Structural and mechanistic studies of novel selective agonists of the M1 muscarinic acetylcholine receptor

The M1 muscarinic acetylcholine receptor (mAChR) is an attractive therapeutic target for the treatment of dysfunctions in cognition and memory, such as those associated with Alzheimer’s disease and schizophrenia. However, like many G protein-coupled receptors, given the homology of the acetylcholine (orthosteric) site between the five subtypes of the mAChR, most drugs developed to target this site are poorly subtype-selective. A burgeoning area in the development of new subtype-selective drugs for GPCRs is the targeting of allosteric sites. Although numerous subtype-selective agonists have been identified for mAChRs, the mechanisms by which ligands engage with allosteric sites and activate the receptor have yet to be fully understood. With the recent appreciation of bitopic ligands that engage with both the orthosteric site and an allosteric site, the purely allosteric mechanisms of action purported of various selective agonists warrant reassessment. This thesis explores the bitopic mechanism of action of the M1 mAChR-selective agonist, TBPB, initially classified as an allosteric agonist. Adoption of a reverse engineering approach enabled analysis of the competitive orthosteric engagement of the receptor by TBPB and its respective fragment derivatives. These findings, along with the dissection of an allosteric binding moiety of TBPB, facilitated the redefinition of the agonist as a bitopic ligand. Subsequent comprehensive mutagenesis studies of the M1 mAChR, examining the role of amino acid residues of the orthosteric binding site and putative allosteric domains, provided further insight into the bitopic mode of action of TBPB. Alternate structural determinants of the binding and function of bitopic ligands, such as TBPB, in comparison to typical orthosteric ligands were identified and yielded a predicted mode of binding to the M1 mAChR. Overall, this thesis provides insight into the mechanisms of action of bitopic ligands, their detection and highlights the need to reassess the potential bitopic mechanisms of action of other putatively allosteric ligands.