Novel, Selective Amylin Agonists as Therapeutic Tools to Treat Diabetes

Abstract

Amylin is a 37 amino acid (AA) peptide hormone that is co-secreted with insulin from pancreatic β-cells after food ingestion. Amylin displays glucoregulatory effects, and is also involved in the reduction of body weight through inducing satiety and decreasing body adiposity. These biological actions mean that amylin offers significant potential for the treatment of obesity and diabetes. The aggregative properties of human amylin (hAmylin) make it an unsuitable therapeutic. The non-aggregating rat amylin (rAmylin) peptide sequence has been exploited to produce an FDA-approved amylin mimetic, pramlintide, for insulin-requiring diabetics. Pramlintide differs from hAmylin in that AAs 25 (Ala), 28 (Ser), and 29 (Ser) were replaced with Pro. This prevents the peptide from forming cytotoxic amyloid fibrils, and renders it a useful therapeutic agent. Despite this, pramlintide has a number of shortcomings as a drug. For instance, pramlintide has a short half-life (t1/2) of 60 min, and is formulated as a subcutaneous thrice daily injection. Hence, there is still room for improvement in designing amylin mimetic drug therapies. Amylin's actions are mediated by amylin receptors, which are comprised of the core family B G protein-coupled receptor (GPCR), the calcitonin receptor (CTR), in complex with one of three receptor activity-modifying proteins (RAMPs). As the C-terminus of amylin is predicted to bind to the extracellular domain (ECD) of the receptor we predicted this region would contain sites that could tolerate modification to the peptide sequence. However, there is little information on how individual C-terminal AAs contribute to receptor activity, binding, and selectivity. Accordingly, this thesis aimed to investigate the role of the C-terminal AAs of the hAmylin peptide, in receptor binding, activity, and selectivity. This information was then used to synthesise a library of peptidomimetics of pramlintide, sharing 83% sequence similarity to hAmylin in this C-terminal region of the peptide. Peptide analogues were then tested in in vitro cell based assays at three amylin-responsive receptors. In Chapter 2, an Ala scan was conducted on the C-terminal 12 residues of hAmylin, combined with exchanging the C-terminal residue with related hormones calcitonin (CT) and CT gene related peptide (CGRP), and substituting the C-terminal amide for an acid. This resulted in the identification of key residues of hAmylin for binding, activity, and selectivity. Furthermore, residues unimportant for receptor activity and binding were discovered to allow for the synthesis of peptidomimetics of pramlintide. Using the information provided from Chapter 2, libraries of peptidomimetics of pramlintide were created, modified by glycosylation (Chapter 3) and lipidation (Chapter 4). In Chapter 5, the successful synthesis of dual agonists of the GLP-1 and amylin receptors (DAGARs) was carried out. All of the analogues from chapters 3 through 5 were tested for receptor activity at one or more receptor(s) and were found to retain high potency amylin receptor agonism. The research described in this thesis provides new information about the contribution of C-terminal AAs on hAmylin receptor binding, activity, and selectivity. Furthermore, the synthesis and biological characterisation of several novel amylin mimetics was carried out. These modified amylin mimetic peptides are expected to improve the pharmacokinetic properties of pramlintide, and thus have the potential to become treatments for metabolic disease, or help guide the future discovery of amylin mimetic peptides.