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

模拟突变 PfCRT 介导的药物转运以预测耐哌喹恶性疟原虫疟疾的出现

• 批准号: 10326368
• 负责人: Laura Marie Hagenah
• 金额: $ 4.68万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10326368
• 关键词: Affect; Africa; African; African American; Alleles; Amino Acid Substitution; Amodiaquine; Anti-malarial drug resistance; Antimalarials; Area; Asia; Asian; Binding; Biological Assay; Biophysics; Blood; Cessation of life; Charge; Child; Chloroquine; Chloroquine resistance; Clinical; Coculture Techniques; Combined Modality Therapy; Cryoelectron Microscopy; Data; Drug Efflux; Drug Transport; Drug resistance; Engineering; Exhibits; Falciparum Malaria; Flow Cytometry; Genes; Genetic; Genetic Engineering; Geographic Locations; Geography; Globin; Goals; Growth; Haplotypes; Hemoglobin; In Vitro; Kinetics; Knowledge; Malaria; Malaria Vaccines; Mediating; Methods; Modeling; Molecular; Morbidity - disease rate; Mutation; Organelles; Parasites; Peptide Transport; Peptides; Persons; Pharmaceutical Preparations; Physiological; Plasmodium falciparum; Predisposition; Property; Protein Biosynthesis; Protein Isoforms; Recombinant Proteins; Regimen; Reporter; Research; Resistance; Resolution; Risk; Role; Site; South America; South American; Southeastern Asia; Structure; Susceptibility Gene; System; Techniques; Testing; Therapeutic; Time; Treatment Protocols; Universities; Vaccines; Vacuole; Variant; asexual; base; chemotherapy; combat; cost; disorder control; fitness; global health; insight; mortality; mutant; new combination therapies; nonsynonymous mutation; novel; resistance mutation; resistant Plasmodium falciparum; resistant strain; socioeconomics; therapy design; therapy development; treatment strategy; Affect; Africa; African; African American; Alleles; Amino Acid Substitution; Amodiaquine; Anti-malarial drug resistance; Antimalarials; Area; Asia; Asian; Binding; Biological Assay; Biophysics; Blood; Cessation of life; Charge; Child; Chloroquine; Chloroquine resistance; Clinical; Coculture Techniques; Combined Modality Therapy; Cryoelectron Microscopy; Data; Drug Efflux; Drug Transport; Drug resistance; Engineering; Exhibits; Falciparum Malaria; Flow Cytometry; Genes; Genetic; Genetic Engineering; Geographic Locations; Geography; Globin; Goals; Growth; Haplotypes; Hemoglobin; In Vitro; Kinetics; Knowledge; Malaria; Malaria Vaccines; Mediating; Methods; Modeling; Molecular; Morbidity - disease rate; Mutation; Organelles; Parasites; Peptide Transport; Peptides; Persons; Pharmaceutical Preparations; Physiological; Plasmodium falciparum; Predisposition; Property; Protein Biosynthesis; Protein Isoforms; Recombinant Proteins; Regimen; Reporter; Research; Resistance; Resolution; Risk; Role; Site; South America; South American; Southeastern Asia; Structure; Susceptibility Gene; System; Techniques; Testing; Therapeutic; Time; Treatment Protocols; Universities; Vaccines; Vacuole; Variant; asexual; base; chemotherapy; combat; cost; disorder control; fitness; global health; insight; mortality; mutant; new combination therapies; nonsynonymous mutation; novel; resistance mutation; resistant Plasmodium falciparum; resistant strain; socioeconomics; therapy design; therapy development; treatment strategy

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.

项目摘要 原生动物寄生虫恶性疟原虫每年导致超过40万疟疾死亡，主要是在年轻人 非洲儿童。没有有效的疫苗，化疗仍然是疟疾治疗和 控制。消除疟疾的努力受到了对一线抗疟药的抗性的兴起 东南亚，包括Piperaquine（PPQ，与二氢二甲蛋白结合使用）。 Piperaquine 耐药性（PPQ-R）和氯喹的耐药性主要是由恶性疟原虫中的突变介导的 氯喹抗性转运蛋白（PFCRT）。突变PFCRT将药物从其作用部位移走 无性血液寄生虫的消化真空吸尘器。这种酸性细胞器降解内吞宿主血红蛋白 并挤出了全球蛋白衍生的宠物用于寄生虫蛋白质的合成。但是，不了解的是如何确定 氨基酸取代会议会议这种外排机制及其如何影响天然转运蛋白的功能。到 研究这些突变的效果，我在目标1中提出了净化当代PPQ-R PFCRT同工型和 用PPQ，其他临床使用的抗疟药进行结合和运输测定 肽作为拟议的天然底物。这些研究将提供全面的功能表征 目前缺乏的PPQ-R PFCRT预测PPQ-R如何扩散或出现也很重要 在其他疟疾内在地区。在东南亚亚洲发现的PFCRT突变尚未在非洲和 南美背景；但是，PPQ在这些领域中使用，包括一线治疗或用于 简单的疟疾。为了预测SE亚洲中看到的当代氨基酸取代是否可以 在其他地区出现以实现PPQ-R，我提出了AIM 2的建议，以将这些突变设计到非洲和 来自这些地区的恶性疟原虫的南美PFCRT同工型。测定将量化 这些线对PPQ和其他抗疟药的敏感性，并确定每条线的相对适应性。这 赋予抵抗力的程度，并且施加的适应性成本以及区域药物方案将是 对于确定哪种PFCRT等位基因占主导地位以及哪些可以在PPQ区域出现和扩散的重要 使用。这些目的预测了（1）PFCRT中PPQ-R突变改变药物转运的假设 和（2）PPQ-R在非洲和南美可以通过单一的出现而出现（2）PPQ-R 在突变体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恢复了氯喹的寄生虫敏感性，如SE亚洲的大多数突变所示， 为新组合疗法创造治疗机会。这些研究有望产生重要 对抗疟药耐药性的分子基础的新见解，并利用知道预测的知识 不同地理区域中新型PPQ-R PFCRT同工型的出现。这将指导发展 减少耐药疟疾的全球影响和传播的治疗策略。