Forskolin-free cAMP assay for G_i-coupled receptors

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• 作者：Julie Gilissen^a ; ^b ; Pierre Geubelle^a ; Nadine Dupuis^a ; Cé ; line Laschet^a ; Bernard Pirotte^b ; Julien Hanson^a ; ^b ; ^{j.hanson@ulg.ac.be" class="auth_mail" title="E-mail the corresponding author}
• 关键词：AC ; adenylate cyclase ; AUC ; area under the curve ; cAMP ; cyclic adenosine monophosphate ; ERK ; extracellular signal regulated kinases ; FSK ; forskolin ; GPCRs ; G-protein coupled receptors ; PTX ; pertussis toxin ; SA ; succinic acid
• 刊名：Biochemical Pharmacology
• 出版年：2015
• 出版时间：1 December 2015
• 年：2015
• 卷：98
• 期：3
• 页码：381-391
• 全文大小：915 K

文摘

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 G_i-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 G_i-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 G_i-coupled receptors agonists compatible with high-throughput screening.

This method will facilitate the study and screening of G_i-coupled receptors for active ligands.