title: Time-resolved FRET binding assay to investigate hetero-oligomer binding properties: proof of concept with dopamine D1/D3 heterodimer.
abstract: G protein-coupled receptors (GPCRs) have been described to form hetero-oligomers. The importance of these complexes in physiology and pathology is considered crucial, and heterodimers represent promising new targets to discover innovative therapeutics. However, there is a lack of binding assays to allow the evaluation of ligand affinity for GPCR hetero-oligomers. Using dopamine receptors and more specifically the D1 and D3 receptors as GPCR models, we developed a new time-resolved FRET (TR-FRET) based assay to determine ligand affinity for the D1/D3 heteromer. Based on the high-resolution structure of the dopamine D3 receptor (D3R), six fluorescent probes derived from a known D3R partial agonist (BP 897) were designed, synthesized and evaluated as high affinity and selective ligands for the D3/D2 receptors, and for other dopamine receptor subtypes. The highest affinity ligand 21 was then employed in the development of the D1/D3 heteromer assay. The TR-FRET was monitored between a fluorescent tag donor carried by the D1 receptor (D1R) and a fluorescent acceptor D3R ligand 21. The newly reported assay, easy to implement on other G protein-coupled receptors, constitutes an attractive strategy to screen for heteromer ligands.
authors.affiliation: CNRS UMR 5203, and INSERM U661, and Université Montpellier I et II, Institut de Génomique Fonctionnelle , Département de Pharmacologie Moléculaire, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France.
authors.forename: Candide
authors.initials: C
authors.lastname: Hounsou
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Margathe
authors.forename: Nadia
authors.initials: N
authors.lastname: Oueslati
authors.forename: Abderazak
authors.initials: A
authors.lastname: Belhocine
authors.forename: Elodie
authors.initials: E
authors.lastname: Dupuis
authors.forename: Cécile
authors.initials: C
authors.lastname: Thomas
authors.forename: André
authors.initials: A
authors.lastname: Mann
authors.forename: Brigitte
authors.initials: B
authors.lastname: Ilien
authors.forename: Didier
authors.initials: D
authors.lastname: Rognan
authors.forename: Eric
authors.initials: E
authors.lastname: Trinquet
authors.forename: Marcel
authors.initials: M
authors.lastname: Hibert
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pin
authors.forename: Dominique
authors.initials: D
authors.lastname: Bonnet
authors.forename: Thierry
authors.initials: T
authors.lastname: Durroux
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D008958:Models, Molecular; D015394:Molecular Structure; D010879:Piperazines / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017447:Receptors, Dopamine D1; D050637:Receptors, Dopamine D3; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D010879:Piperazines; D017447:Receptors, Dopamine D1; D050637:Receptors, Dopamine D3; C120565:BP 897