The Function of Antimalarial Drug Resistance Proteins

点击翻译按钮获取中文摘要

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.