Abstract The enzyme phenylethanolamine N-methyltransferase (PNMT) catalyses the biosynthesis of adrenaline. Although adrenaline is a significant central nervous system (CNS) neurotransmitter, and has ...been associated with various physiological processes such as the control of blood pressure and the neurodegeneration observed in Alzheimer’s disease, its exact role in the CNS is unclear. As part of an international collaborative effort, this project aimed to develop PNMT inhibitors suitable for probing the role of CNS adrenaline, and to generate novel drug leads. Towards the goal of developing potent and selective PNMT inhibitors, this thesis utilised three general approaches. The first approach involved classical structure-guided drug discovery using X-ray crystallography, and is described in Chapter 2. Characterisation of the PNMT pharmacophore provided results that led to a new understanding of how PNMT recognises inhibitors. Structures described in this thesis revealed a cryptic binding pocket that is only revealed on binding of inhibitors that were predicted to be too large to interact with PNMT. The findings therefore demonstrated an extraordinary degree of flexibility inherent to the PNMT binding pocket, and emphasise the need to include greater protein flexibility in inhibitor design strategies. Secondly, this thesis investigated the catalytic mechanism of PNMT, described in Chapters 3 and 4. This research characterised the binding of substrates to wild type and variant PNMT, including the physiological substrate noradrenaline, and model substrates as well as substrate-analogue inhibitors of the enzyme. PNMT catalyses the methylation of a range of substrates. However, differential substitutions to these substrates can dictate the ligand binding position and thereby determine whether methyl transfer will occur. Additionally, the results provided new lessons for the routine use of point mutations in the study of enzymes, because changes are not always simply an indication of the difference in the residue functionality. I found, for example, that single site mutations can induce large movements in enzyme. Therefore structural characterisation of enzyme variants is an important addition to kinetic studies to enable a comprehensive examination of catalytic function. Finally, I have implemented a fragment based screening (FBS) approach to the discovery of novel lead compounds that inhibit PNMT, described in Chapters 5 and 6. The FBS approach has many advantages over existing drug discovery methods including higher hit rates, higher efficiency hits, and the ability to sample a larger range of chemical space. This thesis describes the application of FBS by X-ray crystallography to PNMT. The approach was used to screen a library of 384 compounds yielding 12 novel PNMT fragment leads. Furthermore, chemical elaboration and kinetic evaluation of these hits was performed in Chapter 7. In summary, this thesis has made a significant contribution to our understanding of the chemistry, kinetics and structure of PNMT. This understanding will be important in ongoing efforts to develop potent, selective, and CNS-active inhibitors of the enzyme.
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