The Function of Antimalarial Drug Resistance Proteins

项目摘要 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 伴侣药物（特别是哌喹[PPQ]、阿莫地喹[AQ]和本芴醇[LF]），并受 恶性疟原虫转运蛋白PfCRT的突变。在发现的61种已知不同的PfCRT蛋白亚型中， 自2000年以来，一些介导的新兴或进化的表型选择的相对最近的变化， 线抗疟药物治疗。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。这些赋予了在常见CQ-100中观察到的CQ IC 50的约10倍变化中的大部分。 耐药（CQR）菌株，对其他药物的耐药性较轻，在这些菌株中经常观察到可变的耐药性。 然而，只有少数的异构体已在分子深度研究。Dd 2（亚洲/非洲）和7 G8 （S. America）亚型均赋予CQR，但7 G8 PfCRT赋予较低的CQR，并且还介导AQR。最近 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，转运生理学，反向遗传学， 和结构生物学，利用Roepe（乔治敦），Fidock（哥伦比亚）， 张（加州大学圣地亚哥分校）和斯托威尔（加州大学，博尔德）实验室，以全面界定新的突变异构体 PfCRT影响目前正在演变的抗疟药物耐药性。 我们有三个目标：1）我们将使用新的和以前验证的抗疟疾药物探针，工程酵母 菌株和纯化的重组蛋白，以明确地定义表达于 进化中的恶性疟原虫对喹啉类药物（氯喹; CQ，哌喹; PPQ）和青蒿素都有抗药性 - 2）我们将表征表达PfCRT同种型的恶性疟原虫的反向工程菌株 在特定的恶性疟原虫遗传背景下产生，以分离和表征精确的 PPQR相关PfCRT功能对进化表型的贡献，以及3）我们将利用我们的 PfCRT的纯化，PfCRT特异性纳米抗体的高通量生产， cryoEM方法，分子动力学和其他物理化学方法，以显着扩大最近的 在定义PfCRT同种型的原子水平结构方面的合作成功。

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.