Novel fatty chain-modified GLP-1R G-protein biased agonist exerts prolonged anti-diabetic effects through targeting receptor binding sites.

Here, we design and evaluate novel long-lasting GLP-1R G-protein-biased agonists with promising pharmacological virtues. Firstly, six GLP-1R G-protein-biased peptides (named PX01-PX06), screened by using a previous reported high-throughput autocrine-based method, were fused to the N-terminus of GLP-1(9-37) to generate six fusion peptides (PX07-PX012). In vitro surface plasmon resonance (SPR) measurements showed that PX09 exerts the highest binding affinity for both human and mouse GLP-1R extracellular domains (ECD). We further used the PX09 as a starting point to conduct site-specific modifications yielding twelve lysine-modified conjugates, termed PX13-PX24. Of these conjugates, PX17 retained relatively better in vitro GLP-1R activation potency and plasma stability compared with other ones. Preclinical studies in db/db mice demonstrated that acute treatment of PX17 exerts enhanced hypoglycemic and insulinotropic activities in a dosage dependent model within the range of 0.1-0.9 mg kg-1. Similarly, prolonged glucose-lowering abilities were exhibited in modified multiple oral glucose tolerance tests (OGTTs) and a hypoglycemic duration test. Apparently prolonged in vivo half-lives of ∼96 and ∼141 h were observed after a single subcutaneous administration of PX17 at 0.1 and 0.3 mg kg-1, respectively, in healthy cynomolgus monkeys. In addition, twice-weekly treatment of PX17 in db/db mice for 8 weeks obviously improved the hemoglobin A1C (HbA1C), and was more effective at improving the insulin resistance, glucose tolerance as well as function of pancreatic beta cells compared with Semaglutide. Furthermore, subcutaneously dosed PX17 in diet induced obese (DIO) mice achieved long-term beneficial effects on food intake and body weight control, HbA1C and inflammation-related factor level lowering. The above results indicate that PX17, as a novel GLP-1R G-protein-biased agonist, may be a promising candidate for antidiabetic therapies.