title: Fragment screening at adenosine-A(3) receptors in living cells using a fluorescence-based binding assay.
abstract: G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane proteins. For GPCR drug discovery, it is important that ligand affinity is determined in the correct cellular environment and preferably using an unmodified receptor. We developed a live cell high-content screening assay that uses a fluorescent antagonist, CA200645, to determine binding affinity constants of competing ligands at human adenosine-A(1) and -A(3) receptors. This method was validated as a tool to screen a library of low molecular weight fragments, and identified a hit with submicromolar binding affinity (K(D)). This fragment was structurally unrelated to substructures of known adenosine receptor antagonists and was optimized to show selectivity for the adenosine-A(3) receptor. This technology represents a significant advance that will allow the determination of ligand and fragment affinities at receptors in their native membrane environment.
authors.affiliation: Institute of Cell Signalling, School of Biomedical Science, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK.
authors.forename: Leigh A
authors.initials: LA
authors.lastname: Stoddart
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Vernall
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Denman
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Briddon
authors.forename: Barrie
authors.initials: B
authors.lastname: Kellam
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hill
mesh_terms: D000818:Animals; D016466:CHO Cells; D002470:Cell Survival; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D006801:Humans; D008024:Ligands; D008163:Luminescent Measurements / Q000379:methods*; D018613:Microscopy, Confocal; D015394:Molecular Structure; D011485:Protein Binding; D043684:Receptor, Adenosine A3 / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D008024:Ligands; D043684:Receptor, Adenosine A3; D054852:Small Molecule Libraries