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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的疟疾仍然是一个重大的公共卫生问题。恶性疟原虫种寄生虫感染是最具杀伤力。对当前抗疟治疗的耐药性不断增加使得新药开发势在必行。疟原虫寄生虫是专性细胞内嘌呤营养缺陷型生物。因此，嘌呤回收途径是抗疟药物开发的良好目标。平衡核苷转运蛋白 PfENT1 是主要的核苷转运蛋白恶性疟原虫摄取嘌呤的途径。然而，有证据表明有一条次要途径可以导入AMP作为嘌呤源。该途径仍有待表征。在目标 #1 中，我将描述通过嘌呤放射性标记底物摄取抑制实验将 AMP 嘌呤转运至恶性疟原虫中和潜在的抑制剂。我将测试 AMP 转运是否是通过钠偶联或质子介导的耦合过程。这些实验的结果将为恶性疟原虫生物学提供新的见解。之前在实验室中使用高通量筛选的工作鉴定了 PfENT1 抑制剂。命中阻止转运并杀死培养物中的疟原虫。然而，这些最初的打击还不足以成为药物。在目标#2，我将参与从命中到先导的药物化学过程，以优化这些 PfENT1 抑制剂命中并确定这些化合物的结构-活性关系。我将评估它们在寄生虫中的效力使用人肝癌 HepG2 细胞毒性测定进行细胞毒性测定及其人类细胞毒性。这该过程将使我们能够确定哪些化学特征可以为药物提供潜在的机会转化为候选药物。在目标#3中，我将确定是否自然发生的非同义单一恶性疟原虫野外分离株基因组测序中发现的核苷酸多态性 (SNP) 会影响疗效我们正在开发作为潜在抗疟药物的 PfENT1 抑制剂。其功效有可能是 PfENT1 抑制剂可能会受到 PfENT1 中 SNP 存在的影响。我们已经确定了 13 个非 PlasmoDB 数据库中 100 多个序列中的同义 SNP。我将表达 PfENT1-SNP 使用 PfENT1-PCM189 酵母表达载体在酿酒酵母中进行表达。我会确定是否 SNP 影响嘌呤输入和 PfENT1 抑制剂的功效。可用的 PfENT1-SNP 表达寄生虫还将使用抑制剂的寄生虫细胞毒性测定来测试分离株。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产生耐药性。这些实验将推动药物的发展 PfENT1 抑制剂的开发过程，并希望能够成功开发抗疟药。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。它将为我提供一个为生物医学研究事业奠定良好的基础。