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

恶性疟原虫(PF)的抗药性，这是最致命的疟疾寄生虫，威胁着近一半的人 世界人口，一直与来自野外分离株的特定寄生虫等位基因的遗传变化有关。 Pf无性血液期(ABS)寄生虫对以前和现在的抗性蛋白 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蛋白(PfCRT)。CQ，PPQ和ADQ，所有4-氨基喹啉，消除 通过抑制寄生虫介导的宿主血红素的解毒作用实现对药物敏感的PF ABS寄生虫 在消化液泡(DV)内的血红蛋白降解。位于DV膜上的PfCRT被认为是 通过药物外排介导CQ耐药。我们在理解PfCRT如何工作方面的进展，以及 PfCRT介导的耐药的分子基础因缺乏原子而受到严重阻碍 这辆运输车的型号。使用抗原结合片段(FAB)技术和单粒子冷冻电子 显微镜下，我们已经确定了抗CQ的全长7G8突变异构体的结构。 跨膜蛋白的分辨率为3.2？，呈向内开放的构象。这些初步数据是 结合在纳米盘和脂质体中使用纯化蛋白的功能分析，以及 用pfcrt修饰的线条进行寄生虫检测。在本应用程序中，我们建议压缩定义PfCRT 结构和功能，并利用这一点来预测PfCRT如何进一步 演变以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 全球变异异构体的PfCRT结构，包括与抗疟疾药物CQ，PPQ的络合物 ADQ和生理底物。在目标2中，我们将使用重组生物物理方法 蛋白质阐明PfCRT的自然功能并建立PfCRT介导的底物和药物模型 运输。在目标3中，我们将应用经过验证的基因编辑方法来预测PfCRT介导的新出现 并阐明其在寄生虫中的功能影响。基因编辑研究将集中在#年的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 耐药特征，包括在耐药的非洲高传播环境中存在的异构体 到目前为止，疟疾的影响最大。这项协调一致的研究工作结合了三所哥伦比亚大学 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、膜转运的生物能量学和PF生物学，包括抗疟疾药物的作用机制 分别为抗药性。这个高度集成的项目有可能改变我们对如何 PfCRT通过在破译PfCRT结构和 功能，提供新的抗药性生物标记物，并确定抗疟疾组合 可用于地区性有效治疗抗药性疟疾。

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.