title: A Time-Resolved FRET Cell-Based Binding Assay for the Apelin Receptor.
abstract: Analogues of apelin-13 carrying diverse spacers and an ad hoc DY647-derived fluorophore were designed and synthesized by chemoselective acylation of α-hydrazinopeptides. The resulting probes retain very high affinity and efficacy for both the wild-type and SNAP-tagged apelin receptor (ApelinR). They give a time-resolved FRET (TR-FRET) signal with rare-earth lanthanides used as donor fluorophores grafted onto the SNAP-tagged receptor. This specific signal allowed the validation of a binding assay with a high signal-to-noise ratio. In such an assay, the most potent sub-nanomolar fluorescent probe was found to be competitively displaced by the endogenous apelin peptides with binding constants similar to those obtained in a classical radioligand assay. We have thus validated the first TR-FRET cell-based binding assay for ApelinR with potential high-throughput screening applications.
authors.affiliation: Université de Strasbourg, CNRS, Plateforme de Chimie Biologie Intégrative, PCBIS, UMS3286, Labex MEDALIS, 6700, Strasbourg, France.
authors.forename: Christel
authors.initials: C
authors.lastname: Valencia
authors.affiliation: Cisbio Bioassays, Parc Marcel Boiteux, BP 84175, 30200, Codolet, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Dujet
authors.affiliation: Université de Strasbourg, CNRS, Laboratoire d'Innovation Thérapeutique (LIT), UMR7200, Labex MEDALIS, 6700, Strasbourg, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Margathe
authors.affiliation: Neuropeptides Centraux et Régulations Hydrique et cardiovasculaire, UMR 7241/Inserm U1050, CIRB, Collège de France, 11 place Marcelin Berthelot, 75231, Paris Cedex 05, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Iturrioz
authors.affiliation: Cisbio Bioassays, Parc Marcel Boiteux, BP 84175, 30200, Codolet, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Roux
authors.affiliation: Cisbio Bioassays, Parc Marcel Boiteux, BP 84175, 30200, Codolet, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Trinquet
authors.affiliation: Université de Strasbourg, CNRS, Plateforme de Chimie Biologie Intégrative, PCBIS, UMS3286, Labex MEDALIS, 6700, Strasbourg, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Villa
authors.affiliation: Université de Strasbourg, CNRS, Laboratoire d'Innovation Thérapeutique (LIT), UMR7200, Labex MEDALIS, 6700, Strasbourg, France.
authors.forename: Marcel
authors.initials: M
authors.lastname: Hibert
authors.affiliation: Cisbio Bioassays, Parc Marcel Boiteux, BP 84175, 30200, Codolet, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Dupuis
authors.affiliation: Neuropeptides Centraux et Régulations Hydrique et cardiovasculaire, UMR 7241/Inserm U1050, CIRB, Collège de France, 11 place Marcelin Berthelot, 75231, Paris Cedex 05, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Llorens-Cortes
authors.affiliation: Université de Strasbourg, CNRS, Laboratoire d'Innovation Thérapeutique (LIT), UMR7200, Labex MEDALIS, 6700, Strasbourg, France.
authors.forename: Dominique
authors.identifier: 0000-0002-8252-9199
authors.initials: D
authors.lastname: Bonnet
mesh_terms: D000075244:Apelin Receptors; D001665:Binding Sites / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D028581:Lanthanoid Series Elements / Q000737:chemistry / Q000494:pharmacology*; D008024:Ligands; D015394:Molecular Structure; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D043562:Receptors, G-Protein-Coupled / Q000819:agonists*; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: C104575:APLNR protein, human; D000075244:Apelin Receptors; D028581:Lanthanoid Series Elements; D008024:Ligands; D009942:Organometallic Compounds; D043562:Receptors, G-Protein-Coupled
keywords: G protein-coupled receptors; TR-FRET assays; apelin; chemical ligation; fluorescent probes