Inhibition of Endocannabinoid Biosynthesis via Diacylglycerol Lipase

描述（由申请人提供）：本提案的主要目标是了解二酰基甘油脂肪酶-a（DAGL）在内源性大麻素2-花生四烯酸甘油生物合成中的作用，以及该酶在内源性大麻素信号传导和与食欲、药物滥用和相关健康影响相关的生理反应中的作用。第一个目标涉及DAGL抑制剂的系统合成，其结构衍生自DAGL底物（1，2-二酰基-sn-甘油）和选择性抑制剂四氢利普他汀（THL），其具有较差的溶解性和吸收特性。提出的结构特征包括：（a）靶向DAGL酶活性位点的缓慢可逆反应性官能团（包括氨基甲酸酯、环己基肟基羰基氨基、三氟甲基和b-内酰胺），和（B）导致选择性结合DAGL所需的其他结构特征。第二个主要目标是开发DAGL抑制的高通量测定，以取代目前使用的薄层色谱法。最后，有效DAGL抑制剂的选择性然后可以通过测定先导化合物的其他脂肪酶活性来评估。将测定这些新合成的DAGL抑制剂对胰脂肪酶（TAGL）、乙酰胆碱酯酶（AChE）、二酰基甘油激酶和内源性大麻素蛋白质（单酰基甘油脂肪酶（MAGL）、脂肪酸酰胺水解酶（FAAH）、推定的内源性大麻素转运蛋白系统）的抑制作用，以及对大麻素受体CB 1和CB 2的亲和力。此外，在静脉内或口服给药后，将评估具有大于五倍选择性作为DAGL抑制剂的潜在候选药物在小鼠中的体内血浆和脑浓度，利用定量质谱分析来筛选其分布穿过血脑屏障（BBB）的能力。不存在具有适当溶解度、吸收和分布性质的高度特异性DAGL抑制剂。类似于缺乏特定的MAGL抑制剂，这代表了可用于解剖内源性大麻素系统并研究其与脂质代谢，钙离子转运，炎症，细胞信号传导和大脑中奖励机制的微妙平衡的研究工具中的一个重大空白。通过这种新的药物疗法降低2-AG浓度应有效拮抗CB受体并影响内源性大麻素信号传导，以及CB受体再摄取、可塑性和串扰。内源性大麻素系统的下调对于与大麻素受体的过度刺激相关的医学病症特别重要，包括从物质滥用、肥胖和运动障碍中恢复。内源性大麻素系统参与影响疼痛、饥饿和对滥用药物的渴望的细胞信号。许多目前流行的药物拮抗内源性大麻素受体蛋白，但这种提出的替代方法试图通过选择性抑制其生物合成来调节内源性信号分子（2-花生四烯酰甘油）的水平。内源性大麻素信号的这种下调可能对肥胖、成瘾恢复和细胞通讯途径变化引起的运动障碍症状产生有益影响。

DESCRIPTION (provided by applicant): The primary goal of this proposal is an understanding of the role of diacyglycerol lipase-a (DAGL) in the biosynthesis of the endogeneous cannabinoid 2-arachidonoylglycerol and the role of this enzyme in endocannabinoid signaling and physiological responses related to appetite, substance abuse, and related health effects. The first objective involves a systematic synthesis of DAGL inhibitors structurally derived from DAGL substrate (1,2-diacyl-sn-glycerol) and the selective inhibitor tetrahydrolipstatin (THL), which has poor solubility and absorption characteristics. The proposed structural features include: (a) slowly reversible reactive functional groups (including carbamate, cyclohexyloximinocarbonylamino, trifluoromethyl, and b-lactam) targeted at the active site of DAGL enzyme, and (b) other structural characteristics necessary to result in selective binding to DAGL. The second major objective is to develop a highthroughput assay for DAGL inhibition, to replace the thin layer chromatography assay that is currently used. Finally, the selectivity of potent DAGL inhibitors can then be evaluated by assaying lead compounds for other lipase activities. These newly synthesized DAGL inhibitors will be assayed for inhibition of pancreatic lipase (TAGL), acetylcholineesterase (AChE), diacylglycerol kinase, and the endocannabinoid proteins: monoacylglycerol lipase (MAGL), fatty acid amide hydrolase (FAAH), the putative endocannabinoid transporter system, as well as for affinity to the cannabinoid receptors CB1 and CB2. Also, potential drug candidates with greater than fivefold selectivity as DAGL inhibitors will be evaluated for their in vivo blood plasma and brain concentrations in mouse, following intravenous or oral dosing, utilizing quantitative mass spectrometric analysis to screen for their ability to distribute across the blood-brain barrier (BBB). There are no highly specific DAGL inhibitors having appropriate solubility, absorption, and distribution properties. Analogously to the lack of specific MAGL inhibitors, this represents a significant void in the research tools available to dissect the endocannabinoid system and study its delicate balance with lipid metabolism, calcium ion transport, inflammation, cell signaling, and reward mechanisms in the brain. Lowering 2-AG concentrations by this novel pharmacotherapy should effectively antagonize CB receptors and affect endocannabinoid signaling, as well as CB receptor reuptake, plasticity, and crosstalk. The downregulation of the endocannabinoid system is particularly important for medical conditions related to overstimulation of the cannabinoid receptor including recovery from substance abuse, obesity, and movement disorders. The endocannabinoid system is involved in cell signalings which affect pain, hunger, and cravings for drugs of abuse. A number of currently popular medications antagonize the endocannabinoid receptor proteins, but this proposed alternative approach seeks to modulate the levels of the endogeneous signaling molecule (2- arachidonoylglycerol) by selectively inhibiting its biosynthesis. This downregulation of endocannabinoid signaling may have a benefical effect on obesity, recovery from addiction, and movement disorder symptoms from the resulting changes in cell communication pathways.