A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening

Abstract

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 β 2 -adrenoreceptor (β 2 AR) antagonists and agonists in intact human embryonic kidney HEK293 i cells overexpressing human β 2 -adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β 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 µM for metaproterenol and terbutaline, respectively. The QRET technique with β 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. (Journal of Biomolecular Screening 2009:936-943)

A Homogeneous Single-Label Time-Resolved Fluorescence cAMP Assay

“…this has been studied in a cell-based Qret method earlier, and a stable signal up to 72 hours has been reported. 11 the feasibility of the developed method was proven by comparing the assay performance with the commercial homogeneous tr-Fret assay (lAnce cAMp kit from perkinelmer). the choice of the reference method is justified because the two techniques share the same detection features: excitation at 340 nm and time-resolved fluorescence detection above 600 nm with a temporal resolution leading to reduced background fluorescence.…”

“…the cell-based Qret method has also been shown to tolerate dMso well in a receptor-ligand binding assay. 11 Hts applications also require stable signal to enable off-line detection. this has been studied in a cell-based Qret method earlier, and a stable signal up to 72 hours has been reported.…”

“…dIScuSSIon previously, the potential of the Qret technique has been demonstrated for receptor-ligand and Gtp binding studies on cells and cell membranes. 11,13 Here we have developed a cellbased homogeneous assay for the second messenger, cAMp, using a time-resolved fluorescence single-label approach. the cAMp assay was developed using a stably transfected HeK293 i cell line overexpressing human β 2 Ars.…”

“…We have previously demonstrated that receptor-ligand and Gtp binding assays can be constructed on the Qret concept. 11,13 Fluorescence polarization is another known single-label approach for drug-screening purposes. However, the drawback of this method is the use of conventional fluorescence having 3-4 orders of magnitude less sensitive detection limit than timeresolved fluorescence 24 and a low s/B ratio, 4,12,25,26 reducing the applicability of the method for many target molecules.…”

“…the assay is based on the quenching resonance energy transfer (Qret) technique [11][12][13] using a single-label approach and soluble quenchers affecting the fluorescence of the unbound labeled binding component. less fluorescence quenching is observed when the labeled component binds to an antibody.…”