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：
8859480
负责人：
Myles H. Akabas
金额：
$ 43.06万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
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 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8859480
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 Inhibitory Concentration 50 Inosine Invaded Knock-out Libraries Life Cycle Stages Liver Malaria Mass Spectrum Analysis Mediating Modeling Molecular Conformation Mus Muscle Form Glycogen Phosphorylase Nucleoside Transporter Nucleosides Nucleotides Oocysts Oral Parasites Pathway interactions Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacotherapy Phase Phosphorylases Physiological Plasmodium Plasmodium falciparum Play Process Proteins Purine Antagonist Purines RNA chemical synthesis Relative (related person)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 mutant novel novel strategies nucleobase prevent public health relevance purine resistance mechanism scaffold small molecule structural biology 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（由申请人提供）：单细胞真核疟原虫寄生虫感染导致恶性疟原虫，目前，青蒿素联合疗法（ACT）是感染者的首选治疗方法。东南亚的恶性疟原虫使得开发新的抗疟药物势在必行。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 合成等所需的核苷酸。在人体血液中发现的嘌呤浓度 (<10 µM) 下，PfENT1 敲除寄生虫在培养物中无法存活。因此，PfENT1 抑制剂可能作为有效的抗疟药物，该项目的目标是探索抑制 PfENT1 将杀死疟疾寄生虫的治疗假设，并为抗疟药物的开发提供新的靶点。测定并在高通量筛选 (HTS) 中使用它来鉴定 PfENT1 抑制剂 (5-FUrd) 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体 fui1Δ 酵母对 5-FUrd 转运 5-FUrd 的抵抗力高出 100 倍。在 125 µM 5-FUrd 存在的情况下，表达 PfENT1 的 fui1Δ 酵母只会生长。 PfENT1 抑制剂可防止 384 孔板中 5-FUrd 的摄取。变化是 <6.2%，信号窗口 > 12，Z' 分数 > 0.80，表明我们筛选了 64,500 个化合物库，并在一系列二级分析中测试了 9 个最匹配的结果。抑制表达 PfENT1 的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（五种不同的化学支架）不会杀死酵母，但会杀死培养物中的恶性疟原虫，IC50 值在 5 - 55 µM 范围内。该应用的目的是：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对寄生虫生物学和不同生命周期生长的影响；阶段；3）测试抑制剂在小鼠疟疾模型中的功效；4）确定抑制剂结合位点和抑制剂结合的 PfENT1 构象，该项目的成功完成将确定靶向 PfENT1 的抗疟效用。药物开发并可以识别适合进一步开发的化合物。