DAGLβ inhibition perturbs a lipid network involved in macrophage inflammatory responses

Abstract

The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLβ. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here, we report a series of in vivo-active 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLβ in living systems. Optimized inhibitors showed excellent selectivity for DAGLβ over other serine hydrolases, including DAGLα (~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 DAGLβ 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 (PLA2G4A). We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-α release. These findings indicate that DAGLβ is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.

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

“…1A), serve as useful inactive control probes that display greatly attenuated inhibition of DAGLs, while maintaining activity against common off-targets like ABHD6 (27). Based on this precedent, we synthesized DO53 (8), a 2-phenyl piperazine analog of DO34, and found that this agent exhibited ∼100-fold lower activity against DAGLα compared with DO34 or DH376 as measured by DAG substrate hydrolysis ( Fig.…”

“…The development of DAGL inhibitors has been historically hindered by a dearth of assays for monitoring the activity of these enzymes in native biological systems. We have recently introduced tailored activity-based probes that enable the independent and concurrent monitoring of DAGLα and DAGLβ activities in the brain and other tissue/cell types (27,28). Guided by competitive activitybased protein profiling (ABPP) methods (29), we converted one of these tailored probes into a series of potent 1,2,3-triazole urea (1,2,3-TU) inhibitors of DAGLβ that displayed peripheral, but not central activity in vivo (27) [e.g., KT172 (1)] (Fig.…”

“…ABPP of mouse brain and substrate assays of transfected cell lysates were performed as described previously (27,30). Metabolomic and proteomic analysis, as well as cytokine measurement from mouse brain homogenates were performed as described previously (60) and in SI Appendix, Supporting Experimental Procedures.…”

Serine Hydrolase Inhibitors Block Necrotic Cell Death by Preventing Calcium Overload of the Mitochondria and Permeability Transition Pore Formation

“…The PLA 2 inhibitor Wyeth-1 was produced as previously described (36). The abhydrolase domain containing 6 (ABHD6) inhibitor KT195 and BODIPY-labeled probe HT-01 used for activity based protein profiling (ABPP) assays were synthesized as previously described (37).…”

“…were treated with KT195 and pyrrophenone, followed by preparation of membrane proteomes that were probed with BODIPY-labeled HT-01 to detect ABHD6 (37). The effect of the inhibitors added directly to the proteome in vitro was also tested.…”