AstraZeneca Pharmaceuticals, Wilmington, Delaware 19850, United States.
Biochemistry. 2011 Aug 16;50(32):6867-78. doi: 10.1021/bi200552p. Epub 2011 Jul 22.
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.
脂肪酸酰胺水解酶（FAAH）已成为开发镇痛药、抗焦虑药、抗抑郁药、助眠药和抗炎药的潜在靶点，人们已经做出了巨大的努力来发现有效的、选择性的 FAAH 抑制剂。迄今为止，大多数已知的强效 FAAH 抑制剂都采用共价机制，要么可逆地，要么不可逆地抑制酶。最近，一种苯并噻唑基类似物（1）被描述为对 FAAH 具有高活性，但其结构特征缺乏先前已知与 FAAH 共价相互作用的特征。然而，1 对 FAAH 的共价抑制尚未完全排除，而且可逆性问题尚未得到解决。与之前的报道一致，1 对重组人 FAAH（rhFAAH）具有高抑制活性，IC502 nM。它表现出明显的非竞争性和不可逆抑制作用，在正常测定时间内滴定 rhFAAH 化学计量。抑制作用似乎是时间依赖性的，但时间依赖性仅略微提高了活性（从8 到~2 nM）。然而，反应混合物的质谱分析未能揭示任何裂解产物或共价加合物，并显示出母体化合物的完全恢复，从而排除了共价、不可逆抑制。透析显示，在较长时间（>10 h）内从酶-抑制剂复合物中恢复了酶活性，证明 1 确实是 FAAH 的一种可逆抑制剂，尽管其解离速度较慢。分子对接表明，1 的磺酰胺基团可以与几个参与催化的残基形成氢键，从而模拟过渡态。1 表现出的长停留时间似乎并非完全来自于热力学活性，而是与阻止快速解离的增加的动力学能垒一致。
