Synthesis and pharmacological investigation of allosteric and bitopic ligands for the M₁ muscarinic acetylcholine receptor

This thesis describes the development of allosteric and bitopic molecular tools for the M₁ muscarinic acetylcholine receptor (M₁ mAChR), a potential therapeutic target for the treatment of cognitive deficits experienced in central nervous system (CNS) disorders. Chapter 1 presents a comprehensive review article that provides a rationale for targeting the M₁ mAChR and an account of the M₁ mAChR-selective allosteric and bitopic ligands that have emerged to date. The perspective at the end of Chapter 1 highlights the ongoing complexities encountered in the advancement of allosteric and bitopic ligand drug discovery. This perspective serves as a basis for the research undertaken in this thesis, as we contend that the development of molecular tools that are able to serve as structural and functional probes may contribute to the resolution of these complexities. Chapters 2 and 3 describe the synthesis and pharmacological evaluation of a panel of putative irreversible allosteric ligands based on the structure of benzyl quinolone carboxylic acid (BQCA), the first reported orally-bioavailable M₁ mAChR allosteric ligand exhibiting absolute subtype selectivity. From this research, an electrophilic irreversible allosteric ligand MIPS1262 (11, Chapter 2) and a photoactivatable irreversible allosteric ligand MIPS1455 (4, Chapter 3), were developed, affording novel molecular tools for further probing allosteric interactions at the M1 mAChR. Chapter 4 details the development of novel putative bitopic ligands for the M1 mAChR, including the selection of the constituent ligands (iperoxo and BQCA), the design and synthetic incorporation of linkers into their respective structures, and the synthesis and preliminary pharmacology of two putative bitopic ligands (33a and 44, Chapter 4). Such ligands may prove useful in investigating specific structural and functional hypotheses at the M1 mAChR, in addition to presenting as a prospective therapeutic approach for the treatment of cognitive deficits. In Chapter 5, known BQCA analogues exhibiting improved properties compared to the parent molecule were synthesized and subjected to more detailed pharmacological techniques and analysis than have been previously reported. Briefly, we demonstrate that it may be possible to introduce structural modifications into allosteric ligands to specifically ‘fine-tune’ individual pharmacological properties and use such molecules as in vitro and in vivo tools to delineate the requirements for optimal therapeutic effect without side effects. A diverse toolbox of ligands were synthesized and pharmacologically validated for use in future studies of the M₁ mAChR. A discussion of potential directions for future research is provided in Chapter 6

Synthesis and pharmacological investigation of allosteric and bitopic ligands for the M₁ muscarinic acetylcholine receptor

This thesis describes the development of allosteric and bitopic molecular tools for the M₁ muscarinic acetylcholine receptor (M₁ mAChR), a potential therapeutic target for the treatment of cognitive deficits experienced in central nervous system (CNS) disorders. Chapter 1 presents a comprehensive review article that provides a rationale for targeting the M₁ mAChR and an account of the M₁ mAChR-selective allosteric and bitopic ligands that have emerged to date. The perspective at the end of Chapter 1 highlights the ongoing complexities encountered in the advancement of allosteric and bitopic ligand drug discovery. This perspective serves as a basis for the research undertaken in this thesis, as we contend that the development of molecular tools that are able to serve as structural and functional probes may contribute to the resolution of these complexities. Chapters 2 and 3 describe the synthesis and pharmacological evaluation of a panel of putative irreversible allosteric ligands based on the structure of benzyl quinolone carboxylic acid (BQCA), the first reported orally-bioavailable M₁ mAChR allosteric ligand exhibiting absolute subtype selectivity. From this research, an electrophilic irreversible allosteric ligand MIPS1262 (11, Chapter 2) and a photoactivatable irreversible allosteric ligand MIPS1455 (4, Chapter 3), were developed, affording novel molecular tools for further probing allosteric interactions at the M1 mAChR. Chapter 4 details the development of novel putative bitopic ligands for the M1 mAChR, including the selection of the constituent ligands (iperoxo and BQCA), the design and synthetic incorporation of linkers into their respective structures, and the synthesis and preliminary pharmacology of two putative bitopic ligands (33a and 44, Chapter 4). Such ligands may prove useful in investigating specific structural and functional hypotheses at the M1 mAChR, in addition to presenting as a prospective therapeutic approach for the treatment of cognitive deficits. In Chapter 5, known BQCA analogues exhibiting improved properties compared to the parent molecule were synthesized and subjected to more detailed pharmacological techniques and analysis than have been previously reported. Briefly, we demonstrate that it may be possible to introduce structural modifications into allosteric ligands to specifically ‘fine-tune’ individual pharmacological properties and use such molecules as in vitro and in vivo tools to delineate the requirements for optimal therapeutic effect without side effects. A diverse toolbox of ligands were synthesized and pharmacologically validated for use in future studies of the M₁ mAChR. A discussion of potential directions for future research is provided in Chapter 6

