Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a devastating disease that affects millions of people in the poor nations of sub-Saharan Africa. There is a great need for new treatments of this disease, as only four drugs are currently available, and all are associated with toxicity and other problems. The diamidine, pentamidine, has long been used for the treatment of trypanosomiasis, despite its toxicity and lack of knowledge of its mechanism of action. Drug development efforts have recently focused on creating less toxic, more potent diamidine derivatives of pentamidine with an orally bioavailable formulation. DB289, the prodrug of DB75, is in Phase III clinical trials as an oral treatment of HAT. Almost 2000 diamidines and related compounds have been synthesized, many with potent anti-trypanosomal activity. Although DB75 and other diamidines are potent DNA binders, the overall mechanism of action of this series of compounds is unknown. The focus of this research is to investigate diamidine accumulation and distribution in trypanosomes in vitro and in vivo. Diamidines such as DB75 accumulate to high concentrations in vitro and in vivo in trypanosomes, and localize to the kinetoplast and nucleus, which contain DNA, as well as the acidocalcisomes. The increase in treatment failures of anti-trypanosomal compounds has been a major concern in recent years, especially with melarsoprol. Several trypanosome lines have been developed which are resistant to DB75 in the laboratory, and methods developed to investigate intracellular accumulation and distribution were extended to these lines. These lines lack the P2 transporter, thought to be the main uptake transporter for DB75. DB75 accumulation was lower in the resistant lines, with intracellular distribution also altered. Total accumulation, however, was substantial in these lines, despite lack of the major uptake transporter described for this compound. Benzofuran and benzimidazole diamidines were also investigated. The benzofuran series was found to accumulate in non-DNA containing organelles, which may represent a new mechanism of action for these diamidines. Together, the studies in this dissertation have proven that diamidines selectively accumulate in trypanosomes and that there is likely more than one mechanism of action for diamidines
