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

恶性疟原虫（Pf）的抗药性，这是威胁近一半的疟疾寄生虫中最致命的一种。 世界人口中，已与遗传变化的特定寄生虫等位基因从外地分离。 负责Pf无性血阶段（ABS）寄生虫对以前和现在的抗性的蛋白质 使用的一线抗疟药氯喹（CQ）、哌喹（PPQ）和阿莫地喹（ADQ）是48-kDa的P。 恶性疟原虫氯喹抗性转运体（PfCRT）。CQ、PPQ和ADQ，所有4-氨基喹啉，消除 通过抑制宿主血红素的解毒作用，一种寄生虫介导的 血红蛋白降解，在他们的消化泡（DV）。位于DV膜中的PfCRT被认为是 通过药物外排介导CQ抗性。我们在理解PfCRT功能方面的进展，以及 PfCRT介导的耐药性的分子基础，由于缺乏原子 这个运输机的型号。利用抗原结合片段（Fab）技术和单粒子冷冻电子 显微镜下，我们已经确定了全长CQ抗性7 G8突变体同种型的结构，这10- 跨膜蛋白的3.2倍分辨率，在向内开放的构象。这些初步数据是 本发明的方法，连同使用纳米盘和脂质体中的纯化蛋白的功能测定，以及 用PFCRT修饰的细胞系进行基于寄生虫的测定。在这个应用中，我们提出压缩定义PfCRT 结构和功能，并利用这一点来实验测试预测PfCRT如何能够进一步 在疟疾流行地区推动新的多药耐药性模式。在目标1中，我们将解决 PfCRT结构的整体变异亚型，包括与抗疟药物CQ，PPQ的复合物 和ADQ以及生理底物。在目标2中，我们将实施生物物理方法， 蛋白阐明PfCRT的天然功能，并开发PfCRT介导的底物和药物模型 运输在目标3中，我们将应用经验证的基因编辑方法来预测新出现的PfCRT介导的 抗性，并阐明其在寄生虫中的功能影响。基因编辑研究将重点关注PPQ和ADQ， 主要全球PfCRT变体的背景，作为预测突变PfCRT如何演变新药的一种方式 耐药性状，包括在高传播非洲环境中存在的同种型， 疟疾的影响最大。这项协调的研究工作结合了哥伦比亚大学的三个 由Mancia，Quick和Fidock博士领导的小组，他们带来了膜蛋白生物化学方面的专业知识， 结构、膜转运生物能量学和Pf生物学，包括抗疟药物机制 阻力，分别。这个高度集成的项目有可能改变我们对如何 PfCRT介导多药耐药性，通过在破译PfCRT结构和 功能，提供新的耐药性生物标志物，并确定抗疟组合， 可在区域范围内用于有效治疗耐药Pf疟疾。

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.