A new simple cell-based homogeneous time-resolved fluorescence QRET technique for receptor-ligand interaction screening.
In this article, a single-label separation-free fluorescence technique is presented as a potential screening method for cell-based receptor antagonists and agonists.The time-resolved fluorescence technique, quenching resonance energy transfer (QRET), relies on a single-labeled binding partner in combination with a soluble quencher. The quencher efficiently suppresses the luminescence of the unbound labeled ligand, whereas the luminescence of the bound fraction is not affected. This approach allows the development of cell-based screening assays in a simple and cost-effective manner. The authors have applied the technique to the screening of beta(2)-adrenoreceptor (beta(2)AR) antagonists and agonists in intact human embryonic kidney HEK293(i) cells overexpressing human beta(2)-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for beta(2)AR were investigated in a displacement assay using europium(III)-labeled pindolol ligand. The assay Z' values ranged from 0.68 to 0.78, the coefficient of variation was less than 10%, and the K(i) values were 19 nM for propranolol and alprenolol and 14 and 5.9 microM for metaproterenol and terbutaline, respectively. The QRET technique with beta(2)AR was also applied to LOPAC compound library screening, yielding nearly error-free recognition of known binders. This simple and cost-effective technique can be readily adapted to laboratory and industrial-scale screening.
Härmä
Harri
H
Laboratory of Biophysics, University of Turku, Turku, Finland. harri.harma@utu.fi
Rozwandowicz-Jansen
Anita
A
Martikkala
Eija
E
Frang
Heini
H
Hemmilä
Ilkka
I
Sahlberg
Niko
N
Fey
Vidal
V
Perälä
Merja
M
Hänninen
Pekka
P
D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D000318:Adrenergic beta-Agonists / Q000302:isolation & purification / Q000493:pharmacokinetics; D000319:Adrenergic beta-Antagonists / Q000302:isolation & purification / Q000493:pharmacokinetics; D000526:Alprenolol / Q000493:pharmacokinetics; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005063:Europium / Q000493:pharmacokinetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008024:Ligands; D008163:Luminescent Measurements; D009921:Metaproterenol / Q000493:pharmacokinetics; D008954:Models, Biological; D011433:Propranolol / Q000493:pharmacokinetics; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013726:Terbutaline / Q000493:pharmacokinetics
D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D008024:Ligands; D011956:Receptors, Cell Surface; D005063:Europium; D009921:Metaproterenol; D000526:Alprenolol; D011433:Propranolol; D013726:Terbutaline