Inhibitors of Purine Import into Plasmodium falciparum Kill Malaria Parasites

嘌呤输入恶性疟原虫的抑制剂可杀死疟疾寄生虫

基本信息

批准号：
9000003
负责人：
Myles H. Akabas
金额：
$ 68.26万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-02-01 至 2020-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9000003
关键词：
5 fluorouridine Accounting Adenosine Affinity Antimalarials Artemisinins Binding Binding Sites Biochemical Biological Assay Biological Availability Biology Blood Cell membrane Cell physiology Cessation of life Chemicals Combination Drug Therapy Combined Modality Therapy Cysteine DNA DNA biosynthesis Data Development Drug Kinetics Drug Targeting Economic Burden Endoplasmic Reticulum Equilibrative Nucleoside Transporter 1 Erythrocytes Genetic Genome Goals Growth Guanine Human Hypoxanthines Individual Infection Inosine Invaded Knock-out Libraries Life Cycle Stages Liver Malaria Mass Spectrum Analysis Mediating Modeling Molecular Conformation Mus Nucleoside Transporter Nucleosides Nucleotides Oocysts Oral Parasites Pathway interactions Pharmaceutical Chemistry Pharmaceutical Preparations Phase Phosphorylases Physiological Plasmodium Plasmodium falciparum Play Process Proteins Purine Antagonist Purines RNA chemical synthesis Resistance Resistance development Role Rupture Saccharomyces cerevisiae Series Signal Transduction Site Southeastern Asia Specificity Sporozoites Staging Starvation Structure-Activity Relationship Testing Therapeutic Time Toxic effect Uridine Variant Virulent Work Xanthines Yeasts adenosine deaminase artemisinine base cell growth cytotoxic drug development efficacy testing enzyme pathway high throughput screening human disease improved inhibitor/antagonist killings malaria infection mutant novel novel strategies nucleobase prevent public health relevance resistance mechanism scaffold small molecule inhibitor structural biology targeted treatment treatment choice uptake

项目摘要

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.

描述（由应用提供）：单细胞真核质子寄生虫的感染会导致疟疾。恶性疟原虫会导致疟疾最毒的形式。目前，青蒿素联合疗法（ACT）是感染个体的选择治疗。东南亚抗厄半米斯蛋白抗性恶性疟原虫的兴起使得必须开发新的抗疟药。疟疾寄生虫是纯粹的合子营养。他们通过恶性疟原虫平衡的核苷转运蛋白1（PFENT1）将嘌呤前体从宿主的红细胞从宿主的红细胞传输到寄生虫中。在寄生虫中，嘌呤挽救途径酶修饰了嘌呤前体，形成了RNA和DNA合成和其他细胞过程所需的核萝卜。在人类血液（<10 µm）中发现的嘌呤浓度下，Pfent1基因敲除寄生虫在培养中不可行。这是Pfent1抑制剂可能起作用的潜在抗疟药。该项目的目的是探讨治疗性假设，即抑制PFENT1将杀死疟疾寄生虫，并为抗疟药发育提供新的目标。我们已经开发了一种简单，健壮的酵母细胞生长测定法，并将其用于高吞吐量筛选（HTS）来识别Pfent1抑制剂。 5-氟尿嘧啶（5-富裕）杀死了酿酒酵母的野生型糖果。缺乏内源性质膜购买/尿苷核端转运蛋白的突变FUI1δ酵母对5-粉的耐药性高100倍。在存在125 µm 5-furd的情况下，只有在存在Pfent1抑制剂以防止5-富摄入量时，表达Pfent1的FUI1Δ酵母才会生长。在384个井板中，变化系数为 <6.2％，信号窗口> 12和z'得分> 0.80，表明具有高度健壮的测定法。我们筛选了一个64,500个复合库，并确定了171次命中。我们在一系列辅助测定中测试了9个最高命中率。所有九个抑制[3H]腺苷摄取均表达Pfent1的酵母，并进入无红细胞的滋养体阶段。恶性疟原虫，IC50值在2-40 nm范围内。九种化合物，五种不同的化学支架，不会杀死酵母，而是在5-55 µm范围内具有IC50值的培养物中杀死恶性疟原虫寄生虫。本应用的目标是1）通过医学化学提高PFENT1抑制剂的效力和选择性； 2）定义抑制剂的作用机理及其对各种生命周期阶段的寄生虫生物学和生长的影响； 3）测试小鼠疟疾模型抑制剂的效率； 4）确定抑制剂结合位点和抑制剂结合的Pfent1的构象。该项目的成功完成将确定靶向PFENT1用于抗疟药开发的实用性，并可能确定适合进一步开发的化合物。