Background and Aims: We have sought to develop novel GLP-1R agonists (RA) with improved insulinotropic effects on the pancreatic beta cell, and extended duration of action. Here, we have explored the affinity for the receptor, and action on signalling and insulin secretion, of three novel compounds.
Methods: Agonist dissociation kinetics were assessed by time-resolved FRET using SNAP-tagged GLP-1R-expressing HEK293 cells and fluorescent exendin-(9-39)-FITC. Signal bias was quantified by comparing the generation of cAMP and beta-arrestin recruitment to the GLP-1R. Recycling was measured in INS-1(832/3) cells expressing SNAP-GLP-1R, and time-lapse confocal imaging on a Zeiss LSM-780 inverted microscope. Insulin secretion was measured in vitro in islets isolated from lean C57BL6 J mice. Time-resolved FRET was used to quantify released and total hormone. Semaglutide (sema.) was supplied by Sun Pharma.
Results: GL0001, GL0028 and GL0034 showed similar binding but slower dissociation kinetics at the GLP-1R, leading to a higher binding affinity (pKd ca. 7.6) vs. semaglutide (pKd ca. 7.4). A slightly higher bias (1.3-, 1.5- and 1.5-fold, respectively, n=5) towards beta-arrestin vs. cAMP signalling was also apparent for each RA vs. sema). Coupling to cAMP generation (pEC50-pKd) was lower (1.7-, 2.0- and 1.8-fold, respectively) than sema. for each RA. Whereas endocytosis of SNAP-GLP-1R was essentially complete in 5 min. after RA (100 nM) addition for sema. and GL0034, uptake of labelled receptor was barely detectable even at 10 min. for GL0028. Efficacy in potentiating insulin secretion from isolated mouse islets at 11 mM glucose, assessed after 30 min., was similar for GL0028 and sema.
Conclusion: The novel agents GL0001 and GL0034 show similar binding properties, and impact on GLP-1R uptake, compared to sema. The more limited receptor uptake for GL0028 vs. other RA may influence the balance of central vs. pancreatic actions on insulin secretion and glucose homeostasis.
G. Carrat: None. M. Nguyen-Tu: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. R. Thennati: None. B. Jones: None. A. Tomas: None. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd.
Medical Research Council (R022259/1); UK Wellcome Trust (212625/Z/18/Z); European Union (115881)