Dye-Labeled Benzodiazepines:  Development of Small Ligands for Receptor Binding Studies Using Fluorescence Correlation Spectroscopy

Abstract

To investigate benzodiazepine receptor binding studies by fluorescence correlation spectroscopy (FCS), the four fluorophores fluorescein, tetramethylrhodamine, Oregon Green 488, and Alexa 532 were coupled to the benzodiazepine Ro 07-1986/602 (Ro). Binding assays to polyclonal antibodies to benzodiazepines and at the native benzodiazepine receptor on the membrane of rat hippocampal neurons were established to examine the dye-labeled ligands for their benzodiazepine character and their binding behavior. Both the fluorescein and the Oregon Green488 moiety led to a loss of the benzodiazepine receptor binding of the corresponding Ro derivatives. Antibody recognition and interactions to the receptor were observed for the tetramethylrhodamine derivative (K(D) = 96.0 +/- 9.5 nM) but with a high amount of nonspecific binding at the cell membrane of about 50%. In saturation experiments a K(D) value of 97.2 +/- 8.5 nM was found for the Alexa Fluor 532 derivative-antibody interaction. Investigation of the binding of this ligand to the benzodiazepine receptor in FCS cell measurements led to confirmation of high specific binding behavior with a K(D) value of 9.9 +/- 1.9 nM. A nonspecific binding of <10% was observed after coincubation with 1 microM of midazolam. The different properties of the labeled benzodiazepine derivatives and the requirements of the fluorophore in small dye-labeled ligands in FCS binding studies, at the membrane of living cells, are discussed.

Pharmacology under the microscope: the use of fluorescence correlation spectroscopy to determine the properties of ligand–receptor complexes

“…Ligand-binding affinities, receptor densities and off rates for a number of different ligand–receptor combinations have all been determined successfully. In each case, even in heterogeneous cell populations from primary sources, useful quantitative information about ligand–receptor interactions was obtained, supporting the applicability of such methodology to studying pharmacology in biopsy samples [18,19,22–25] . In the case of the insulin C-peptide [18,22,23] , kavain [25] and glucocorticoid receptors [26,27] , the FCS data provided the first direct evidence for the existence of specific cell-surface GPCRs for these ligands.…”

A Förster Resonance Energy Transfer‐Based Ratiometric Sensor with the Allosteric Transcription Factor TetR

“…[ 6,7 ] Our aTF‐based FRET sensors utilize the analyte‐responsive unbinding of the donor‐labeled aTF and its acceptor‐labeled cognate DNA sequence to effect a large change in the donor–acceptor distance. Thus, these FRET sensors eliminate the need to dye‐label ligands, which can change their binding behavior, [ 12 ] while enabling the substantial signal changes produced through complete dissociation of the donor and acceptor fluorophores ( Figure ).…”

Ligands for Fluorescence Correlation Spectroscopy on G Protein-Coupled Receptors

“…Recently, the influence of the dye moiety on the receptor binding and signaling of fluorescently labeled ligands has been demonstrated in several publications [31,32].…”

“…Hegener et al showed for example that coupling Alexa532 to the Therefore, predictions about the binding affinities and the pharmacological properties of fluorescently labeled ligands are not possible and have to be validated. Examples for such series of experiments investigating the influence of different fluorophores and/or linkers on the pharmacology of receptor ligands were published by Baker et al[30] and Hegener et al[31].…”