Mechanism of inhibition of fatty acid amide hydrolase by sulfonamide-containing benzothiazoles: long residence time derived from increased kinetic barrier and not exclusively from thermodynamic potency.

Fatty acid amide hydrolase (FAAH) has emerged as a potential target for developing analgesic, anxiolytic, antidepressant, sleep-enhancing and anti-inflammatory drugs, and tremendous efforts have been made to discover potent and selective inhibitors of FAAH. Most known potent FAAH inhibitors described to date employ covalent mechanisms, inhibiting the enzyme either reversibly or irreversibly. Recently, a benzothiazole-based analogue (1) has been described possessing a high potency against FAAH yet lacking a structural feature previously known to interact with FAAH covalently. However, covalent inhibition of FAAH by 1 has not been fully ruled out, and the issue of reversibility has not been addressed. Confirming previous reports, 1 inhibited recombinant human FAAH (rhFAAH) with high potency with IC50 similar to 2 nM. It displayed an apparently noncompetitive and irreversible inhibition, titrating rhFAAH stoichiometrically within normal assay times. The inhibition appeared to be time dependent, but the time dependence only improved potency by a small degree (from similar to 8 to similar to 2 nM). However, mass spectrometric analyses of the reaction mixture failed to reveal any cleavage product or covalent adduct and showed full recovery of the parent compound, ruling out covalent, irreversible inhibition. Dialysis revealed recovery of enzyme activity from enzyme-inhibitor complex over a prolonged time (>10 h), demonstrating that 1 is indeed a reversible, albeit slowly dissociating inhibitor of FAAH. Molecular docking indicated that the sulfonamide group of 1 could form hydrogen bonds with several residues involved in catalysis, thereby mimicking the transition state. The long residence time displayed by 1 does not appear to derive exclusively from great thermodynamic potency and is consistent with an increased kinetic energy barrier that prevents dissociation from happening quickly.