Cell-based β2-adrenergic receptor–ligand binding assay using synthesized europium-labeled ligands and time-resolved fluorescence

High-sensitivity, high-throughput, and user-friendly lanthanide-based assays for receptor–ligand interactions provide an attractive alternative to the traditional radioligand displacement assays. In this study, three small-molecule pindolol ligand derivatives were synthesized and their binding properties were tested in a radioligand displacement assay. The ligand derivatives were further labeled with fluorescent europium(III) chelate for β2-adrenergic receptor–ligand binding assay. The europium-labeled pindolol ligands having no spacer (C0) or a 12-carbon spacer (C12) arm bound to the human β2-adrenergic receptors overexpressed in human embryonic kidney HEK293i cells. Europium ligand with a 6-carbon spacer arm (C6) showed no binding. Competitive binding assays were developed with the functional labeled ligands. The IC50 values for β2-adrenergic antagonist propranolol were 60 and 37 nM, the Z′ values were 0.51 and 0.77, and the signal-to-background ratios were 5.5 and 16.0 for C0 and C12, respectively. This study shows that functional time-resolved fluorescent assays can be constructed using fluorescent lanthanide chelates conjugated to small-molecule ligands.