The function of antimalarial drug resistance proteins

描述（由申请人提供）：耐药疟疾每年杀死数百万人。要扭转发病率和死亡率方面的可怕趋势，需要在疫苗和药物研究方面采取平衡的做法，并在现场努力控制病媒种群和感染率。当前和未来对现有的许多不同耐药疟疾菌株的治疗需要对多种基因型和表型有更全面的了解。我们绝不能被目前基于青蒿素（ART）的疗法所提供的（暂时）安全的错误感觉所迷惑；黑市抗逆转录病毒疗法已经在流通，并产生了对抗逆转录病毒疗法的抵制。防治耐药疟疾的斗争仍在继续，必须继续进行；否则，我们就没有从过去50年的经验教训中吸取教训，只能眼睁睁地看着CQ和其他药物的失败。我们必须“走在耐药性曲线的前面”，确定指导正在进行的药物和疫苗研究的分子机制。我们的实验室在使用异源表达系统对PfCRT和PfMDR1蛋白进行分子水平分析方面处于领先地位。在这一竞争更新时期，我们将：目标1)通过酵母中的异种表达和纯化膜、ISOV和PL制剂的分析，继续确定PfCRT亚型的结合功能。我们将使用最新开发的技术和化学探针来研究药物、氨基酸和离子的结合和运输。我们还将合成用于PfCRT功能的其他探针（例如AzB-MQ， AzBCQ侧链长度变体，AzB-QN）。这些探针也将在Aim 3中使用。目的2)继续使用ISOV和PLs以及无线电标记和荧光（如NBD CQ）探针确定PfCRT亚型的药物转运功能。我们还将使用先前合成的中间体和与成功合成NBD-CQ相似的化学反应合成其他探针（例如，NBD-MQ, NBD-QN）。目的3)采用类似的方法测试PfMDR1功能的假设，并使用我们开发和发表的基于高通量板的atp酶测定[93,93b]。我们将研究不寻常的（相对于其他ABCB转运体）药物影响的PfMDR1两个对称半体之间的“通信”[93]。我们将分析含有各种PfCRT和PfMDR1蛋白的已知比例的ISOV和PLs的结合、运输和atp酶特性，以测试两种转运蛋白之间的相互作用。

DESCRIPTION (provided by applicant): Drug resistant malaria kills millions annually. Reversing horrific trends in incidence and mortality requires a balanced approach in vaccine and drug research, as well as field based efforts to control vector populations and infection rates. Current and future treatment of the many different strains of drug resistant malaria that now exist requires a more complete understanding of multiple genotypes and phenotypes. We must not be lulled into a false sense of (temporary) security provided by current artemisinin (ART) based therapies; black market ART is already circulating and generating ART resistance. The struggle against drug resistant malaria is ongoing and must be met continuously; else we have learned nothing from the past 50 years while watching CQ and other drugs fail. We must "stay ahead of the resistance curve" and define molecular mechanisms that guides ongoing drug and vaccine research. Our laboratory has helped to lead the field in molecular level analysis of PfCRT and PfMDR1 proteins using heterologous expression systems. In this competitive renewal period we will: Aim 1) Continue to define binding functions of PfCRT isoforms via heterlogous expression in yeast and analysis of purified membrane, ISOV, and PL preparations harboring these proteins. We will use recently developed techniques and chemical probes for drug, amino acid, and ion binding and transport. We will also synthesize additional probes (e.g.,AzB-MQ, AzBCQ side chain length variants, AzB-QN) for PfCRT function. These probes will also be used in Aim 3. Aim 2) Continue to define drug transport functions of PfCRT isoforms using ISOV and PLs and radio labeled and fluorescent (e.g. NBD CQ) probes. We will also synthesize additional probes (e.g., NBD-MQ, NBD-QN) using previously synthesized intermediates and similar chemistry relative to successful synthesis of NBD-CQ. Aim 3) Test hypotheses for function of PfMDR1 following a similar approach, and also using high throughput plate based ATPase assays we have developed and published [93, 93B]. We will investigate the unusual (relative to other ABCB transporters) drug - influenced "communication" between the two symmetrical halves of PfMDR1 [93]. We will analyze binding, transport and ATPase properties of ISOV and PLs harboring known ratios of various PfCRT and PfMDR1 proteins to test for interactions between the two transporters. PUBLIC HEALTH RELEVANCE: Drug resistant malaria continues to both evolve and spread, and globally causes over 1 million deaths annually. This project aims to define, at a molecular level, how mutated proteins cause that drug resistance. Such information is central to development of new drugs and other therapies to combat drug resistant malaria.