title: Development of a homogeneous high-throughput live-cell G-protein-coupled receptor binding assay.
abstract: The measurement of ligand receptor binding parameters for G-protein-coupled receptors is indispensable in the drug discovery process. Traditional ligand receptor binding assays require scale-up of cells and membrane preparations, which is an expensive and time-consuming process. In this report, the authors describe the development of a homogeneous live-cell binding assay for GPCRs using a fluorophore-labeled nonpeptide ligand. The model assay used Cy3B-labeled telenzepine and Chinese hamster ovary cells expressing M1 muscarinic acetylcholine receptors. This homogeneous live-cell fluorescence binding assay format is superior to the traditional binding methods because it measures binding of a ligand to intact receptors on living cells. The assay requires no washing or separation steps, thereby allowing a real-time kinetic readout for the determination of ligand association and dissociation from the intact receptors. The results also suggest that miniaturization is feasible without compromising the data quality.
authors.affiliation: Chemistry Research and Discovery, Amgen, Inc., Thousand Oaks, California 91320, USA. paull@amgen.com
authors.forename: Paul H
authors.initials: PH
authors.lastname: Lee
authors.forename: Steven C
authors.initials: SC
authors.lastname: Miller
authors.forename: Carlo
authors.initials: C
authors.lastname: van Staden
authors.forename: Evan F
authors.initials: EF
authors.lastname: Cromwell
mesh_terms: D000818:Animals; D001285:Atropine / Q000378:metabolism; D001557:Benzenesulfonates / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D002232:Carbocyanines / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000378:metabolism; D008024:Ligands; D008904:Miniaturization; D018727:Muscarinic Antagonists / Q000737:chemistry / Q000378:metabolism; D010276:Parasympatholytics / Q000378:metabolism; D010890:Pirenzepine / Q000031:analogs & derivatives / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000737:chemistry / Q000378:metabolism; D043643:Receptor, Muscarinic M1 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*
chemical_list: D001557:Benzenesulfonates; D002232:Carbocyanines; C495965:Cy3B N-hydroxysuccinimide ester; D005456:Fluorescent Dyes; D008024:Ligands; D018727:Muscarinic Antagonists; D010276:Parasympatholytics; D043643:Receptor, Muscarinic M1; D043562:Receptors, G-Protein-Coupled; C046338:telenzepine; D010890:Pirenzepine; D011813:Quinuclidinyl Benzilate; D001285:Atropine