title: Forskolin-free cAMP assay for Gi-coupled receptors.
abstract: 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.
authors.affiliation: 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.
authors.forename: Julie
authors.initials: J
authors.lastname: Gilissen
authors.affiliation: Laboratory of Molecular Pharmacology, GIGA-Signal Transduction Unit, University of Liège, 11, Avenue de l'hôpital, 4000 Liège, Belgium.
authors.forename: Pierre
authors.initials: P
authors.lastname: Geubelle
authors.affiliation: Laboratory of Molecular Pharmacology, GIGA-Signal Transduction Unit, University of Liège, 11, Avenue de l'hôpital, 4000 Liège, Belgium.
authors.forename: Nadine
authors.initials: N
authors.lastname: Dupuis
authors.affiliation: Laboratory of Molecular Pharmacology, GIGA-Signal Transduction Unit, University of Liège, 11, Avenue de l'hôpital, 4000 Liège, Belgium.
authors.forename: Céline
authors.initials: C
authors.lastname: Laschet
authors.affiliation: Laboratory of Medicinal Chemistry, Centre for Interdisciplinary Research on Medicines (CIRM), University of Liège, 15, Avenue Hippocrate, 4000 Liège, Belgium.
authors.forename: Bernard
authors.initials: B
authors.lastname: Pirotte
authors.affiliation: 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. Electronic address: j.hanson@ulg.ac.be.
authors.forename: Julien
authors.initials: J
authors.lastname: Hanson
mesh_terms: D002118:Calcium / Q000378:metabolism; D005576:Colforsin / Q000032:analysis*; D000242:Cyclic AMP / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D043562:Receptors, G-Protein-Coupled / Q000032:analysis*
chemical_list: D043562:Receptors, G-Protein-Coupled; D005576:Colforsin; D000242:Cyclic AMP; D002118:Calcium
keywords: Forskolin; GPCR; GPR91; SUCNR1; cAMP