Exploring aspects of allosteric modulation and bias signalling at the glucagon-like peptide-1 receptor

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of insulin biosynthesis and secretion, and is one of the key therapeutic targets in the management of type II diabetes mellitus and obesity. Like most GPCRs, the GLP-1R is pleiotropically coupled, to physiologically relevant signalling pathways including cAMP formation, intracellular calcium (iCa2+) mobilization and phosphorylation of extracellular signal regulated kinases 1 and 2 (pERK1/2). The GLP-1R is a class B G protein-coupled receptor (GPCR) that has the ability to be activated by multiple endogenous ligands including four variants of GLP-1 (the predominant form being GLP-1(7-36)NH2) and oxyntomodulin. This receptor is also activated by the exogenous peptide exendin-4 and allosteric ligands such as the Novo Nordisk Compound 2 and Eli Lily 4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP). These allosteric ligands also have unique properties compared to orthosteric exogenous ligands including the ability to alter the signalling of the GLP-1R in response to orthosteric ligands. These effects can be different depending on which orthosteric ligand is co bound to the receptor, a effect known as probe-dependence. This thesis identifies a novel case of probe dependence, the ability of allosteric ligands to modify the signalling mediated by metabolites of endogenous ligands that were previously considered to be ‘inert’ breakdown products that may open up new avenues for allosteric drug discovery. It is widely accepted that insulin secretion downstream of GLP-1R activation is critically dependent on cAMP formation, but recent evidence is also emerging for an essential role of regulatory proteins such as β-arrestins and G protein-coupled receptor kinases (GRK). The canonical role of these regulatory proteins is to terminate GPCR signalling and promote receptor internalization. However, more recently, roles as scaffolding proteins that can regulate G protein-independent signalling have emerged. Consequently, the studies comprising this thesis illustrate distinct recruitment profiles of regulatory proteins to the GLP-1R in response to multiple endogenous and exogenous ligands. This thesis identifies differential actions of allosteric modulators on GLP-1R peptide ligands (‘probe dependence’), thus demonstrating differential responses of receptor signalling with respect to both orthosteric and allosteric ligands, highlighting the ability for both ligand- and pathway-specific effects (‘biased signalling’). Collectively, this work further demonstrates the potential benefits of biased signalling and allosteric modulation, but may also influence the approaches and precautions that must be considered in the design, identification and development of small molecules for therapeutic use.