Leveraging PfCRT Structure to Discern Function and Predict Emergence of Drug-Resistant Malaria

利用 PfCRT 结构识别功能并预测耐药性疟疾的出现

基本信息

批准号：
10653063
负责人：
David A Fidock
金额：
$ 69.46万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10653063
关键词：
4-aminoquinoline Africa African Alleles Amino Acids Amodiaquine Anti-malarial drug resistance Antimalarials Asia Asian Binding Bioenergetics Biological Assay Biological Markers Biology Blood Cambodia Cessation of life Chloroquine Chloroquine resistance Clinical Trials Combined Modality Therapy Complex Cryoelectron Microscopy DNA Sequence DNA delivery Data Dependence Drug Efflux Drug Metabolic Detoxication Drug Transport Drug resistance Engineering Fab Immunoglobulins Falciparum Malaria Genes Genetic Genetic Markers Genetic Variation Geography Glutathione Goals Haplotypes Heme Hemoglobin In Vitro Infection Integral Membrane Protein Investigation Ions Kinetics Length Life Lipids Liposomes Malaria Mediating Mediator Membrane Membrane Proteins Modeling Molecular Molecular Conformation Multi-Drug Resistance Mutate Mutation Parasite resistance Parasites Pattern Peptides Pharmaceutical Preparations Physiological Plasmodium falciparum Population Predisposition Principal Investigator Property Protein Biochemistry Protein Isoforms Proteins Recombinant Proteins Recombinants Reporting Research Resistance Resistance profile Resolution Role South American Structure System Technology Testing Transmembrane Transport Treatment Failure Universities Vacuole Variant asexual biophysical techniques driving force drug-sensitive effective therapy experimental study genetic resistance insight monomer mortality mutant nanodisk novel novel therapeutics particle pressure protein purification protein structure resistant Plasmodium falciparum trait transmission process

项目摘要

Drug resistance in Plasmodium falciparum (Pf), the deadliest of the malaria parasites that threatens almost half the world’s population, has been associated with genetic changes in specific parasite alleles from field isolates. The protein responsible for Pf asexual blood stage (ABS) parasite resistance to both previously and currently used first-line antimalarials, chloroquine (CQ) piperaquine (PPQ) and amodiaquine (ADQ), is the 48-kDa P. falciparum chloroquine resistance transporter (PfCRT). CQ, PPQ and ADQ, all 4-aminoquinolines, eliminate drug-sensitive Pf ABS parasites by inhibiting the detoxification of host heme, a product of parasite-mediated hemoglobin degradation, inside their digestive vacuole (DV). PfCRT, situated in the DV membrane, is thought to mediate CQ resistance via drug efflux. Our progress in understanding how PfCRT functions, and the molecular basis of PfCRT-mediated drug resistance, has been seriously hampered by the lack of an atomic model of this transporter. Using antigen-binding fragment (Fab) technology and single-particle cryo-electron microscopy, we have determined the structure of the full-length CQ-resistant 7G8 mutant isoform of this 10- transmembrane protein to 3.2 Å resolution, in an inward-open conformation. These preliminary data are presented herein, together with functional assays using purified protein in nanodiscs and in liposomes, and parasite-based assays with pfcrt-modified lines. In this application, we propose to compressively define PfCRT structure and function and leverage this into experimentally testable predictions of how PfCRT can further evolve to drive new patterns of multidrug resistance across malaria-endemic regions. In Aim 1, we will solve the PfCRT structure for globally variant isoforms, including complexes with the antimalarial drugs CQ, PPQ and ADQ, and physiologic substrates. In Aim 2, we will implement biophysical approaches with recombinant protein to elucidate the natural function of PfCRT and develop a model of PfCRT-mediated substrate and drug transport. In Aim 3, we will apply validated gene-editing approaches to predict emerging PfCRT-mediated resistance and elucidate its functional impact in parasites. Gene-editing studies will focus on PPQ and ADQ in the context of major global PfCRT variants, as a way to anticipate how mutant PfCRT could evolve new drug resistance traits, including with isoforms present in high-transmission African settings where drug-resistant malaria exerts by far its greatest impact. This coordinated research effort combines three Columbia University groups led by Drs. Mancia, Quick and Fidock, who bring expertise in membrane protein biochemistry and structure, bioenergetics of membrane transport, and Pf biology including mechanisms of antimalarial drug resistance, respectively. This highly integrated project has the potential to transform our understanding of how PfCRT mediates multidrug resistance, by providing powerful advances in deciphering PfCRT structure and function, delivering new biomarkers of emerging resistance, and identifying antimalarial combinations that could be used regionally to effectively treat drug-resistant Pf malaria.

恶性疟原虫（PF）中的耐药性，疟疾中最致命的寄生虫威胁近一半世界的人口与现场分离株的特定寄生虫等位基因的遗传变化有关。负责PF无性血液阶段（ABS）寄生虫抗性的蛋白质对以前和目前的抗性使用的一线抗疟药，氯喹（CQ）piperaquine（PPQ）和amodiaquine（ADQ）是48 kDa P. 恶性氯喹抗性转运蛋白（PFCRT）。 CQ，PPQ和ADQ，所有4-氨基喹啉，都消除了药物敏感的PF ABS寄生虫通过抑制寄生虫介导的产物宿主血红素的排毒血红蛋白降解，内部消化效果（DV）。 PFCRT位于DV膜中，被认为通过药物外介导CQ耐药性。我们了解PFCRT的功能以及 PFCRT介导的耐药性的分子基础，由于缺乏原子而受到严重阻碍使用抗原结合片段（FAB）技术和单粒子冷冻电子显微镜，我们已经确定了该10-的全长耐CQ 7G8突变体同工型的结构跨膜蛋白在向内开启构象中分辨率为3.2Å。这些初步数据是本文介绍，以及使用纳米散发和脂质体中纯化蛋白质的功能测定，以及基于PFCRT修饰线的寄生虫测定法。在此应用程序中，我们建议复杂定义PFCRT 结构和功能，并将其用于实验测试的预测PFCRT如何进一步的预测进化以驱动跨疟疾 - 内态区域的多药耐药性的新模式。在AIM 1中，我们将解决全球变体同工型的PFCRT结构，包括抗疟药CQ，PPQ的复合物和ADQ和生理基板。在AIM 2中，我们将通过重组实施生物物理方法蛋白质阐明PFCRT的自然功能并开发PFCRT介导的底物和药物的模型运输。在AIM 3中，我们将采用验证的基因编辑方法来预测新兴的PFCRT介导抗性并阐明其在寄生虫中的功能影响。基因编辑研究将集中于PPQ和ADQ 主要全球PFCRT变体的背景，以预测突变体PFCRT如何进化新药抵抗性状，包括在非洲抗药性的高转移环境中存在的同工型疟疾的影响最大。这项协调的研究工作结合了三所哥伦比亚大学由博士领导的小组。曼西亚（Mancia），快速和菲达克（Fidock），他带来了膜蛋白生物化学方面的专业知识结构，膜传输的生物能学和PF生物学，包括抗疟药机制电阻分别。这个高度集成的项目有可能改变我们对 PFCRT通过在解密PFCRT结构和功能，提供新的抗性生物标志物，并确定抗疟疾组合可以在区域用来有效治疗耐药的PF疟疾。