A fluorescent ligand-binding alternative using Tag-lite® technology.
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.
Cisbio Bioassays, Bagnols-sur-Cèze, France.
Jurriaan M
JM
Zwier
Thomas
T
Roux
Martin
M
Cottet
Thierry
T
Durroux
Stephanie
S
Douzon
Sara
S
Bdioui
Nathalie
N
Gregor
Emmanuel
E
Bourrier
Nadia
N
Oueslati
Ludovic
L
Nicolas
Norbert
N
Tinel
Chloe
C
Boisseau
Paul
P
Yverneau
Fabienne
F
Charrier-Savournin
Michel
M
Fink
Eric
E
Trinquet
D000818:Animals; D006224:Cricetinae; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005453:Fluorescence; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008024:Ligands; D043886:Receptor, Cholecystokinin A / Q000378:metabolism; D043887:Receptor, Cholecystokinin B / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism*; D011957:Receptors, Opioid / Q000378:metabolism
D008024:Ligands; D043886:Receptor, Cholecystokinin A; D043887:Receptor, Cholecystokinin B; D019718:Receptors, CXCR4; D043562:Receptors, G-Protein-Coupled; D011957:Receptors, Opioid