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

 描述(申请人提供)：以青蒿素为基础的联合疗法在大幅减轻全球恶性疟疾负担方面发挥了关键作用。PF寄生虫对青蒿素的抗药性最初是通过K13基因突变在柬埔寨出现的，这给其合作伙伴的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治疗双氢青蒿素+哌喹(PPQ)的临床治疗失败。法属圭亚那也出现了对PPQ的抵抗。由于缺乏完全有效的替代品，因此需要确定PPQ抗性的分子基础，以此作为遏制其传播的一种手段。全基因组序列(WGS)分析和对PPQ抗性柬埔寨分离株的表型研究已经确定了候选基因，其可能的功能与寄生虫的消化液泡(DV)中的血红蛋白(Hb)代谢和溶质运输有关。我们的中心假设是，PPQ抵抗是由DV中Hb代谢的改变以及调节DV生理的转运蛋白的伴随变化所介导的。在目标1中，我们解决了柬埔寨分离株主要通过扩增血浆蛋白2和3获得抗药性的假设，血浆蛋白2和3编码HB消化的蛋白酶。我们还将对涉及HB多肽酶DPAP1和DV溶质以及多药转运蛋白PfCRT和PfMDR1突变的证据进行遗传学评估。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来进行工程设计。耐药性的变化将通过PPQ存活率和剂量反应分析来检查。在目标2中，我们通过与人源化FRG-NOD小鼠进行基因杂交来解决法属圭亚那新出现的PPQ抗性问题，这些FRG-NOD小鼠接受PF肝脏和血液阶段。WGS和对后代的表型分析将确定与抗性相关的基因座。WGS的平行研究 PPQ抗性分离株将有助于在选择耐药候选菌株方面趋同，其作用将通过转基因进行评估。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疾药物的影响。利用临床分离株和重组株，我们将评估PPQ积累和血红素结合减少是否是耐药的标志。我们还将评估抗药性是否会导致有毒的游离血红素水平增加 Hb代谢。代谢组学研究将阐明PPQ抗性和Hb消化之间的关系。最后，我们将评估耐药性对其他与血红素结合的抗疟疾药物的影响，重点是确定保留甚至增加其对PPQ耐药寄生虫的活性的药物。我们认为，该项目为阐明PPQ抗性的分子基础、产生遗传标记以监测其出现和传播以及确定最佳治疗方法提供了强大的科学创新方法。该项目还将为Hb新陈代谢提供重要的新见解，Hb新陈代谢将继续为未来的临床使用提供丰富的抗疟疾药物来源。

 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.