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J Med Chem. 2005 Dec 1;48(24):7847-59.

On the use of nonfluorescent dye labeled ligands in FRET-based receptor binding studies.

Author information

1
Laboratoire de Pharmacochimie de la Communication Cellulaire, Faculté de Pharmacie, UMR CNRS/ULP 7081, 74 route du Rhin, BP 24, 67401 Illkirch, France.

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.

PMID:
16302823
DOI:
10.1021/jm050459+
[Indexed for MEDLINE]

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