title: A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors.
abstract: Cyclic adenosine 3',5'-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein-coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or Gi-coupled GPCRs, which leads to increased or decreased cAMP levels, respectively. Here we describe a real-time Förster resonance energy transfer (FRET)-based cAMP high-throughput screening (HTS) assay for identification and characterization of Gs-coupled GPCR ligands and phosphodiesterase (PDE) inhibitors in living cells. We used the β2-adrenergic receptor (β(2)AR) as a representative Gs-coupled receptor and characterized two cell lines with different expression levels. Low receptor expression allowed detection of desensitization kinetics and delineation of partial agonism, whereas high receptor expression resulted in prolonged signaling and enabled detection of weak partial agonists and/or ligands with low potency, which is highly advantageous in large HTS settings and hit identification. In addition, the assay enabled detection of β(2)AR inverse agonists and PDE inhibitors. High signal-to-noise ratios were also observed for the other representative Gs-coupled GPCRs tested, GLP-1R and GlucagonR. The FRET-based cAMP biosensor assay is robust, reproducible, and inexpensive with good Z factors and is highly applicable for HTS.
authors: Department of Drug Design and Pharmacology, Faculty of Medical and Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors: Line
authors: L
authors: Vedel
authors: Department of Drug Design and Pharmacology, Faculty of Medical and Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors: Hans
authors: H
authors: Bräuner-Osborne
authors: Department of Drug Design and Pharmacology, Faculty of Medical and Health Sciences, University of Copenhagen, Copenhagen, Denmark jmm@zealandpharma.com.
authors: Jesper Mosolff
authors: JM
authors: Mathiesen
mesh_terms: D015374:Biosensing Techniques; D000242:Cyclic AMP / Q000378:metabolism*; D055808:Drug Discovery / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D008024:Ligands; D010726:Phosphodiesterase Inhibitors; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP
keywords: FRET; G protein–coupled receptor (GPCR); Gs-coupled receptor activation; cAMP biosensor; high-throughput screening (HTS)