Inhibition of Endocannabinoid Biosynthesis via Diacylglycerol Lipase

描述（由申请人提供）：本申请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-花生四烯醇甘油的生物合成中的作用，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。第一个目标是系统合成从DAGL底物（1,2-二酰基-锡甘油）和选择性抑制剂四氢利普他汀（THL）结构上衍生的DAGL抑制剂，该抑制剂具有较差的溶解性和吸收特性。提出的结构特征包括：(a)针对DAGL酶活性位点的缓慢可逆反应官能团（包括氨基甲酸酯、环己胺羰基胺、三氟甲基和b-内酰胺），以及(b)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一种高通量测定DAGL抑制的方法，以取代目前使用的薄层色谱法。最后，有效的DAGL抑制剂的选择性可以通过测定其他脂肪酶活性的先导化合物来评估。这些新合成的DAGL抑制剂将被检测对胰脂肪酶（TAGL）、乙酰胆碱酯酶（AChE）、二酰基甘油激酶、内源性大麻素蛋白：单酰基甘油脂肪酶（MAGL）、脂肪酸酰胺水解酶（FAAH）、内源性大麻素转运系统的抑制作用，以及对大麻素受体CB1和CB2的亲和力。此外，在静脉或口服给药后，将评估具有5倍以上选择性的潜在候选药物在小鼠体内的血浆和脑浓度，利用定量质谱分析来筛选它们在血脑屏障（BBB）上分布的能力。没有高度特异性的DAGL抑制剂具有适当的溶解度、吸收和分布特性。与缺乏特定的MAGL抑制剂类似，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、钙离子运输、炎症、细胞信号传导和大脑奖励机制之间微妙平衡的研究工具的重大空白。通过这种新型药物疗法降低2-AG浓度，可以有效拮抗CB受体，影响内源性大麻素信号传导，以及CB受体的再摄取、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过度刺激相关的医疗状况尤其重要，包括从药物滥用、肥胖和运动障碍中恢复过来。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，但这一提出的替代方法旨在通过选择性抑制其生物合成来调节内源性信号分子（2-花生四烯醇甘油）的水平。这种内源性大麻素信号的下调可能对肥胖、成瘾恢复和运动障碍症状有有益的影响，这些症状源于细胞通信途径的改变。

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.