Abstract
N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme that inhibits the degradation of palmitoylethanolamide (PEA), an endogenous lipid that induces analgesic, anti-inflammation, and anti-multiple sclerosis through PPARα activation. Only a few potent NAAA inhibitors have been reported to date, which is mainly due to the restricted substrate-binding site of NAAA. Here, we established a high-throughput fluorescence-based assay for NAAA inhibitor screening. Several new classes of NAAA inhibitors were discovered from a small library of natural products. One of these is atractylodin, a polyethylene alkyne compound from the root of Atractylodes lancea (Thunb) DC., which significantly inhibits NAAA activity and has an IC50 of 2.81 µM. Kinetic analyses and dialysis assays suggested that atractylodin engages in competitive inhibition via reversible reaction to the enzyme. Docking assays revealed that atractylodin occupies the catalytic cavity of NAAA, where the atractylodin furan head group has a hydrophobic-related interaction with the backbone of the Trp181 and Leu152 residues of human NAAA. Further investigation indicated that atractylodin significantly increases PEA and OEA levels and dose-dependently inhibits LPS-induced nitrate, TNF-α, IL-1β, and IL-6 pro-inflammatory cytokine release in BV-2 microglia. Our results show that atractylodin elevates cellular PEA levels and inhibits microglial activation by inhibiting NAAA activity, which in turn could contribute to NAAA functional research.
Highlights
N-Acylethanolamines (NAEs) are a class of functional long-chain lipids that are ubiquitous in animal tissues including arachidonyl ethanolamide, oleoylethanolamide (OEA), and N-palmitoylethanolamide (PEA) (Iannotti et al, 2016)
Several compounds have been discovered as N-acylethanolamine-hydrolyzing acid amidase (NAAA) inhibitors, reports on natural derivatives are limited
PAMCA was metabolized to the fluorescent 7-amino-4-methyl coumarin (AMC) and palmitic acid by NAAA at pH 4.5 (Figure 1A)
Summary
N-Acylethanolamines (NAEs) are a class of functional long-chain lipids that are ubiquitous in animal tissues including arachidonyl ethanolamide (anandamide, AEA), oleoylethanolamide (OEA), and N-palmitoylethanolamide (PEA) (Iannotti et al, 2016). NAEs are widely involved in modulating various physiological responses ranging from inflammation, pain, neuroprotection, Atractylodin Inhibits Microglial Activation appetite, and lipid metabolism (Kaur et al, 2016). NAAA is mainly distributed in lysosomes and shows no homology to fatty acid amide hydrolase (FAAH), another important NAE degradation enzyme (Tsuboi et al, 2007b; Ren et al, 2020). Several series of compounds targeting NAAA inhibition have been reported to have potential therapeutic use for inflammatory and neuropathic pain (Yang et al, 2015; Petrosino et al, 2017; Ren et al, 2017), multiple sclerosis (Migliore et al, 2016), allergic dermatitis (Sasso et al, 2018), arthritis (Bonezzi et al, 2016), and inflammatory bowel diseases (Alhouayek et al, 2015)
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