The Function of Antimalarial Drug Resistance Proteins

项目概要 疟疾寄生虫对氯喹（CQ）以及重要的青蒿素联合疗法（ACT）的敏感性 伙伴药物（特别是哌喹 [PPQ]、阿莫地喹 [AQ] 和苯芴醇 [LF]），并受到 恶性疟原虫转运蛋白 PfCRT 发生突变。已发现 61 种已知的不同 PfCRT 蛋白亚型 自2000年以来，一些介导了由相对最近的变化选择的新兴或演变的表型 行抗疟药物治疗。在耐药寄生虫中，PfCRT 蛋白含有 4 至 10 个氨基酸 相对于野生型的取代。这些赋予了常见 CQ- 所见 CQ IC50 的大部分约 10 倍的变化 耐药（CQR）菌株，对这些菌株中经常观察到的其他药物具有较温和、可变的耐药性。 然而，只有少数异构体得到了分子深度的研究。 Dd2（亚洲/非洲）和 7G8 （南美）亚型均赋予 CQR，但 7G8 PfCRT 赋予较低的 CQR，并介导 AQR。最近 数据显示，一些罕见的 PfCRT 突变体可能根本不产生耐药性。 该项目汇集了蛋白质生物化学、转运生理学、反向遗传学、 和结构生物学，利用 Roepe（乔治城）、Fidock（哥伦比亚）的既定专业知识， Chang（加州大学圣地亚哥分校）和 Stowell（加州大学博尔德分校）实验室全面定义新的突变亚型 PfCRT 影响当前不断发展的抗疟药物耐药性。 我们有三个目标：1）我们将使用新颖且先前经过验证的抗疟药物探针、工程酵母 菌株和纯化的重组蛋白，以明确定义 PfCRT 亚型的功能 不断发展的恶性疟原虫对喹啉类药物（氯喹；CQ，哌喹；PPQ）和青蒿素具有抗药性 – 基于药物，2) 我们将表征表达 PfCRT 同种型的恶性疟原虫逆向工程菌株 在特定的恶性疟原虫遗传背景中创建，以便分离和表征精确的恶性疟原虫 PPQR 相关的 PfCRT 功能对进化表型的贡献，3）我们将利用我们的 在 PfCRT 的纯化、PfCRT 特异性纳米抗体的高通量生产方面取得了重大进展， 冷冻电镜方法、分子动力学和其他物理化学方法显着扩展了最近的研究 合作成功定义 PfCRT 同种型的原子级结构。

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.