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

项目总结(见说明)： 抗疟疾耐药性一再阻碍全球治疗和控制疟疾的努力。氯喹， 这种曾经的黄金标准药物，早些时候死于携带疟原虫突变的寄生虫菌株 恶性疟原虫氯喹抗性转运蛋白PfCRT。现在，基于青蒿素的联合疗法已经 在东南亚失败了，部分原因是新型PfCRT变种给一线合作伙伴带来了阻力 毒品哌喹。对其他伙伴药物，包括阿莫地喹、鲁米芬和甲氟喹的耐药性可能 由PfCRT的变种形式或ABC转运体PfMDR1授予。我们的R37 AI050234，自 2001年，在这一领域进行了大量先进的研究，包括：发表了PfCRT的确凿证据 突变是氯喹和哌喹耐药性的主要决定因素；将耐药性归因于 PfCRT介导的药物外排；解决PfCRT结构；定义健身成本和生理效应 影响抗性等位基因的传播；在全球范围内定位pfcrt和pfmdr1变异；以及 PfMDR-1介导的多药耐药性的特征。自从我们在2017年8月提交上一份文件以来，我们已经 发表了23篇论文，包括《自然》、《自然微生物》、《柳叶刀感染疾病》和《细胞化学生物学》。我们的R37 功德延期申请提出了四个新的具体目标来推进这一点。目标1：定义是否 突变体PfCRT可介导非洲菌株对哌喹的耐药性。我们将确定PfCRT是否 在亚洲寄生虫中导致哌喹抗药性的突变，在非洲也同样会产生抗药性。 具有区域性不同PfCRT亚型的菌株。我们还将评估这些异构体对其他 抗疟疾药物和寄生虫健康。目标2：定义与PfCRT上位性作用的基因决定因素 来调停抵抗。利用我们最新的恶性疟原虫基因杂交，我们将定位并鉴定 在氯喹和/或奎宁抗性后代中与突变体PfCRT共遗传的基因并确定 无论这些决定因素是增加了抵抗力，还是已经进化到抵消了健身成本。目标3： 确定可以逆转耐药性的PfCRT特异性抑制剂。我们建议筛选不同的化学品 鉴定抗药性寄生虫中阻断氯喹或哌喹外流的化合物的文库 台词。用pfcrt条件基因敲除寄生虫和重组pfCRT进行检测将检测出 可以结合这个转运蛋白，阻止外流，恢复药物效力。目标4：解决PfMDR1结构和 确定其与PfCRT相比的药物外排特性。继我们最近在PfCRT上取得成功后，我们 提出用低温电子显微镜解决PfMDR1结构的问题。我们还将执行绑定和 用纯化重组蛋白转运法鉴定PfMDR1的抗性特性 变种。这项提案与NIAID支持抗微生物药物研究的优先事项一致 耐药性，将大大促进我们对抗疟疾耐药性和产量生物标志物的理解 以及对抗抗药性疟疾的新战略。

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.