Roles for the Trypanosoma brucei P2 transporter in DB75 uptake and resistance.
A novel trypanocide, 2,5-bis(4-amidinophenyl)furan (DB75), in its prodrug amidoxime-derivative form, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (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 occurred via a high-affinity (K(m app), 3.2 microM) carriermediated 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 Deltatbat1 knockout cell line were greatly reduced 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 Deltatbat1-null parasites or in the parasites selected for resistance to DB75.
Lanteri
Charlotte A
CA
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, The Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
Stewart
Mhairi L
ML
Brock
Janice M
JM
Alibu
Vincent P
VP
Meshnick
Steven R
SR
Tidwell
Richard R
RR
Barrett
Michael P
MP
D000241:Adenosine / Q000378:metabolism; D000818:Animals; D001550:Benzamidines / Q000493:pharmacokinetics*; D001692:Biological Transport; D004351:Drug Resistance; D005260:Female; D005453:Fluorescence; D007553:Isotope Labeling; D008297:Male; D051379:Mice; D009154:Mutation / Q000235:genetics; D033703:Nucleoside Transport Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000378:metabolism*
D001550:Benzamidines; D033703:Nucleoside Transport Proteins; C486446:P2 transporter, Trypanosoma brucei; C033286:furamidine; D000241:Adenosine