Novel Antitumoral Strategies Inducing Membrane Lipid and DNA Damage:Artificial Chemical Nucleases & the ‘ClickGene’ Project

This PhD project was carried out within the frame of a Marie Curie Network with the acronym ‘ClickGene’, which focuses on the development of next-generation gene silencing therapeutics, such as the artificial metallo-nucleases (AMNs). So far, the molecular basis of the drugs mechanism has focused primarily on DNA damage, but recently the novel antitumoral strategies highlighted the cell membranes as a relevant site of the drug’s multitarget reactivity towards the unsaturated lipids. The PhD thesis is devoted to the study of the oxidation mechanisms of both lipids and nucleic acids induced by free radicals in the presence of AMN. The work first aimed to investigate the lipid reactivity in the presence of a metallo-drug. To simulate the cell membrane, a biomimetic model of liposomes was designed by different mono- and poly-unsaturated fatty acid moieties in order to examine the reactivity in the presence of the novel synthesized AMN and a reducing agent. A variety of conditions was tested giving an interesting mechanistic picture of the membrane-drug interaction. Connected with the fatty acid transformations, the novel synthesis and the full analytical characterization of the six mono-trans isomers of docosahexaenoic acid (DHA) was performed. Two different synthetic approaches were combined to obtain a full identification of the six mono-trans isomers and the analysis was based on GC and NMR for building-up a molecular reference library. In the second part of the project, the work focused on AMN ability for precise cleavage of DNA. In these experiments, the damage profiles were studied using major groove recognition elements and spin-trapping scavengers of ROS. The DNA damage fragments were purified and enzymatically digested to single nucleosides. Finally, the analytical protocol established by our group was applied to identify the drug effect regarding the formation of oxidative lesions, in double strand DNA fragments.