High content analysis of primary macrophages hosting proliferating Leishmania amastigotes: application to anti-leishmanial drug discovery.
High content analysis of primary macrophages hosting proliferating Leishmania amastigotes: application to anti-leishmanial drug discovery.
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DOI：
10.1371/journal.pntd.0002154
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发表时间：
2013
影响因子：
3.8
通讯作者：
Prina E
中科院分区：
文献类型：
--
作者：
Aulner N;Danckaert A;Rouault-Hardoin E;Desrivot J;Helynck O;Commere PH;Munier-Lehmann H;Späth GF;Shorte SL;Milon G;Prina E
人类利什曼病是引起严重发病率和死亡率的寄生虫病。没有可用的疫苗，许多因素限制了目前治疗方法的使用。因此，迫切需要创新举措，以确定对细胞内利什曼原虫无尾线虫具有选择性活性的新化学型，这些无尾线虫在巨噬细胞内发育和增殖，从而导致利什曼病的病理。我们已经开发了一种生物学上合理的高含量分析方法，基于使用携带亚马逊利什曼原虫的小鼠原代巨噬细胞的同质群体。与传统的基于promastigotes的筛选相比，我们的试验更接近地模拟了细胞内无纺锤体在宿主细胞的酸性寄生液泡内生长的环境。这项多参数分析提供了定量数据，可以准确监测无尾线虫宿主巨噬细胞培养物的寄生负荷，以发现利什曼尼化合物，以及它们对宿主巨噬细胞的潜在毒性作用。我们通过使用一小部分利什曼尼类药物的化合物和最近发表的化学实体来验证我们的方法。根据其巨噬细胞内利什曼尼菌活性及其对宿主细胞的毒性，化合物被归类为不相关或相关的药物发现管道进入下一步。我们的试验代表了一个新的筛选平台，克服了抗利什曼原虫药物发现的几个限制。首先，检测原代巨噬细胞毒性的能力允许发现能够穿过巨噬细胞、液泡和无尾线虫膜的化合物，从而加速选择性靶向细胞内寄生虫的化合物的开发过程。其次，我们的实验允许发现干扰寄生虫发育和生长所需巨噬细胞生物学功能的抗利什曼动物，例如细胞器运输/酸化或杀微生物效应物的产生。因此，这些数据验证了一种新的表型筛选方法，该方法使用生长在原代巨噬细胞内的强毒利什曼原虫来鉴定具有真正药物潜力的新化学实体。利什曼病是由利什曼属原生动物寄生虫引起的被忽视的疾病。没有针对任何形式的利什曼病的疫苗，大多数现有的抗利什曼病药物都有严重的副作用。目前发现新的利什曼尼分子的策略主要是使用不适当类型的宿主细胞或使用不相关的昆虫特异性寄生虫阶段。我们在此提出了一种基于哺乳动物巨噬细胞宿主细胞和利什曼原虫致病性无尾线虫的创新和生物学相关的药物筛选试验。我们已经建立了视觉荧光试验，目的是筛选各种小分子，包括化学物质和天然化合物，这些化合物选择性地靶向巨噬细胞内的无尾线虫，而不对巨噬细胞宿主细胞显示毒性。本文介绍了一种依赖于生物材料和荧光成像探针自动处理、图像采集、数据存储和分析的小型试验的验证。基于稳健的统计方法和质量控制指标，数据分析管道允许根据治疗三天后观察到的对寄生虫和巨噬细胞存活的影响对化合物进行分类。
Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis. We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline. Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential. Leishmaniases are neglected diseases caused by protozoan parasites that belong to the genus Leishmania. No vaccine exists against any form of leishmaniasis and most of the existing anti-leishmanial drugs have serious side effects. Current strategies for discovering new leishmanicidal molecules are largely using inappropriate types of host cells or employing the irrelevant insect-specific parasite stage. We presented here an innovative and biologically relevant drug screening assay based on the use of the mammalian macrophage host cells and Leishmania pathogenic amastigotes. Our visual fluorescence assay has been established with the objective to screen diverse small-molecules including chemicals and natural compounds that selectively target intra-macrophagic amastigotes without displaying toxicity for the macrophage host cells. The validation of a miniaturized assay, relying on automated handling of biological materials and fluorescent imaging probes, image acquisition, data storage and analysis is presented. Based on robust statistical methods and quality control metrics, the data analysis pipeline allows for the classification of compounds based on their effect on parasite and macrophage survival observed after three days of treatment.
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DOI：
10.4269/ajtmh.1981.30.566
发表时间：
1981-01-01
影响因子：
3.3
作者：
BERMAN, JD
通讯作者：
BERMAN, JD
影响因子：
4.9
作者：
El Fadili, Karima;Imbeault, Michael;Ouellette, Marc
通讯作者：
Ouellette, Marc
影响因子：
30.3
作者：
Jaramillo, Maritza;Gomez, Maria Adelaide;Sonenberg, Nahum
通讯作者：
Sonenberg, Nahum
影响因子：
6.4
作者：
Lira, R;Sundar, S;Sacks, D
通讯作者：
Sacks, D
影响因子：
3.3
作者：
Gradoni, Luigi;Soteriadou, Ketty;Dujardin, Jean-Claude
通讯作者：
Dujardin, Jean-Claude