Franchini Luca, Porter Joseph J, Lueck John D, Orlandi Cesare
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY, 14642, USA.
Nat Commun. 2025 May 15;16(1):4521. doi: 10.1038/s41467-025-59850-8.
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 family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce GESTY, a sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of G-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 GESTY components. GESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, and responses from endogenous GPCRs. Notably, with GESTY 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蛋白偶联受体（GPCRs）是关键的药理学靶点，但由于激活机制和配体未知，许多靶点仍未得到充分利用。孤儿GPCRs缺乏已确定的天然配体，是研究的重点，因为确定它们的配体将有助于理解其功能和作为药物靶点的潜力。大多数GPCRs，包括孤儿GPCRs，与G家族成员偶联，然而目前检测其激活的方法有限，阻碍了配体鉴定工作。我们引入了GESTY，这是一种基于细胞的灵敏检测方法，以易于交付的形式开发，旨在研究G偶联GPCRs的药理学并协助解除孤儿受体身份。我们优化了检测条件，并开发了一种一体化载体，采用克隆方法确保GESTY组件的正确表达比例。GESTY成功评估了配体激活的GPCRs文库的激活情况，检测到完全激动和部分激动作用以及内源性GPCRs的反应。值得注意的是，通过GESTY我们确定了脑提取物中GPR176和GPR37的内源性配体的存在，验证了其在解除孤儿受体身份工作中的应用。该检测方法增强了寻找孤儿GPCRs配体的能力，扩展了GPCR药理学家的工具包。
