Characterizing and targeting malaria parasite purine uptake pathways to generate novel antimalarial drugs

表征和靶向疟原虫嘌呤摄取途径以产生新型抗疟药物

• 批准号: 9899196
• 负责人: Yvett D Sosa
• 金额: $ 3.31万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-01 至 2020-08-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9899196
• 关键词: Adenine Nucleotides; Affect; Affinity; Antimalarials; Artemisinins; Biological Assay; Biology; Biomedical Research; Blood; Cations; Cell membrane; Cells; Cessation of life; Characteristics; Chemicals; Chemistry; Combined Modality Therapy; Coupled; Databases; Development; Drug Targeting; Drug resistance; Erythrocytes; Foundations; Goals; HepG2; Human; Infection; Knock-out; Lead; Learning; Light; Malaria; Measures; Mediating; Monovalent Cations; Nucleoside Transporter; Nucleosides; Nucleotides; Parasites; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Physiological; Physiology; Plasmodium; Plasmodium falciparum; Point Mutation; Primary carcinoma of the liver cells; Process; Property; Protons; Public Health; Purines; Resistance; Saccharomyces cerevisiae; Single Nucleotide Polymorphism; Sodium; Source; Southeastern Asia; Specificity; Structure-Activity Relationship; Testing; Toxic effect; Transmembrane Transport; Virulent; Work; World Health; Yeasts; base; career; clinical efficacy; cytotoxicity; drug candidate; drug development; drug efficacy; experimental study; expression vector; extracellular; genome sequencing; high throughput screening; inhibitor/antagonist; insight; lead optimization; mutant; new therapeutic target; novel; novel therapeutics; nucleobase; obligate intracellular parasite; process optimization; radiotracer; success; symporter; uptake

Abstract Malaria remains a major public health issue. Infection with Plasmodium falciparum species parasites is most lethal. Increasing resistance to current antimalarial treatments makes new drug development imperative. Plasmodium parasites are obligate intracellular purine auxotrophs. Purine salvage pathways are therefore good targets for antimalarial drug development. An equilibrative nucleoside transporter, PfENT1, is the primary pathway for purine uptake by P. falciparum. However, there has been evidence of a secondary pathway that can import AMP as a purine source. This pathway remains to be characterized. In Aim #1 I will characterize the AMP purine transport into P. falciparum through radiolabel substrate uptake inhibition experiments with purines and potential inhibitors. I will test whether AMP transport is mediated through a sodium-coupled or proton- coupled process. Results from these experiments will provide novel insight into P. falciparum biology. Previous work in the lab using a high throughput screen identified PfENT1 inhibitors. The hits block the transporter and kill malaria parasites in culture. However, these initial hits are not potent enough to be drugs. In Aim #2, I will participate in the hit-to-lead medicinal chemistry process to optimize these PfENT1 inhibitor hits and determine the structure-activity relationships for these compounds. I will evaluate their potency in parasite cytotoxicity assays and their human cell toxicity using human hepatoma HepG2 cytotoxicity assays. This process will allow us to determine what chemical characteristics provide drug hits with the potential to be converted into drug candidates. In Aim #3, I will determine whether naturally occurring non-synonymous single nucleotide polymorphisms (SNP) identified in genome sequencing of P. falciparum field isolates affect efficacy of the PfENT1 inhibitors that we are developing as potential antimalarial drugs. It is possible that the efficacy of PfENT1 inhibitors can be affected by the presence of SNPs in PfENT1. We have identified 13 non- synonymous SNPs in the more than 100 sequences in the PlasmoDB database. I will express PfENT1-SNPs in Saccharomyces cerevisiae by using a PfENT1-pCM189 yeast expression vector. I will determine whether the SNPs affect purine import and PfENT1 inhibitor efficacy. Available PfENT1-SNP expressing parasite isolates will also be tested using parasite cytotoxicity assays with inhibitors. These experiments will help us determine if PfENT1 SNPs could cause resistance to our inhibitors. These experiments will advance the drug development process for the PfENT1 inhibitors and hopefully lead to the development of successful antimalarial drugs. Participation in this project will provide me with the opportunity to learn about membrane transport physiology, parasite biology and the early stage drug development process. It will provide me with an excellent foundation for a career in biomedical research.

摘要 疟疾仍然是一个重大的公共卫生问题。感染恶性疟原虫属寄生虫是 最致命的。对当前抗疟治疗的耐药性日益增加，使得新药开发势在必行。 疟原虫是专性细胞内嘌呤营养缺陷型。因此，嘌呤补救途径 抗疟疾药物开发的良好目标。平衡型核苷转运蛋白PfENT1是主要的 恶性疟原虫摄取嘌呤的途径。然而，有证据表明， 可以输入AMP作为嘌呤来源。该途径仍有待表征。在目标#1中，我将描述 通过嘌呤抑制放射性标记底物摄取实验研究AMP嘌呤转运入恶性疟原虫 和潜在的抑制剂。我将测试AMP转运是否通过钠偶联或质子介导- 耦合过程这些实验的结果将为恶性疟原虫生物学提供新的见解。 先前在实验室中的工作使用高通量筛选鉴定了PfENT1抑制剂。点击阻止 运输和杀死疟疾寄生虫。然而，这些最初的打击并不足以成为药物。在 目标#2，我将参与药物化学过程，以优化这些PfENT1抑制剂的命中率 并确定这些化合物的结构-活性关系。我会评估它们在寄生虫中的效力 细胞毒性测定和使用人肝癌HepG2细胞毒性测定的它们的人细胞毒性。这 这一过程将使我们能够确定哪些化学特性为药物命中提供了潜在的可能性， 转化为候选药物。在目标#3中，我将确定自然发生的非同义单 恶性疟原虫田间分离株基因组测序中发现的核苷酸多态性（SNP）影响疗效 PfENT1抑制剂，我们正在开发作为潜在的抗疟疾药物。有可能是因为 PfENT1抑制剂可能受到PfENT1中SNP存在的影响。我们发现了13个非- PlasmoDB数据库中超过100个序列的同义SNP。我将表达PfENT1-SNP 通过使用PfENT1-pCM 189酵母表达载体在酿酒酵母中表达。我将决定 SNP影响嘌呤输入和PfENT1抑制剂功效。可用的PfENT1-SNP表达寄生虫 还将使用具有抑制剂的寄生虫细胞毒性测定来测试分离物。这些实验将帮助我们 确定PfENT1 SNP是否会导致对我们的抑制剂的耐药性。这些实验将促进药物 PfENT1抑制剂的开发过程，并有望导致成功的开发 抗疟疾药物。参与这个项目将为我提供了解膜的机会 运输生理学、寄生虫生物学和早期药物开发过程。它将为我提供一个 在生物医学研究中有着良好的职业基础。