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. 2012 Apr 6;3(5):422-6.
doi: 10.1021/ml300056y. eCollection 2012 May 10.

β-Lactones Inhibit N-acylethanolamine Acid Amidase by S-Acylation of the Catalytic N-Terminal Cysteine

Andrea Armirotti  1 , Elisa Romeo  1 , Stefano Ponzano  1 , Luisa Mengatto  1 , Mauro Dionisi  1 , Claudia Karacsonyi  1 , Fabio Bertozzi  1 , Gianpiero Garau  1 , Glauco Tarozzo  1 , Angelo Reggiani  1 , Tiziano Bandiera  1 , Giorgio Tarzia  2 , Marco Mor  3 , Daniele Piomelli  4
Affiliations

β-Lactones Inhibit N-acylethanolamine Acid Amidase by S-Acylation of the Catalytic N-Terminal Cysteine

Andrea Armirotti et al. ACS Med Chem Lett. .

Abstract

The cysteine amidase N-acylethanolamine acid amidase (NAAA) is a member of the N-terminal nucleophile class of enzymes and a potential target for anti-inflammatory drugs. We investigated the mechanism of inhibition of human NAAA by substituted β-lactones. We characterized pharmacologically a representative member of this class, ARN077, and showed, using high-resolution liquid chromatography-tandem mass spectrometry, that this compound forms a thioester bond with the N-terminal catalytic cysteine in human NAAA.

Keywords: NAAA; covalent inhibitors; cysteine amidase; high resolution mass spectrometry; proteomics.

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Figures

Figure 1
Figure 1
Chemical structures of β-lactone NAAA inhibitors, (S)-N-(2-oxo-3-oxetanyl)-3-phenylpropanamide [(S)-OOPP], N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl] carbamate (ARN077), and N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]-7-phenyl-heptanamide (ARN768).
Figure 2
Figure 2
Inhibition of human NAAA by ARN077. (A) Concentration–response curve for inhibition of recombinant hNAAA by ARN077 after 30 min of incubation. (B) Michaelis–Menten analysis of ARN077 (vehicle, ●; 10 nM ARN077, ■; and 30 nM ARN077, ▲). (C) Preincbation time course of ARN077 (100 nM). (D) Reversibility study of ARN077 (vehicle, open bars; ARN077, black bars). *p < 0.05 ARN077 postdialysis vs vehicle postdialysis; **p < 0.01 ARN077 postdialysis vs ARN077 predialysis; and ***p < 0.001 ARN077 predialysis vs vehicle predialysis.
Figure 3
Figure 3
(A) Extracted ion chromatograms of the native (540.26 m/z) and acylated (685.43 m/z) peptide (for clarity purposes, peaks have not been normalized). (B and C) MS/MS spectra of m/z 540.26 and m/z 685.43. In both spectra, the y fragment ions series perfectly match a CTSIVAQDSR sequence (bearing a side chain adduct on the cysteine in the second case).
Scheme 1
Scheme 1. Possible Nucleophylic Attacks of Cysteine on ARN077
Path a, thioether adduct; path b, thioester adduct; and path c, hydrolysis of the carbamate, with two possible adducts (c and c′). R = (CH2)5-Ph.
Figure 4
Figure 4
Zoomed MS/MS spectra of peptide CTSIVAQDSR acylated by ARN077 and ARN768. Cysteine side chain fragment ions are indicated along with the y2 fragment (262.15 m/z). S-Acylation diagnostic fragment ion at 262.18 m/z is reported in the inset.
See this image and copyright information in PMC

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