The function of antimalarial drug resistance proteins

描述(申请人提供)：抗药性疟疾每年导致数百万人死亡。要扭转发病率和死亡率的可怕趋势，需要在疫苗和药物研究方面采取平衡的方法，并以实地为基础努力控制媒介人口和感染率。目前和未来对目前存在的许多不同抗药性疟疾菌株的治疗需要对多种基因和表型有更全面的了解。我们不能被当前基于青蒿素(ART)的疗法提供的(暂时)安全感所迷惑；黑市上的艺术品已经在流通，并产生了艺术品抵抗。与抗药性疟疾的斗争正在进行中，必须持续进行；否则，我们在过去50年里看着CQ和其他药物失败时，什么也学不到。我们必须“保持在耐药性曲线的领先地位”，并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。在这一竞争更新期，我们将：目标1)通过在酵母中异源表达和分析含有PfCRT亚型的纯化膜、ISOV和PL制剂，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进行药物、氨基酸和离子的结合和运输。我们还将为PfCRT功能合成额外的探针(例如，Azb-MQ，AzBCQ侧链长度变体，Azb-QN)。这些探针也将用于AIM 3。AIM 2)继续使用ISOV和PLS以及放射性标记和荧光(如NbD CQ)探针来确定PfCRT异构体的药物转运功能。我们还将使用以前合成的中间体和与成功合成NBD-CQ类似的化学方法合成其他探针(例如NBD-MQ、NBD-QN)。目的3)按照类似的方法测试PfMDR1的功能假设，并使用我们开发和发表的基于高通量平板的ATPase分析[93，93B]。我们将调查不寻常的(相对于其他ABCB转运体)药物影响的PfMDR1的两个对称部分之间的“通讯”[93]。我们将分析含有不同比例的PfCRT和PfMDR1蛋白的ISOV和PLS的结合、运输和ATPase特性，以测试这两种转运蛋白之间的相互作用。 公共卫生相关性：抗药性疟疾继续演变和传播，全球每年造成100多万人死亡。这个项目的目的是在分子水平上定义突变的蛋白质是如何导致耐药性的。这些信息对于抗击抗药性疟疾的新药和其他疗法的开发至关重要。

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.