Platform Reagents Enable Synthesis of Ligand-Directed Covalent Probes: Study of Cannabinoid Receptor 2 in Live Cells.
Platform Reagents Enable Synthesis of Ligand-Directed Covalent Probes: Study of Cannabinoid Receptor 2 in Live Cells.
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DOI：
10.1021/jacs.2c13629
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发表时间：
2023-07
影响因子：
15
通讯作者：
Miroslav Kosar;D. Sykes;Alexander E G Viray;R. Vitale;Roman C. Sarott;Rudolf L Ganzoni;D. Onion;Janelle M Tobias;Philipp Leippe;C. Ullmer;Elisabeth A. Zirwes;W. Guba;U. Grether;J. A. Frank;D. Veprintsev;E. Carreira
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作者：
Miroslav Kosar;D. Sykes;Alexander E G Viray;R. Vitale;Roman C. Sarott;Rudolf L Ganzoni;D. Onion;Janelle M Tobias;Philipp Leippe;C. Ullmer;Elisabeth A. Zirwes;W. Guba;U. Grether;J. A. Frank;D. Veprintsev;E. Carreira
大麻素2型受体(CB2R)的药理调节有望用于治疗神经炎症性疾病，如阿尔茨海默病。尽管CB2R很重要，但在疾病和组织特有的背景下，其表达和下游信号转导还不够清楚。在这里，我们报道了首次通过一种新的合成策略和平台试剂的应用来实现CB2R的配体导向共价(LDC)标记。LDC修饰允许可视化和研究CB2R，同时保持其在邻位位置与其他配体结合的能力。我们利用电子对接和分子动力学模拟来指导探针设计和评估CB2R的LDC标记的可行性。我们展示了选择性的，共价标记的CB2R的外周赖氨酸残基，通过利用荧光邻硝基苯并恶二唑(O-NBD)功能化的探针在TRRET分析中。O-NBD探针的快速概念验证激发了适用于活细胞实验的先进亲电剂的加入。为此，开发了新的合成策略，以合成N-磺酰基吡啶酮(N-SP)和N-酰基-N-烷基磺酰胺(NASA)LDC探针，使其能够共价传递适合于细胞研究的荧光团。通过放射性配基结合实验和TRRET实验对LDC探针进行了表征。此外，这些探针被用于在常规和成像流式细胞术以及使用过表达和内源性表达的小胶质细胞的共聚焦荧光显微镜中特异性地显示CB2R。
Pharmacological modulation of cannabinoid receptor type 2 (CB2R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CB2R, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific contexts. Herein, we report the first ligand-directed covalent (LDC) labeling of CB2R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB2R while maintaining its ability to bind other ligands at the orthosteric site. We employed in silico docking and molecular dynamics simulations to guide probe design and assess the feasibility of LDC labeling of CB2R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB2R by exploiting fluorogenic O-nitrobenzoxadiazole (O-NBD)-functionalized probes in a TR-FRET assay. The rapid proof-of-concept validation with O-NBD probes inspired incorporation of advanced electrophiles suitable for experiments in live cells. To this end, novel synthetic strategies toward N-sulfonyl pyridone (N-SP) and N-acyl-N-alkyl sulfonamide (NASA) LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular studies. The LDC probes were characterized by a radioligand binding assay and TR-FRET experiments. Additionally, the probes were applied to specifically visualize CB2R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.