Development of a HiBiT Peptide-Based NanoBRET Ligand Binding Assay for Galanin Receptor 1 in Live Cells

Galanin is a neuroendocrine peptide that regulates a wide range of physiological functions, including feeding and energy homeostasis, mood and anxiety, and modulation of pain. The function of the galanin peptide is mediated through its three galanin receptors, namely, GALR1, GALR2, and GALR3, which belong to the G protein-coupled receptor family. To measure the interaction of ligands with galanin receptor 1 (GALR1) in living cells, we developed a novel HiBiT peptide-based NanoBRET ligand binding assay. We generated six bioluminescence resonance energy transfer (BRET) tracers composed of modified and truncated galanin peptide derivatives tagged with a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) acceptor fluorophore. The fluorophore-tagged peptide tracers were evaluated in cells expressing GALR1 tagged with HiBiT, an 11-amino acid subunit of NanoLuc, which, upon high affinity complementation with the cell-impermeable subunit LgBiT, reconstituted a functional NanoLuc luciferase. Addition of the furimazine substrate induced BRET to the BODIPY fluorophore acceptor component of the galanin-derived peptide tracers and produced a fluorescence signal output. Using this BRET assay, we characterized the binding affinity and binding kinetics of tracers with GALR1 in both equilibrium and real time. To validate our assay, we evaluated the binding affinity and function of a panel of unmodified galanin-derived peptide ligands through the competitive displacement of bound fluorescent galanin tracers. Our data showed that the binding affinity of these galanin peptide ligands correlated well with their rank order in β-arrestin recruitment and internalization functional assays. This study demonstrates that the HiBiT peptide-based NanoBRET ligand binding assay is a valuable system for studying the ligand engagement of GALR1 in living cells, offering an alternative to neuropeptide radioligand binding assays.