Defining the Role of PfCRT and PfMDR1 as Pleiotropic Mediators of Plasmodium falciparum Multidrug Resistance

定义 PfCRT 和 PfMDR1 作为恶性疟原虫多药耐药性多效性介质的作用

• 批准号: 10445547
• 负责人: David A Fidock
• 金额: $ 54.68万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445547
• 关键词: Africa; Anti-malarial drug resistance; Antimalarials; Combined Modality Therapy; Drug Efflux; Drug resistance; Genes; Instruction; Malaria; Mediating; Mediator; Multi-Drug Resistance; Mutation; Parasite resistance; Parasites; Plasmodium falciparum; Play; Resistance; Risk; Role; cost; drug action; drug efficacy; evidence base; fitness; genetic association; mutant

PROJECT SUMMARY (See instructions): Antimalarial drug resistance has repeatedly thwarted global efforts to treat and control malaria. Chloroquine, the former gold-standard drug, earlier succumbed to parasite strains harboring mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT. Now, artemisinin-based combination therapies have failed in Southeast Asia, driven in part by novel PfCRT variants that confer resistance to the first-line partner drug piperaquine. Resistance to other partner drugs including amodiaquine, lumefantrine, and mefloquine can be conferred by variant forms of PfCRT or the ABC transporter PfMDR1. Our R37 AI050234, funded since 2001, has substantially advanced research in this area including: publishing definitive evidence that PfCRT mutations are the primary determinant of chloroquine and piperaquine resistance; attributing resistance to PfCRT-mediated drug efflux; solving the PfCRT structure; defining fitness costs and physiological effects that impact the spread of resistant alleles; globally mapping pfcrt and pfmdr1 variants across the globe; and characterizing PfMDR 1-mediated multidrug resistance. Since our prior submission in August 2017 we have published 23 articles including in Nature, Nature Microbiol, Lancet Infect Dis, and Cell Chem Biol. Our R37 MERIT Extension application proposes to advance this with four new Specific Aims. Aim 1: Define whether mutant PfCRT can mediate piperaquine resistance in African strains. We will determine whether PfCRT mutations that cause piperaquine resistance in Asian parasites will similarly confer resistance in African strains with regionally distinct PfCRT isoforms. We will also assess the impact of these isoforms on other antimalarials and on parasite fitness. Aim 2: Define genetic determinants that function epistatically with PfCRT to mediate resistance. Using our recent P. falciparum genetic cross, we will positionally map and characterize genes that are coinherited with mutant PfCRT in chloroquine- and/or quinine-resistant progeny and determine whether these determinants augment resistance or have evolved to offset fitness costs. Aim 3: Identify PfCRT-specific inhibitors that can reverse drug resistance. We propose to screen diverse chemical libraries to identify compounds that block the efflux of chloroquine or piperaquine in drug-resistant parasite lines. Assays with pfcrt-conditional knockdown parasites and recombinant PfCRT will test for compounds that can bind this transporter, block efflux, and restore drug potency. Aim 4: Solve the PfMDR1 structure and define its drug efflux properties compared with PfCRT. Following our recent success with PfCRT, we propose to solve the PfMDR1 structure using cryo-electron microscopy. We will also perform binding and transport assays with purified recombinant protein to characterize the resistance properties of PfMDR1 variants. This proposal, which aligns with the NIAID priority of supporting research on antimicrobial drug resistance, will substantially advance our understanding of antimalarial resistance and yield biomarkers and new strategies to counter drug-resistant malaria.

项目摘要（参见说明）： 抗疟药物耐药性一再阻碍全球治疗和控制疟疾的努力。氯喹， 以前的金标准药物，早些时候死于携带疟原虫突变的寄生虫菌株 恶性疟原虫氯喹耐药转运蛋白，PfCRT。现在，基于青蒿素的联合疗法已 在东南亚失败，部分原因是新颖的 PfCRT 变体对一线合作伙伴产生了抵抗力 药物哌喹。对其他药物（包括阿莫地喹、本芴醇和甲氟喹）的耐药性可能会导致 由 PfCRT 或 ABC 转运蛋白 PfMDR1 的变体形式赋予。我们的 R37 AI050234，资助自 2001 年，在该领域取得了实质性进展，包括： 发表了 PfCRT 的明确证据 突变是氯喹和哌喹耐药性的主要决定因素；将阻力归因于 PfCRT介导的药物外流；求解PfCRT结构；定义健身成本和生理效应 影响耐药等位基因的传播；在全球范围内绘制 pfcrt 和 pfmdr1 变体的全球地图；和 表征 PfMDR 1 介导的多药耐药性。自 2017 年 8 月提交以来，我们已经 在Nature、Nature Microbiol、Lancet Infect Dis、Cell Chem Biol等杂志上发表文章23篇。我们的R37 MERIT Extension 申请建议通过四个新的具体目标来推进这一目标。目标 1：定义是否 突变体 PfCRT 可以介导非洲菌株的哌喹耐药性。我们将确定是否 PfCRT 导致亚洲寄生虫对哌喹产生耐药性的突变同样会导致非洲寄生虫产生耐药性 具有区域性不同 PfCRT 同种型的菌株。我们还将评估这些亚型对其他亚型的影响 抗疟药和寄生虫健康。目标 2：定义与 PfCRT 发挥上位作用的遗传决定因素 来调解抵抗力。利用我们最近的恶性疟原虫遗传杂交，我们将绘制位置图并表征 在氯喹和/或奎宁抗性后代中与突变型 PfCRT 共同遗传的基因，并确定 这些决定因素是否会增强抵抗力，或者是否已经进化到抵消健身成本。目标 3： 鉴定可逆转耐药性的 PfCRT 特异性抑制剂。我们建议筛选不同的化学物质 鉴定可阻断耐药寄生虫中氯喹或哌喹外流的化合物的文库 线。使用 pfcrt 条件击倒寄生虫和重组 PfCRT 进行的检测将测试以下化合物： 可以结合该转运蛋白，阻止外流并恢复药物效力。目标 4：解析 PfMDR1 结构并 与 PfCRT 相比，定义其药物流出特性。继最近 PfCRT 取得成功之后，我们 建议使用冷冻电子显微镜解析 PfMDR1 结构。我们还将进行绑定和 使用纯化的重组蛋白进行转运测定，以表征 PfMDR1 的抗性特性 变种。该提案符合 NIAID 支持抗菌药物研究的优先事项 耐药性，将大大增进我们对抗疟药耐药性的理解并产生生物标志物 以及对抗耐药性疟疾的新策略。