Detection of novel functional selectivity at M3 muscarinic acetylcholine receptors using a Saccharomyces cerevisiae platform

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Abstract

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.
Original languageEnglish
Pages (from-to)365 - 375
Number of pages11
JournalACS Chemical Biology
Volume5
Issue number4
Publication statusPublished - 2010

Cite this

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title = "Detection of novel functional selectivity at M3 muscarinic acetylcholine receptors using a Saccharomyces cerevisiae platform",
abstract = "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.",
author = "Stewart, {Gregory D} and Patrick Sexton and Arthur Christopoulos",
year = "2010",
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pages = "365 -- 375",
journal = "ACS Chemical Biology",
issn = "1554-8929",
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TY - JOUR

T1 - Detection of novel functional selectivity at M3 muscarinic acetylcholine receptors using a Saccharomyces cerevisiae platform

AU - Stewart, Gregory D

AU - Sexton, Patrick

AU - Christopoulos, Arthur

PY - 2010

Y1 - 2010

N2 - 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.

AB - 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.

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