In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes. MMV085203 and GNF-Pf-3600 are two structurally related napthoquinone phenotypic screening hits that kill both blood- and sexual-stage P. falciparum parasites in the low nanomolar to low micromolar range. In order to understand their mechanism of action, parasites from two different genetic backgrounds were exposed to sublethal concentrations of MMV085203 and GNF-Pf-3600 until resistance emerged. Whole genome sequencing revealed all 17 resistant clones acquired nonsynonymous mutations in the gene encoding the orphan apicomplexan transporter PF3D7_0312500 (pfmfr3) predicted to encode a member of the major facilitator superfamily (MFS). Disruption of pfmfr3 and testing against a panel of antimalarial compounds showed decreased sensitivity to MMV085203 and GNF-Pf-3600 as well as other compounds that have a mitochondrial mechanism of action. In contrast, mutations in pfmfr3 provided no protection against compounds that act in the food vacuole or the cytosol. A dihydroorotate dehydrogenase rescue assay using transgenic parasite lines, however, indicated a different mechanism of action for both MMV085203 and GNF-Pf-3600 than the direct inhibition of cytochrome bc1. Green fluorescent protein (GFP) tagging of PfMFR3 revealed that it localizes to the parasite mitochondrion. Our data are consistent with PfMFR3 playing roles in mitochondrial transport as well as drug resistance for clinically relevant antimalarials that target the mitochondria. Furthermore, given that pfmfr3 is naturally polymorphic, naturally occurring mutations may lead to differential sensitivity to clinically relevant compounds such as atovaquone.

中文翻译：

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PfMFR3：恶性疟原虫的多药耐药调节剂

在疟疾中，化学遗传学是一种将功能分配给未表征基因的强大方法。MMV085203 和 GNF-Pf-3600 是两种结构相关的萘醌表型筛选结果，可杀​​死低纳摩尔至低微摩尔范围内的血液和性阶段恶性疟原虫寄生虫。为了了解它们的作用机制，来自两种不同遗传背景的寄生虫暴露于亚致死浓度的 MMV085203 和 GNF-Pf-3600 直到出现抗药性。全基因组测序显示，所有 17 个抗性克隆在编码孤儿顶复门转运蛋白 PF3D7_0312500 ( pfmfr3 ) 的基因中获得了非同义突变，该基因预测编码主要促进子超家族 (MFS) 的成员。pfmfr3中断对一组抗疟化合物的测试表明，对 MMV085203 和 GNF-Pf-3600 以及其他具有线粒体作用机制的化合物的敏感性降低。相反，pfmfr3的突变对作用于食物液泡或胞质溶胶的化合物没有提供保护。然而，使用转基因寄生虫系的二氢乳清酸脱氢酶拯救测定表明，MMV085203 和 GNF-Pf-3600 的作用机制与直接抑制细胞色素 bc1 不同。PfMFR3 的绿色荧光蛋白 (GFP) 标记显示它定位于寄生虫线粒体。我们的数据与 PfMFR3 在线粒体转运中发挥作用以及对靶向线粒体的临床相关抗疟药的耐药性一致。此外，鉴于pfmfr3是天然多态性的，天然发生的突变可能导致对临床相关化合物（如 atovaquone）的不同敏感性。