Singh Praveen K, Markwick Rachel, Howell Fiona V, Williams Gareth, Doherty Patrick
Wolfson Centre for Age-Related Diseases, King's College London, SE1 9RT, U.K.
Biosci Rep. 2016 May 6;36(3). doi: 10.1042/BSR20160073. Print 2016 Jun.
Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.
二酰甘油脂肪酶α（DAGLα）在中枢神经系统中将二酰甘油（DAG）水解生成主要的内源性大麻素（eCB）2-花生四烯酸甘油酯（2-AG）。依赖DAGLα的大麻素（CB）信号传导参与了许多过程，包括轴突生长与导向、成体神经发生以及突触处的逆向信号传导。最近的研究表明，DAGLα是包括疼痛和肥胖在内的多种疾病的新兴药物靶点。活性测定对于药物发现过程至关重要；然而，使用其天然底物测量二酰甘油脂肪酶（DAGL）活性通常涉及低通量的质谱技术。已经报道了一些使用替代底物的相对高通量的基于膜的测定方法，但这些方法没有考虑到药物跨细胞膜能力通常所伴随的限速效应。在本研究中，我们报告了一种用于测量DAGLα活性的活细胞测定方法的开发。使用稳定表达人DAGLα转基因的人细胞系，评估了两种先前报道的DAGLα替代底物对硝基苯基丁酸酯（PNPB）和6,8-二氟-4-甲基伞形酮辛酸酯（DiFMUO）在活细胞测定中检测DAGLα活性的能力。经过优化，与荧光底物DiFMUO相比，小分子显色底物PNPB具有更低的背景活性以及转染细胞与亲本细胞之间更大的信号窗口，被证明更具优势。使用已确立的DAGL抑制剂对该测定方法进行了进一步验证。总之，本文报道的活细胞DAGLα测定方法提供了一种经济便捷的形式，可作为药物发现计划的一部分筛选新型抑制剂，并且补充了先前报道的基于膜的高通量DAGL测定方法。
