Human African trypanosomiasis (HAT), a lethal disease caused by infection with Trypanosoma brucei rhodesiense or Trypanosoma brucei gambiense, affects a significant number of people in sub-Saharan Africa. Related trypanosome species are also responsible for veterinary trypanosomiasis in many species of domestic and wild animals, causing disruption to agricultural and economic development in one of the world’s poorest areas. Current control and management of the disease mainly relies on a handful of trypanocides that have been in use for over 50 years. The treatment is associated with numerous of limitations such as drug toxicity, affordability and, most worryingly, increasing rates of treatment failure due to spread of parasites resistant to the existing drugs. Therefore, development of new drugs against Human African Trypanosomiasis is much needed. Trypanosomes are incapable of de novo synthesis of the purine rings, and since purines are essential for many cellular functions (e.g. nucleic acid synthesis, energy metabolism) this means the parasites have an absolute requirement for exogenous purines. Several nucleoside and nucleobase transporters have been identified in T. brucei brucei, including the P1 and P2 adenosine. These transporters have recently received a great deal of interest as potential drug targets as possible determinants of drug resistance. The P2 nucleoside transporter is one of the best-studied transporters and it has been shown to facilitate entry of arsenical and diamidine trypanocides, such as melarsoprol and pentamidine. Loss of this transporter appears to confer resistance to some of these drugs in vitro. It has been shown that pentamidine is taken up by two additional transporters named high affinity pentamidine transporter (HAPT1) and low affinity pentamidine transporter (LAPT1). These transporters have been studied based on their kinetic role of drug uptake and neither has been characterised at the molecular level. The main aims of the project were to study the HAPT1 transporter in detail, including identification of substrate recognition determinants, involvement in transport of diamidine trypanocides other than pentamidine, and the cloning of the genes coding for HAPT1 and /or LAPT1 from T. b. brucei. It was found that the main veterinary drug, the diamidine diminazene, was also transported by HAPT1, albeit much less efficiently than pentamidine. This provided new insights into the causes of drug resistance for African trypanosomes, in particular, the well-documented cross-resistance between melaminophenyl arsenical drugs and diamidines. A large number of diamidine analogues and other compounds were tested for inhibition of HAPT1 and their inhibition constants were determined. This identified the essential characteristics for high affinity interaction between substrate and the transporter-binding pocket. This study will aid the design of new ligands and inhibitors for this drug transporter. Furthermore, the gene for HAPT1 was identified as AT-E, a close analogue of TbAT1, which encodes for the P2 transporter. Two distinct alleles, named AT-E1 and AT-E2, were identified in wild-type T. b. brucei, probably representing a single gene locus. Knockdown of AT-E expression using RNAi significantly decreased HAPT1 in both bloodstream forms and procyclics. Expression of AT-E was reduced in the B48 pentamidine resistant line that lacks HAPT-mediated drug uptake. This project has greatly increased our understanding of the biochemical and molecular nature of HAPT1 transporters in T. b. brucei.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:533598 |
| Date | January 2011 |
| Creators | Teka, Ibrahim |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/2640/ |
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