惡性瘧原蟲轉運體PfCRT的結構獲解析
作者：
小柯機器人發布時間：2019/11/28 13:21:26
美國哥倫比亞大學歐文醫學中心Filippo Mancia、David A. Fidock和Matthias Quick團隊合作，解析了惡性瘧原蟲轉運蛋白PfCRT的結構與耐藥性。這一研究成果11月27日在線發表在國際學術期刊《自然》上。
研究人員使用單粒子冷凍電鏡和抗原結合片段的技術，解析了抗氯奎（CQ）、哌喹(PPQ)的南美7G8寄生蟲PfCRT亞型的結構，其解析度為3.2 Å。導致CQ和PPQ抗性的突變主要位於中間帶負電荷腔的不同螺旋上中等保守位點，這表明該腔是與帶正電荷CQ和PPQ相互作用的主要位點。
結合和轉運研究表明7G8亞型對這兩種藥物有相似的結合親和力，並且這些藥物具有相互競爭性。7G8異構體以膜電位和pH依賴的方式運輸CQ，這與導致CQ抗性但不運輸PPQ的主動外排機制一致。對與亞洲和南美洲PPQ敏感性降低相關的新出現的PfCRT F145I和C350R突變的功能研究發現，它們介導7G8變異蛋白中PPQ的轉運並在基因編輯的寄生蟲中賦予抗性。結構、功能和計算機分析表明，介導PfCRT變體對CQ和PPQ的抗性具有不同的機制。這些數據在原子級別為失敗的抗瘧疾治療提供了分子機制。
研究人員表示，耐藥性惡性瘧原蟲的出現和擴散阻礙了全球控制和消除瘧疾的努力。幾十年來，瘧疾的治療一直依靠CQ，直到在東南亞和南美出現耐藥株並在世界範圍內傳播；CQ來源於安全且廉價的4-氨基喹啉，對紅細胞內無性血液階段的寄生蟲非常有效。然而，當前一線聯合藥物PPQ的臨床耐藥性已經在一些區域出現，從而降低了其療效。惡性瘧原蟲CQ抗性轉運蛋白PfCRT中不同的點突變與CQ和PPQ的抗藥性相關，PfCRT屬於大小為49 kDa的藥物/代謝物轉運蛋白超家族的成員之一，橫穿寄生蟲的酸性消化液膜。
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