The function of antimalarial drug resistance proteins

描述（由申请人提供）：抗药性疟疾每年造成数百万人死亡。要扭转发病率和死亡率的可怕趋势，就需要在疫苗和药物研究方面采取平衡的办法，并在实地努力控制病媒种群和感染率。目前和未来对现有的许多不同抗药性疟疾菌株的治疗需要更全面地了解多种基因型和表型。我们绝不能被目前基于青蒿素的疗法提供的（暂时）安全的错误感觉所迷惑;黑市ART已经在流通并产生ART耐药性。对抗抗药性疟疾的斗争正在进行，必须持续不断地进行;否则，我们在看着CQ和其他药物失败的同时，从过去50年中什么也没学到。我们必须“保持在耐药曲线的前面”，并确定指导正在进行的药物和疫苗研究的分子机制。我们的实验室已经帮助领导使用异源表达系统在PfCRT和PfMDR 1蛋白的分子水平分析领域。在这一竞争性更新期，我们将：目的1）通过在酵母中的异源表达和分析含有这些蛋白的纯化膜、ISOV和PL制剂，继续确定PfCRT同种型的结合功能。我们将使用最近开发的技术和化学探针进行药物、氨基酸和离子结合和转运。我们还将合成额外的探针（例如，AzB-MQ、AzBCQ侧链长度变体、AzB-QN）用于PfCRT功能。这些探头也将用于目标3。目的2）继续使用ISOV和PL以及放射性标记和荧光（例如NBD）来确定PfCRT异构体的药物转运功能 CQ）探针。我们还将合成额外的探针（例如，NBD-MQ、NBD-QN），使用先前合成的中间体和与NBD-CQ的成功合成类似的化学。目的3）按照类似的方法测试PfMDR 1功能的假设，并使用我们开发并发表的基于高通量板的ATP酶测定法[93，93 B]。我们将研究PfMDR 1的两个对称部分之间不寻常的（相对于其他ABCB转运蛋白）药物影响的“通信”[93]。我们将分析ISOV和PL的结合、转运和ATP酶性质，这些ISOV和PL含有已知比例的各种PfCRT和PfMDR 1蛋白，以测试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。该项目旨在从分子水平上确定突变蛋白质如何导致耐药性。这些信息对于开发新药物和其他治疗方法以对抗抗药性疟疾至关重要。

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.