DAGLβ inhibition perturbs a lipid network involved in macrophage inflammatory responses

The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGL alpha and DAGL beta. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here we report a series of 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGL beta in living systems. Optimized inhibitors showed high selectivity for DAGL beta over other serine hydrolases, including DAGL alpha (similar to 60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb(-/-) mice, we show that DAGL beta inactivation lowers 2-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A2. We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-alpha release. These findings indicate that DAGL beta is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.