This thesis focuses on the study of α-conotoxins, a class of disulfide rich peptides found in the venom of carnivorous marine snails from the genus Conus.
Possessing an ability to specifically antagonise nicotinic acetylcholine receptors, not only in Conus
prey species but also in mammals, these peptides have potential as therapeutics for the treatment of a plethora of disease states. These include, for example, nicotine-related diseases of the lungs, such as small cell lung carcinoma, and neurological disorders, such as epilepsy, depression, addiction and chronic pain. This thesis is concerned with structural studies of α-conotoxins using 1
H nuclear magnetic resonance spectroscopy. Fundamental questions regarding α-conotoxin structure and the suitability of various modifications for improving the therapeutic potential of α-conotoxins are addressed.
Satisfying the first aim of this work, the structure of α-conotoxin AuIB is presented. This peptide has different disulfide spacing from all other α-conotoxins for which structures had been determined previously. As a result, determination of the structure of AuIB completed the set of α-conotoxin structures and allowed a thorough examination of the effects of disulfide bond spacing on α-conotoxin structure. Further to this study, the disulfide bond isomers of AuIB were synthesised and the structure of the most stable isomer determined. The structure of the first loop of this isomer, the "ribbon" isomer, was very similar to the corresponding region of the native toxin. Remarkably, the ribbon isomer was found by a collaborator to exhibit greater antagonistic activity at rat parasympathetic neurons than native AuIB. This was the first time that the activity of a conotoxin had been shown to increase as a result of altering disulfide bond connectivity. Attempts are made in this thesis to explain this unexpected result in structural terms.
One of the common problems with using peptides as drugs is their intrinsic susceptibility to degradation in the blood and gastrointestinal tract. A cyclic peptide backbone is believed to be an important factor in conferring the exceptional biological stability of a class of plant peptides known as the cyclotides. Taking inspiration from this peptide family, the structural effects of cyclisation are examined for four cyclic analogues of α-conotoxin 1ml. This was the first time that α-conotoxins had been cyclized and structural studies of these peptides has highlighted a number of design considerations that will allow further development of this strategy in the future. Another limitation for the use of α-conotoxins, and peptides in general, as drugs is that they have a poor ability to cross the blood brain barrier. A study of the structural effects of adding a lipoamino acid group to α-conotoxin Mil is presented. One of the analogues shows particular potential, as it exhibited no significant structural change from the native peptide.
While α-conotoxins have a range of possible medical uses they also have prospective use as agrochemicals, as some are active at the nicotinic acetylcholine receptors in insects. In addition, there is potential for grafting the active sites from larger antimicrobial or insecticidal proteins onto the α-conotoxin framework. In the last chapter of this thesis the idea of producing transgenic plants encoding cyclic analogues of α-conotoxins or α-conotoxin based peptides is contemplated. As the production of recombinant cyclic peptides in vivo has only been achieved in bacterial systems it is of interest to investigate the natural process of cyclic peptide production in plants. Prior to the study presented in this thesis it had been established by collaborators that in Oldenlandia affinis the plant cyclotides were gene encoded. cDNA clones had not been isolated from other species. In this study cDNA clones were isolated from a distantly related plant Viola odorata. Comparison of the predicted translations of the O. affinis and V. odorata clones allowed the sites of processing of the cyclotide sequences from the precursor protein to be identified. The insights from this study allowed a basic model of how the cyclotides are processed in vivo to be determined.
In summary, this thesis explores the effects of disulfide bonding on α-conotoxin structure and their considerable potential as therapeutics and agrochemicals. The findings of this thesis form a basis for the further development of cyclic backbone and lipid-modified α-conotoxins and bring us closer to developing technologies that will facilitate the expression of cyclic analogues of α-conotoxins in plants.