Inhibitors of Purine Import into Plasmodium falciparum Kill Malaria Parasites

 描述(申请人提供)：感染单细胞真核疟原虫类寄生虫可引起疟疾。恶性疟原虫导致最致命的疟疾形式。目前，青蒿素联合疗法(ACT)是感染者的首选治疗方法。随着抗青蒿素恶性疟原虫在东南亚的兴起，开发新的抗疟疾药物势在必行。疟疾寄生虫是嘌呤营养缺乏症。它们通过恶性疟原虫平衡核苷转运蛋白1(PfENT1)将嘌呤前体从宿主红细胞输送到寄生虫中。在寄生虫中，嘌呤挽救途径酶修饰嘌呤前体，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(&lt；10微米)，PfENT1基因敲除寄生虫在培养中不能存活。因此，PfENT1抑制剂可能作为有效的抗疟疾药物发挥作用。该项目的目标是探索抑制PfENT1将杀死疟疾寄生虫的治疗假说，并为抗疟疾药物开发提供一个新的靶点。我们开发了一种简单、健壮的酵母细胞生长试验，并将其用于高通量筛选(HTS)以鉴定PfENT1抑制剂。5-氟尿苷(5-FURD)可杀死野生型酿酒酵母。缺乏内源质膜嘌呤/尿苷核苷转运蛋白的突变株FUI1Δ对5-FURD的抗性提高了100倍。PfENT1转运5-FURD。在125uM5-FURD存在的情况下，只有在存在PfENT1抑制剂以阻止5-FURD摄取的情况下，表达PfENT1的FUI1Δ酵母才能生长。在384个井板中，变异系数为 &lt；6.2%、信号窗口&gt；12和Z‘Score&gt；0.80，这表明检测非常可靠。我们筛选了64,500个化合物库，确定了171个匹配项。我们在一系列二次化验中测试了九个最热门的项目。所有9种药物均抑制PfENT1表达酵母和无红细胞滋养体阶段恶性疟原虫对[~3H]腺苷的摄取，IC50值在2-40 NM范围内。这九种化合物是五种不同的化学支架，它们不会杀死酵母菌，但会在IC50值在5-55微米范围内的培养中杀死恶性疟原虫。这项应用的目标是1)通过药物化学提高PfENT1抑制剂的效力和选择性；2)确定抑制剂的作用机制及其在不同生命周期阶段对寄生虫生物学和生长的影响；3)在小鼠疟疾模型中测试抑制剂的有效性；以及4)确定抑制剂结合部位和抑制剂与PfENT1结合的构象。该项目的成功完成将确定以PfENT1为靶点进行抗疟疾药物开发的效用，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.