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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Mechoulam, R. et al. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem. Pharmacol. 50, 83–90 (1995).
Sugiura, T. et al. 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem. Biophys. Res. Commun. 215, 89–97 (1995).
Devane, W.A. et al. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258, 1946–1949 (1992).
Mackie, K. Cannabinoid receptors as therapeutic targets. Annu. Rev. Pharmacol. Toxicol. 46, 101–122 (2006).
Ledent, C. et al. Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice. Science 283, 401–404 (1999).
Zimmer, A., Zimmer, A.M., Hohmann, A.G., Herkenham, M. & Bonner, T.I. Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc. Natl. Acad. Sci. USA 96, 5780–5785 (1999).
Cravatt, B.F. et al. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature 384, 83–87 (1996).
Dinh, T.P. et al. Brain monoglyceride lipase participating in endocannabinoid inactivation. Proc. Natl. Acad. Sci. USA 99, 10819–10824 (2002).
Blankman, J.L., Simon, G.M. & Cravatt, B.F. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem. Biol. 14, 1347–1356 (2007).
Kathuria, S. et al. Modulation of anxiety through blockade of anandamide hydrolysis. Nat. Med. 9, 76–81 (2003).
Ahn, K. et al. Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain. Chem. Biol. 16, 411–420 (2009).
Long, J.Z. et al. Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects. Nat. Chem. Biol. 5, 37–44 (2009).
Nomura, D.K. et al. Endocannabinoid hydrolysis generates brain prostaglandins that promote neuroinflammation. Science 334, 809–813 (2011).
Bisogno, T. et al. Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain. J. Cell Biol. 163, 463–468 (2003).
Gao, Y. et al. Loss of retrograde endocannabinoid signaling and reduced adult neurogenesis in diacylglycerol lipase knock-out mice. J. Neurosci. 30, 2017–2024 (2010).
Tanimura, A. et al. The endocannabinoid 2-arachidonoylglycerol produced by diacylglycerol lipase α mediates retrograde suppression of synaptic transmission. Neuron 65, 320–327 (2010).
Gregg, L.C. et al. Activation of type 5 metabotropic glutamate receptors and diacylglycerol lipase-α initiates 2-arachidonoylglycerol formation and endocannabinoid-mediated analgesia. J. Neurosci. 32, 9457–9468 (2012).
Hoover, H.S., Blankman, J.L., Niessen, S. & Cravatt, B.F. Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling. Bioorg. Med. Chem. Lett. 18, 5838–5841 (2008).
Ortar, G. et al. Tetrahydrolipstatin analogues as modulators of endocannabinoid 2-arachidonoylglycerol metabolism. J. Med. Chem. 51, 6970–6979 (2008).
Bisogno, T. et al. Development of the first potent and specific inhibitors of endocannabinoid biosynthesis. Biochim. Biophys. Acta 1761, 205–212 (2006).
Pedicord, D.L. et al. Molecular characterization and identification of surrogate substrates for diacylglycerol lipase α. Biochem. Biophys. Res. Commun. 411, 809–814 (2011).
Long, J.Z. & Cravatt, B.F. The metabolic serine hydrolases and their functions in mammalian physiology and disease. Chem. Rev. 111, 6022–6063 (2011).
Adibekian, A. et al. Click-generated triazole ureas as ultrapotent in vivo–active serine hydrolase inhibitors. Nat. Chem. Biol. 7, 469–478 (2011).
Cravatt, B.F., Wright, A.T. & Kozarich, J.W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77, 383–414 (2008).
Bachovchin, D.A. et al. Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors. Proc. Natl. Acad. Sci. USA 108, 6811–6816 (2011).
Oudin, M.J., Hobbs, C. & Doherty, P. DAGL-dependent endocannabinoid signalling: roles in axonal pathfinding, synaptic plasticity and adult neurogenesis. Eur. J. Neurosci. 34, 1634–1646 (2011).
Di Marzo, V. Endocannabinoid signaling in the brain: biosynthetic mechanisms in the limelight. Nat. Neurosci. 14, 9–15 (2011).
Jung, K.-M. A key role for diacylglycerol lipase-α in metabotropic glutamate receptor-dependent endocannabinoid mobilization. Mol. Pharmacol. 72, 612–621 (2007).
Jessani, N. et al. A streamlined platform for high-content functional proteomics of primary human specimens. Nat. Methods 2, 691–697 (2005).
