title: Design and validation of a homogeneous time-resolved fluorescence cell-based assay targeting the ligand-gated ion channel 5-HT3A.
abstract: Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries.
authors.affiliation: Cisbio Bioassays, 30200 Codolet, France; Institut de Génomique Fonctionnelle, Département de Pharmacologie Moléculaire, CNRS UMR 5203, INSERM U661, Université Montpellier I et II, 34094 Montpellier Cedex 5, France.
authors.forename: Emilie
authors.initials: E
authors.lastname: Blanc
authors.affiliation: Laboratoire d'Innovation Thérapeutique, Faculté de Pharmacie, UMR 7200 CNRS/Université de Strasbourg, 67401 Illkirch, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Wagner
authors.affiliation: Sanofi R&D, Research Center of Vitry/Alfortville, 94403 Vitry-sur-Seine, France.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Plaisier
authors.affiliation: Laboratoire d'Innovation Thérapeutique, Faculté de Pharmacie, UMR 7200 CNRS/Université de Strasbourg, 67401 Illkirch, France.
authors.forename: Martine
authors.initials: M
authors.lastname: Schmitt
authors.affiliation: Institut de Génomique Fonctionnelle, Département de Pharmacologie Moléculaire, CNRS UMR 5203, INSERM U661, Université Montpellier I et II, 34094 Montpellier Cedex 5, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Durroux
authors.affiliation: Laboratoire d'Innovation Thérapeutique, Faculté de Pharmacie, UMR 7200 CNRS/Université de Strasbourg, 67401 Illkirch, France.
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Bourguignon
authors.affiliation: Sanofi R&D, Research Center of Vitry/Alfortville, 94403 Vitry-sur-Seine, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Partiseti
authors.affiliation: Cisbio Bioassays, 30200 Codolet, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Dupuis
authors.affiliation: Laboratoire d'Innovation Thérapeutique, Faculté de Pharmacie, UMR 7200 CNRS/Université de Strasbourg, 67401 Illkirch, France. Electronic address: fbihel@unistra.fr.
authors.forename: Frederic
authors.initials: F
authors.lastname: Bihel
mesh_terms: D001681:Biological Assay / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008904:Miniaturization; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D044406:Receptors, Serotonin, 5-HT3
keywords: 5-HT3A-R; Fluorescent probes; HTRF; Ligand-gated ion channels; Tag-lite