Biased agonists with less glucagon-like peptide-1 receptor-mediated endocytosis prolong hypoglycaemic effects.

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.