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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 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 high 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. 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.

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Figure 1: Optimization of 1,2,3-TU inhibitors for DAGLβ.
Figure 2: Development of an activity-based probe tailored for profiling DAGLβ.
Figure 3: In situ activity of DAGLβ inhibitors in Neuro2A cells.
Figure 4: Profiling DAGLβ activity in macrophages from inhibitor-treated mice.
Figure 5: Metabolic effects of DAGLβ inactivation in macrophages.

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Acknowledgements

We thank M. Niphakis, H.-C. Lee and G. Simon for helpful discussions and C. Joslyn for technical assistance. We thank M. Watanabe (Hokkaido University School of Medicine) for providing the antibody to DAGLα. This work was supported by the US National Institutes of Health (DA009789, DA033760, MH084512) and a Hewitt Foundation Postdoctoral Fellowship (K.-L.H.).

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Authors

Contributions

K.-L.H. and B.F.C. designed the experiments; K.-L.H., K.T. and A.A. did the experiments; H.P. and K.M. assisted with experiments; K.-L.H. and B.F.C. analyzed data; K.-L.H. and B.F.C. wrote the manuscript.

Corresponding author

Correspondence to Benjamin F Cravatt.

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Competing interests

B.F.C. is a cofounder and advisor for a biotechnology company interested in developing inhibitors for serine hydrolase as therapeutic targets.

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Mass spec characterization of peptide sequences (XLSX 1628 kb)

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Hsu, KL., Tsuboi, K., Adibekian, A. et al. DAGLβ inhibition perturbs a lipid network involved in macrophage inflammatory responses. Nat Chem Biol 8, 999–1007 (2012). https://doi.org/10.1038/nchembio.1105

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