A Fluorescent Ligand-Binding Alternative Using Tag-lite (R) Technology

作者：Zwier Jurriaan M; Roux Thomas; Cottet Martin; Durroux Thierry; Douzon Stephanie; Bdioui Sara; Gregor Nathalie; Bourrier Emmanuel; Oueslati Nadia; Nicolas Ludovic; Tinel Norbert; Boisseau Chloe; Yverneau Paul; Charrier Savournin Fabienne; Fink Michel; Trinquet Eric^*

来源：Journal of Biomolecular Screening, 2010, 15(10): 1248-1259.

DOI：10.1177/1087057110384611

• Tag-lite (R)
• ligand binding assay
• HTRF (R)
• opioid
• chemokine and cholecystokinin receptors
• GPCR

G-protein-coupled receptors (GPCRs) are crucial cell surface receptors that transmit signals from a wide range of extracellular ligands. Indeed, 40% to 50% of all marketed drugs are thought to modulate GPCR activity, making them the major class of targets in the drug discovery process. Binding assays are widely used to identify high-affinity, selective, and potent GPCR drugs. In this field, the use of radiolabeled ligands has remained so far the gold-standard method. Here the authors report a less hazardous alternative for high-throughput screening (HTS) applications by the setup of a nonradioactive fluorescence-based technology named Tag-lite (R). Selective binding of various fluorescent ligands, either peptidic or not, covering a large panel of GPCRs from different classes is illustrated, particularly for chemokine (CXCR4), opioid (delta, mu, and kappa), and cholecystokinin (CCK1 and CCK2) receptors. Affinity constants of well-known pharmacological agents of numerous GPCRs are in line with values published in the literature. The authors clearly demonstrate that the Tag-lite binding assay format can be successfully and reproducibly applied by using different cellular materials such as transient or stable recombinant cells lines expressing SNAP-tagged GPCR. Such fluorescent-based binding assays can be performed with adherent cells or cells in suspension, in 96- or 384-well plates. Altogether, this new technology offers great advantages in terms of flexibility, rapidity, and user-friendliness; allows easy miniaturization; and makes it completely suitable for HTS applications. (Journal of Biomolecular Screening 2010:1248-1259)

• 出版日期2010-12