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 IC(50) ~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 ~8 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.

Biochemical characterization and in vitro activity of AZ513, a noncovalent, reversible, and noncompetitive inhibitor of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) hydrolyzes several bioactive lipids including the endocannabinoid anandamide. Synthetic FAAH inhibitors are being generated to help define the biological role(s) of this enzyme, the lipids it degrades in vivo, and the disease states that might benefit from its pharmacological modulation. AZ513 inhibits human FAAH (IC(50)=551 nM), is 20-fold more potent against rat FAAH (IC(50)=27 nM), and is inactive at 10 µM against the serine hydrolases acetylcholinesterase, thrombin, and trypsin. In contrast to most other potent FAAH inhibitors, AZ513 showed no evidence of covalently modifying the enzyme and displayed reversible inhibition. In an enzyme cross-competition assay, AZ513 did not compete with OL-135, an inhibitor that binds to the catalytic site in FAAH, which indicates that AZ513 does not bind to the catalytic site and is therefore noncompetitive with respect to substrate. AZ513 has good cell penetration as demonstrated by inhibition of anandamide hydrolysis in human FAAH-transfected HEK293 cells (IC(50)=360 nM). AZ513 was tested in a rat spinal cord slice preparation where CB(1) activation reduces excitatory post-synaptic currents (EPSCs). In this native tissue assay of synaptic activity, AZ513 reduced EPSCs, which is consistent with inhibiting endogenous FAAH and augmenting endocannabinoid tone. AZ513 has a unique biochemical profile compared with other published FAAH inhibitors and will be a useful tool compound to further explore the role of FAAH in various biological processes.

Neurokinin-3 receptor-specific antagonists talnetant and osanetant show distinct mode of action in cellular Ca2+ mobilization but display similar binding kinetics and identical mechanism of binding in ligand cross-competition.

Talnetant and osanetant, two structurally diverse antagonists of neurokinin-3 receptor (NK3), displayed distinct modes of action in Ca2+ mobilization. Although talnetant showed a normal Schild plot with a slope close to unity and a Kb similar to its Ki value in binding, osanetant presented an aberrant Schild with a steep slope (3.3 +/- 0.5) and a Kb value (12 nM) significantly elevated compared with its Ki value (0.8 nM) in binding. Kinetic binding experiments indicated a simple one-step binding mechanism with relatively fast on- and off-rates for both antagonists, arguing against slow onset of antagonism as the reason for abnormal Schild. This conclusion was supported by prolonged preincubation of antagonist that failed to improve the observed aberrant Schild. In ligand cross-competition binding, both talnetant and osanetant displayed linear reciprocal plots of identical slope when [MePhe7]neurokinin B (NKB) was used as the other competition partner with 125I-[MePhe7]NKB as the radioligand, indicating competitive binding of either antagonist with regard to [MePhe7]NKB. Similar patterns were obtained when talnetant was tested against osanetant, indicating competitive binding between the two antagonists as well. These results were reproduced when [3H]4-quinolinecarboxamide (SB222200), a close derivative of talnetant, was used as the radioligand. Taken together, these data strongly suggest binding of both talnetant and osanetant at the orthosteric binding site with similar kinetic properties and do not support the hypothesis that the aberrant Schild observed in functional assays for osanetant is derived from differences in the mechanism of binding for these NK3 antagonists.