DataSheet1_Discovering monoacylglycerol lipase inhibitors by a combination of fluorogenic substrate assay and activity-based protein profiling.docx

The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly metabolized by monoacylglycerol lipase (MAGL) in the brain. Selective inhibitors of MAGL provide valuable insights into the role of 2-AG in a variety of (patho)physiological processes and are potential therapeutics for the treatment of diseases such as neurodegenerative disease and inflammation, pain, as well as cancer. Despite a number of MAGL inhibitors been reported, inhibitors with new chemotypes are still required. Here, we developed a substrate-based fluorescence assay by using a new fluorogenic probe AA-HNA and successfully screened a focused library containing 320 natural organic compounds. Furthermore, we applied activity-based protein profiling (ABPP) as an orthogonal method to confirm the inhibitory activity against MAGL in the primary substrate-based screening. Our investigations culminated in the identification of two major compound classes, including quinoid diterpene (23, cryptotanshinone) and β-carbolines (82 and 93, cis- and trans-isomers), with significant potency towards MAGL and good selectivity over other 2-AG hydrolases (ABHD6 and ABHD12). Moreover, these compounds also showed antiproliferative activities against multiple cancer cells, including A431, H1975, B16-F10, OVCAR-3, and A549. Remarkably, 23 achieved complete inhibition towards endogenous MAGL in most cancer cells determined by ABPP. Our results demonstrate the potential utility of the substrate-based fluorescence assay in combination with ABPP for rapidly discovering MAGL inhibitors, as well as providing an effective approach to identify potential targets for compounds with significant biological activities.

内源大麻素2-花生四烯酰甘油（2-AG）在脑部主要由单酰甘油酯脂肪酶（MAGL）进行代谢。针对MAGL的选择性抑制剂为我们揭示了2-AG在多种（病理）生理过程中的作用，并成为治疗神经退行性疾病、炎症、疼痛以及癌症等疾病的潜在疗法。尽管已有多种MAGL抑制剂被报道，但仍有需求开发具有新化学结构的抑制剂。本研究中，我们利用新的荧光探针AA-HNA构建了一种基于底物的荧光分析测试，并成功筛选了一个包含320种天然有机化合物的针对性化合物库。此外，我们还应用了基于活性的蛋白质谱分析（ABPP）作为互补方法，以确认在基于底物的初步筛选中针对MAGL的抑制活性。我们的研究最终确定了两种主要化合物类别，包括喹啉二萜（23，隐丹参酮）和β-咔啉（82和93，顺式和反式异构体），它们对MAGL表现出显著的活性，并对其他2-AG水解酶（ABHD6和ABHD12）具有良好的选择性。此外，这些化合物还对多种癌细胞表现出抗增殖活性，包括A431、H1975、B16-F10、OVCAR-3和A549。值得注意的是，23在大多数癌细胞中通过ABPP实现了对内源性MAGL的完全抑制。我们的研究结果表明，基于底物的荧光分析测试与ABPP相结合，具有快速发现MAGL抑制剂的潜力，同时也为具有显著生物活性的化合物潜在靶点的识别提供了一种有效的方法。