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

项目摘要 原生动物寄生虫恶性疟原虫每年导致40多万人死于疟疾，其中大多数是年轻人。 非洲儿童。由于没有有效的疫苗，化疗仍然是疟疾治疗的基石， 控制2010年，一线抗疟药物耐药性上升，阻碍了根除疟疾的努力。 东南亚，包括哌喹（PPQ，与双氢青蒿素联合使用）。哌喹 抗多效唑（PPQ-R）和氯喹抗性主要由恶性疟原虫中的突变介导 氯喹抗性转运蛋白（PfCRT）。突变PfCRT转运药物远离其作用位点， 无性血液期寄生虫的消化泡。这种酸性细胞器降解内吞的宿主血红蛋白 并挤出珠蛋白衍生肽用于寄生虫蛋白质合成。然而，人们不太了解的是， 氨基酸取代赋予这种外排机制以及它们如何影响天然转运蛋白功能。到 为了研究这些突变的影响，我在目标1中提出纯化当代PPQ-R PfCRT亚型， 与PPQ、其他临床使用的抗疟药和正电荷药物进行结合和转运试验 肽作为建议的天然底物。这些研究将提供一个全面的功能表征 PPQ-R PfCRT目前缺乏。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突变还没有在非洲和 南美背景;但是，PPQ正在这些领域使用，包括作为一线治疗或 单纯性疟疾为了预测在东南亚发现的当代氨基酸替换是否可以 为了实现PPQ-R，我在Aim 2中建议将这些突变工程化到非洲和非洲的基因组中， 来自这些地区的恶性疟原虫中的南美洲PfCRT亚型。分析将量化 这些品系对PPQ和其他抗疟药的敏感性，并确定每个品系的相对适合度。的 赋予的抵抗程度和强加的健身成本，沿着区域药物方案，将是 重要的是确定哪些pfcrt等位基因占优势，哪些可以出现和传播的地区PPQ 使用.这些目标是基于以下假设：（1）PfCRT中的PPQ-R突变改变药物的转运 PPQ-R可以在非洲和南美洲通过出现单一的 在东南亚突变PfCRT中观察到的氨基酸取代。这些数据还将确定是否获得 PPQ-R恢复了寄生虫对氯喹的易感性，正如在东南亚的大多数突变所见， 为新的组合疗法创造治疗机会。这些研究预计将产生重要的 对抗疟药物耐药性的分子基础的新见解，并利用这些知识来预测 在不同的地理区域出现新的PPQ-R PfCRT同种型。这将指导发展 因此，我们需要制定治疗战略，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.