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.
Highlights
20:4Δ),[5,8,11,14] which is preferentially inactivated by fatty acid amide hydrolase (FAAH).[14,15] Anandamide is an endogenous agonist for cannabinoid receptors and participates in the control of stress-coping responses and pain initiation,[16] among other functions, while PEA and OEA regulate pain, inflammation, and energy balance primarily by ligating peroxisome proliferator-activated receptor-α (PPAR-α), a member of the nuclear receptor superfamily.[11,17−19]
N-Acylethanolamine acid amidase (NAAA) is a cysteine hydrolase that belongs to the N-terminal nucleophile (Ntn) family of enzymes.[1−3] It is localized to lysosomes and bears a significant degree of sequence homology with the bacterial choloylglycine hydrolases, which are characterized by the ability to cleave nonpeptide amide bonds.[4]
Like other Ntn enzymes, NAAA is activated by autoproteolysis at acidic pH, which generates a catalytically competent form of the enzyme.[5]
Summary
20:4Δ),[5,8,11,14] which is preferentially inactivated by fatty acid amide hydrolase (FAAH).[14,15] Anandamide is an endogenous agonist for cannabinoid receptors and participates in the control of stress-coping responses and pain initiation,[16] among other functions, while PEA and OEA regulate pain, inflammation, and energy balance primarily by ligating peroxisome proliferator-activated receptor-α (PPAR-α), a member of the nuclear receptor superfamily.[11,17−19]. We used a maleimidecontaining probe (2, see Supporting Information), incubated it with wt HEK293 cells, and applied the CC-ABPP procedure to cytosol and membrane fractions.
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