A fluorescent ligand-binding alternative using Tag-lite® technology.
J Biomol Screen
; 15(10): 1248-59, 2010 Dec.
Article
in En
| MEDLINE
| ID: mdl-20974902
ABSTRACT
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Receptors, G-Protein-Coupled
/
High-Throughput Screening Assays
Limits:
Animals
/
Humans
Language:
En
Journal:
J Biomol Screen
Journal subject:
BIOLOGIA MOLECULAR
Year:
2010
Document type:
Article
Affiliation country:
France