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

Abstract

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.

Rapid and profound rewiring of brain lipid signaling networks by acute diacylglycerol lipase inhibition

“…Various forms of synaptic plasticity are regulated by 2-AG signaling, including depolarizationinduced suppression of excitation (DSE) and inhibition (DSI) (20), both of which are abolished in DAGLα −/− mice (13,14). Interestingly, however, conflicting findings have emerged about whether the retrograde signaling 2-AG is biosynthesized by DAGLα on-demand or, alternatively, presynthesized and stored within neurons before stimulus-induced release (21)(22)(23)25). As noted by others (21), these differences may reflect the poor physicochemical properties of the DAGL inhibitors used in past studies, as the high lipophilicity of these molecules could limit their penetration into brain tissue preparations used to measure DSI and DSE, resulting in incomplete inhibition of DAGLs.…”

“…Unfortunately, selective and in vivo active inhibitors are not yet available for many lipid biosynthetic enzymes. Known inhibitors for DAGLs, for example, have been used to study the function of 2-AG as a retrograde messenger in neuronal cell and brain slice preparations (20)(21)(22)(23)(24)(25), but these inhibitors lack the selectivity (26), potency, and chemical properties (21) required for central activity in vivo.…”

Assay and Inhibition of the Purified Catalytic Domain of Diacylglycerol Lipase Beta

“…RHC-80267) as well as recently discovered novel DAGL inhibitors in this "clean" and homogenous assay will not only further validate the assay but also potentially provide useful information regarding their inhibition kinetics and enable comparisons with their DAGLα inhibition profiles 2,13,34,44,45 . In general, the DAGLβ assays described here also have particular utility as selectivity assays.…”

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

“…We have successfully applied this homology model to guide the design of new DAGLα inhibitors, which led to in the identification of 6-phenyl-1-(6-( p -tolyl)oxazolo[4,5- b ]pyridin-2-yl)hexan-1-one (LEI105) as a highly selective, reversible and dual DAGLα/DAGL-β inhibitor that was active in cells and reduced cannabinoid CB 1 -receptor-dependent synaptic plasticity. 13 Our current efforts are directed towards optimizing the physicochemical properties of the α-ketoheterocycles to improve their pharmacokinetic properties. Of note, the reversible character of the α-ketoheterocycle inhibitors may have less probability to induce idiosyncratic toxic side effects, which may be associated with covalent irreversible inhibitors.…”