title: On the use of nonfluorescent dye labeled ligands in FRET-based receptor binding studies.
abstract: The efficiency of fluorescence resonance energy transfer (FRET) is dependent upon donor-acceptor proximity and spectral overlap, whether the acceptor partner is fluorescent or not. We report here on the design, synthesis, and characterization of two novel pirenzepine derivatives that were coupled to patent blue VF and pinacyanol dyes. These nonfluorescent compounds, when added to cells stably expressing enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors, promote EGFP fluorescence extinction in a time-, concentration-, and atropine-dependent manner. They display nanomolar affinity for the muscarinic receptor, determined using either FRET or classical radioligand binding conditions. We provide evidence that these compounds behave as potent acceptors of energy from excited EGFP with quenching efficiencies comparable to those of analogous fluorescent bodipy or rhodamine red pirenzepine derivatives. The advantages they offer over fluorescent ligands are illustrated and discussed in terms of reliability, sensitivity, and wider applicability of FRET-based receptor binding assays.
authors.affiliation: Laboratoire de Pharmacochimie de la Communication Cellulaire, Faculté de Pharmacie, UMR CNRS/ULP 7081, 74 route du Rhin, BP 24, 67401 Illkirch, France.
authors.forename: Chouaib
authors.initials: C
authors.lastname: Tahtaoui
authors.forename: Fabrice
authors.initials: F
authors.lastname: Guillier
authors.forename: Philippe
authors.initials: P
authors.lastname: Klotz
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Galzi
authors.forename: Marcel
authors.initials: M
authors.lastname: Hibert
authors.forename: Brigitte
authors.initials: B
authors.lastname: Ilien
mesh_terms: D001557:Benzenesulfonates / Q000138:chemical synthesis* / Q000737:chemistry; D001570:Benzodiazepinones / Q000138:chemical synthesis* / Q000737:chemistry; D001896:Boron Compounds; D002460:Cell Line; D004396:Coloring Agents / Q000138:chemical synthesis* / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D010890:Pirenzepine / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry; D011806:Quinolinium Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D011869:Radioligand Assay; D043643:Receptor, Muscarinic M1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C507726:1-ethyl-2-(3-(1-ethylquinolin-2(1H)-ylidene)prop-1-enyl)-6-((6-oxo-6-((2-(2-(2-(4-(2-oxo-2-(6-oxo-5,6-dihydro-11H-pyrido(2,3-b)(1,4)benzodiazepin-11-yl)ethyl)piperazin-1-yl)ethoxy)ethoxy)ethyl)amino)hexanoyl)amino)quinolinium; C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001557:Benzenesulfonates; D001570:Benzodiazepinones; D001896:Boron Compounds; D004396:Coloring Agents; D008024:Ligands; C507725:PAT(17)PZ compound; D011806:Quinolinium Compounds; D043643:Receptor, Muscarinic M1; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D010890:Pirenzepine