Hay, DBrimble, MBower, Rebekah2017-02-272017http://hdl.handle.net/2292/31980Amylin is a 37 amino-acid neuroendocrine peptide hormone produced in the pancreatic β-cells alongside insulin. Food ingestion stimulates the release of both amylin and insulin for the swift restoration of blood-glucose homeostasis. In addition to its glucoregulatory effects, amylin is also a satiety-inducing hormone, stimulating the perception of fullness and causing the discontinuation of feeding. The physiological activities of amylin are mediated by amylin receptors, which are comprised of the family B G protein-coupled receptor (GPCR), the calcitonin receptor (CTR), in complex with one of three single transmembrane receptor activity-modifying proteins (RAMPs) 1, 2 or 3. The beneficial biological activity of amylin prompted investigation into is clinical potential as an anti-diabetic and anti-obesity therapy and led to the development of the amylin analogue pramlintide, which is now an FDAapproved drug for the treatment of insulin-requiring diabetes. However, there is still room for improvement on amylinmimetic drug therapies. In order to realise the full potential of the amylin system for optimal drug design it is important to investigate how the peptides structure translates to its biological function to help guide drug development efforts. The paradigm for family B GPCR peptide ligands for receptor binding and activation is called the 2-domain model. In this model, the C-terminus of the peptide is required for receptor binding, and the Nterminus stimulates receptor activation. Based on this model, this thesis aimed to investigate the contribution of important N-terminal structural elements and the role of the first 17 N-terminal residues of human amylin peptide on receptor activation and binding. This was done by employing peptide synthesis and purification, combined with biological analysis of these peptide analogues in cell-based assays in vitro at three amylin-responsive receptors. In Chapter 3, the role of the N-terminal disulphide loop of human amylin was interrogated for its impact on receptor activity by synthesising and testing broken-ring and truncated analogues. In Chapter 4, individual residues within the N-terminal disulphide loop were substituted with alanine or glycine and tested for biological activity to ascertain their contribution to receptor activation and binding. In Chapter 5, native residues beyond the loop within the proposed 8-17 alpha helical region of human amylin were individually substituted with alanine or glycine to discern their contributions on potency and binding at amylin-responsive receptors. Chapter six included the synthesis of more targeted peptide modifications including a single-residue replacement to investigate potential selectivity residue between related peptides. In Chapter 6, a truncated 1-17 N-terminal fragment was also synthesised and tested as well as a pramlintide analogue, based on results from Chapter 5. The research in this thesis presents novel information on the molecular contribution of N-terminal residues of human amylin to receptor activation and binding. The N-terminal disulphide loop structure was important but not critical for function. Truncated and broken-loop analogues still activated all three amylin-responsive receptors, although with greatly reduced potency and affinity. Only one specific residue within the loop was important for receptor activity, partially as a result of reduced binding affinity at all three receptors. Residues beyond the Nterminal disulphide loop were also important with reduced activity and/or binding at all three receptors for many analogues. However, some single alanine analogues unexpectedly increased the potency and/or affinity of the peptide at one or more of the three receptors tested. The information presented herein can help guide better informed amylinmimetic drug development strategies for treatments of metabolic disease.Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher.https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htmhttp://creativecommons.org/licenses/by-nc-sa/3.0/nz/ThesisCopyright: The authorhttp://purl.org/eprint/accessRights/OpenAccessQ112932017