The Function of Antimalarial Drug Resistance Proteins

项目摘要 疟疾寄生虫对氯喹的敏感性(CQ)以及重要的青蒿素联合疗法(ACT) 伙伴药物(特别是哌喹[PPQ]、阿莫地喹[AQ]和鲁米芬[LF])，受 恶性疟原虫转运蛋白PfCRT的突变。在已发现的61种不同的PfCRT蛋白亚型中 自2000年以来，一些中介新出现或进化的表型是通过前部相对较新的变化而选择的- 一线抗疟疾药物治疗。在耐药寄生虫中，PfCRT蛋白含有4到10个氨基酸 相对于野生型的替换。这些提供了常见CQ-IC50中~10倍的移位- 耐药(CQR)菌株，对其他药物的耐药性较温和，在这些菌株中经常观察到。 然而，只有少数几种异构体被分子深度研究。DD2(亚洲/非洲)和7G8 美国的7G8 PfCRT既有CQR，也有CQR，但7G8 PfCRT有较低的CQR，也有AQR。近期 数据显示，一些罕见的突变PfCRT可能根本不会产生耐药性。 该项目汇集了蛋白质生物化学、转运蛋白生理学、反向遗传学、 和结构生物学，通过利用Roepe(乔治敦)、Fidock(哥伦比亚)、 Chang(UCSD)和Stowell(加州大学博尔德分校)实验室全面定义新的突变异构体 PfCRT的影响目前正在演变中的抗疟疾耐药性。 我们有三个目标：1)我们将使用新的和以前验证过的抗疟疾药物探针，工程酵母 菌株和纯化的重组蛋白明确定义PfCRT异构体的功能 恶性疟原虫对喹啉类(氯喹；氯喹；哌喹；PPQ)和青蒿素的抗药性演变 -基于药物，2)我们将表征表达PfCRT亚型的恶性疟原虫反向工程菌株 是在特定的恶性疟原虫遗传背景下创建的，以便分离和表征准确的 PPQR相关的PfCRT功能对进化表型的贡献，以及3)我们将利用我们的 在PfCRT的纯化、高通量生产PfCRT特定的纳米体方面取得了相当大的进展， 低温电子显微镜方法、分子动力学和其他物理化学方法显著扩展了最近的 在确定PfCRT异构体的原子级结构方面取得合作成功。

Project Summary Malarial parasite sensitivity to chloroquine (CQ) as well as important artemisinin combination therapy (ACT) partner drugs (notably piperaquine [PPQ], amodiaquine [AQ], and lumefantrine [LF]) and are affected by mutations in the P. falciparum transporter PfCRT. Of the 61 known distinct PfCRT protein isoforms discovered since 2000, some mediate emerging or evolving phenotypes selected for by relatively recent changes in front- line antimalarial drug therapy. In drug resistant parasites, PfCRT protein harbors 4 to 10 amino acid substitutions relative to wild type. These confer most of the ~ 10-fold shift in CQ IC50 seen for common CQ- resistant (CQR) strains, with milder, variable resistance to other drugs often observed in these strains. However, only a handful of the isoforms have been studied in molecular depth. The Dd2 (Asia/Africa) and 7G8 (S. America) isoforms both confer CQR, but 7G8 PfCRT confers lower CQR and also mediates AQR. Recent data shows some rare mutant PfCRTs may not confer drug resistance at all. This project brings together expertise in protein biochemistry, transporter physiology, reverse genetics, and structural biology by leveraging the established expertise of the Roepe (Georgetown), Fidock (Columbia), Chang (UCSD), and Stowell (U.C.,Boulder) laboratories to comprehensively define how new mutant isoforms of PfCRT influence currently evolving antimalarial drug resistance. We have three aims: 1) we will use novel and previously validated antimalarial drug probes, engineered yeast strains and purified recombinant protein to unambiguously define function of PfCRT isoforms expressed in evolving P. falciparum resistant to both quinoline – based (chloroquine; CQ, piperaquine; PPQ) and Artemisinin – based drugs, 2) we will characterize reverse engineered strains of P. falciparum expressing PfCRT isoforms that are created in specific P. falciparum genetic backgrounds, in order to isolate and characterize the precise contribution of PPQR-associated PfCRT function to evolving phenotypes, and 3) we will leverage our considerable progress with purification of PfCRT, highthroughput production of PfCRT specific nanobodies, cryoEM methods, molecular dynamics, and other physical chemical approaches to significantly expand recent collaborative success at defining atomic level structure of PfCRT isoforms.