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Title: Investigating the mode of action of AN5568, a novel therapeutic against African trypanosomiasis
Author: Steketee, Pieter Christiaan
ISNI:       0000 0004 5917 2237
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2016
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The protozoan parasite Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT) and Nagana disease in mammals. These diseases present a major socioeconomic burden to large areas of sub-Saharan Africa. Current therapeutics involve complex and toxic regimens which can lead to fatal side-effects. In addition, there is evidence for drug resistance emerging in the field. Hence, there is a desperate need for novel therapies. Benzoxaboroles are a novel class of boron-containing compounds under development for use against a wide spectrum of diseases. AN5568 is a lead compound for the treatment of HAT, which has demonstrated effective clearance of both early- and late-stage trypanosomiasis in a murine model, and is currently undergoing clinical trials. However, the mechanism by which AN5568 kills T. brucei is elusive. In this study we sought to use 'omics'-based techniques to investigate the mode of action of AN5568 in a laboratory strain of Trypanosoma brucei brucei. Cells treated with the benzoxaborole showed significant perturbations in methionine metabolism. In particular, there were increases in S-adenosyl-L-methionine, an essential methyl group donor involved in methyltransferase reactions. These changes were similar to those elicited by the nonspecific methyltransferase inhibitor sinefungin. Changes were also observed in lipid metabolism, sugar nucleotide metabolism and glycophosphatidylinositol biosynthesis. Further analyses were carried out to investigate the effect of AN5568 on cellular stress responses and cell morphology. To further probe the mechanics of AN5568-treatment, a drug-resistant cell line was generated. This cell line showed cross-resistance with sinefungin, further supporting similar modes of action for these two drugs. Interestingly, the AN5568-resistant cell line exhibited upregulation of procyclic form-specific genes, as well as downregulation of blood-stream form-specific genes, which led to the hypothesis that the cell line had undergone a differentiation event. However, microscopy analysis showed that overall morphology of the cells still resembled those of bloodstream forms, despite them having acquired a procyclic-like metabolic physiology. A secondary aim of this project was to elucidate the metabolic changes that lead to increased growth rates in T. brucei cells undergoing loss-of-heterozygosity on chromosome 10. This phenomenon, whereby a significant portion of the chromosome is lost, has been observed independently on multiple occasions in lab adapted T. brucei strains, yet how this alteration affects intracellular metabolism was hitherto unknown. Using two procyclic T. brucei cell lines, this study was able to show that the increased growth rates are glucose-dependent with a potential intracellular alteration in succinate and acetate production. These data have important implications for the field, where LOH has been observed in the clonally expanding T.b. gambiense type I.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology