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

 描述（由申请人提供）：基于青蒿素的联合疗法在实现恶性疟原虫（Pf）疟疾全球负担的重大减少方面至关重要。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，这给其伙伴药物带来了更大的选择压力。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。PPQ耐药性也在法属圭亚那出现。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，以此作为遏制其传播的手段。全基因组序列（WGS）分析和PPQ耐药柬埔寨分离株的表型研究已经确定了候选基因，其推定的功能涉及血红蛋白（Hb）代谢和溶质运输在寄生虫的消化泡（DV）。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生理学的转运蛋白的伴随变化介导的。在目标1中，我们解决了柬埔寨分离株主要通过扩增编码Hb消化蛋白酶的plasmepsin 2和3来实现耐药性的假设。我们还将从遗传学角度评估涉及Hb肽酶DPAP 1和DV溶质和多药转运蛋白PfCRT和PfMDR 1突变的证据。为了验证这一假设，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工程改造。将使用PPQ存活和剂量反应试验检查耐药性变化。在目标2中，我们通过与接受Pf肝脏和血液阶段的人源化FRG-NOD小鼠进行遗传杂交来解决法属圭亚那出现的PPQ抗性问题。后代的WGS和表型分析将鉴定与抗性相关的基因座。平行WGS研究 PPQ抗性分离株将有助于集中在抗性候选物的选择上，其作用将使用转染进行评估。在目标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株系，我们将评估PPQ蓄积和血红素结合减少是否是耐药的标志。我们还将评估耐药性是否会导致毒性游离血红素水平的增加， 血红蛋白代谢。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疟药的影响，重点是确定保留甚至增加其对PPQ耐药寄生虫活性的药物。我们相信，该项目提供了强大的和科学的创新方法来阐明PPQ抗性的分子基础，产生遗传标记来监测其出现和传播，并确定最佳的治疗方法。该项目还将为Hb代谢提供重要的新见解，这将继续为未来的临床使用提供丰富的抗疟药物来源。

 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.