A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs
A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs
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DOI：
10.1021/acsinfecdis.5b00141
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发表时间：
2017-02-01
影响因子：
5.3
通讯作者：
Davioud-Charvet, Elisabeth
中科院分区：
文献类型：
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作者：
Jida, Mouhamad;Sanchez, Cecilia P.;Davioud-Charvet, Elisabeth
恶性疟原虫氯喹抗性转运蛋白（PfCRT）的突变变化与对广谱生物活性化合物（包括当前和以前的喹啉和喹啉样抗疟药物）的不同反应有关。Pf CRT通过充当运输系统，将药物从寄生虫的消化泡排出，从而改变药物反应性，这些药物在消化泡中发挥至少部分抗疟原虫活性。为了保持这些宝贵的药物的疗效，需要新的功能工具进行流行病学调查的寄生虫菌株携带突变PfCRT变体和药物开发计划，旨在抑制或规避PfCRT的行动。在这里，我们报告了一种pH敏感的荧光氯喹类似物的合成和表征，该类似物由荧光染料7-硝基苯并呋咱（NBD）官能化的7-氯-N-{2-[（丙-2-基）氨基]乙基）喹啉-4-胺组成（以下称为Fluo-COJ）。在寄生虫中，Fluo-CQ在消化泡中积累，产生强烈的荧光信号，但仅在携带野生型PfCRT的寄生虫中。在携带突变PfCRT的寄生虫中，Fluo-CQ不会蓄积。荧光探针的差异处理，结合活细胞成像，提供了一种诊断工具，用于快速检测携带Pf CRT变体的恶性疟原虫菌株，该变体与喹啉和喹啉样抗疟药物的反应性改变相关。与蓄积研究相反，当在各种CQ敏感和耐药寄生虫菌株中检测化学探针时，观察到氯喹（CQ）耐药寄生虫对Fluo-CQ交叉耐药。使用基于高铁血红蛋白和来自恶性疟原虫的NADPH依赖性谷胱甘肽还原酶（GR）的偶联测定，发现NBD衍生物充当两个基本靶标的氧化还原循环剂。这种氧化还原活性被认为有助于Fluo-CQ对氧化还原平衡和高铁血红蛋白还原的双重作用，通过PfCRT介导的药物在胞质溶胶中流出，然后在食物泡和胞质溶胶之间连续的氧化还原依赖性穿梭。考虑到这些物理化学特性，提出了一个模型来解释Fluo-CQ抗疟作用，涉及PfCRT介导的转运，高铁血红蛋白还原，血红素结合和NBD还原活性催化的PfGR在CQ抗性与CQ敏感的贡献：寄生虫。因此，在药物中引入NBD荧光团不是惰性的，并且在药物转运和成像研究中应考虑。
Mutational changes in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) have been associated with differential responses to a wide spectrum of biologically active compounds including current and former quinoline and quinoline-like antimalarial drugs. Pf CRT confers altered drug responsiveness by acting as a transport system, expelling drugs from the parasite's digestive vacuole where these drugs exert, at least part of, their antiplasmodial activity. To preserve the efficacy of these invaluable drugs, novel functional tools are required for epidemiological surveys of parasite strains carrying mutant PfCRT variants and for drug development programs aimed at inhibiting or circumventing the action of PfCRT. Here we report the synthesis and characterization of a pH-sensitive fluorescent chloroquine analogue consisting of 7-chloro-N-{2-[(propan-2-yl)amino]ethyl)quinolin-4-amine functionalized with the fluorochrome 7-nitrobenzofurazan (NBD) (henceforth termed Fluo-COJ. In the parasite, Fluo-CQ accumulates in the digestive vacuole, giving rise to a strong fluorescence signal but only in parasites carrying the wild type PfCRT. In parasites carrying the mutant PfCRT, Fluo-CQ does not accumulate. The differential handling of the fluorescent probe, combined with live cell imaging, provides a diagnostic tool for quick detection of those P. falciparum strains that carry a Pf CRT variant associated with altered responsiveness to quinoline and quinoline-like antimalarial drugs. In contrast to the accumulation studies, chloroquine (CQ)-resistant parasites were observed cross-resistant to Fluo-CQwhen the chemical probe was tested in various CQ:sensitive and-resistant parasite strains. NBD derivatives were found to act as redox cyclers of two essential targets, using a coupled assay based on methemoglobin and the NADPH-dependent glutathione reductase (GRs) from P. falciparum. This redox activity is proposed to contribute to the dual action of Fluo-CQ on redox equilibrium and methemoglobin reduction via PfCRT-mediated drug efflux in the cytosol and then continuous redox-dependent shuttling between food vacuole and cytosol. Taking into account these physicochemical characteristics, a model was proposed to explain Fluo-CQ antimalarial effects involving the contribution of PfCRT-mediated transport, methemoglobin reduction, hematin binding, and NBD reduction activity catalyzed by Pf GR in CQresistant versus CQ:sensitive parasites. Therefore, introduction of NBD fluorophore in drugs is not inert and should be taken into account in drug transport and imaging studies.