Laboratory of Molecular Pharmacology, GIGA-Signal Transduction Unit, University of Liège, 11, Avenue de l'hôpital, 4000 Liège, Belgium; Laboratory of Medicinal Chemistry, Centre for Interdisciplinary Research on Medicines (CIRM), University of Liège, 15, Avenue Hippocrate, 4000 Liège, Belgium.
Laboratory of Molecular Pharmacology, GIGA-Signal Transduction Unit, University of Liège, 11, Avenue de l'hôpital, 4000 Liège, Belgium.
Biochem Pharmacol. 2015 Dec 1;98(3):381-91. doi: 10.1016/j.bcp.2015.09.010. Epub 2015 Sep 16.
G protein-coupled receptors (GPCRs) represent the most successful receptor family for treating human diseases. Many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays. Measurement of intracellular cyclic AMP (cAMP) levels is a validated strategy for measuring GPCRs activation. However, agonist ligands for Gi-coupled receptors are difficult to track because inducers such as forskolin (FSK) must be used and are sources of variations and errors. We developed a method based on the GloSensor system, a kinetic assay that consists in a luciferase fused with cAMP binding domain. As a proof of concept, we selected the succinate receptor 1 (SUCNR1 or GPR91) which could be an attractive drug target. It has never been validated as such because very few ligands have been described. Following analyses of SUCNR1 signaling pathways, we show that the GloSensor system allows real time, FSK-free detection of an agonist effect. This FSK-free agonist signal was confirmed on other Gi-coupled receptors such as CXCR4. In a test screening on SUCNR1, we compared the results obtained with a FSK vs FSK-free protocol and were able to identify agonists with both methods but with fewer false positives when measuring the basal levels. In this report, we validate a cAMP-inducer free method for the detection of Gi-coupled receptors agonists compatible with high-throughput screening. This method will facilitate the study and screening of Gi-coupled receptors for active ligands.
G 蛋白偶联受体（GPCRs）是治疗人类疾病最成功的受体家族之一。许多 GPCRs 的特征尚未被充分阐明，仅有少数配体被报道，或者仍然完全是孤儿受体。因此，人们越来越需要筛选兼容和敏感的测定方法。测量细胞内环磷酸腺苷（cAMP）水平是测量 GPCR 激活的一种经过验证的策略。然而，Gi 偶联受体的激动剂配体很难追踪，因为必须使用诱导剂如forskolin（FSK），而 FSK 是产生变异和误差的来源。我们开发了一种基于 GloSensor 系统的方法，该系统是一种动力学测定法，由与 cAMP 结合域融合的荧光素酶组成。作为概念验证，我们选择了琥珀酸受体 1（SUCNR1 或 GPR91），它可能是一个有吸引力的药物靶点。由于很少有配体被描述，因此从未对此进行过验证。在对 SUCNR1 信号通路进行分析后，我们表明 GloSensor 系统允许实时、无需 FSK 检测激动剂的作用。这种无需 FSK 的激动剂信号在其他 Gi 偶联受体（如 CXCR4）上得到了证实。在对 SUCNR1 的测试筛选中，我们比较了使用 FSK 与无 FSK 方案获得的结果，并用两种方法都能识别出激动剂，但在测量基础水平时假阳性较少。在本报告中，我们验证了一种无需 cAMP 诱导剂即可检测 Gi 偶联受体激动剂的方法，该方法与高通量筛选兼容。该方法将有助于研究和筛选 Gi 偶联受体的活性配体。
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