Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay
Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay
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DOI：
10.1186/s12936-015-0962-2
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发表时间：
2015-11-05
期刊：
影响因子：
3
通讯作者：
Gamo, Francisco-Javier
中科院分区：
文献类型：
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作者：
Linares, Maria;Viera, Sara;Gamo, Francisco-Javier
背景资料：恶性疟原虫对青蒿素的抗药性的出现有可能破坏以青蒿素为基础的抗疟复方疗法的效力。开发替代青蒿素的合适药物需要鉴定显示快速杀寄生虫动力学的新化合物。然而，目前还没有完全满足要求的方法来筛选大型化合物库的候选人与这些属性。本研究描述了一种体外寄生虫活力快速测定法的开发和验证，用于快速鉴定杀寄生虫的抗疟drugs.Methods：寄生虫杀灭动力学通过首先将未标记的红细胞与恶性疟原虫在抗疟药物存在下培养24或48 h来确定。除去药物后，将样品加入到用细胞内染料预标记的红细胞中，以允许其随后的鉴定。在标记寄生虫DNA后，可以通过双色流式细胞术检测活寄生虫在标记的红细胞中重新建立感染的能力。因此，双染红细胞（预先标记的细胞内染料和寄生虫DNA染料）仅在存活寄生虫建立新感染后产生。测试抗疟疾药物的能力，以消除可行的寄生虫在24或48小时内，因此，可以determined.Results：寄生虫生存能力快速测定可以在48小时内完成药物治疗后，区分快速杀寄生虫抗疟疾药物与那些行动更慢。该测定法进行了验证，对10个标准的抗疟疾药物与已知的属性和结果相关，以及与建立的方法。一个简短的测定，适合于适应中高通量筛选，进行了验证，并适用于一组20个化合物检索的药物疟疾风险'疟疾Box'.Conclusion：新的感染，以确定寄生虫生存能力的量化提供了重要的优势，超过现有的方法，并适合中高通量筛选。特别是，寄生虫活力快速测定允许快速杀寄生虫的抗疟疾候选物的歧视。
Background: The emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs.Methods: Parasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined.Results: The parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium-high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture 'Malaria Box'.Conclusion: The quantification of new infections to determine parasite viability offers important advantages over existing methods, and is amenable to medium-high throughput screening. In particular, the parasite viability fast assay allows discrimination of rapidly parasiticidal anti-malarial candidates.