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Development of a Multiplexed Activity-Based Protein Profiling Assay to Evaluate Activity of Endocannabinoid Hydrolase Inhibitors.
ACS Chemical Biology ( IF 4 ) Pub Date : 2018-09-12 , DOI: 10.1021/acschembio.8b00534
Antonius P A Janssen 1 , Daan van der Vliet 1 , Alexander T Bakker 1 , Ming Jiang 1 , Sebastian H Grimm 1 , Giuseppe Campiani 2 , Stefania Butini 2 , Mario van der Stelt 1
Affiliation  

Endocannabinoids, an important class of signaling lipids involved in health and disease, are predominantly synthesized and metabolized by enzymes of the serine hydrolase superfamily. Activity-based protein profiling (ABPP) using fluorescent probes, such as fluorophosphonate (FP)-TAMRA and β-lactone-based MB064, enables drug discovery activities for serine hydrolases. FP-TAMRA and MB064 have distinct, albeit partially overlapping, target profiles but cannot be used in conjunction due to overlapping excitation/emission spectra. We therefore synthesized a novel FP-probe with a green BODIPY as a fluorescent tag and studied its labeling profile in mouse proteomes. Surprisingly, we found that the reporter tag plays an important role in the binding potency and selectivity of the probe. A multiplexed ABPP assay was developed in which a probe cocktail of FP-BODIPY and MB064 visualized most endocannabinoid serine hydrolases in mouse brain proteomes in a single experiment. The multiplexed ABPP assay was employed to profile endocannabinoid hydrolase inhibitor activity and selectivity in the mouse brain.

中文翻译:

评估基于内源性大麻素水解酶抑制剂活性的基于活性的多重蛋白分析方法的开发。

内源性大麻素是涉及健康和疾病的重要一类信号脂质,主要由丝氨酸水解酶超家族的酶合成和代谢。使用荧光探针的基于活性的蛋白质谱(ABPP),例如氟代磷酸酯(FP)-TAMRA和基于β-内酯的MB064,可实现丝氨酸水解酶的药物发现活性。FP-TAMRA和MB064具有不同的目标轮廓(尽管部分重叠),但由于重叠的激发/发射光谱而无法结合使用。因此,我们合成了带有绿色BODIPY作为荧光标签的新型FP探针,并研究了其在小鼠蛋白质组中的标记概况。令人惊讶地,我们发现报告子标签在探针的结合力和选择性中起重要作用。开发了一种多重ABPP测定法,其中在单个实验中,FP-BODIPY和MB064的探针混合物使小鼠脑蛋白质组中的大多数内源性大麻素丝氨酸水解酶可视化。采用多重ABPP测定法分析小鼠大脑中内源性大麻素水解酶抑制剂的活性和选择性。
更新日期:2018-09-10
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