Development of a Photoactivatable Allosteric Ligand for the M₁ Muscarinic Acetylcholine Receptor

The field of G protein-coupled receptor drug discovery has benefited greatly from the structural and functional insights afforded by photoactivatable ligands. One G protein-coupled receptor subfamily for which photoactivatable ligands have been developed is the muscarinic acetylcholine receptor family, though, to date, all such ligands have been designed to target the orthosteric (endogenous ligand) binding site of these receptors. Herein we report the synthesis and pharmacological investigation of a novel photoaffinity label, MIPS1455 (<b>4</b>), designed to bind irreversibly to an allosteric site of the M₁ muscarinic acetylcholine receptor; a target of therapeutic interest for the treatment of cognitive deficits. MIPS1455 may be a valuable molecular tool for further investigating allosteric interactions at this receptor

Synthesis and Pharmacological Evaluation of Analogues of Benzyl Quinolone Carboxylic Acid (BQCA) Designed to Bind Irreversibly to an Allosteric Site of the M₁ Muscarinic Acetylcholine Receptor

Activation of the M₁ muscarinic acetylcholine receptor (mAChR) is a prospective treatment for alleviating cognitive decline experienced in central nervous system (CNS) disorders. Current therapeutics indiscriminately enhance the activity of the endogenous neurotransmitter ACh, leading to side effects. BQCA is a positive allosteric modulator and allosteric agonist at the M₁ mAChR that has high subtype selectivity and is a promising template from which to generate higher affinity, more pharmacokinetically viable drug candidates. However, to efficiently guide rational drug design, the binding site of BQCA needs to be conclusively elucidated. We report the synthesis and pharmacological validation of BQCA analogues designed to bind irreversibly to the M₁ mAChR. One analogue in particular, <b>11</b>, can serve as a useful structural probe to confirm the location of the BQCA binding site; ideally, by co-crystallization with the M₁ mAChR. Furthermore, this ligand may also be used as a pharmacological tool with a range of applications

Adopting an active learning approach to teaching in a research-intensive higher education context transformed staff teaching attitudes and behaviours

The conventional lecture has significant limitations in the higher education context, often leading to a passive learning experience for students. This paper reports a process of transforming teaching and learning with active learning strategies in a research-intensive educational context across a faculty of 45 academic staff and more than 1000 students. A phased approach was used, involving nine staff in a pilot phase during which a common vision and principles were developed. In short, our approach was to mandate a move away from didactic lectures to classes that involved students interacting with content, with each other and with instructors in order to attain domain-specific learning outcomes and generic skills. After refinement, an implementation phase commenced within all first-year subjects, involving 12 staff including three from the pilot group. The staff use of active learning methods in classes increased by sixfold and sevenfold in the pilot and implementation phases, respectively. An analysis of implementation phase exam questions indicated that staff increased their use of questions addressing higher order cognitive skills by 51%. Results of a staff survey indicated that this change in practice was caused by the involvement of staff in the active learning approach. Fifty-six percent of staff respondents indicated that they had maintained constructive alignment as they introduced active learning. After the pilot, only three out of nine staff agreed that they understood what makes for an effective active learning exercise. This rose to seven out of nine staff at the completion of the implementation phase. The development of a common approach with explicit vision and principles and the evaluation and refinement of active learning were effective elements of our transformational change management strategy. Future efforts will focus on ensuring that all staff have the time, skills and pedagogical understanding required to embed constructively aligned active learning within the approach