Fluorescent GLP1R/GIPR dual agonist probes reveal cell targets in the pancreas and brain

Abstract

Dual agonists targeting the glucagon-like peptide-1 receptor (GLP1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are breakthrough treatments for type 2 diabetes and overweight. Compared to GLP1R agonists, dual agonists show superior efficacy for glucose lowering and weight reduction. However, delineation of dual agonist cell targets remains challenging. Here, we develop and test Tirzepa3 and Tirzepa5, fluorescent GLP1R/GIPR dual agonist probes. Tirzepa3 and Tirzepa5 have similar pharmacological profiles to tirzepatide, but advantageously show less functional selectivity for mouse GLP1R over mouse GIPR. Both probes specifically label GLP1R and GIPR in cells and tissue. Tirzepa3 and Tirzepa5 label all major rodent and human pancreatic islet cells, with signal intensity beta cells > alpha cells = delta cells. Systemic administration of Tirzepa5 strongly labels the median eminence, area postrema and other circumventricular organs characterized by an incomplete blood-brain barrier, but does not readily penetrate into the brain beyond this. Upon intracerebroventricular administration in the brain, Tirzepa5 co-localizes widely with GLP1R + and GIPR + neurons, with evidence of uptake by ventricle-lining cells in the 3rd ventricle. At the single molecule level, Tirzepa5 targets endogenous GLP1R-GIPR nanodomains, which differ in organization and composition to those targeted by single agonist. Tirzepa3 and Tirzepa5 thus reveal dual agonist targets in the pancreas and brain, and further inform the different modes of action of dual agonists versus single agonists.