A Potent Systemically Active N-Acylethanolamine Acid Amidase Inhibitor that Suppresses Inflammation and Human Macrophage Activation

Abstract

Fatty acid ethanolamides such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are lipid-derived mediators that potently inhibit pain and inflammation by ligating type-α peroxisome proliferator-activated receptors (PPAR-α). These bioactive substances are preferentially degraded by the cysteine hydrolase, N-acylethanolamine acid amidase (NAAA), which is highly expressed in macrophages. Here, we describe a new class of β-lactam derivatives that are potent, selective, and systemically active inhibitors of intracellular NAAA activity. The prototype of this class deactivates NAAA by covalently binding the enzyme's catalytic cysteine and exerts profound anti-inflammatory effects in both mouse models and human macrophages. This agent may be used to probe the functions of NAAA in health and disease and as a starting point to discover better anti-inflammatory drugs.

Figure 1

The endogenous N-acylethanolamine acid amidase (NAAA) substrates, palmitoylethanolamide (PEA) (1) and oleoylethanolamide (OEA) (2)

Figure 2

Chemical structures of current N-acylethanolamine acid amidase (NAAA) inhibitors.

Figure 3

Syntheses of compounds 6 (ARN726) and 7. Reaction conditions: a) 4-Dimethylaminopyridine (DMAP), di-2-pyridyl carbonate (2-DPC), dry CH₂Cl₂, room temperature, 16 h. b) (S)-11 or (R)-12, N,N-Diisopropylethylamine (DIPEA), dry CH₂Cl₂, room temperature, 16 h.

Figure 4

Compound 6 inhibits N-acylethanolamine acid amidase (NAAA) activity. A) Concentration-response curve for inhibition of h-NAAA by 6 (red circle) and its (R)-enantiomer 7 (black square). B) Time-course of h-NAAA inhibition by 6 (100 nM). C) Kinetic analysis of h-NAAA inhibition by 6 (vehicle, black circle; 10 nM, red square; 30 nM, red triangle). D) Effects of centrifugation dialysis on the inhibition of h-NAAA by 6. E) Effects of 6 (30 mg kg⁻¹, oral) on ex vivo NAAA activity and F) palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) levels in mouse lungs (vehicle, open bars; 6 red bars); saline (Sal) and carrageenan (Carr). Data are represented as mean ± SEM; * P<0.05; ** P<0.01; *** P<0.001.

Figure 5

Compound 6 inhibits N-acylethanolamine acid amidase (NAAA) via S-acylation of the enzyme's catalytic cysteine. A) Nucleophilic attack of Cys131 on 6 can theoretically yield the three adducts 1–3. B) Extracted ion chromatograms of doubly-charged ion (m/z = 795.40) corresponding to the formation of adducts 1 of r-NAAA N-terminus with 6 (red trace) or 7 (black trace). C). MS/MS spectrum of adduct 1 of 6 with r-NAAA. The mass increase introduced by 6 (*) is carried by Cys131, confirming the proposed inhibition mechanism. D) MS/MS spectrum of the same adduct from rat lung, 3 h after intravenous administration of 6.

Figure 6

Compound 6 blunts inflammatory responses in mouse lungs in vivo and human macrophages ex vivo. Effects of 6 (1–30 mg kg⁻¹, oral), its (R)-enantiomer 7 (30 mg kg⁻¹, oral), or dexamethasone (0.5 mg kg⁻¹, i.p.) on carrageenan (Carr)-induced inflammation assessed as A) myeloperoxidase (MPO) activity in lung tissue and B) tumor necrosis factor (TNF)-α levels in pleural exudate. Peroxisome proliferator-activated receptor (PPAR)-α deletion eliminates the anti-inflammatory effects of 6: C) MPO activity in lungs and D) TNF-α levels in pleural exudate of PPAR-α-null mice or C57BL6/J wild-type (WT) littermates. E) N-acylethanolamine acid amidase (NAAA) expression in human granulocytes (CD15+), monocytes (CD14+), T-lymphocytes (CD3+), B-lymphocytes (CD19+), and NK cells (CD56+). F) NAAA expression in monocytes and monocyte-derived macrophages. G) Concentration-dependent effects of 6 on inducible nitric oxide synthase (iNOS) and H) TNF-α expression and I) TNF-α release by LPS-activated human macrophages. (A–D) Data are represented as mean ± SEM; # P<0.05 vs saline-treated mice (open bar; same genotype); * P<0.05, ** P<0.01, and *** P<0.0001 vs carrageenan-treated mice (shaded bar; same genotype); °°° P<0.0001 indicates differences in the responses between C57BL/6J WT and PPAR-α-null mice. (E–I) MFI=mean fluorescence intensity; # P<0.05 vs control macrophages (open bar) and * P<0.05 vs LPS-stimulated macrophages (shaded bar).