Sandler Center for Basic Research in Parasitic Diseases and Department of Pathology, University of California, San Francisco, UCSF-MC 2550, Room 501E, 1700 4th St., San Francisco, CA 94158-2330, USA.
Antimicrob Agents Chemother. 2010 Aug;54(8):3326-34. doi: 10.1128/AAC.01777-09. Epub 2010 Jun 14.
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.
恰加斯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病因。经典临床表现源于心肌细胞感染，导致进行性心肌病。为改善疾病，化疗必须消灭寄生虫。目前的药物无效且有毒，因此急需新疗法。为加速治疗这种被忽视疾病的药物筛选，我们开发并验证了一种基于细胞的高通量检测方法，可用于多种未转染的克氏锥虫分离株和宿主细胞，并能同时测量对细胞内无鞭毛体阶段的疗效和对宿主细胞的毒性。用测试化合物孵育感染了克氏锥虫的肌肉细胞。在 96 孔板中，根据 DAPI（4'，6-二脒基-2-苯基吲哚）染色的宿主细胞核与寄生虫动基体之间的大小差异，自动对检测板进行成像和分析。宿主细胞中克氏锥虫数量与寄生虫生长抑制的定量测量呈反比，而宿主细胞核数量的减少则表明化合物的毒性。该检测方法用于筛选临床批准药物库，发现 55 种具有抗克氏锥虫活性的化合物。该检测方法设计灵活，可用于各种寄生虫株，包括具有不同生物学特性（如组织嗜性和药物敏感性）的临床分离株，以及广泛的宿主细胞，甚至可能适应于筛选针对其他细胞内病原体的抑制剂。这种高通量检测方法将对寄生虫药物发现产生重要影响。
