Fluorescence Cross-Correlation Spectroscopy Yields True Affinity and Binding Kinetics of Plasmodium Lactate Transport Inhibitors.
Fluorescence Cross-Correlation Spectroscopy Yields True Affinity and Binding Kinetics of Plasmodium Lactate Transport Inhibitors.
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DOI：
10.3390/ph14080757
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发表时间：
2021-08-02
期刊：
影响因子：
--
通讯作者：
Hannus S
中科院分区：
文献类型：
--
作者：
Jakobowska I;Becker F;Minguzzi S;Hansen K;Henke B;Epalle NH;Beitz E;Hannus S
阻断寄生性原生动物恶性疟原虫的乳酸输出是一种对抗疟疾的新策略。我们发现了类似药物的小分子，可以抑制唯一的疟原虫乳酸转运蛋白PfFNT，并杀死培养物中的寄生虫。五氟-3-羟基-顺-2-烯-1-酮BH 296以纳摩尔效率阻断PfFNT，但体外选择的PfFNT G107 S突变赋予对该药物的抗性。我们通过将氮原子作为氢键受体位点引入抑制剂的芳环中来规避突变，从而产生BH 267. meta。目前的PfFNT抑制剂效率值来自基于酵母的乳酸转运测定，但直接亲和力和结合动力学数据缺失。这里，我们在人胚肾细胞中表达了与绿色荧光蛋白融合的PfFNT，并产生了抑制剂BH 296和BH 267的荧光衍生物。使用共聚焦成像，我们证实了建议的结合位点在胞质转运蛋白进入位点的位置。然后，我们进行了荧光互相关光谱测量，以分配真正的Ki值，以及kon和koff速率常数的抑制剂结合PfFNT野生型和G107 S突变体。BH 296和BH267.meta给出了与PfFNT野生型结合的相似速率常数。BH 296对PfFNT G107 S无活性，而BH267.meta结合突变蛋白，尽管与PfFNT野生型相比具有较弱的亲和力。最终，使用一组PfFNT抑制剂化合物，我们发现了生物物理FCCS结合测定的结果与功能转运测定的抑制数据的稳健相关性。
Blocking lactate export in the parasitic protozoan Plasmodium falciparum is a novel strategy to combat malaria. We discovered small drug-like molecules that inhibit the sole plasmodial lactate transporter, PfFNT, and kill parasites in culture. The pentafluoro-3-hydroxy-pent-2-en-1-one BH296 blocks PfFNT with nanomolar efficiency but an in vitro selected PfFNT G107S mutation confers resistance against the drug. We circumvented the mutation by introducing a nitrogen atom as a hydrogen bond acceptor site into the aromatic ring of the inhibitor yielding BH267.meta. The current PfFNT inhibitor efficiency values were derived from yeast-based lactate transport assays, yet direct affinity and binding kinetics data are missing. Here, we expressed PfFNT fused with a green fluorescent protein in human embryonic kidney cells and generated fluorescent derivatives of the inhibitors, BH296 and BH267.meta. Using confocal imaging, we confirmed the location of the proposed binding site at the cytosolic transporter entry site. We then carried out fluorescence cross-correlation spectroscopy measurements to assign true Ki-values, as well as kon and koff rate constants for inhibitor binding to PfFNT wildtype and the G107S mutant. BH296 and BH267.meta gave similar rate constants for binding to PfFNT wildtype. BH296 was inactive on PfFNT G107S, whereas BH267.meta bound the mutant protein albeit with weaker affinity than to PfFNT wildtype. Eventually, using a set of PfFNT inhibitor compounds, we found a robust correlation of the results from the biophysical FCCS binding assay to inhibition data of the functional transport assay.
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影响因子：
7.3
作者：
Walloch, Philipp;Henke, Bjoern;Beitz, Eric
通讯作者：
Beitz, Eric
影响因子：
6.7
作者：
Hapuarachchi SV;Cobbold SA;Shafik SH;Dennis AS;McConville MJ;Martin RE;Kirk K;Lehane AM
通讯作者：
Lehane AM
影响因子：
3.4
作者：
Schwille, P;MeyerAlmes, FJ;Rigler, R
通讯作者：
Rigler, R
影响因子：
16.6
作者：
Wu, Binghua;Rambow, Janis;Beitz, Eric
通讯作者：
Beitz, Eric
影响因子：
3.4
作者：
Walloch P;Hansen C;Priegann T;Schade D;Beitz E
通讯作者：
Beitz E