Elucidating the molecular basis of piperaquine resistance in Plasmodium falciparum

项目总结 恶性疟疾病例最近激增，2020年估计有62.7万人死亡， 主要发生在非洲儿童身上。减轻非洲疾病负担的努力包括化学预防 保护弱势人群和用有效的青蒿素治疗症状性感染的战略-- 以综合疗法为基础。临床研究已证实哌喹(PPQ)是一种理想的化学预防方法 和治疗伙伴药物，因为它对无性血液期寄生虫有效，血浆半衰期长，以及 儿童和孕妇的安全状况良好。然而，这两种药物在一线组合中 双氢青蒿素-PPQ在东南亚遇到了广泛的耐药性。考虑到最近的 在卢旺达和乌干达出现了对青蒿素具有抗药性的PF寄生虫，迫切需要进行调查 PPQ抗药性能否在非洲出现和传播，确定其遗传媒介，并开发新的方法 来中和抵抗。在目标1中，我们的研究将集中在在以下背景下出现的新突变 Pf氯喹抗性转运蛋白PfCRT的氯喹抗性亚型及其扩增 血浆蛋白2和3(PM2/3)，这两种蛋白都与PPQ耐药和东南地区的治疗失败有关 亚洲。利用基因编辑和过度表达的方法，我们将确定PPQ抗性是否可以 通过将单个突变引入非洲特有的PfCRT亚型并评估 PM2/3拷贝数增加是否会增强抵抗力。我们还将评估PM2/3的扩增 为PfCRT突变寄生虫提供适合性益处，这可能有助于建立和保持对PPQ的抗性 高传播率的非洲环境。在目标2中，我们将研究抑制血红素解毒和 浓度依赖的药物外排分别在PPQ作用和耐药性中起作用。我们的研究还将测试 假设PPQ抗性PfCRT亚型扰乱细胞内血红蛋白衍生肽水平，这是 可通过PM2/3扩增进行校正。在目标3中，我们提出了直接靶向PfCRT介导的 耐药性代表了保持PPQ效力的一种高价值方法。利用关于反对的洞察力 选择压力，我们将测试PPQ是否与其他基于喹啉的药物(氯喹， 阿莫地喹或甲氟喹)可消除PPQ耐药寄生虫，防止耐药寄生虫复发 变种。我们还将筛选通过突变的PfCRT阻止PPQ外流来逆转耐药性的抑制剂。 检测将包括ZY19489，一种人体临床试验中的抗疟疾药物，可抑制PfCRT介导的药物外排和 使PF寄生虫对PPQ过敏。同时，我们将进行高通量筛查，旨在识别 新型PPQ抗性逆转剂。这项建议与NIAID支持研究的优先事项一致 关于抗菌素耐药性，旨在主动预测非洲出现PPQ耐药性 并确定可以中和耐药性并帮助维持这种基本药物的临床疗效的抑制剂。

PROJECT SUMMARY Plasmodium falciparum (Pf) malaria cases have recently surged, with an estimated 627,000 deaths in 2020, mostly in young African children. Efforts to reduce the burden of disease in Africa include both chemoprevention strategies to protect vulnerable populations and treatment of symptomatic infections with effective artemisinin- based combination therapies. Clinical studies have identified piperaquine (PPQ) as an ideal chemoprevention and treatment partner drug due to its potency against asexual blood stage parasites, long plasma half-life, and good safety profile in children and pregnant women. However, both drugs in the first-line combination of dihydroartemisinin-PPQ have encountered widespread resistance in Southeast Asia. Given the recent emergence in Rwanda and Uganda of artemisinin-resistant Pf parasites, there is a pressing need to investigate whether PPQ resistance can emerge and spread in Africa, identify its genetic mediators, and develop new ways to neutralize resistance. In Aim 1, our studies will focus on novel mutations that emerged on the background of chloroquine-resistant isoforms of the Pf chloroquine resistance transporter PfCRT, as well as the amplification of plasmepsins 2 and 3 (pm2/3), both of which contribute to PPQ resistance and treatment failure in Southeast Asia. Using gene editing and overexpression approaches, we will determine whether PPQ resistance can be generated in African lines by introducing individual mutations into African-specific PfCRT isoforms and assessing whether increased pm2/3 copy numbers augment resistance. We will also assess whether pm2/3 amplifications afford a fitness benefit to PfCRT mutant parasites, which could help establish and maintain PPQ resistance in high-transmission African settings. In Aim 2, we will examine the role of inhibition of heme detoxification and concentration-dependent drug efflux in PPQ action and resistance, respectively. Our studies will also test the hypothesis that PPQ-resistant PfCRT isoforms perturb intracellular hemoglobin-derived peptide levels, which may be corrected by pm2/3 amplification. In Aim 3, we propose that directly targeting PfCRT-mediated resistance represents a high-value approach to retaining PPQ efficacy. Leveraging insights regarding opposing selective pressures, we will test whether combining PPQ with other quinoline-based drugs (chloroquine, amodiaquine or mefloquine) can eliminate PPQ-resistant parasites and prevent the recrudescence of resistant variants. We will also screen for inhibitors that reverse resistance by blocking PPQ efflux via mutant PfCRT. Assays will include ZY19489, an antimalarial in human clinical trials that inhibits PfCRT-mediated drug efflux and hypersensitizes Pf parasites to PPQ. Concurrently, we will conduct a high-throughput screen designed to identify novel PPQ resistance reversal agents. This proposal, which aligns with NIAID’s priority of supporting research on antimicrobial drug resistance, is designed to proactively predict the emergence of PPQ resistance in Africa and to identify inhibitors that can neutralize resistance and help sustain the clinical efficacy of this essential drug.