On the use of nonfluorescent dye labeled ligands in FRET-based receptor binding studies
Résumé
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.
Mots clés
Benzenesulfonates/*chemical synthesis/chemistry
Benzodiazepinones/*chemical synthesis/chemistry
Boron Compounds
Cell Line
Coloring Agents/*chemical synthesis/chemistry
Comparative Study
Fluorescence Resonance Energy Transfer
Green Fluorescent Proteins/genetics/*metabolism
Humans
Ligands
Pirenzepine/*analogs & derivatives/*chemical synthesis/chemistry
Quinolinium Compounds/*chemical synthesis/chemistry
Radioligand Assay
Receptor
Muscarinic M1/genetics/*metabolism
Recombinant Fusion Proteins/genetics/*metabolism
Research Support
Non-U.S. Gov't