Bouillon Anthony, Gorgette Olivier, Mercereau-Puijalon Odile, Barale Jean-Christophe
Unité d'Immunologie Moléculaire des Parasites, Institut Pasteur, Paris, France.
Methods Mol Biol. 2013;923:523-34. doi: 10.1007/978-1-62703-026-7_36.
Drug discovery programs heavily rely on assays adequately monitoring the activity of the drug on the -parasite stage targeted. So far, assays used to screen molecules active against Plasmodium falciparum parasites have mostly been based on measuring growth inhibition of asexual blood stages. We have developed a robust protocol allowing for monitoring parasite egress at the late schizont stage and subsequent erythrocyte invasion. This cytometry-based methodology uses nucleic acid labelling by the dye YOYO-1 and synchronized in vitro culture of P.falciparum exposed to inhibitors during the late phase of the intraerythrocytic cycle and the reinvasion process. This cytometry-based method is quick, accurate and allows for distinguishing egress from reinvasion on thousands of events. The throughput is also increased, as the assay can be scaled up for medium throughput screening for compounds that inhibit either the egress of merozoites or their entry into host erythrocytes.
药物研发项目严重依赖于能够充分监测药物对目标寄生虫阶段活性的检测方法。到目前为止，用于筛选对恶性疟原虫寄生虫有活性的分子的检测方法大多基于测量无性血液阶段的生长抑制。我们已经开发出一种可靠的方案，可用于监测晚期裂殖体阶段的寄生虫逸出以及随后的红细胞入侵。这种基于细胞计数的方法利用YOYO-1染料进行核酸标记，并在红细胞内周期后期和再入侵过程中对暴露于抑制剂的恶性疟原虫进行体外同步培养。这种基于细胞计数的方法快速、准确，能够在数千个事件中区分逸出和再入侵。由于该检测方法可以扩大规模用于中等通量筛选抑制裂殖子逸出或其进入宿主红细胞的化合物，因此通量也有所提高。
