title: Image-based high-throughput drug screening targeting the intracellular stage of Trypanosoma cruzi, the agent of Chagas' disease.
abstract: 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.
authors.affiliation: 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. juan.engel@ucsf.edu
authors.forename: Juan C
authors.initials: JC
authors.lastname: Engel
authors.forename: Kenny K H
authors.initials: KK
authors.lastname: Ang
authors.forename: Steven
authors.initials: S
authors.lastname: Chen
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Arkin
authors.forename: James H
authors.initials: JH
authors.lastname: McKerrow
authors.forename: Patricia S
authors.initials: PS
authors.lastname: Doyle
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D014355:Chagas Disease / Q000188:drug therapy / Q000469:parasitology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D022781:Hepatocytes / Q000166:cytology / Q000469:parasitology* / Q000648:ultrastructure; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018482:Muscle, Skeletal / Q000166:cytology / Q000469:parasitology* / Q000648:ultrastructure; D021261:Parasitic Sensitivity Tests; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000254:growth & development
chemical_list: D014344:Trypanocidal Agents