Elucidating the molecular basis of piperaquine resistance and the role of altered hemoglobin metabolism in Plasmodium falciparum

阐明恶性疟原虫哌喹耐药性的分子基础以及血红蛋白代谢改变的作用

• 批准号: 9212775
• 负责人: David A Fidock
• 金额: $ 53.58万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9212775
• 关键词: Address; Amino Acids; Antimalarials; Artemisinins; Asia; Binding; Biological Assay; Blood; Cambodia; Cambodian; Candidate Disease Gene; Cell Death; Clinical; Clinical Treatment; Combined Modality Therapy; Crystallization; Cytosol; Data; Digestion; Dipeptidyl Peptidases; Dose; Drug Efflux; Drug Transport; Drug resistance; Drug usage; Engineering; Face; Falciparum Malaria; French Guiana; Future; Genes; Genetic; Genetic Crosses; Genetic Markers; Globin; Heme; Hemoglobin; Hemoglobin A; Inbred NOD Mice; Link; Liver; Malaria; Maps; Measures; Mediating; Mediator of activation protein; Membrane Proteins; Metabolism; Modeling; Molecular; Monitor; Multi-Drug Resistance; Mutation; Parasite resistance; Parasites; Peptide Fragments; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Phenotype; Physiology; Plasmodium falciparum; Policies; Process; Protein Isoforms; Proteolysis; Recombinants; Report (document); Resistance; Role; Sequence Analysis; Source; South America; Testing; Transfection; Treatment Failure; Vacuole; base; cost; experimental study; fight against; fitness; genome sequencing; hemozoin; innovation; insight; metabolomics; molecular marker; multi drug transporter; novel; novel therapeutics; plasmepsin; pressure; prevent; public health relevance; resistant strain; response; solute; transgene expression; whole genome; zinc finger nuclease

 DESCRIPTION (provided by applicant): Artemisinin-based combination therapies have been pivotal in achieving major reductions in the global burden of Plasmodium falciparum (Pf) malaria. Pf parasite resistance to artemisinin, which first emerged in Cambodia through mutations in K13, has placed increased selection pressure on its partner drugs. Recent reports document clinical treatment failures with the first-line therapy dihydroartemisinin+piperaquine (PPQ) in Cambodia. PPQ resistance is also emerging in French Guiana. The absence of fully effective alternatives underscores the need to define the molecular basis of PPQ resistance as a means to curtail its spread. Whole-genome sequence (WGS) analysis and phenotypic studies of PPQ-resistant Cambodian isolates have identified candidate genes whose putative functions relate to hemoglobin (Hb) metabolism and solute transport in the parasite's digestive vacuole (DV). Our central hypothesis is that PPQ resistance is mediated by altered Hb metabolism in the DV and accompanying changes in transporters that regulate DV physiology. In Aim 1 we address the hypothesis that Cambodian isolates achieve resistance primarily via amplification of plasmepsins 2 and 3, which encode Hb-digesting proteases. We will also genetically assess the evidence implicating mutations in the Hb peptidase DPAP1 and the DV solute and multidrug transporters PfCRT and PfMDR1. To test this hypothesis, parasites will be engineered through transgene expression and Cas9 or zinc-finger nuclease-mediated gene editing. Changes in resistance will be examined using PPQ survival and dose-response assays. In Aim 2 we tackle the emerging problem of PPQ resistance in French Guiana by implementing genetic crosses with humanized FRG-NOD mice that are receptive to Pf liver and blood stages. WGS and phenotypic analysis of the progeny will identify loci linked to resistance. Parallel WGS studies on PPQ-resistant isolates will help converge on the selection of resistance candidates, whose role will be assessed using transfection. In Aim 3 we define the functional basis of PPQ resistance and its impact on other antimalarials. Using clinical isolates and recombinant lines, we will assess whether reduced PPQ accumulation and heme binding is a hallmark of resistance. We will also evaluate whether resistance results in increased levels of toxic free heme resulting from Hb metabolism. Metabolomic studies will delineate the relationship between PPQ resistance and Hb digestion. Finally, we will assess the impact of resistance on other heme-binding antimalarials, with an emphasis on identifying drugs that retain or even increase their activity against PPQ-resistant parasites. We believe that this project provides powerful and scientifically innovative approaches to elucidate the molecular basis of PPQ resistance, yield genetic markers to monitor its emergence and spread, and identify optimal means of treatment. This project will also provide important new insights into Hb metabolism, which continues to provide a rich source of antimalarials for future clinical use.

 描述（由应用程序提供）：基于青蒿素的组合疗法在实现全球恶性疟原虫（PF）疟疾的全球燃烧大大减少方面至关重要。 PF寄生虫对青蒿素的耐药性是通过K13中的突变在柬埔寨出现的，对其伴侣药物的选择压力增加了。最近的报道记录了柬埔寨的一线治疗二氢甲酸二氢甲酸（PPQ）的临床治疗失败。 PPQ抗性也在法属圭亚那出现。缺乏完全有效的替代方案强调了定义PPQ抗性分子基础的必要性，以减少其扩散。全基因组序列（WGS）分析和对PPQ抗药性柬埔寨分离株的表型研究已经确定了候选基因，其假定功能与寄生虫消化液（DV）中的假定功能与血红蛋白（HB）代谢和可溶性转运有关。我们的中心假设是，PPQ抗性是由DV中HB代谢的改变以及调节DV生理的转运蛋白的参与变化所介导的。在AIM 1中，我们解决了以下假设：柬埔寨分离株通过对血浆蛋白2和3的扩增实现抗性原发性，该血浆蛋白2和3编码HB消化蛋白酶。我们还将从基因评估HB Pepperase DPAP1以及DV溶质和多药转运蛋白PFCRT和PFMDR1中的隐式突变。为了检验该假设，将通过转化表达和CAS9或锌指核酸酶介导的基因编辑来设计寄生虫。将使用PPQ存活和剂量反应刺客检查电阻的变化。在AIM 2中，我们通过使用人源化的FRG-NOD小鼠实施遗传杂交来解决法国圭亚那的PPQ耐药性问题，这些小鼠接受PF肝脏和血液阶段。后代的WGS和表型分析将确定与抗性有关的地方。平行WGS研究 耐PPQ的分离株将有助于融合候选抗性的选择，其作用将使用转换评估。在AIM 3中，我们定义了PPQ耐药性的功能基础及其对其他抗疟药的影响。使用临床分离株和重组线，我们将评估降低的PPQ积累和血红素结合是抗性的标志。我们还将评估抗性是否导致因 HB代谢。代谢组学研究将描述PPQ抗性与HB消化之间的关系。最后，我们将评估耐药性对其他血红素结合抗菌素的影响，重点是鉴定保留甚至增加其对耐PPQ抗性寄生虫活性的药物。我们认为，该项目提供了强大而科学的创新方法来阐明PPQ抗性的分子基础，产生遗传标记以监测其出现和传播，并确定最佳治疗方法。该项目还将为HB代谢提供重要的新见解，该见解继续为未来的临床用途提供丰富的抗疟药来源。