Pharmacological and structure-function studies of M₁ muscarinic acetylcholine receptor allosteric modulation

The M₁ muscarinic acetylcholine receptor (mAChR) is predominantly expressed in the brain where it plays a major role in mediating cognitive processes such as learning and memory. As a result, it has been implicated in diseases where such processes are impaired, such as Alzheimer’s disease and schizophrenia. Drug discovery efforts aimed at developing selective ligands for this receptor, both as therapeutics and as experimental tools, have largely failed as they focused on targeting the acetylcholine (ACh) binding site, which is identical in all five mAChR subtypes. The discovery of benzyl quinolone carboxylic acid (BQCA), the first positive allosteric modulator (PAM) with high selectivity for the M₁ mAChR, has lead to a renaissance in selective targeting of this receptor family. In chapter 2 we exploit the unique “two-state” pharmacology of BQCA to investigate allosteric modulation at a chemogenetically modified M1 mAChR, developed as an alternative means to achieve selective receptor targeting in vivo. This study demonstrates that such an approach may not be valid, as chemogenetic modification of the M₁ mAChR leads to changes in the allosteric behaviour of BQCA that are not reminiscent of its behaviour at the native receptor. As a consequence, caution must be exercised when interpreting studies of allosteric modulation using chemogenetically modified receptors in vivo. Despite the unique pharmacology of BQCA, the molecular mechanisms of its binding and function and the structural basis of its M₁ mAChR selectivity remain poorly defined. Such knowledge would enable the design of novel M₁ mAChR PAMs with improved pharmacological profiles. Chapters 3 and 4 comprise studies focussed on identifying the amino acid residues that form the allosteric binding pocket at the M₁ mAChR and/or play a role, either directly or indirectly, in the transmission of cooperativity with the orthosteric (ACh) binding site. Deeper mechanistic insights into allosteric modulation at the M₁ mAChR are further afforded by the use of benzoquinazolinone 12, a high affinity structural derivative of BQCA. The experimental findings are contextualised using molecular models, and collectively, the results suggest that many of the key residues that form the allosteric binding pocket at the M₁ mAChR are structurally conserved in other mAChR subtypes. The findings in this thesis challenge the common assumption that allosteric ligands achieve subtype selectivity through binding to allosteric sites that are less conserved between subtypes and propose that the selectivity of BQCA and benzoquinazolinone 12 arises from selective cooperativity with ACh at the M₁ mAChR. The information herein may guide the rational design of M₁ mAChR positive and/or negative allosteric ligands with increased therapeutic potential