Snake venom proteins are potential sources for novel drug design both for treatment of snake bites and for human genetic disorders. To achieve these, the basic sequence, structure and functional relationships of venom proteins should be understood. Proteolytic enzymes such as metallo and serine proteases are the major components of venom in vipers and are responsible for local and systematic envenomation effects. We have purified, partially sequenced and functionally characterised a serine protease, rhinocerase from the venom of Bitis gabonica rhinoceros. Rhinocerase is a multifunctional enzyme with kininogenase, clotting and defibrase activities.

Each of the 500 known Conus species has its own distinctive, complex and peptide rich venom. Approximately 100 different peptide toxins, so called conotoxins can be expressed with little similarity between the species. An overwhelming majority of conotoxins are probably targeted selectively to specific ion channels. Because conotoxins can discriminate between closely related subtypes of ion channels, many of the characterised conotoxins are used as pharmacological agents in ion channel research. Several have direct diagnostic and therapeutic potential. Although the venoms have been studied for the last two decades, only a miniscule fraction of the postulated conotoxins have been identified. The studies presented in this dissertation were set out with the aim to further characterise the relative unknown venom of Conus ixntricosus and to explore the protein content of Conus venoms in general. In total five different venoms were investigated. Chapter three describes the analysis of protein content in various Conus venoms. Although both one and two-dimensional gel electrophoresis was applied, no known toxin precursors were found. Proteins identified are mainly housekeeping proteins, such as actin. One of the strategies applied for finding novel conotoxins involved a multi dimensional liquid chromatography experiment where a trypsin digest was performed in between two separations before mass spectrometric analysis. One putative novel partial conotoxin was found. Chapters five and six describe the mass spectrometric analysis of Conus ventricosus and textile venom after a two-dimensional liquid chromatography separation. With the methods applied, besides verifying a number of putative sequences, several novel conotoxins were found, in particular a number of so called contryphans. One of these contryphans found contained a number of post-translational modifications, amongst them hydroxyproline and the very rare brominated tryptophan.

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The term disintegrin was coined in the early 90s to describe a class of proteins found in snake venoms. The same term is used today for molecules that can recognise and bind to integrins: membrane receptors that are responsible for the activation of many vital pathways that lead to cell proliferation. By binding integrins, disintegrins can stop the internal cellular signalling, which can result in apoptosis, induced cell death. Laminin is a trimeric glycoprotein, found in the extracellular matrix, formed from three different chains: α, β and γ and is also involved in the binding of integrins. Recently it was shown that the N-terminal region of the β-chain of laminin incorporates a conserved sequence: YIGSR, located in a turn rich region, where its conformation is fixed by disulphide bridges, responsible to impose a biological conformation. This epitope was found to promote cell attachment, proliferation and migration. In this work we have looked into creating more stable and pronounced conformations of YIGSR mimetics. This has been achieved designing and synthesising the constructs in a cyclic fashion. The different cyclo-peptides obtained were than screened using celladhesion and migration assays, revealing that turn-like structures exhibit stronger activities. The secondary structure of these mimetics was probed by circular dichroism and NMR spectroscopy and was visualised by MD simulations, collectively suggesting the presence of a type II β-turn conformation proving that the latter is essential for the activity. The mimetics inhibited cell attachment and migration of several cancers lines indicating their potential as antimetastatic agents. In parallel, the potential use of these mimetics in regenerative medicine has been also investigated biomimetic matrices. These scaffolds assemble from polypeptide chains that fold into coiled coil structures, to give stable materials with fibrils that range from nano- to micrometres in length and 20- 50 nm in diameter. Introduction of the disintegrins-like peptides into such fibrous matrices, as cell-adhesion baits, stimulated cell proliferation and migration suggesting their use in tissue engineering and wound healing.

Scorpion venoms are rich sources of bioactive polypeptides. They are classified into two major groups: peptides with or without disulfide bridges. Toxins with disulfide bridges are responsible for toxicity of venom by targeting ion channels on cell membranes, while peptides without disulfide bridges are diverse in both structures and bioactivities, rendering them interesting to study. This project aimed to discover novel bioactive peptides from the venom of a South American scorpion, Tityus serrulatus. There are four highlights in this study. Firstly, two putative antimicrobial peptides (TsAP-l and TsAP-2) were identified in scorpion venom, by shotgun molecular cloning and RP-HPLC fractionation of venom. Their synthetic replicates were subjected to a series of bioactivity assays. TsAP- l exhibited low potency against all three test organisms, whereas TsAP-2 exhibited high potency against Gram-positive bacterium, Staphylococcus aureus, and the yeast, Candida albicans. Meanwhile, haemolytic and anti-proliferative activities of TsAP-l were low, while those of TsAP-2 were considerably higher. Secondly, two analogous peptides (TsAP-Sl and TsAP-S2), in which positive net charges and a-helicity increased, were designed by site substitutions in natural antimicrobial peptides. They both exhibited dramatic enhancement in antimicrobial, haemolytic and anti-proliferative activities, indicating the significance of cationicity and a-helical structure in bioactivities of antimicrobial peptides. Thirdly, two putative bradykinin-potentiating peptides (TsHpt-I and QUB 1397) were also identified in venom, and synthesised. In vitro pharmacological assays revealed that they could enhance bradykinin-induced hypotension on rat vascular tissues at micromole levels, although neither of them produced any significant effects on the same tissue. Finally, six cDNAs encoding putative classical disulphide-bridged neurotoxin precursors were also cloned from the same venom-derived cDNA library and were structurally-analysed. These extend knowledge of scorpion venom neurotoxins. All these findings suggest the value of scorpion venom in searching and designing potential drug candidates.

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they eo-encode bradykinin-potentiating peptides and in venomous lizards, some eo-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum) and the Gila Monster (Heloderma suspectum) and the structures of the novel peptides, helokinestatin-6, helokinestatin-7S and helokinestatin-7H, that have inhibitory effects on bradykinin responses in vascular smooth muscle. Snakes of the Elapidae and Viperidae families have evolved to produce venoms with specific well- defined modes of action that lead to prey immobilisation and ultimately death. The venoms of elapids contain neurotoxic components that attack both the peripheral and central nervous systems. In this project, dendrotoxin-K/I sequences from black mamba (Dendroaspis polylepis) venom were obtained, and these have inhibitory effects on the proteases, trypsin and chymotrypsin. The structures were obtained through use of a special primer in the 'shotgun cloning' of a venom- derived cDNA library. By this approach, the structure and precursor sequence of cardiotoxin-3 from the venom of the King Cobra, (Ophiophagus hannah) was also obtained. In contrast, viperid snake venoms contain components that are mainly haemorrhagic in nature or act upon the vascular system in other ways. Using the same molecular cloning strategy as adopted for elapid snakes, the structure of a novel C-type lectin was obtained from Puff Adder (Bitis arietans) venom and that of a novel PLA2 from the venom of the Cantil (Agkistrodon bilineatus).