An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages
An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages
复制标题
DOI：
10.1371/journal.pntd.0001671
复制
发表时间：
2012-06-01
影响因子：
3.8
通讯作者：
Freitas-Junior, Lucio H.
中科院分区：
文献类型：
--
作者：
Siqueira-Neto, Jair L.;Moon, Seunghyun;Freitas-Junior, Lucio H.
利什曼病是一种热带疾病，威胁着流行地区的3.5亿人。现有的治疗药物不足，存在严重副作用、寄生虫抗药性或费用高昂等局限性。在这种对新药需求的推动下，我们开发了一种高含量、高通量的基于图像的筛选试验，靶向感染的人巨噬细胞中不同种类利什曼原虫的细胞内无鞭毛体阶段。体外感染方案适用于384孔板格式，使得能够通过自动共聚焦显微镜获得大量读数。阅读方法基于DNA染色，需要开发定制算法来分析图像，这使得能够使用未修饰的寄生虫。自动化分析生成用于量化化合物活性的参数，包括感染率以及细胞内无鞭毛体寄生虫的数量，并基于宿主细胞的数量产生细胞毒性信息。该测定与使用前鞭毛体形式筛选26，500种化合物的测定的比较显示，针对细胞内无鞭毛体选择的命中中的50%在前鞭毛体筛选中未被选择。这些数据证实了这样的想法，即细胞内无鞭毛体形式的寄生虫是最适合用于初级筛选试验利什曼原虫。
Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania.