A fluorescence anisotropy assay for the muscarinic M1 G-protein-coupled receptor
| Autor: | Kurt W. Vogel, Therese De Rosier, Kristin G Huwiler, Bonnie J. Hanson |
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| Rok vydání: | 2010 |
| Předmět: |
Boron Compounds
Fluorescence Polarization Muscarinic Antagonists Nicotinic Antagonists Ligands Fluorescence Cell Line Receptors G-Protein-Coupled Radioligand Assay Drug Discovery Muscarinic acetylcholine receptor medicine Humans Receptor G protein-coupled receptor Fluorescent Dyes Chromatography Gallamine Triethiodide Chemistry Cell Membrane Receptor Muscarinic M1 Muscarinic acetylcholine receptor M1 Pirenzepine Guanosine 5'-O-(3-Thiotriphosphate) Biophysics Molecular Medicine Fluorescence anisotropy medicine.drug |
| Zdroj: | Assay and drug development technologies. 8(3) |
| ISSN: | 1557-8127 |
| Popis: | In the search for new chemical entities that interact with G-proteincoupled receptors (GPCRs), assays that quantify efficacy and affinity are employed. Traditional methods for measuring affinity involve radiolabeled ligands. To address the need for homogeneous biochemical fluorescent assays to characterize orthosteric ligand affinity and dissociation rates, we have developed a fluorescence anisotropy (FA) assay for the muscarinic M1 receptor that can be conducted in a 384-well plate. We used membranes from a muscarinic M1 cell line optimized for high-throughput functional assays and the previously characterized fluorescent antagonist BODIPY FL pirenzepine. The affinities of reference compounds were determined in the competitive FA assay and compared with those obtained with a competitive filter-based radioligand-binding assay using [(3)H] N-methylscopolamine. The IC(50) values produced from the FA assay were well-correlated with the radioligand-binding K(i) values (R(2) = 0.98). The dissociation of the BODIPY FL pirenzepine was readily monitored in real time using the FA assay and was sensitive to the presence of the allosteric modulator gallamine. This M1 FA assay offers advantages over traditional radioligandbinding assays as it eliminates radioactivity while allowing investigation of orthosteric or allosteric muscarinic M1 ligands in a homogeneous format. |
| Databáze: | OpenAIRE |
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