Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, California 92093, United States.
VA San Diego Healthcare System, Medical and Research Sections, La Jolla, California 92161, United States.
ACS Infect Dis. 2021 Apr 9;7(4):811-825. doi: 10.1021/acsinfecdis.0c00676. Epub 2021 Mar 14.
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 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 () predicted to encode a member of the major facilitator superfamily (MFS). Disruption of 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 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 is naturally polymorphic, naturally occurring mutations may lead to differential sensitivity to clinically relevant compounds such as atovaquone.
在疟疾中，化学遗传学是一种将未鉴定基因功能进行分配的强大方法。MMV085203 和 GNF-Pf-3600 是两种结构相关的萘醌表型筛选命中物，能够以低纳摩尔至低微摩尔范围杀死血期和有性期寄生虫。为了了解它们的作用机制，用亚致死浓度的 MMV085203 和 GNF-Pf-3600 暴露于来自两种不同遗传背景的寄生虫，直到出现抗性。全基因组测序揭示了所有 17 个抗性克隆都获得了编码孤儿顶复门转运蛋白 PF3D7_0312500（）的基因中的非同义突变，该基因预测编码主要易化因子超家族（MFS）的成员。和对一组抗疟化合物的测试表明， 和对 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。相比之下，突变 并没有为作用于食物泡或细胞质的化合物提供保护。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）标记表明它定位于寄生虫线粒体。我们的数据与 PfMFR3 在参与线粒体运输以及对针对线粒体的临床相关抗疟药物的耐药性中发挥作用一致。此外，鉴于 是自然多态性的，自然发生的突变可能导致对临床相关化合物（如阿托伐醌）的敏感性不同。
