title: 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.
authors.affiliation: †Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Marc P
authors.initials: MP
authors.lastname: Baggelaar
authors.affiliation: ‡Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam 1000 GG, The Netherlands.
authors.forename: Pascal J P
authors.initials: PJ
authors.lastname: Chameau
authors.affiliation: §Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Vasudev
authors.initials: V
authors.lastname: Kantae
authors.affiliation: †Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Jessica
authors.initials: J
authors.lastname: Hummel
authors.affiliation: ∥Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, United States.
authors.forename: Ku-Lung
authors.initials: KL
authors.lastname: Hsu
authors.affiliation: †Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Freek
authors.initials: F
authors.lastname: Janssen
authors.affiliation: †Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Tom
authors.initials: T
authors.lastname: van der Wel
authors.affiliation: †Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Marjolein
authors.initials: M
authors.lastname: Soethoudt
authors.affiliation: †Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Hui
authors.initials: H
authors.lastname: Deng
authors.affiliation: †Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: den Dulk
authors.affiliation: ⊥Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Pozzuoli 80078, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Allarà
authors.affiliation: †Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Bogdan I
authors.initials: BI
authors.lastname: Florea
authors.affiliation: ⊥Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Pozzuoli 80078, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Di Marzo
authors.affiliation: ‡Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam 1000 GG, The Netherlands.
authors.forename: Wytse J
authors.initials: WJ
authors.lastname: Wadman
authors.affiliation: ‡Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam 1000 GG, The Netherlands.
authors.forename: Chris G
authors.initials: CG
authors.lastname: Kruse
authors.affiliation: †Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Herman S
authors.initials: HS
authors.lastname: Overkleeft
authors.affiliation: §Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hankemeier
authors.affiliation: ‡Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam 1000 GG, The Netherlands.
authors.forename: Taco R
authors.initials: TR
authors.lastname: Werkman
authors.affiliation: ∥Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, United States.
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Cravatt
authors.affiliation: †Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden 2300 RA, The Netherlands.
authors.forename: Mario
authors.initials: M
authors.lastname: van der Stelt
mesh_terms: D000818:Animals; D002460:Cell Line; D055808:Drug Discovery; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D006571:Heterocyclic Compounds / Q000737:chemistry / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects / Q000502:physiology; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology*; D009435:Synaptic Transmission / Q000187:drug effects
chemical_list: D004791:Enzyme Inhibitors; D006571:Heterocyclic Compounds; D008071:Lipoprotein Lipase