Les secrétions de peau des grenouilles néo tropicales et sud américaines contiennent un large éventail de molécules biologiquement actives et notamment les peptides ayant des propriétés antimicrobiennes. Disposant de sécrétions de peau de la grenouille sud américaine du genre Phyllomedusa bicolor, nous avons recherché la présence molécules ayant des activités antitumoraleet angiostatique. Deux peptides cationiques antimicrobiens membres de la famille des dermaseptine (Drs) ont été identifies comme possédant ces activités: les dermaseptines B2 et B3 (Drs B2 et Drs B3). Ces deux peptides sont ainsi capables d’inhiber la prolifération et la formation de colonies de différentes lignées de cellules tumorales, la prolifération des cellules endothéliales ainsi que la formation de pseudo-capillaires in vitro. D’autre part, la Drs B2 est également capable d’inhiber la croissance des cellules tumorales dans un modèle in vivo dexénogreffe. L’exploration du mécanisme d’action de la Drs B2 sur les cellules tumorales PC3 nous a permis de mettre en évidence un rapide re-largage de la LDH cytosolique, une absence d’activation des caspases-3, -9 et -8 ainsi que l’absence de modification du potentiel de membrane mitochondriale. L’ensemble de ces données semble indiquer que la Drs B2 n’induit pas la mort de cellules tumorales par un mécanisme d’apoptose mais plus probablement par une fixation à la surface de cellules entraînant une lyse rapide de la membrane plasmique des cellules conduisant à une mort par nécrose. En conclusion, la Drs B2 est une molécule qui cible préférentiellement les cellules tumorales pour des concentrations efficaces de l’ordre du micro molaire, ce qui en fait un outil pharmacologique potentiellement intéressant pour le traitement du cancer. / The skin secretions of neotropical and South American frogs contains large amounts of a widerange of biological active molecules. Commonly studied are peptides with antimicrobialactivities. In this study we have postulated that the skin secretions from the South Americanfrog Phyllomedusa bicolor contain molecules with antitumor and angiostatic activities. Twowell known cationic alpha helical antimicrobial peptides of the dermaseptin (Drs) family wereidentified to have these activities: Drs B2 and Drs B3. Both peptides inhibited proliferationand colony formation of various tumor cell lines, and the proliferation and capillary formationof endothelial cell in vitro. Furthermore, Drs B2 inhibited tumor growth in a PC3 xenograftmodel in vivo.Research on the mechanism of action of Drs B2 on tumor cells PC3 demonstrated a rapidincreasing amount of cytosolic LDH, no activation of caspase-3, -9 or -8, and no changes inmitochondrial membrane potential. These data together indicate that Drs B2 does not act byapoptosis but possibly could fix to the tumor cell surface, disrupt the cellular plasmamembrane leading to its death by necrosis.In conclusion, Drs B2 could be an new interesting and promising pharmacological leadermolecule for the treatment of cancer. Its antitumor and angiostatic activities, especially itsselective targeting of tumoral cells with micro molar concentrations propose Drs B2 as anpotential candidate for the development of a new efficient targeting therapy against cancer.

This thesis deals with the analysis of natural peptide libraries using mass spectrometry. In the course of the study, both ribosomal and non-ribosomal classes of peptides have been investigated. Microheterogeneity, post-translational modifications (PTM), isobaric amino acids and disulfide crosslinks present critical challenges in routine mass spectral structure determination of natural peptides. These problems form the core of this thesis. Chapter 2 describes an approach where chemical derivatization, in unison with high resolution LC-MSn experiments, resulted in deconvolution of a microheterogenous peptide library of B. subtilis K1. Chapter 3 describes an approach for distinction between isobaric amino acids (Leu/Ile/Hyp), by the use of combined ETD-CID fragmentation, through characteristic side chain losses. Chapters 4-6 address a long standing problem in structure elucidation of peptide toxins; the determination of disulfide connectivity. Through the use of direct mass spectral CID fragmentation, a methodology has been proposed for determination of the S-S pairing schemes in polypeptides. Further, an algorithm DisConnect has been developed for a rapid and robust solution to the problem. This general approach is applicable to both peptides and proteins, irrespective of the size and the number of disulfide bonds present. The method has been successfully applied to a large number of peptide toxins from marine cone snails, conotoxins, synthetic foldamers and proteins. Chapter 7 describes an attempt to integrate next generation sequencing (NGS) data with mass spectrometric analysis of the crude venom. This approach couples rapidly generated cDNA sequences, with high-throughput LC-ESI-MS/MS analysis, which provides mass spectral fragmentation information. An algorithm has been developed that allows the construction of a putative conus peptide database from the NGS data, followed by a protocol that permits rapid annotation of tandem MS data. The approach is exemplified by an analysis of the peptide components present in the venom of Conus amadis, yielding 225 chemically unique sequences, with identification of more than 150 sites of PTMs. In summary, this thesis presents different methodologies that address the existing limitations of de novo mass spectral structure determination of natural peptides and presents new methodologies that permit for rapid and efficient analysis of complex mixtures.