A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here, we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system.

Diacylglycerol lipases (DAGLα and DAGLβ) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLα is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network.

Diacylglycerol lipase (DAGL)-α and -β 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 β-lactone-based probes led to the discovery of α-ketoheterocycle LEI105 as a potent, highly selective and reversible dual DAGL-α/DAGL-β 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.

Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent context. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease and to the detection of CB2R in human breast cancer cells.

In recent years, lipids have come to the foreground as signaling mediators in the central nervous system (CNS) 1,2 . While classical neurotransmitters are stored in synaptic vesicles and released on fusion with the plasma membrane of neurons, due to their lipophilic nature, lipids readily diffuse through membranes and are not stored in vesicles. It is, therefore, generally accepted that signaling lipids are produced 'on demand' and are rapidly metabolized to terminate their biological action 3 . In particular, NAEs, including N-palmitoylethanolamine (PEA), N-oleoylethanolamine (OEA) and the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA) have emerged as key lipid signaling molecules. Genetic deletion or pharmacological inhibition of the main NAE hydrolytic enzyme, fatty acid amide hydrolase (FAAH), revealed elevated anandamide, PEA and OEA levels in brain and implicated these molecules in the modulation of various physiological processes such as pain, stress, anxiety, appetite, cardiovascular function and inflammation [4][5][6][7] . The physiological effects resulting from perturbation of the production of anandamide and other NAEs in living systems are, however, poorly studied, partly because of a lack of pharmacological tools to modulate their biosynthetic enzymes 8 . NAPE-PLD is generally considered a principal NAE biosynthetic enzyme 9,10 . Biochemical and structural studies have demonstrated that NAPE-PLD is a membrane-associated, constitutively active zinc hydrolase with a metallo-β-lactamase fold 11 . The enzyme generates a broad range of NAEs by hydrolysis of the phosphodiester bond between the phosphoglyceride and the NAE in N-acylphosphatidylethanolamines (NAPEs) 12 . Knockout (KO) studies have shown that the Ca 2+ -dependent conversion of NAPE to NAEs bearing both saturated and polyunsaturated fatty acyl groups are fivefold reduced in brain lysates from mice that genetically lack Napepld 13 . In accordance, reduced levels of saturated and mono-unsaturated NAEs were observed in the brains of NAPE-PLD KO mice [13][14][15] . Anandamide levels were not reduced in the transgenic model reported by Leung et al., which suggested the presence of compensatory mechanisms 13 . Indeed, multiple alternative biosynthetic pathways for anandamide have been discovered since 10 .