Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons.
Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons.
复制标题
DOI：
10.1021/jacs.5b04883
复制
发表时间：
2015-07-15
影响因子：
15
通讯作者：
van der Stelt M
中科院分区：
文献类型：
--
作者：
Baggelaar MP;Chameau PJ;Kantae V;Hummel J;Hsu KL;Janssen F;van der Wel T;Soethoudt M;Deng H;den Dulk H;Allarà M;Florea BI;Di Marzo V;Wadman WJ;Kruse CG;Overkleeft HS;Hankemeier T;Werkman TR;Cravatt BF;van der Stelt M
二酰基甘油脂肪酶 (DAGL)-α 和 -β 是负责内源性大麻素 2-花生四烯酰甘油 (2-AG) 生物合成的酶。需要选择性和可逆的抑制剂来以急性和暂时的方式研究 DAGL 在神经元细胞中的功能，但目前还缺乏这种抑制剂。在这里，我们描述了使用结构引导和化学蛋白质组学策略来鉴定高选择性 DAGL 抑制剂，以表征复杂蛋白质组中抑制剂的选择性。这种方法成功的关键是使用基于比较和竞争活性的蛋白质组分析 (ABPP)，其中将广谱和定制的基于活性的探针相结合，报告蛋白质家族在其天然环境中的抑制情况。具有广谱氟磷酸盐探针和特异性 β-内酯探针的竞争性 ABPP 发现了 α-酮杂环 LEI105，它是一种有效、高选择性和可逆的双重 DAGL-α/DAGL-β 抑制剂。 LEI105 不影响参与内源性大麻素代谢的其他酶，包括含抗解酶结构域的蛋白 6、含抗解酶结构域的蛋白 12、单酰基甘油脂肪酶和脂肪酸酰胺水解酶，并且不显示对大麻素 CB1 受体的亲和力。靶向脂质组学显示，LEI105 浓度依赖性地降低 Neuro2A 细胞中的 2-AG 水平，但不降低 anandamide 水平。我们发现 LEI105 可以降低小鼠海马切片模型中大麻素 CB1 受体介导的短期突触可塑性。因此，我们开发了一种高度选择性的 DAGL 抑制剂，并提供了新的药理学证据来支持 2-AG 的“按需生物合成”负责逆行信号传导的假设。
Diacylglycerol lipase (DAGL)-α and -β are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes. Key to the success of this approach is the use of comparative and competitive activity-based proteome profiling (ABPP), in which broad-spectrum and tailor-made activity-based probes are combined to report on the inhibition of a protein family in its native environment. Competitive ABPP with broad-spectrum fluorophosphonate-based probes and specific β-lactone-based probes led to the discovery of α-ketoheterocycle LEI105 as a potent, highly selective and reversible dual DAGL-α/DAGL-β inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metabolism including abhydrolase domain-containing protein 6, abhydrolase domain-containing protein 12, monoacylglycerol lipase and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concentration-dependently reduced 2-AG levels, but not anandamide levels, in Neuro2A cells. We show that cannabinoid CB1-receptor-mediated short-term synaptic plasticity in a mouse hippocampal slice model can be reduced by LEI105. Thus, we have developed a highly selective DAGL inhibitor and provide new pharmacological evidence to support the hypothesis that ‘on demand biosynthesis’ of 2-AG is responsible for retrograde signaling.