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（由申请人提供）：基于青蒿素的联合疗法在大幅减少全球恶性疟原虫（Pf）疟疾负担方面发挥了关键作用，Pf寄生虫对青蒿素的耐药性首先在柬埔寨通过 K13 突变出现，并增加了疟疾的耐药性。最近的报告记录了柬埔寨一线疗法双氢青蒿素+哌喹（PPQ）的临床治疗失败。法属圭亚那也出现了 PPQ 耐药性，由于缺乏完全有效的替代品，因此需要确定 PPQ 耐药性的分子基础，以此作为遏制 PPQ 耐药性柬埔寨病毒传播的手段。分离株已鉴定出候选基因，其假定的功能与寄生虫消化液泡（DV）中的血红蛋白（Hb）代谢和溶质转运相关。我们的中心假设是PPQ抗性是由介导的。 DV 中 Hb 代谢的改变以及调节 DV 生理学的转运蛋白的变化在目标 1 中，我们提出了这样的假设：柬埔寨分离株主要通过编码 Hb 消化蛋白酶的 plasmepsins 2 和 3 的扩增来实现耐药性。涉及 Hb 肽酶 DPAP1 和 DV 溶质以及多药物转运蛋白 PfCRT 和 PfMDR1 突变的证据来测试这一点。假设，寄生虫将通过转基因表达和 Cas9 或锌指核酸酶介导的基因编辑进行改造。在目标 2 中，我们将使用 PPQ 存活和剂量反应测定来检查耐药性的变化。通过与接受 Pf 肝脏和血液阶段的人源化 FRG-NOD 小鼠进行遗传杂交，对后代的表型分析将识别与耐药性相关的位点。并行 WGS 研究 PPQ 耐药菌株将有助于集中选择耐药候选者，其作用将通过转染进行评估。在目标 3 中，我们将使用临床菌株和重组株来评估 PPQ 耐药性的功能基础及其对其他抗疟药的影响。 PPQ积累和血红素结合减少是否是耐药性的标志我们还将评估耐药性是否会导致无毒血红素水平增加。 代谢组学研究将描述 PPQ 耐药性和 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疟药的影响，重点是确定保留甚至增强抗 PPQ 寄生虫活性的药物。我们相信，该项目提供了强大且科学创新的方法来阐明 PPQ 耐药性的分子基础，产生遗传标记来监测其出现和传播，并确定最佳治疗手段。该项目还将提供重要的新见解。进入血红蛋白代谢，继续为未来的临床使用提供丰富的抗疟药来源。