title: Cell-based beta2-adrenergic receptor-ligand binding assay using synthesized europium-labeled ligands and time-resolved fluorescence.
abstract: 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 beta(2)-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 beta(2)-adrenergic receptors overexpressed in human embryonic kidney HEK293(i) cells. Europium ligand with a 6-carbon spacer arm (C6) showed no binding. Competitive binding assays were developed with the functional labeled ligands. The IC(50) values for beta(2)-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.
authors.affiliation: Laboratory of Biophysics, University of Turku, FI-20520 Turku, Finland.
authors.forename: Eija
authors.initials: E
authors.lastname: Martikkala
authors.forename: Mirva
authors.initials: M
authors.lastname: Lehmusto
authors.forename: Minna
authors.initials: M
authors.lastname: Lilja
authors.forename: Anita
authors.initials: A
authors.lastname: Rozwandowicz-Jansen
authors.forename: Jenni
authors.initials: J
authors.lastname: Lunden
authors.forename: Takenori
authors.initials: T
authors.lastname: Tomohiro
authors.forename: Pekka
authors.initials: P
authors.lastname: Hänninen
authors.forename: Ulla
authors.initials: U
authors.lastname: Petäjä-Repo
authors.forename: Harri
authors.initials: H
authors.lastname: Härmä
mesh_terms: D000319:Adrenergic beta-Antagonists / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D005063:Europium / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D010869:Pindolol / Q000031:analogs & derivatives / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D019076:Transgenes / Q000235:genetics
chemical_list: D000319:Adrenergic beta-Antagonists; D002614:Chelating Agents; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D005063:Europium; D010869:Pindolol