Homogeneous time-resolved fluorescence-based assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a
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- 作者：Jean-Philippe Leyris ; Thomas Roux ; Eric Trinquet ; Pascal Verdié ; Jean-Alain Fehrentz ; Nadia Oueslati ; Sté ; phanie Douzon ; Emmanuel Bourrier ; Laurent Lamarque ; Didier Gagne ; Jean-Claude Galleyr
- 关键词：G protein-coupled receptor ; Growth hormone secretagogue receptor type 1a ; Ghrelin ; Tag-lite ; HTRF ; SNAP-tag ; Ligand-binding ; TR-FRET
- 刊名：Analytical Biochemistry
- 出版年：2011
- 出版时间：15 January 2011
- 年：2011
- 卷：408
- 期：2
- 页码：253-262
- 全文大小：792 K
文摘
The growth hormone secretagogue receptor type 1a (GHS-R1a) belongs to class A G-protein-coupled receptors (GPCR). This receptor mediates pleiotropic effects of ghrelin and represents a promising target for dysfunctions of growth hormone secretion and energy homeostasis including obesity. Identification of new compounds which bind GHS-R1a is traditionally achieved using radioactive binding assays. Here we propose a new fluorescence-based assay, called Tag-lite binding assay, based on a fluorescence resonance energy transfer (FRET) process between a terbium cryptate covalently attached to a SNAP-tag fused GHS-R1a (SNAP-GHS-R1a) and a high-affinity red fluorescent ghrelin ligand. The long fluorescence lifetime of the terbium cryptate allows a time-resolved detection of the FRET signal. The assay was made compatible with high-throughput screening by using prelabeled cells in suspension under a 384-well plate format. K<sub>isub> values for a panel of 14 compounds displaying agonist, antagonist, or inverse agonist properties were determined using both the radioactive and the Tag-lite binding assays performed on the same batches of GHS-R1a-expressing cells. Compound potencies obtained in the two assays were nicely correlated. This study is the first description of a sensitive and reliable nonradioactive binding assay for GHS-R1a in a format amenable to high-throughput screening.