It is well established that chloroquine, a quinoline antimalarial, inhibits hemozoin formation in the malaria parasite. Counterintuitively, this archetypal antimalarial is also used in the treatment of diseases in which hemozoin biocrystallization does not play a role. Hence, we decided to investigate whether chloroquine possesses binding targets other than Fe(III) protoporphyrin IX in blood stage Plasmodium falciparum parasites and whether these are related to sites of accumulation within the parasite other than the digestive vacuole. A 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled fluorescent derivative of chloroquine, especially sensitive to regions outside the digestive vacuole and retaining the antiplasmodial pharmacophore, was synthesized to investigate subcellular localization in the parasite. Super-resolution microscopy revealed association with membranes including the parasite plasma membrane, the endoplasmic reticulum, and possibly also the mitochondrion. A drug-labeled affinity matrix was then prepared to capture protein binding targets of chloroquine. SDS-PAGE revealed a single prominent band between 200 and 250 kDa from the membrane-associated fraction. Subsequent proteomic analysis revealed that this band corresponded to P. falciparum multidrug resistance-associated protein (PfMRP1). Intrigued by this finding, we demonstrated pull-down of PfMRP1 by matrices labeled with Cinchona alkaloids quinine and quinidine. While PfMRP1 has been implicated in resistance to quinolines and other antimalarials, this is the first time that these drugs have been found to bind directly to this protein. Based on previous reports, PfMRP1, the only prominent protein found to bind to quinolines in this work, is likely to modulate the activity of these antimalarials in P. falciparum rather than act as a drug target.

中文翻译：

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化学蛋白质组学和超分辨率成像显示氯喹与恶性疟原虫多药耐药相关蛋白和脂质相互作用

众所周知，喹啉抗疟药氯喹可以抑制疟原虫中的血红蛋白形成。与直觉相反，这种典型的抗疟药也用于治疗血红素生物结晶不起作用的疾病。因此，我们决定调查在血液阶段恶性疟原虫中，氯喹是否具有除Fe（III）原卟啉IX以外的结合靶标寄生虫以及这些寄生虫是否与除消化液以外的其他寄生虫内的积累位点有关。合成了7-硝基苯-2-氧杂-1,3-二唑（NBD）标记的氯喹荧光衍生物，尤其对消化液泡以外的区域敏感并保留了抗疟原虫药效团，以研究该寄生虫中的亚细胞定位。超分辨率显微镜显示与膜相关，包括寄生虫质膜，内质网以及线粒体。然后制备药物标记的亲和基质以捕获氯喹的蛋白质结合靶标。SDS-PAGE显示与膜相关的部分在200至250 kDa之间有一条明显的条带。随后的蛋白质组学分析表明，该条带对应于恶性疟原虫多药耐药相关蛋白（PfMRP1）。这项发现引起了我们的兴趣，我们证明了用金鸡纳生物碱奎宁和奎尼丁标记的基质对PfMRP1的下拉作用。虽然PfMRP1与喹啉和其他抗疟药具有抗药性，但这是首次发现这些药物直接与该蛋白结合。根据以前的报道，在这项工作中发现唯一与喹啉结合的重要蛋白质PfMRP1可能会调节恶性疟原虫中这些抗疟药的活性，而不是充当药物靶标。