The role of the P2 purine transporter in drug uptake and resistance in trypanosomes

Stewart, Mhairi L (2003) The role of the P2 purine transporter in drug uptake and resistance in trypanosomes. PhD thesis, University of Glasgow.

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Abstract

Human African Trypanosomiasis (HAT), caused by the parasitic trypanosomatids T. b. gambiense and T. b. rhodesiense, is endemic in over 36 countries in sub-Saharan Africa. Estimates place the numbers of infected individuals at around 500,000 with a further 60 million at risk. Treatment for HAT is available, although all the drugs in use have problems with availability, administration and disease stage specificity, and most cause very severe side effects. The arsenical based trypanocides are extremely toxic, causing death due to treatment in up to 10% of patients. Reports of drug resistance in the field are wide spread. A few reports have suggested the involvement of the P2 transporter in the development of drug resistance. Point mutations within TbAT1, which encodes the P2 transporter, have been linked to drug resistant isolates in the field, and the loss of function of P2, through various molecular processes are a feature of laboratory derived drug resistant cell lines. However, the loss of P2 transport in a DeltaTbat1 cell line does not produce the high levels of resistance seen in laboratory derived drug resistant cell lines, and many isolates from relapse patients have shown no difference in TbAT1 indicating that the situation in the field regarding resistance and P2 transport is very complicated, and that other routes to resistance exist. Nonetheless, an exponential increase in the prevalence and distribution of HAT, coupled with an increased incidence of reported drug resistance, makes the development of new trypanotoxic compounds vital. The exploitation of the P2 purine transporter as a parasite-specific entry point for new trypanocidal compounds has been investigated in this thesis. The P2 transporter is ideal for this approach to drug design due to its high affinity for substrates carrying the primary recognition motif, H2N-C(R1)=N-R2, the lack of any similar motif on mammalian purine transporters and its natural substrates being at very low concentrations within the bloodstream of the host. Melamine and benzamidine groups, which carry the recognition motif for the P2 transporter, were attached to potentially cytotoxic moieties (polyamine analogues, HIV gp120 binding mimetics, nitrofuran and nitroimidazole compounds) in order to target them specifically to the parasite transporter and hence the parasite. All compounds carrying a melamine or benzamidine group were able to block the uptake of radiolabelled adenosine (a natural substrate of P2) to some degree. Toxicity varied, but some compounds were extremely trypanotoxic in vitro, and one nitrofuran is active in vivo and could represent a promising lead compound. In vitro toxicity experiments using a DeltaTbat1 (P2 knockout) cell line, reveal no innate resistance to P2 recognition motif bearing compounds in these cells, suggesting that the P2 transporter is not crucial for the toxic actions of these compounds, allaying fears over the development of resistance and cross resistance to other trypanocides through the loss of P2. The role of the P2 transporter in the development of drug resistance was investigated in a T. equiperdum cell line (P) and a Berenil resistant cell line (PBR) derived from it that had been reported to have a P2 transport component with greatly reduced affinity for adenosine. Sequencing results indicate that TbAT1 in T. equiperdum P exists as two alleles, whereas the T. equiperdum PBR cell line only contains sequence corresponding to one. Additionally, the T. equiperdum PBR cell line lacks any TbAT1 transcript detectable by Northern blot, which suggests that this loss of heterozygosity in T. equiperdum PBR has resulted in a lack of transcription of the gene, and a loss of P2 transport activity. An adenine sensitive adenosine transport component was identifiable in this cell line as well as in the DeltaTbat1 cell line, suggesting the presence of an, as yet unidentified, low affinity adenosine transporter in trypanosomes.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Mike Barrett
Keywords: Pharmacology, Parasitology
Date of Award: 2003
Depositing User: Enlighten Team
Unique ID: glathesis:2003-71210
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71210

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