(1267-D) Development of a HiBit peptide-based nanoBRET ligand binding assay for galanin receptor 1 in the live cell
Tuesday, February 6, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: Galanin is a neuroendocrine peptide regulating a wide range of physiological functions, including feeding and energy homeostasis, mood and anxiety, and modulation of pain. The function of galanin peptide is mediated through its three galanin receptors, GalR1, GalR2 and GalR3, which belong to the G protein coupled receptor family. To measure the interaction of ligands and galanin receptor 1 (GALR1) in living cells, we developed a novel HiBit peptide-based NanoBRETâ„¢ ligand binding assay. We generated a series of six modified and truncated galanin derivatives tagged with a BODIPY acceptor fluorophore. The fluorescent-tagged peptide tracers were evaluated in living cells that express a GalR1 receptor tagged with HiBiT at the cell surface. We characterized the binding affinity and the binding kinetics of our tracers on GALR1 receptor in both equilibrium and real time. We also evaluated the binding affinity and function of a panel of unmodified galanin peptide ligands to validate our assay. Our data showed that the binding affinity of these galanin peptide ligands were well correlated with their potency in the functional activation of the receptors. This study demonstrates that the HiBit peptide-based NanoBRET ligand binding assay is a valuable system for studying the target engagement of ligands and GALR1 receptor in living cells and represents an alternative to radioligand binding assays.