Long, J.Z., Nomura, D.K. & Cravatt, B.F. Characterization of monoacylglycerol lipase inhibition reveals differences in central and peripheral endocannabinoid metabolism. Chem. Biol. 16, 744–753 (2009).
Bachovchin, D.A. et al. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening. Proc. Natl. Acad. Sci. USA 107, 20941–20946 (2010).
Uozumi, N. et al. Role of cytosolic phospholipase A2 in allergic response and parturition. Nature 390, 618–622 (1997).
Bonventre, J.V. et al. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature 390, 622–625 (1997).
Rouzer, C.A. et al. Cyclooxygenase-1-dependent prostaglandin synthesis modulates tumor necrosis factor-α secretion in lipopolysaccharide-challenged murine resident peritoneal macrophages. J. Biol. Chem. 279, 34256–34268 (2004).
Watanabe, S., Kobayashi, T. & Okuyama, H. Regulation of lipopolysaccharide-induced tumor necrosis factor α production by endogenous prostaglandin E2 in rat resident and thioglycollate-elicited macrophages. J. Lipid Mediat. Cell Signal. 10, 283–294 (1994).
Rouzer, C.A. et al. RAW264.7 cells lack prostaglandin-dependent autoregulation of tumor necrosis factor-α secretion. J. Lipid Res. 46, 1027–1037 (2005).
Carrasco, S. & Merida, I. Diacylglycerol, when simplicity becomes complex. Trends Biochem. Sci. 32, 27–36 (2007).
Chevaleyre, V., Takahashi, K.A. & Castillo, P.E. Endocannabinoid-mediated synaptic plasticity in the CNS. Annu. Rev. Neurosci. 29, 37–76 (2006).
Rouzer, C.A. & Marnett, L.J. Endocannabinoid oxygenation by cyclooxygenases, lipoxygenases, and cytochromes P450: cross-talk between the eicosanoid and endocannabinoid signaling pathways. Chem. Rev. 111, 5899–5921 (2011).
Buczynski, M.W., Dumlao, D.S. & Dennis, E.A. Thematic Review Series: Proteomics. An integrated omics analysis of eicosanoid biology. J. Lipid Res. 50, 1015–1038 (2009).
Parameswaran, N. & Patial, S. Tumor necrosis factor-α signaling in macrophages. Crit. Rev. Eukaryot. Gene Expr. 20, 87–103 (2010).
Bondeson, J. The mechanisms of action of disease-modifying antirheumatic drugs: a review with emphasis on macrophage signal transduction and the induction of proinflammatory cytokines. Gen. Pharmacol. 29, 127–150 (1997).
Lin, H.I., Chu, S.J., Wang, D. & Feng, N.H. Pharmacological modulation of TNF production in macrophages. J. Microbiol. Immunol. Infect. 37, 8–15 (2004).
Hashimotodani, Y. et al. Phospholipase Cβ serves as a coincidence detector through its Ca2+ dependency for triggering retrograde endocannabinoid signal. Neuron 45, 257–268 (2005).
Hashimotodani, Y., Ohno-Shosaku, T., Watanabe, M. & Kano, M. Roles of phospholipase Cβ and NMDA receptor in activity-dependent endocannabinoid release. J. Physiol. (Lond.) 584, 373–380 (2007).
Qiu, Z.H., Gijon, M.A., de Carvalho, M.S., Spencer, D.M. & Leslie, C.C. The role of calcium and phosphorylation of cytosolic phospholipase A2 in regulating arachidonic acid release in macrophages. J. Biol. Chem. 273, 8203–8211 (1998).
Turunen, P.M., Jantti, M.H. & Kukkonen, J.P. OX1 orexin/hypocretin receptor signaling through arachidonic acid and endocannabinoid release. Mol. Pharmacol. 82, 156–167 (2012).
Burstein, S.H. & Zurier, R.B. Cannabinoids, endocannabinoids, and related analogs in inflammation. AAPS J. 11, 109–119 (2009).
Kunos, G. & Batkai, S. Novel physiologic functions of endocannabinoids as revealed through the use of mutant mice. Neurochem. Res. 26, 1015–1021 (2001).
Marrs, W.R. et al. The serine hydrolase ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors. Nat. Neurosci. 13, 951–957 (2010).
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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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.
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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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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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DOI: https://doi.org/10.1038/nchembio.1105
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