Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 211198, PR China.
Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 211198, PR China.
Eur J Pharmacol. 2021 Sep 15;907:174203. doi: 10.1016/j.ejphar.2021.174203. Epub 2021 May 26.
Receptor endocytic trafficking entails targeting receptors and ligands to endocytic sites, followed by internalization and sorting to recycling or degradative compartments. Thus, membrane receptor-mediated signalling pathways not only contribute to the efficacy of the drugs but also play a crucial role in the metabolic elimination of peptide drugs. Glucagon-like peptide-1 (GLP-1) receptor is the crucial target for type 2 diabetes mellitus. We mainly focused on the characteristics, early evaluation of GLP-1 receptor endocytosis and effects of optimization for endocytosis on druggability. The GLP-1 receptor endocytosis characteristics of agonists were analysed by a multifunction microplate reader, flow cytometer and confocal microscope. The intracellular cyclic adenosine monophosphate (cAMP) activation of agonists was analysed based on a reporter gene assay, and intracellular β-arrestin recruitment detection was detected based on a Tango assay. We established quantitative evaluation methods of endocytosis based on fluorescently labelled agonist and receptor trafficking and used them to screen agonists with less endocytosis. Sprague-Dawley rats were used for pharmacokinetic analyses, and the hypoglycaemic activity was evaluated by intraperitoneal glucose tolerance tests (IPGTT). Our results showed that GLP-1 receptor-mediated endocytosis, as a manner of elimination, was clathrin-dependent. More importantly, we found that agonists biased towards the G protein pathway were less endocytosed by GLP-1 receptor. We screened an analogue of Exendin-4 M4, which was biased toward the G protein pathway with less endocytosis by the GLP-1 receptor. M4, which shows prolonged hypoglycaemic activities and a long half-life, can be used as a lead compound for type 2 diabetes mellitus treatment.
受体的内吞转运涉及将受体和配体靶向内吞部位，随后进行内化和分拣到再循环或降解隔室。因此，膜受体介导的信号通路不仅有助于药物的疗效，而且在肽类药物的代谢消除中也起着至关重要的作用。胰高血糖素样肽-1 (GLP-1) 受体是 2 型糖尿病的关键靶点。我们主要关注 GLP-1 受体内吞的特征、早期评估以及内吞优化对成药性的影响。通过多功能微孔板读数仪、流式细胞仪和共聚焦显微镜分析激动剂的 GLP-1 受体内吞特征。基于报告基因测定分析激动剂的细胞内环磷酸腺苷 (cAMP) 激活，基于 Tango 测定检测细胞内β-arrestin 募集检测。我们建立了基于荧光标记激动剂和受体转运的内吞定量评估方法，并使用它们筛选内吞作用较弱的激动剂。使用 Sprague-Dawley 大鼠进行药代动力学分析，并通过腹腔葡萄糖耐量试验 (IPGTT) 评估降血糖活性。我们的结果表明，GLP-1 受体介导的内吞作用作为一种消除方式，是网格蛋白依赖性的。更重要的是，我们发现偏向 G 蛋白途径的激动剂被 GLP-1 受体的内吞作用较少。我们筛选出一种 Exendin-4 M4 的类似物，它偏向 G 蛋白途径，被 GLP-1 受体的内吞作用较少。M4 具有延长的降血糖活性和较长的半衰期，可用作 2 型糖尿病治疗的先导化合物。
