Allosteric control of monoacylglyceride lipase (MGL) activity

项目摘要 内源性大麻素2-花生四烯酸-sn-甘油（2-AG）从突触后棘释放并激活 轴突末梢上的CB 1受体调节离子通道活性和神经递质释放。单甘油酯 脂肪酶（MGL）--一种将2-AG水解成花生四烯酸和甘油的突触前水解酶--阻止了这一过程 逆行信号传导过程靶向MGL的小分子抑制剂提供了对 2-AG的功能，最近已进入临床开发阶段。目前的药物开发工作 在这一领域的重点是不可逆或可逆地与MGL的催化位点相互作用的试剂。那里 然而，这些方法中的任何一种都有严重的缺点：不可逆抑制剂引起过量的2- AG积累和随后的CB 1脱敏，而可逆的正构抑制剂必须竞争 与2-AG和其他单酰基甘油的活性位点结合，其在细胞中可达到高微摩尔浓度。 相关的生物相（例如，突触前膜）。我们已经证明，可逆的过氧化物依赖性 MGL中C201和C208的亚磺酰化稳定了酶的催化失活构象，因此 增强CB 1受体上2-AG介导的信号传导。在此修订的R21应用程序中，我们建议开发 靶向该调节区的变构MGL抑制剂，通过这样做，可能能够绕过底物 竞争特别地，我们鉴定了一类抑制MGL的苯并异噻唑啉-3（2 H）-酮衍生物， 通过与调节性半胱氨酸C201和C208以可逆的方式相互作用而具有高效力的活性。 我们建议使用实验和计算方法的结合-分子建模， 构效关系研究、定点突变、质谱和体内药理学 - 以修饰这些先导分子并产生有效的和全身活性的变构MGL抑制剂。这些 化合物将通过测试2-AG是否 失活-这是内源性大麻素信号的核心-可能是由生理调节 以及影响这种状态的病理因素，例如局部缺血。它们也可以作为 发现治疗疼痛、物质使用障碍和其他人类疾病的新疗法。

PROJECT SUMMARY The endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) is released from postsynaptic spines and activates CB1 receptors on axon terminals to regulate ion-channel activity and neurotransmitter release. Monoglyceride lipase (MGL) – a presynaptic hydrolase that hydrolyzes 2-AG into arachidonic acid and glycerol – stops this retrograde signaling process. Small-molecule inhibitors that target MGL have provided insights into the functions of 2-AG and have been recently advanced to clinical development. Current drug development efforts in this area are focused on agents that interact irreversibly or reversibly with the catalytic site of MGL. There are, however, serious disadvantages to either of these approaches: irreversible inhibitors cause excessive 2- AG accumulation and consequent CB1 desensitization, while reversible orthosteric inhibitors must compete for active-site binding with 2-AG and other monoacylglycerols, which may reach high micromolar concentrations in the relevant biophase (e.g., presynaptic membranes). We have shown that the reversible peroxide-dependent sulfenylation of C201 and C208 in MGL stabilizes a catalytically inactive conformation of the enzyme, and thus enhances 2-AG-mediated signaling at CB1 receptors. In this revised R21 application, we propose to develop allosteric MGL inhibitors that target this regulatory region and, by doing so, may be able to bypass substrate competition. In particular, we identified a class of benzisothiazolin-3(2H)-one derivatives that inhibit MGL activity with high potency by interacting in a reversible manner with the regulatory cysteines C201 and C208. We propose to use a combination of experimental and computational approaches – molecular modeling, structure-activity relationship studies, site-directed mutagenesis, mass spectrometry, and in vivo pharmacology – to modify these lead molecules and generate potent and systemically active allosteric MGL inhibitors. These compounds will advance our understanding of 2-AG-mediated signaling by allowing us to test whether 2-AG deactivation – which lies at the very core of endocannabinoid signaling – might be modulated by physiological and pathological factors that affect such status, such as ischemia. They might also serve as starting point for the discovery of novel therapeutics for pain, substance use disorders and other human diseases.