小柯机器人

美国哥伦比亚大学欧文医学中心Filippo Mancia、David A. Fidock和Matthias Quick团队合作，解析了恶性疟原虫转运蛋白PfCRT的结构与耐药性。这一研究成果11月27日在线发表在国际学术期刊《自然》上。

研究人员使用单粒子冷冻电镜和抗原结合片段的技术，解析了抗氯奎（CQ）、哌喹(PPQ)的南美7G8寄生虫PfCRT亚型的结构，其分辨率为3.2 Å。导致CQ和PPQ抗性的突变主要位于中间带负电荷腔的不同螺旋上中等保守位点，这表明该腔是与带正电荷CQ和PPQ相互作用的主要位点。

结合和转运研究表明7G8亚型对这两种药物有相似的结合亲和力，并且这些药物具有相互竞争性。7G8异构体以膜电位和pH依赖的方式运输CQ，这与导致CQ抗性但不运输PPQ的主动外排机制一致。对与亚洲和南美洲PPQ敏感性降低相关的新出现的PfCRT F145I和C350R突变的功能研究发现，它们介导7G8变异蛋白中PPQ的转运并在基因编辑的寄生虫中赋予抗性。结构、功能和计算机分析表明，介导PfCRT变体对CQ和PPQ的抗性具有不同的机制。这些数据在原子级别为失败的抗疟疾治疗提供了分子机制。

研究人员表示，耐药性恶性疟原虫的出现和扩散阻碍了全球控制和消除疟疾的努力。几十年来，疟疾的治疗一直依靠CQ，直到在东南亚和南美出现耐药株并在世界范围内传播；CQ来源于安全且廉价的4-氨基喹啉，对红细胞内无性血液阶段的寄生虫非常有效。然而，当前一线联合药物PPQ的临床耐药性已经在一些区域出现，从而降低了其疗效。恶性疟原虫CQ抗性转运蛋白PfCRT中不同的点突变与CQ和PPQ的抗药性相关，PfCRT属于大小为49 kDa的药物/代谢物转运蛋白超家族的成员之一，横穿寄生虫的酸性消化液膜。

附：英文原文

Title: Structure and drug resistance of the Plasmodium falciparum transporter PfCRT

Author: Jonathan Kim, Yong Zi Tan, Kathryn J. Wicht, Satchal K. Erramilli, Satish K. Dhingra, John Okombo, Jeremie Vendome, Laura M. Hagenah, Sabrina I. Giacometti, Audrey L. Warren, Kamil Nosol, Paul D. Roepe, Clinton S. Potter, Bridget Carragher, Anthony A. Kossiakoff, Matthias Quick, David A. Fidock, Filippo Mancia

Issue&Volume: 2019-11-27

Abstract: The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite39. Here we present the structure, at 3.2  resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures. Structural, functional and in silico analyses of the chloroquine-resistance transporter PfCRT of Plasmodium falciparum suggest that distinct mechanistic features mediate the resistance to chloroquine and piperaquine in drug-resistant parasites.

DOI: 10.1038/s41586-019-1795-x

Source:https://www.nature.com/articles/s41586-019-1795-x

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