Inhibitors of Purine Import into Plasmodium falciparum Kill Malaria Parasites

 描述（由申请人提供）：单细胞真核疟原虫属寄生虫感染引起疟疾。恶性疟原虫是最致命的疟疾。目前，青蒿素联合疗法是受感染者的首选治疗方法。抗青蒿素恶性疟原虫在东南亚的兴起使得开发新的抗疟药物势在必行。疟疾寄生虫是嘌呤营养缺陷型。它们通过恶性疟原虫平衡核苷转运蛋白1（PfENT 1）将嘌呤前体从宿主红细胞转运到寄生虫中。在寄生虫中，嘌呤补救途径酶修饰嘌呤前体以形成RNA和DNA合成以及其他细胞过程所需的核苷酸。在人类血液中发现的嘌呤浓度（<10 µM）下，PfENT 1敲除寄生虫在培养物中不能存活。因此，PfENT 1抑制剂可以作为有效的抗疟疾药物发挥作用。该项目的目标是探索治疗假说，即抑制PfENT 1将杀死疟疾寄生虫，并为抗疟药物开发提供新的靶点。我们已经开发了一种简单，稳健的酵母细胞生长测定法，并将其用于高通量筛选（HTS）以鉴定PfENT 1抑制剂。5-氟尿苷（5-FUrd）杀死野生型酿酒酵母。缺乏内源性质膜嘌呤/尿苷核苷转运蛋白的突变型fui 1 Δ酵母对5-FUrd的抗性高100倍。PfENT 1转运5-FUrd。在存在125 µM 5-FUrd的情况下，只有当存在PfENT 1抑制剂以阻止5-FUrd摄取时，表达PfENT 1的fui 1 Δ酵母才会生长。在384孔板中，变异系数为 <6.2%，信号窗口> 12，并且Z'得分> 0.80，表明高度稳健的测定。我们筛选了64，500个化合物库，并鉴定了171个命中。我们在一系列的二次检测中检测了9个最高的命中率。所有9种药物均抑制PfENT 1表达酵母和无红细胞滋养体阶段恶性疟原虫的[3 H]腺苷摄取，IC 50值在2 - 40 nM范围内。这九种化合物，五种不同的化学支架，不杀死酵母，但确实杀死培养物中的恶性疟原虫寄生虫，IC 50值在5 - 55 μM范围内。本申请的目的是1）通过药物化学提高PfENT 1抑制剂的效力和选择性; 2）确定抑制剂的作用机制及其对寄生虫生物学和在不同生命周期阶段生长的影响; 3）在小鼠疟疾模型中测试寄生虫的功效;和4）鉴定抑制剂结合位点和抑制剂结合的PfENT 1构象。该项目的成功完成将确定靶向PfENT 1用于抗疟药物开发的效用，并可能确定适合进一步开发的化合物。

 DESCRIPTION (provided by applicant): Infection with unicellular eukaryotic Plasmodium species parasites causes malaria. P. falciparum causes the most virulent form of malaria. Currently, artemisinin combination therapy (ACT) is the treatment of choice for infected individuals. The rise of artemisinin resistant P. falciparum in Southeast Asia makes it imperative to develop new antimalarial drugs. Malaria parasites are purine auxotrophs. They transport purine precursors from the host erythrocyte into the parasite via the P. falciparum Equilibrative Nucleoside Transporter 1 (PfENT1). In the parasite, purine salvage pathway enzymes modify the purine precursors to form the nucleotides needed for RNA and DNA synthesis and other cellular processes. At purine concentrations found in human blood (<10 µM), PfENT1 knockout parasites are not viable in culture. Thus, PfENT1 inhibitors may function as potent antimalarial drugs. The goal of this project is to explore the therapeutic hypothesis that inhibition of PfENT1 will kill malaria parasites and provide a novel target for antimalarial drug development. We have developed a simple, robust yeast cell growth assay and used it in a high throughput screen (HTS) to identify PfENT1 inhibitors. 5-fluorouridine (5-FUrd) kills wild type Saccharomyces cerevisiae. Mutant fui1Δ yeast that lack the endogenous plasma membrane purine/uridine nucleoside transporter are 100 times more resistant to 5-FUrd. PfENT1 transports 5-FUrd. In the presence of 125 µM 5-FUrd, PfENT1-expressing fui1Δ yeast will only grow if a PfENT1 inhibitor is present to prevent 5-FUrd uptake. In 384 well plates, the Coefficient of Variation was <6.2%, Signal Window > 12, and the Z' score > 0.80, indicating a highly robust assay. We screened a 64,500 compound library and identified 171 hits. We tested nine of the top hits in a series of secondary assays. All nine inhibited [3H]adenosine uptake into both PfENT1-expressing yeast and into erythrocyte-free trophozoite stage P. falciparum with IC50 values in the 2 - 40 nM range. The nine compounds, five distinct chemical scaffolds, do not kill yeast but do kill P. falciparum parasites in culture with IC50 values in the 5 - 55 µM range. The goals of this application are 1) to improve the potency and selectivity of the PfENT1 inhibitors through medicinal chemistry; 2) to define the mechanism of action of the inhibitors and their impact on parasite biology and growth at various life cycle stages; 3) to test the efficacy of the inhibitorsin a mouse malaria model; and 4) to identify the inhibitor binding site and the conformation of PfENT1 to which the inhibitors bind. Successful completion of this project will determine the utility of targeting PfENT1 for antimalarial drug development and may identify compounds suitable for further development.