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

被引：16

作者：

Martikkala, Eija ^{[1
]}

Lehmusto, Mirva ^{[1
]}

Lilja, Minna ^{[2
]}

Rozwandowicz-Jansen, Anita ^{[1
]}

Lunden, Jenni ^{[3
]}

Tomohiro, Takenori ^{[4
]}

Hanninen, Pekka ^{[1
]}

Petaja-Repo, Ulla ^{[2
]}

Harma, Harri ^{[1
]}

机构：

[1] Univ Turku, Biophys Lab, FI-20520 Turku, Finland

[2] Univ Oulu, Inst Biomed, Dept Anat & Cell Biol, FI-90014 Oulu, Finland

[3] Wallac Oy PerkinElmer Life & Analyt Sci, FI-20520 Turku, Finland

[4] Toyama Univ, Lab Biorecognit Chem, Toyama 9300194, Japan

来源：

ANALYTICAL BIOCHEMISTRY | 2009年 / 392卷 / 02期

关键词：

beta(2)-Adrenergic receptor; GPCR; G protein-coupled receptor; Time-resolved fluorescence; Cell-based assay; Ligand synthesis; PROTEIN-COUPLED RECEPTORS; LUTEINIZING-HORMONE RECEPTOR; ENDOPLASMIC-RETICULUM; BETA-ADRENOCEPTORS; DRUG DISCOVERY; ANTAGONISTS; ANALOGS; SURFACE; PROBES;

D O I：

10.1016/j.ab.2009.05.022

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 (CO) 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 IC50 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 CO and C12, respectively. This Study shows that functional time-resolved fluorescent assays can be constructed using fluorescent lanthanide chelates conjugated to small-molecule ligands. (C) 2009 Elsevier Inc. All rights reserved.

引用

页码：103 / 109

页数：7

共 23 条

[11] Structural basis for ligand binding and specificity in adrenergic receptors: Implications for GPCR-targeted drug discovery [J].