Function of antimalarial drug resistance proteins

 描述（由申请人提供）：抗药性疟疾每年造成60万人死亡。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恶性疟原虫疟疾菌株的治疗需要更全面地了解多种基因型和表型。防治抗药性疟疾的斗争正在进行，必须持续不断地进行，否则，我们在过去50年里什么也没学到，眼睁睁地看着药物失效。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。我们的实验室在PfCRT和PfMDR1蛋白的分子水平分析领域处于领先地位，这两种转运蛋白对几种形式的抗疟药物和多药耐药性至关重要。最近，已经发现了这些蛋白质的数十种新的同种型，但只有少数尚未被研究。阐明这些同种型的功能和赋予抗性的能力对于定义局部耐药性和选择其功效不受这些耐药性转运蛋白的局部同种型损害的区域特异性和基因型特异性药物是至关重要的。在此期间，我们将：目标1。阐明PfCRT功能多样性的分子基础并定义K76T表型。(1.1)定量比较所有已知的PfCRT同种型（1.2）在恶性疟原虫中进行pfcrt基因编辑实验以测试体外结论，目的2。利用类似的方法来阐明PfMDR 1功能多样性，包括：（2.1）体外药物结合和药物转运，以及（2.2）恶性疟原虫中PfMDR 1基因编辑实验，以在体外测试结论目的3。阐明PfCRT和PfMDR 1蛋白的三维结构。(3.1)使用具有多个CRT直系同源物的猎枪结晶策略，（3.2）利用TransportPDB资源中的工具，以及（3.3）生成紧密结合的纳米抗体（Nbs）以陪伴分子晶格形成。

 DESCRIPTION (provided by applicant): Drug resistant malaria kills 600,000 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 rates of infection. Current and future treatment of the many different strains of drug resistant Plasmodium falciparum malaria that now exist requires a more complete understanding of multiple genotypes and phenotypes. 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 drugs fail. We must "stay ahead of the resistance curve" and define molecular mechanisms that guide ongoing drug and vaccine research. Our laboratory has helped to lead the field in the molecular level analysis of the PfCRT and PfMDR1 proteins, two transporters that are crucial for several forms of antimalarial drug and multidrug resistance. Recently, dozens of new isoforms of these proteins have been discovered, but only a handful have yet been studied. Elucidating function and resistance - conferring ability of these isoforms is crucial for defining local drug resistance and selecting region- and genotype-specific drugs whose efficacy is not compromised by local isoforms of these drug resistance transporters. In this grant period we will: Aim 1. Elucidate the molecular basis of PfCRT functional diversity and define K76T phenotypes. (1.1) Quantitatively compare all known PfCRT isoforms (1.2) Perform pfcrt gene editing experiments in P. falciparum to test in vitro conclusions, and Aim 2. Utilize a similar approach in order to elucidate PfMDR1 functional diversity, including: (2.1) Drug binding and drug transport in vitro, and (2.2) Pfmdr1 gene editing experiments in P. falciparum to test in vitr conclusions Aim 3. Elucidate the three-dimensional structures of PfCRT and PfMDR1 proteins. (3.1) Use a shot-gun crystallization strategy with multiple CRT orthologs, (3.2) Leverage tools within the TransportPDB resource, and (3.3) Generate tight-binding Nanobodies (Nbs) to chaperone crystal lattice formation.