In the search for new chemical entities that interact with G-protein-coupled 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 [3H] N-methylscopolamine. The IC50 values produced from the FA assay were well-correlated with the radioligand-binding Kivalues (R2= 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 radioligand-binding assays as it eliminates radioactivity while allowing investigation of orthosteric or allosteric muscarinic M1 ligands in a homogeneous format.
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