Biochemical and mass spectrometric characterization of human N-acylethanolamine-hydrolyzing acid amidase inhibition.

The mechanism of inactivation of human enzyme N-acylethanolamine-hydrolyzing acid amidase (hNAAA), with selected inhibitors identified in a novel fluorescent based assay developed for characterization of both reversible and irreversible inhibitors, was investigated kinetically and using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 1-Isothiocyanatopentadecane (AM9023) was found to be a potent, selective and reversible hNAAA inhibitor, while two others, 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-Benzyloxycarbonyl-L-serine β-lactone (N-Cbz-serine β-lactone), inhibited hNAAA in a covalent and irreversible manner. MS analysis of the hNAAA/covalent inhibitor complexes identified modification only of the N-terminal cysteine (Cys126) of the β-subunit, confirming a suggested mechanism of hNAAA inactivation by the β-lactone containing inhibitors. These experiments provide direct evidence of the key role of Cys126 in hNAAA inactivation by different classes of covalent inhibitors, confirming the essential role of cysteine for catalysis and inhibition in this cysteine N-terminal nucleophile hydrolase enzyme. They also provide a methodology for the rapid screening and characterization of large libraries of compounds as potential inhibitors of NAAA, and subsequent characterization or their mechanism through MALDI-TOF MS based bottom up-proteomics

MALDI-TOF MS/MS analysis of the hNAAA tryptic peptide T10-β after covalent modification.

<p>Tandem MALDI-TOF MS/MS spectra of the T10-β peptide (sequence: CTSIVAQDSR) demonstrates covalent modification of Cys126 by both AM6701 (Panel (A)) and <i>N-</i>Cbz-serine β-lactone (Panel (B)).</p

Representation of the active site of hNAAA after treatment with <i>N-</i>Cbz-serine β-lactone.

<p>Homology model illustrates acylated catalytic nucleophile Cys126 after treatment with <i>N-</i>Cbz-serine β-lactone.</p

Potencies of hNAAA inhibitors.

<p>The <i>k</i>_inact and <i>K</i>_I values for the covalent inhibitors were obtained as described in the Experimental Procedures. The IC₅₀ values were calculated after 2 hours preincubation of the enzyme and inhibitor before addition of the substrate. Values are averages ± SD of three independent experiments.</p

Representation of the active site of hNAAA after treatment with AM6701.

<p>Homology model illustrates thiocarbamylation of catalytic nucleophile Cys126 after treatment with AM6701.</p

Concentration dependent inhibition of purified hNAAA by three compounds.

<p>hNAAA was incubated with the compounds AM6701 (squares), <i>N-</i>Cbz-serine β-lactone (circles), and AM9023 (diamonds) for two hours in order to reach full inhibition before measuring activity. Panel (A). A radioactivity-based assay with [¹⁴C] PEA as substrate. Panel (B). A fluorescence-based assay with PAMCA as substrate. Representative curves are displayed.</p

Putative mechanism of inhibition of hNAAA for three compounds studied.

<p>Panel (A). Reversible inhibition of hNAAA by AM9023. Panel (B). Irreversible inhibition of hNAAA by AM6701 via thiocarbamylation of Cys126. Panel (C). Irreversible inhibition of hNAAA by <i>N-</i>Cbz-serine β-lactone most likely proceeds via route 2.</p

Mass of tryptic peptide containing Cys126 of hNAAA after covalent modification.

<p>T10-β peptides identified in the tryptic digest of untreated (control) and AM6701 or N-Cbz-serine β-lactone treated hNAAA samples.</p