title: Determination of adenosine A1 receptor agonist and antagonist pharmacology using Saccharomyces cerevisiae: implications for ligand screening and functional selectivity.
abstract: The budding yeast, Saccharomyces cerevisiae, is a convenient system for coupling heterologous G protein-coupled receptors (GPCRs) to the pheromone response pathway to facilitate empirical ligand screening and/or GPCR mutagenesis studies. However, few studies have applied this system to define GPCR-G protein-coupling preferences and furnish information on ligand affinities, efficacies, and functional selectivity. We thus used different S. cerevisiae strains, each expressing a specific human Galpha/yeast Gpa1 protein chimera, and determined the pharmacology of various ligands of the coexpressed human adenosine A(1) receptor. These assays, in conjunction with the application of quantitative models of agonism and antagonism, revealed that (-)-N(6)-(2-phenylisopropyl)adenosine was a high-efficacy agonist that selectively coupled to Gpa/1Galpha(o), Gpa1/Galpha(i1/2), and Gpa1/Galpha(i3), whereas the novel compound, 5'-deoxy-N(6)-(endo-norborn-2-yl)-5'-(2-fluorophenylthio)adenosine (VCP-189), was a lower-efficacy agonist that selectively coupled to Gpa1/Galpha(i) proteins; the latter finding suggested that VCP-189 might be functionally selective. The affinity of the antagonist, 8-cyclopentyl-1,3-dipropylxanthine, was also determined at the various strains. Subsequent experiments performed in mammalian Chinese hamster ovary cells monitoring cAMP formation/inhibition, intracellular calcium mobilization, phosphorylation of extracellular signal-regulated kinase 1 and 2 or (35)S-labeled guanosine 5'-(gamma-thio)triphosphate binding, were in general agreement with the yeast data regarding agonist efficacy estimation and antagonist affinity estimation, but revealed that the apparent functional selectivity of VCP-189 could be explained by differences in stimulus-response coupling between yeast and mammalian cells. Our results suggest that this yeast system is a useful tool for quantifying ligand affinity and relative efficacy, but it may lack the sensitivity required to detect functional selectivity of low-efficacy agonists.
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Stewart
authors.forename: Celine
authors.initials: C
authors.lastname: Valant
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Dowell
authors.forename: Dalibor
authors.initials: D
authors.lastname: Mijaljica
authors.forename: Rodney J
authors.initials: RJ
authors.lastname: Devenish
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Scammells
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
mesh_terms: D058907:Adenosine A1 Receptor Agonists; D058916:Adenosine A1 Receptor Antagonists; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D008024:Ligands; D043682:Receptor, Adenosine A1 / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D014970:Xanthines / Q000737:chemistry / Q000494:pharmacology
chemical_list: D058907:Adenosine A1 Receptor Agonists; D058916:Adenosine A1 Receptor Antagonists; D008024:Ligands; D043682:Receptor, Adenosine A1; D014970:Xanthines; C051360:1,3-dipropyl-8-cyclopentylxanthine