Structure-Function Analysis of Orthosteric, Allosteric and Bitopic Ligand Binding at the Adenosine A1 Receptor

The adenosine A₁ G protein-coupled receptor (A₁AR) is an attractive therapeutic target for a range of cardiovascular and neuronal disorders. However, like many G protein-coupled receptors (GPCR), the A₁AR remains sub-optimally targeted due to the lack of highly subtype-selective agonists and allosteric modulators – despite the fact that this is the first GPCR for which synthetic small molecule allosteric enhancers were discovered over two decades ago. Recent development of bitopic ligands that engage with both the orthosteric site and an allosteric site can offer considerable potential for greater receptor selectivity, greater affinity and promote unique receptor conformations that engender biased agonism. To aid the rational design of more efficacious A₁AR therapeutics, the focus of this thesis is to gain greater structural knowledge of the orthosteric, allosteric sites as well as the mechanisms by which bitopic ligands engage both sites of the A₁AR and to develop an VLS approach to identify novel A₁AR allosteric modulators. In particular, Chapter 2 and 3 combined alanine scanning mutagenesis of the A₁AR-ECL2 with quantitative analytical pharmacology, homology modeling and molecular dynamics simulations to explore the role of this domain on the key underlying molecular properties governing A₁AR orthosteric and allosteric ligand drug actions, namely, affinity, efficacy and cooperativity. With the recent appreciation of VLS as a more direct and rational drug discovery approach to screen large libraries of chemical structures compared with the traditional experimental HTS, Chapter 4 developed an approach to optimize the allosteric pocket which can be used to future large-scale VLS of commercially available compounds to identify novel scaffolds for A₁AR allosteric modulators in the absence of an A₁AR crystal structure. A novel A₁AR bitopic ligand, VCP746, was reported to have a biased agonist profile relative to a number of adenosine-like prototypical A₁AR agonists and promoted A₁AR-mediated cardioprotection in the absence of effects on atrial heart rate at the A₁AR. Chapter 5 provides insight into the molecular determinants involved in the affinity and engagement of signaling of this bitopic ligand.