Topoisomerase-II mediated biochemical mapping of centromeres and nitroreductase mediated drug metabolism in Trypanosomatids

The protozoan parasites Trypanosoma cruzi and Trypanosoma brucei are the causative agents of Chagas disease and Human African sleeping sickness respectively. Existing therapies are toxic and ineffective against the later stages of the two diseases, consequently safer, improved therapies are urgently required. Here, two areas of trypanosome biology are explored. In the first section, the process of cell division is approached from a fundamental biology perspective. Centromeres are the region of DNA where kinetochore structures form, allowing the attachment of microtubules to facilitate chromosome segregation. In T. brucei we have characterized the nature and location of centromeres by exploiting the localized activity of topoisomerase-II, a cancer chemotherapy target, at the centromere. Etoposide mediated DNA cleavage mapping revealed the presence of signature AT-rich repeat regions coupled with adjacent retrotransposons at the centromere. Further experiments demonstrate that of the two nuclear T. brucei topoisomerase-II isoforms, only topoisomerase-IIα is essential and active at the centromere. The second section centres on pro-drug development against a trypanosome type I nitroreductase. This enzyme has previously been implicated in activation of nifurtimox and benznidazole, the two therapies in clinical use against Chagas disease. Initially we have developed a luciferase based drug assay system in the clinically relevant intracellular T. cruzi stage and rapidly screened a range of nitroaromatic based compounds for trypanocidal activity. A series of derived nitrofuryl compounds previously developed against Chagas disease were also screened against T. brucei, where most demonstrate trypanocidal activities of less than 1 μM. Further we show that these compounds are active substrates of nitroreductase, and act as pro-drugs within the parasite by specific activation of nitroreductase to generate cytotoxic moieties.EThOS - Electronic Theses Online ServiceGBUnited Kingdo