Image-Based High-Throughput Drug Screening Targeting the Intracellular Stage of Trypanosoma cruzi, the Agent of Chagas' Disease
Image-Based High-Throughput Drug Screening Targeting the Intracellular Stage of Trypanosoma cruzi, the Agent of Chagas' Disease
复制标题
DOI：
10.1128/aac.01777-09
复制
发表时间：
2010-08-01
影响因子：
4.9
通讯作者：
Doyle, Patricia S.
中科院分区：
文献类型：
--
作者：
Engel, Juan C.;Ang, Kenny K. H.;Doyle, Patricia S.
南美锥虫病由寄生虫克氏锥虫感染引起，是拉丁美洲心力衰竭的主要原因。典型的临床表现是由于心肌细胞感染导致进行性心肌病。为了改善疾病，化疗必须根除寄生虫。目前的药物是无效和有毒的，新的治疗是一个关键的需求。为了加快这种被忽视的疾病的药物筛选，我们已经开发并验证了一种基于细胞的高通量检测方法，该方法可用于各种未转染的T细胞。Cruzi分离物和宿主细胞，并同时测量针对细胞内无鞭毛体阶段的功效和对宿主细胞的毒性。T.将Cruzi感染的肌细胞与测试化合物一起在96孔板中温育。基于DAPI（4 '，6-二脒基-2-苯基吲哚）染色的宿主细胞核和寄生虫动质体之间的大小差异，对测定板进行自动成像和分析。T.每个宿主细胞的Cruzi提供了寄生虫生长抑制的定量测量，而宿主细胞核计数的减少表明化合物毒性。该方法用于筛选临床批准的药物库，并鉴定了55种具有抗T.克鲁兹灵活的测定设计允许使用各种寄生虫菌株，包括具有不同生物学特性的临床分离株（例如，组织嗜性和药物敏感性）和广泛的宿主细胞，甚至可以适于筛选针对其它细胞内病原体的抑制剂。这种高通量检测将在抗寄生虫药物发现中产生重要影响。
Chagas' disease, caused by infection with the parasite Trypanosoma cruzi, is the major cause of heart failure in Latin America. Classic clinical manifestations result from the infection of heart muscle cells leading to progressive cardiomyopathy. To ameliorate disease, chemotherapy must eradicate the parasite. Current drugs are ineffective and toxic, and new therapy is a critical need. To expedite drug screening for this neglected disease, we have developed and validated a cell-based, high-throughput assay that can be used with a variety of untransfected T. cruzi isolates and host cells and that simultaneously measures efficacy against the intracellular amastigote stage and toxicity to host cells. T. cruzi-infected muscle cells were incubated in 96-well plates with test compounds. Assay plates were automatically imaged and analyzed based on size differences between the DAPI (4',6-diamidino-2-phenylindole)-stained host cell nuclei and parasite kinetoplasts. A reduction in the ratio of T. cruzi per host cell provided a quantitative measure of parasite growth inhibition, while a decrease in count of the host nuclei indicated compound toxicity. The assay was used to screen a library of clinically approved drugs and identified 55 compounds with activity against T. cruzi. The flexible assay design allows the use of various parasite strains, including clinical isolates with different biological characteristics (e.g., tissue tropism and drug sensitivity), and a broad range of host cells and may even be adapted to screen for inhibitors against other intracellular pathogens. This high-throughput assay will have an important impact in antiparasitic drug discovery.