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A Fluorescent Ligand-Binding Alternative Using Tag-lite® Technology

2010; Elsevier BV; Volume: 15; Issue: 10 Linguagem: Inglês

10.1177/1087057110384611

2472-5560

Jurriaan M. Zwier, Thomas Roux, Martin Cottet, Thierry Durroux, Stéphanie Douzon, Sara Bdioui, Nathalie Gregor, Emmanuel Bourrier, Nadia Oueslati, Ludovic Nicolas, Norbert Tinel, Chloé Boisseau, Paul Yverneau, Fabienne Charrier‐Savournin, Michel Fink, Eric Trinquet,

Pharmacological Effects and Assays

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(®). 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 (δ, µ, and κ), 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.