Synthesis, Structure and Activity of Disulfide-Rich Conus Peptides

There is great interest in investigating structural and activity relationships between conotoxins and their biological targets. The information obtained using these techniques has added to our knowledge of how these molecules interact with receptors, and allowed us to pursue the synthesis of more selective and/or potent analogues. Chapters 1 and 2 serve as an introduction to the field of conotoxin research, and to the diversity of biological targets for which they are selective. They also give a general introduction to solid-phase peptide synthesis, the powerful tool used for the synthesis of peptides and analogues used in this study. Chapter 3 contains the materials and methods used throughout this thesis. Common methods used throughout the thesis have been included in one chapter to help minimise repetition. A brief introduction to many of the specialised techniques encountered is also given. Chapter 4 involves the SAR study of á-Ctx [A10L] PnIA through alaninescanning mutagenesis. Peptides were synthesised and their affinity for the á7 nAChR evaluated in a rat brain homogenate, and potency was determined with electrophysiological studies. This provides information as to which residues are important for interacting with the á7 subtype of the neuronal nicotinic acetylcholine receptor. Preliminary structural studies were performed to determine whether structural changes accompanied any loss in affinity of the peptide to the receptor, to distinguish important interactions from structural perturbations. Chapter 5 probes the importance that the side-chain of position 10 has on affinity to the á7 nAChR. A series of seventeen point mutations were synthesised, with side chains containing aromatic, aliphatic, polar and charged residues. Previous work demonstrated that the side-chain at position 10 could discriminate between two different subtypes of the nAChR, therefore a functional screen was performed to estimate the activity and selectivity of these mutated peptides at different subtypes of the receptor. Chapter 6 describes the synthesis and structural studies of á-conotoxin ImII, a conotoxin that is functionally active at the á7 nAChR, but does not bind to the classical conotoxin binding site. It is the only á-conotoxin described to date that does not contain a proline residue at position 6, and makes an excellent candidate for structural studies. Chapter 7 investigates a novel method for the synthesis of conotoxins from the O-superfamily. These conotoxins all contain adjacent cysteine residues located between the second and third loops, making them excellent targets for synthesis by native chemical ligation of two peptide segments. This novel strategy was applied to synthesise chimeras of N-type VGCC selective CVID and P/Q-type VGCC selective MVIIC, two peptides that are pharmacologically well characterised. It is hoped that this methodology will enable synthesis of peptides that have either been difficult to synthesise or oxidise in the past. The overall aim of this thesis is to investigate the structure and activity of some disulfide-rich conus peptides, and determine the residues responsible for selectivity and affinity toward their biological target. The information obtained from this study will further our knowledge of how these peptides interact with these receptors and receptor subtypes. Subtype selective ligands can be used as pharmacological tools to dissect and characterise the roles individual subtypes play in the complex mixtures of receptors found in native tissues