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.

Figure 1

Structures of NAAA inhibitors: β-lactone ARN077 and β-lactam ARN726.

Figure 2

(a) Structure of the click chemistry probe 1. (b) General strategy for click chemistry activity-based protein profiling (CC-ABPP). Whole-cells or lysates are incubated with the probe and subjected to click chemistry to introduce the reporter tag. The labeled proteome is visualized by in-gel /western blot analysis before and/or after enrichment on streptavidin beads. Protein identification is carried out by mass spectrometry of the enriched proteome tryptic peptides.

Figure 3

(a): Full MS/MS spectrum of hNAAA peptide CTSIVAQGR acylated by ARN14686. (b): Zoomed range showing b1, b2 and b3 backbone fragment ions indicating that the acylation occurred on the N-terminal cysteine residue. (c): Further zoom of the same mass range, showing two side-chain fragmentations (F1 and F2) that are diagnostic for a thioester formation (F1= [C₁₅H₂₅N₂O₃S]⁺, expected m/z 313.1586, measured m/z 313.1580, 1.9 ppm mass error. F2= [C₁₅H₂₅N₂O₃]⁺, expected m/z 281.1865, measured m/z 281.1860, 1.8 ppm mass error).

Figure 4. Labeling of purified hNAAA

(a) Protein blot analysis of activated recombinant hNAAA incubated with DMSO (−) or compound 1 (+) at pH 4.5 or 7.4. The blotting membranes were probed with streptavidin-HRP conjugate or anti-NAAA antibody (α-NAAA), as indicated. (b) Concentration dependence of the interaction of 1 with NAAA. 1 was incubated at various concentrations with a constant amount of hNAAA (1 μM). (c) Limit of detection of hNAAA by 1. hNAAA was incubated at various concentrations with a constant amount of 1 (10 μM) in the presence of 10 μg of protein extract from HEK293 cells; blotting membrane in panels a and c were probed with streptavidin-HRP conjugate; FL: full-length protein; β: NAAA β–subunit; Pb = Protein blot; C = Coomassie blue staining.

Figure 5. Labeling of hNAAA in lysed and intact NAAA-HEK293 cells

(a) Protein blot analysis of hNAAA-overexpressing HEK293 intact cells (lane 1 and 2) or lysate (lane 3 and 4) incubated with 1 (+) or DMSO (−). (b) hNAAA-HEK intact cells preincubated with ARN726 (lane 3) or ARN077 (lane 4) before addition of 1. Cells incubated with DMSO (lane 1) or 1 only (lane 2) were used as controls. The blotting membranes were probed with a streptavidin-HRP conjugate or an anti-NAAA antibody (α-NAAA) as indicated in the figure. FL: full-length protein; β: NAAA β–subunit.

Figure 6. Probe labeling profile of wild-type HEK293 cells

(a) Protein blot analysis performed on cellular fractions from wt HEK293 cells incubated with 1 (left membrane) or 2 (right membrane). The total cell lysate of NAAA-HEK293 cells incubated with 1 was used as positive control (lane 5, left membrane). (b) Protein blot analysis of 1 incubated samples enriched on streptavidin-agarose and eluted. Blotting membranes were probed with a streptavidin-HRP conjugate. C: cytosol; M: membranes; T: total lysate

Figure 7. In vitro and in vivo probe labeling profile of rat lungs

(a) Protein blot analysis of streptavidin-enriched proteins from rat lung cellular fractions incubated in vitro with 1 (1 μM or 10 μM) or DMSO as negative control (−). (b) Protein blot analysis of streptavidin-enriched proteins of lung lysosomal fractions from rats intravenously injected with ARN726 followed by 1 (lane 3) or with 1 only (lane 4). Treatment with vehicle (lane 1) or ARN726 alone (lane 2) were used as negative controls. Blotting membranes were probed with a streptavidin-HRP conjugate. Signals corresponding to AC and NAAA are indicated. T: total lysate; M: membranes; C: cytosol; L: Lysosomes.