title: Activity-Based Probe for N-Acylethanolamine Acid Amidase.
abstract: N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, α-amino-β-lactone (3-aminooxetan-2-one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl-N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate) and ARN726 (4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate). The former is active in vivo by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the in vivo detection of NAAA. The newly synthesized molecule 1 is an effective in vitro and in vivo click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with 1 confirmed that ARN726 and ARN077 inhibit NAAA in vitro and in vivo. Compound 1 is a useful new tool to identify activated NAAA both in vitro and in vivo and to investigate the physiological and pathological roles of this enzyme.
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Romeo
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Ponzano
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Armirotti
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Summa
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Bertozzi
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy.
authors.forename: Gianpiero
authors.initials: G
authors.lastname: Garau
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy.
authors.forename: Tiziano
authors.initials: T
authors.lastname: Bandiera
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy.|Departments of Anatomy and Neurobiology, Pharmacology and Biological Chemistry, University of California, 3216 Gillespie Neuroscience Facility Irvine, California 92697-4621 (United States).
authors.forename: Daniele
authors.initials: D
authors.lastname: Piomelli
mesh_terms: D000581:Amidohydrolases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D057075:Enzyme Assays / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008168:Lung / Q000201:enzymology; D008297:Male; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism*; D017207:Rats, Sprague-Dawley
chemical_list: D015335:Molecular Probes; D000581:Amidohydrolases; C523108:NAAA protein, human