Modeling mutant PfCRT-mediated drug transport to predict the emergence of piperaquine-resistant Plasmodium falciparum malaria

项目总结 原虫恶性疟原虫每年导致40多万人死于疟疾，其中大部分是年轻人。 非洲儿童。由于没有有效的疫苗，化疗仍然是疟疾治疗和治疗的基石 控制力。年，对一线抗疟疾药物的耐药性上升，阻碍了根除疟疾的努力 东南亚，包括哌喹(PPQ，与双氢青蒿素结合使用)。哌喹 恶性疟原虫的抗药性(PPQ-R)和氯喹抗药性主要由突变所介导 氯喹抗性转运蛋白(PfCRT)。突变体PfCRT将药物从其作用部位转移到 无性血液期寄生虫的消化液泡。这种酸性细胞器降解内吞的宿主血红蛋白。 并分泌珠蛋白衍生的多肽用于寄生虫蛋白质的合成。然而，人们不太清楚的是，有多确定 氨基酸替代提供了这种外流机制，以及它们如何影响天然转运蛋白的功能。至 研究这些突变的影响，我在目标1中建议纯化当代PPQ-R PfCRT亚型和 与PPQ、其他临床使用的抗疟疾药物和带正电荷的药物进行结合和转运分析 多肽作为建议的天然底物。这些研究将提供一个全面的功能特征 目前所缺乏的PPQ-R PfCRT。预测PPQ-R如何传播或出现也很重要 在其他疟疾流行地区。在东南亚发现的PfCRT突变尚未在非洲和 南美背景；然而，PPQ正被用于这些领域，包括作为一线治疗或用于 简单的疟疾。为了预测东南亚当代的氨基酸替代是否可以 在其他地区出现以实现PPQ-R，我在目标2中建议将这些突变工程到非洲和 南美地区恶性疟原虫PfCRT亚型。化验结果会量化 这些品系对PPQ和其他抗疟疾药物的敏感性，并确定每个品系的相对适合度。这个 授予的抗药性程度和强加的适应成本，以及区域药物方案，将是 在确定哪些pfcrt等位基因占优势，以及哪些可能在PPQ地区出现和传播方面很重要 使用。这些目的是基于以下假设：(1)PfCRT中的PPQ-R突变改变了药物的转运 和天然底物，以及(2)PPQ-R可以通过单一的 在东南亚观察到突变型PfCRT的氨基酸替换。这些数据还将确定是否获得了 PPQ-R恢复了寄生虫对氯喹的敏感性，就像东南亚的大多数突变一样，从而 为新的联合疗法创造治疗机会。这些研究预计将产生重要的 对抗疟疾耐药性的分子基础的新见解，并利用这些知识来预测 新的PPQ-R PfCRT亚型在不同的地理区域出现。这将指导我们的发展。 制定治疗战略，以减少抗药性疟疾的全球影响和传播。

PROJECT SUMMARY The protozoan parasite Plasmodium falciparum causes over 400,000 malaria deaths each year, mostly in young African children. With no effective vaccine, chemotherapy remains the cornerstone of malaria treatment and control. Malaria eradication efforts have been hindered by the rise of resistance to first-line antimalarials in Southeast Asia, including piperaquine (PPQ, used in combination with dihydroartemisinin). Piperaquine resistance (PPQ-R) and chloroquine resistance are primarily mediated by mutations in the P. falciparum Chloroquine Resistance Transporter (PfCRT). Mutant PfCRT transports drug away from its site of action in the asexual blood stage parasite’s digestive vacuole. This acidic organelle degrades endocytosed host hemoglobin and extrudes globin-derived peptides for parasite protein synthesis. Less understood, however, is how certain amino acid substitutions confer this efflux mechanism and how they impact native transporter function. To investigate the effect of these mutations, I propose in Aim 1 to purify contemporary PPQ-R PfCRT isoforms and perform binding and transport assays with PPQ, other clinically used antimalarials, and positively-charged peptides as proposed natural substrates. These studies will provide a comprehensive functional characterization of PPQ-R PfCRT that is currently lacking. It is also important to predict how PPQ-R could spread to or emerge in other malaria endemic regions. The PfCRT mutations found in SE Asia have yet to be seen on African and South American backgrounds; however, PPQ is being used in these areas including as first-line treatment or for uncomplicated malaria. To predict whether the contemporary amino acid substitutions seen in SE Asia could emerge in other regions to achieve PPQ-R, I propose in Aim 2 to engineer these mutations onto African and South American PfCRT isoforms in P. falciparum parasites from these regions. Assays will quantify the susceptibility of these lines to PPQ and other antimalarials, and determine the relative fitness of each line. The degree of resistance conferred and the fitness cost imposed, along with the regional drug regimen, will be important in determining which pfcrt alleles predominate and which can emerge and spread in areas of PPQ use. These aims are predicated on the hypotheses that (1) PPQ-R mutations in PfCRT alter transport of drugs and natural substrates and that (2) PPQ-R can arise in Africa and South America via the emergence of single amino acid substitutions observed in SE Asia in mutant PfCRT. These data will also identify whether a gain of PPQ-R restores parasite susceptibility to chloroquine, as has been seen with most mutations in SE Asia, thereby creating therapeutic opportunities for new combination therapies. These studies are expected to yield important new insights into the molecular basis for antimalarial drug resistance, and to leverage that knowledge to predict the emergence of novel PPQ-R PfCRT isoforms in distinct geographic regions. This will guide the development of treatment strategies to reduce the global impact and spread of drug-resistant malaria.