 2-AG. Previously, we identified the α-ketoheterocycles as potent and highly selective DAGLα inhibitors. Here, we present the first comprehensive structure-activity relationship study of α-ketoheterocycles as DAGLα inhibitors. Our findings indicate that the active site of DAGLα is remarkably sensitive to the type of heterocyclic scaffold with oxazolo-4N-pyridines as the most active framework. We uncovered a fundamental substituent effect in which electron-withdrawing meta-oxazole substituents increased inhibitor potency. (C6-C9)-acyl chains with a distal phenyl group proved to be the most potent inhibitors. The integrated SAR data was consistent with the proposed binding pose in a DAGLα homology model. Altogether, our results may guide the design of future DAGLα inhibitors as leads for molecular therapies to treat neuroinflammation, obesity, and related metabolic disorders.

Discovery of glycine sulfonamides as dual inhibitors of sn-1-diacylglycerol lipase α and α/ß-hydrolase domain 6.

sn-1-Diacylglycerol lipase α (DAGL-α) is the main enzyme responsible for the production of the endocannabinoid 2-arachidonoylglycerol in the central nervous system. Glycine sulfonamides have recently been identified by a high throughput screening campaign as a novel class of inhibitors for this enzyme. Here, we report on the first structure-activity relationship study of glycine sulfonamide inhibitors and their brain membrane proteome-wide selectivity on serine hydrolases with activity-based protein profiling (ABPP). We found that (i) DAGL-α tolerates a variety of biaryl substituents, (ii) the sulfonamide is required for inducing a specific orientation of the 2,2-dimethylchroman substituent, and (iii) a carboxylic acid is essential for its activity. ABPP revealed that the sulfonamide glycine inhibitors have at least three off-targets, including α/ß-hydrolase domain 6 (ABHD6). Finally, we identified LEI-106 as a potent, dual DAGL-α/ABHD6 inhibitor, which makes this compound a potential lead for the discovery of new molecular therapies for diet-induced obesity and metabolic syndrome.