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<p>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<sub>i/o/z</sub> family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce G<sub>z</sub>ESTY, a sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of G<sub>i/o/z</sub>-coupled GPCRs and assist in deorphanization. We optimized assay conditions and developed an all-in-one vector employing cloning methods to ensure the correct expression ratio of G<sub>z</sub>ESTY components. G<sub>z</sub>ESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, and responses from endogenous GPCRs. Notably, with G<sub>z</sub>ESTY 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.</p>

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GzESTY as an optimized cell-based assay for initial steps in GPCR deorphanization

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月15日

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  • Luca Franchini,
  • Joseph J. Porter,
  • John D. Lueck,
  • Cesare Orlandi

摘要

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 Gi/o/z family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce GzESTY, a sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of Gi/o/z-coupled GPCRs and assist in deorphanization. We optimized assay conditions and developed an all-in-one vector employing cloning methods to ensure the correct expression ratio of GzESTY components. GzESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, and responses from endogenous GPCRs. Notably, with GzESTY 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.