Elucidating the molecular basis of piperaquine resistance in Plasmodium falciparum

项目摘要 恶性疟原虫（Pf）疟疾病例最近激增，2020年估计有627，000人死亡， 大多数是非洲儿童。减少非洲疾病负担的努力包括化学预防和 制定战略，保护弱势群体，用有效的青蒿素治疗有症状的感染， 基于联合疗法。临床研究已确定哌喹（PPQ）作为一种理想的化学预防 和治疗伴侣药物，因为其对无性血液阶段寄生虫的效力，血浆半衰期长， 在儿童和孕妇中具有良好的安全性。然而，这两种药物在一线联合用药中， 双氢青蒿素-PPQ在东南亚遇到了广泛的耐药性。鉴于最近 由于卢旺达和乌干达出现了对青蒿素具有抗药性的Pf寄生虫，因此迫切需要进行调查 PPQ耐药性是否会在非洲出现和传播，确定其遗传介质，并开发新的方法， 来压制抵抗在目标1中，我们的研究将集中在新出现的突变的背景下， Pf氯喹抗性转运蛋白PfCRT的氯喹抗性同种型，以及Pf氯喹抗性转运蛋白PfCRT的扩增。 plasmepsin 2和3（pm2/3），这两者都有助于PPQ耐药和治疗失败，在东南部 亚洲使用基因编辑和过表达方法，我们将确定PPQ抗性是否可以被抑制。 通过将个体突变引入非洲特异性PfCRT同种型并评估 增加的pm2/3拷贝数是否增加抗性。我们还将评估pm2/3扩增是否 为PfCRT突变寄生虫提供适合性益处，这可以帮助建立和维持PPQ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的作用， PPQ作用和耐药性中的浓度依赖性药物外排。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， 可以通过pm2/3扩增来校正。在目标3中，我们提出直接靶向PfCRT介导的 耐药性代表了保持PPQ疗效的高价值方法。利用关于反对 选择性压力，我们将测试是否将PPQ与其他喹啉类药物（氯喹， 阿莫地喹或甲氟喹）可以消除抗PPQ寄生虫，并防止耐药寄生虫复发。 变体。我们还将筛选通过突变PfCRT阻断PPQ外排逆转耐药性的抑制剂。 试验将包括ZY 19489，一种在人体临床试验中抑制PfCRT介导的药物外排的抗疟药， 使Pf寄生虫对PPQ超敏。同时，我们将进行高通量筛选， 新型PPQ抗性逆转剂。该提案符合NIAID支持研究的优先事项 关于抗菌药物耐药性，旨在积极预测非洲PPQ耐药性的出现 并确定抑制剂，可以中和耐药性，并帮助维持这种基本药物的临床疗效。

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.