Detection of novel functional selectivity at M3 muscarinic acetylcholine receptors using a Saccharomyces cerevisiae platform.
"Functional selectivity", although new to many chemists and biologists only a few years ago, has now become a dominant theme in drug discovery. This concept posits that different ligands engender unique receptor conformations such that only a subset of signaling pathways linked to a given receptor are recruited. However, successful exploitation of the phenomenon to achieve pathway-based selectivity requires the ability to routinely detect it when assessing ligand behavior. We have utilized different strains of the yeast S. cerevisiae, each expressing a specific human Galpha/yeast Gpa1 protein chimera coupled to a MAP kinase-linked reporter gene readout, to investigate the signaling of the M(3) muscarinic receptor, a G protein-coupled receptor (GPCR) for which various antagonists are used clinically. Using this novel platform, we found that the "antagonists", atropine, N-methylscopolamine, and pirenzepine, were inverse agonists for Gpa1/Galpha(q) but low efficacy agonists for Gpa1/Galpha(12.) Subsequent studies with atropine performed in mammalian 3T3 cells validated these findings by demonstrating inverse agonism for G(q/11)-mediated calcium mobilization but positive agonism for G(12)-mediated membrane ruffling. This is the first study to utilize a yeast platform to discover pathway-biased functional selectivity in a GPCR. In addition to the likely applicability of this approach for identifying biased signaling by novel chemical entities, our findings also suggest that currently marketed medications may exhibit hitherto unappreciated functional selectivity.
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences & Department of Pharmacology, Monash University, Parkville, Victoria, Australia 3052.
Gregory D
GD
Stewart
Patrick M
PM
Sexton
Arthur
A
Christopoulos
D016475:3T3 Cells; D000818:Animals; D001285:Atropine / Q000494:pharmacology*; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D018727:Muscarinic Antagonists / Q000494:pharmacology*; D019832:N-Methylscopolamine / Q000494:pharmacology*; D010890:Pirenzepine / Q000494:pharmacology*; D043587:Receptor, Muscarinic M3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D044385:GTP-Binding Protein alpha Subunits; D018727:Muscarinic Antagonists; D043587:Receptor, Muscarinic M3; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D010890:Pirenzepine; D001285:Atropine; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019832:N-Methylscopolamine