Abstract
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.
Highlights
Current pharmacological strategies in drug development targeting the endocannabinoid system are focused on the discovery of therapeutic agents that selectively modulate the cannabinergic signaling for the treatment of human disorders, without being accompanied by undesirable psychotropic side effects [1,2,3]
N-(4-methyl coumarin)palmitamide (PAMCA) substrate We previously described the fluorogenic substrate N-(4-methyl coumarin)palmitamide (PAMCA), which is hydrolyzed by N-acylethanolamine-hydrolyzing acid amidase (NAAA) to the fluorescent compound 7-amino-4-methyl coumarin (AMC) and palmitic acid [20]
We previously introduced the novel fluorogenic compound N(4-methyl coumarin) palmitamide (PAMCA), which has an affinity for hydrolyzing acid amidase (hNAAA) comparable to the native substrate PEA (Km 6.2 mM and 21 mM for PAMCA and PEA, respectively), and which is enzymatically hydrolyzed to the fluorescent 7-amino-4-methyl coumarin (AMC) and palmitic acid [20]
Summary
Current pharmacological strategies in drug development targeting the endocannabinoid system are focused on the discovery of therapeutic agents that selectively modulate the cannabinergic signaling for the treatment of human disorders, without being accompanied by undesirable psychotropic side effects [1,2,3] Toward this goal, two principal endocannabinoid enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL), are promising candidates for drug discovery, by modulating the effects of the two principal endocannabinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG), respectively, at the cannabinoid receptors 1 and 2 (CB1 and CB2) [4,5]. A putative hNAAA catalytic triad of amino acid residues Cys126, Arg142 and Asp145 has been predicted from the highly conserved nature of these residues in the cysteine Ntn hydrolase superfamily and sitedirected mutagenesis experiments, which identified Glu195 as a key determinant of acidic cleavage, and suggested that Asn287 plays an important role in proteolytic zymogen activation [19,21]. A mechanism of catalysis via a zwitterionic N-terminal cysteine in CBAH was proposed based on computational analyses of free energy simulations and suggested that NAAA may cleave its substrates using the same catalytic strategy [22]
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