Gz Enhanced Signal Transduction assaY (GZESTY) for GPCR deorphanization

G protein-coupled receptors (GPCRs) are key pharmacological targets, yet many remain underutilized due to unknown activation mechanisms and ligands. Orphan GPCRs, lacking identified natural ligands, are a high priority for research, as identifying their ligands will aid in understanding their functions and potential as drug targets. Most GPCRs, including orphans, couple to G_i/o/z family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce G_ZESTY, a highly sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of G_i/o/z-coupled GPCRs and assist in deorphanization. We optimized assay conditions and developed an all-in-one vector employing novel cloning methods to ensure the correct expression ratio of G_ZESTY components. G_ZESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, as well as responses from endogenous GPCRs. Notably, with G_ZESTY we established the presence of endogenous ligands for GPR176 and GPR37 in brain extracts, validating its use in deorphanization efforts. This assay enhances the ability to find ligands for orphan GPCRs, expanding the toolkit for GPCR pharmacologists.

G protein‐coupled receptor (GPCR); cell-based assays; high-throughput screening; molecular pharmacology; orphan GPCR.