Roles for the Trypanosoma brucei P2 transporter in DB75 uptake and resistance
Roles for the Trypanosoma brucei P2 transporter in DB75 uptake and resistance
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DOI：
10.1124/mol.106.024653
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发表时间：
2006-11-01
影响因子：
3.6
通讯作者：
Barrett, Michael P.
中科院分区：
文献类型：
--
作者：
Lanteri, Charlotte A.;Stewart, Mhairi L.;Barrett, Michael P.
一种新的锥虫杀灭剂，2，5-双（4-脒基苯基）呋喃（DB 75），其前药偕胺肟衍生物形式，2，5-双（4-脒基苯基）呋喃-双-O-甲基偕胺肟（DB289），作为第一种口服给药的非洲人锥虫药物正在试验中。DB 75是一种联脒。对某些二脒类药物的耐药性与通过P2氨基嘌呤转运蛋白的摄取丧失相关。我们在这里表明，摄取DB 75到布氏锥虫也主要通过P2转运蛋白发生。摄取氚DB 75发生通过高亲和力（Km（应用程序），3.2 μ M）载体介导的途径，抑制腺苷，腺嘌呤，和喷他脒，所有已知的底物的P2转运蛋白。当在受控的体外条件下测量时，缺乏编码P2转运蛋白的TbAT 1基因的锥虫表现出对DB 75的敏感性降低11倍。这些敲除细胞对DB 75的敏感性也低于小鼠中的野生型细胞。与野生型细胞中的速率相比，DB 75进入Delta tbat 1敲除细胞系的初始摄取速率大大降低。体外选择的DB 75抗性的锥虫细胞系显示失去了P2介导的DB 75摄取。TbAT 1基因定位于T.布氏杆菌基因组和DB 75抗性寄生虫显示该基因的两个等位基因都缺失。DB 75处理锥虫的荧光显微镜显示，DB 75荧光迅速定位在野生型锥虫的含DNA的细胞器内，而在Delta tbat 1-null寄生虫或选择对DB 75具有抗性的寄生虫中没有观察到荧光。
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 mu M) 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 Delta tbat1 knockout cell line were greatly reduced compared with rates in wildtype 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 Delta tbat1-null parasites or in the parasites selected for resistance to DB75.