Received for publication March 31, 2006.
Revised August 15, 2006.
Accepted for publication August 15, 2006.

Roles for the Trypanosoma brucei P2 transporter in DB75 uptake and resistance

Abstract

A novel trypanocide, DB75 (2,5-bis(4-amidinophenyl)furan), which in its prodrug amidoxime-derivative form, DB289, is in trials as the first orally administered drug for human African trypanosomiasis. DB75 is a diamidine. Resistance to some diamidines correlates to loss of uptake via the P2 aminopurine transporter. We show here that uptake of DB75 into Trypanosoma brucei also occurs principally via the P2 transporter. Uptake of tritiated DB75 was via a high affinity (K_{m app} of 3.2 µM) carrier mediated route that was inhibited by adenosine, adenine and pentamidine, all known substrates of the P2 transporter. Trypanosomes lacking the TbAT1 gene that encodes the P2 transporter demonstrated an 11-fold reduction in sensitivity to DB75 when measured under controlled in vitro conditions. These knockout cells were also less sensitive to DB75 than wild-type cells in mice. Initial uptake rates of DB75 into the TbAT1 knockout cell line were greatly reduced when compared with rates in wild-type cells. A trypanosome cell line selected in vitro for DB75 resistance was shown to have lost P2-mediated DB75 uptake. The TbAT1 gene was mapped to chromosome V of the T. brucei genome and the DB75-resistant parasites were shown to have deleted both alleles of this gene. Fluorescence microscopy of DB75-treated trypanosomes revealed that DB75 fluorescence localizes rapidly within the DNA-containing organelles of wild-type trypanosomes, whereas no fluorescence was observed in TbAT1-null parasites, nor in the parasites selected for resistance to DB75.

Key words: Nucleoside/Nucleotide, Antiprotozoal drugs

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