Activity-Based Probe for N‑Acylethanolamine Acid Amidase

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.

N-酰乙胺酸酰胺酶（NAAA）是一种溶酶体半胱氨酸水解酶，参与饱和及单不饱和脂肪酸乙酰胺（FAEs）的降解过程。FAEs是一类内源性脂质信号分子家族，包括油酸乙酰胺（OEA）和棕榈酸乙酰胺（PEA）。在已报道的NAAA抑制剂中，α-氨基-β-内酯（3-氨基氧杂环己酮）衍生物已被证明可以阻止FAEs在先天免疫细胞和神经细胞中的水解，并减少对炎症刺激的反应。近期，我们披露了两种高效的、选择性的NAAA抑制剂，化合物ARN077（5-苯基戊基-N-[(2S,3R)-2-甲基-4-氧代氧杂环己烷-3-基]羰酰胺）和ARN726（4-环己基丁基-N-[(S)-2-氧代嘧啶-3-基]羰酰胺）。其中，ARN077在啮齿动物模型的高痛觉过敏和痛觉过敏中通过局部给药表现出活性，而ARN726在肺炎症小鼠模型中产生全身抗炎效果。在本研究中，我们设计并验证了ARN726的衍生物作为第一个基于活性的蛋白质组学探针（ABPP），用于体内NAAA的检测。新合成的分子1是一种有效的体外和体内点击化学活性探针（ABP），能够捕捉在过表达人类NAAA的HEK293细胞以及大鼠肺组织中NAAA的催化活性形式。1与NAAA的竞争性ABPP实验证实了ARN726和ARN077在体外和体内抑制NAAA。化合物1是一种有用的新型工具，可用于在体外和体内识别激活的NAAA，并研究该酶的生理和病理作用。