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) 的耐药性是最致命的疟原虫，威胁着近一半的疟原虫世界人口的变化与野外分离株的特定寄生虫等位基因的遗传变化有关。负责 Pf 无性血期 (ABS) 寄生虫对以前和现在的抵抗力的蛋白质使用的一线抗疟药是氯喹 (CQ)、哌喹 (PPQ) 和阿莫地喹 (ADQ)，是 48 kDa P. 恶性疟原虫氯喹抗性转运蛋白 (PfCRT)，CQ、PPQ 和 ADQ，所有 4-氨基喹啉，均被消除。通过抑制宿主血红素（寄生虫介导的产物）的解毒来抑制药物敏感的 Pf ABS 寄生虫消化液泡 (DV) 内的血红蛋白降解被认为是位于 DV 膜上的。通过药物外流介导 CQ 耐药性我们在了解 PfCRT 如何发挥作用以及 PfCRT 介导的耐药性的分子基础由于缺乏原子该转运蛋白的模型使用抗原结合片段（Fab）技术和单粒子冷冻电子。显微镜下，我们已经确定了该 10-CQ 抗性 7G8 突变同种型的全长结构跨膜蛋白分辨率为 3.2 Å，处于向内开放的构象。本文提出的，以及使用纳米盘和脂质体中的纯化蛋白质的功能测定，以及在此应用中，我们建议压缩定义 PfCRT。结构和功能，并利用其进行可实验测试的预测，以了解 PfCRT 如何进一步在目标 1 中，我们将解决疟疾流行地区的多重耐药性新模式。全球变异异构体的 PfCRT 结构，包括与抗疟药 CQ、PPQ 的复合物在目标 2 中，我们将通过重组实施生物物理方法。蛋白质来阐明 PfCRT 的天然功能并开发 PfCRT 介导的底物和药物模型在目标 3 中，我们将应用经过验证的基因编辑方法来预测新兴的 PfCRT 介导的运输。抗性并阐明其对寄生虫的功能影响基因编辑研究将重点关注 PPQ 和 ADQ。全球主要 PfCRT 变体的背景，作为预测突变 PfCRT 如何进化新药的一种方式耐药性特征，包括在非洲高传播环境中存在的同种型，其中耐药性这项协调一致的研究工作由哥伦比亚大学三所大学联合开展。由 Mancia、Quick 和 Fidock 博士领导的小组，他们带来了膜蛋白生物化学和结构、膜转运的生物能学和 Pf 生物学，包括抗疟药物的机制这个高度集成的项目有可能改变我们对如何抵抗的理解。 PfCRT 通过在破译 PfCRT 结构和功能，提供新出现的耐药性的新生物标志物，并识别抗疟药物组合可在区域内有效治疗耐药性 Pf 疟疾。