Zeghal Manel, Laroche Geneviève, Giguère Patrick M
Department of Biochemistry, Microbiology and Immunology, University of Ottawa.
Department of Biochemistry, Microbiology and Immunology, University of Ottawa; Brain and Mind Research Institute, University of Ottawa;
J Vis Exp. 2020 Mar 10(157). doi: 10.3791/60823.
As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent β-arrestin2 recruitment assay, the universality of β-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango's sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.
作为最大且最具多功能性的基因超家族以及一系列细胞信号通路的介导者，G蛋白偶联受体（GPCRs）是制药行业最具潜力的靶点之一。因此，GPCR配体筛选检测的设计、实施和优化至关重要，因为它们是药物发现以及操控GPCR药理学和结果的远程控制工具。过去，G蛋白依赖性检测是该研究领域的典型方法，用于检测配体诱导的事件并量化第二信使的产生。然而，自从功能选择性出现，以及人们对其他几种G蛋白非依赖性通路的认识增加，同时认识到G蛋白依赖性检测存在的局限性后，现在更倾向于创建替代性的GPCR配体筛选检测方法。为了实现这一目标，我们描述了一种这样的资源即PRESTO-Tango平台的应用，这是一种基于荧光素酶报告基因的系统，能够对人类GPCR组进行平行且同时的检测，这一壮举以前在技术和经济上被认为是不可行的。基于一种G蛋白非依赖性的β-抑制蛋白2募集检测，β-抑制蛋白2在GPCRs上介导的转运和信号传导的普遍性使得PRESTO-Tango成为研究大约300种非嗅觉人类GPCRs（包括大约100种孤儿受体）的合适工具。PRESTO-Tango的灵敏度和稳健性使其适用于使用化合物库进行的初级高通量筛选，用于发现已知药物的新GPCR靶点或为孤儿受体发现新配体。
