| New drugs are urgently needed to treat human African trypanosomiasis (HAT), which is caused by protozoans of the species Trypanosoma brucei spp. Diamidines are a class of compounds that are active against a variety of pathogens, however, their mechanisms of antimicrobial action have yet to be resolved. The trypanocidal compound, 2,5-bis(4-amidinophenyl)furan (DB75), is a structural analogue of the aromatic diamidine drug, pentamidine. DB75 is the metabolite of the orally active prodrug, referred to as DB289, that is undergoing clinical trials as a new therapy for early stage HAT. The major objectives of this dissertation work were to determine mechanisms through which DB75 is trypanocidal and is taken up by bloodstream forms of T. b. brucei. In addition, mechanisms of DB75 resistance development were investigated in trypanosomes. Results using yeast as a model organism to investigate the mechanism of action of DB75 suggest that DB75 inhibits yeast mitochondrial function. For instance, yeast cells relying upon mitochondrial metabolism for energy production are especially sensitive to DB75. Also, DB75 localizes within yeast mitochondria, as evidenced by ultraviolet fluorescence microscopy, and collapses the mitochondrial membrane potential in isolated yeast mitochondria. Furthermore, addition of DB75 to yeast cells or isolated rat liver mitochondria results in immediate uncoupling of oxidative phosphorylation and subsequent inhibition of respiration. Results of experiments conducted in bloodstream-form trypanosomes indicate that the mitochondrion is also a cellular target of the trypanocidal action of DB75. DB75 appears to rapidly distribute within the trypanosome mitochondrion, as indicated by fluorescent colocalization of DB75 with a mitochondrion-specific dye. DB75 inhibits whole cell respiration, and dissipates the mitochondrial membrane potential of trypanosomes. Results of an investigation into the uptake of [3H]DB75 indicate that DB75 enters trypanosomes primarily through uptake mediated by the P2 amino-purine transporter and a secondary uptake route. A laboratory-derived T. b. brucei strain that is resistant to DB75 is defective in DB75 uptake, and is missing the gene encoding the P2 transporter. This work contributes to a better understanding of diamidine drug action, and to the successful development of the prodrug, DB289, as a new HAT therapy. |
