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Pharmacological profiling of G protein-coupled receptors using Saccharomyces cerevisiae

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posted on 06.03.2017, 05:44 authored by Stewart, Gregory David
It is commonly accepted that the nature of drug efficacy is not a linear concept, but rather a multi-dimensional signalling event. This gives rise to the potential for ligands to be functionally selective, which is preferential activation of a set of signalling cascades at the exclusion of others. This idea poses certain problems and provides new approaches for drug discovery programs with regard to choice of assay format and generation of pathway-selective compounds, respectively. In light of this, the majority of drug discovery programs that search for functionally selective ligands, combat this issue by using multiple endpoint assays, which vary depending on the targeted pathway. However, this approach is sometimes difficult to interpret for a number of reasons, such as the fact that many pathways are mediated by more than one upstream effector, the experimental conditions may vary between assay formats, for example, buffer composition and kinetics of activation. Moreover, using many functional endpoints is often expensive and inefficient. In this vein, this thesis discusses the development of a novel approach to screening for functional selective ligands, using a Saccharomyces cerevisiae expression system, which has been hitherto overlooked for this purpose. The ability of this system to detect functional selectivity of ligands within a Gα protein family (Gαi/o), and across multiple Gα proteins (Gαq, Gαi1/2, Gα12), was assessed. There was evidence that both orthosteric and allosteric ligands that bind M3 muscarinic acetylcholine receptors display functional selectivity, which was predicted using the yeast system; whilst the capability of the yeast system to predict selectivity between Gαi/o subunits was comparatively reduced. In addition, the capacity of the yeast system to predict ligand parameters such as affinity and efficacy was also investigated. It was found that from data obtained in yeast, accurate affinity estimates could be generated. Furthermore, a potential use for yeast in estimating conformation-specific affinity values for agonists was revealed. Taken together, the evidence in this thesis suggests that the yeast signalling assays is a valuable and tractable platform for detecting pharmacological characteristics of existing and novel ligands.


Principal supervisor

Arthur Christopoulos

Year of Award


Department, School or Centre

Pharmacology and Monash Institute of Pharmaceutical Sciences

Campus location



Doctor of Philosophy

Degree Type



Faculty of Medicine Nursing and Health Sciences