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. 2010 Aug;54(8):3326-34.
doi: 10.1128/AAC.01777-09. Epub 2010 Jun 14.

Image-based High-Throughput Drug Screening Targeting the Intracellular Stage of Trypanosoma Cruzi, the Agent of Chagas' Disease

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Free PMC article

Image-based High-Throughput Drug Screening Targeting the Intracellular Stage of Trypanosoma Cruzi, the Agent of Chagas' Disease

Juan C Engel et al. Antimicrob Agents Chemother. .
Free PMC article

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.

Figures

FIG. 1.
FIG. 1.
(A) Amplified fluorescence image obtained in the IN Cell Analyzer 1000 (10×) of BESM nuclei (N) and T. cruzi kDNA (k) stained with DAPI. (B) Image segmentation of BESM nuclei (blue outline) and T. cruzi kDNA (yellow outline) using the Developer Toolbox software. Each size bar is 10 μm.
FIG. 2.
FIG. 2.
Representative images collected with the IN Cell 1000 microscope of BESM cells infected with T. cruzi for 88 h (10×). (A) Untreated control. Similar T. cruzi-infected cells are amplified in Fig. 1. (B) Culture treated with 20 μM K11777 for 72 h. Arrows indicate intracellular parasites also highlighted in yellow by IN Cell software. Inserts show the relative fluorescence of DAPI-stained parasite kDNA (k) and nucleic (n) DNA and host cell nucleus (N) (40×).
FIG. 3.
FIG. 3.
(A) K11777 prevents T. cruzi development in BESM cells. Mean numbers of parasites/cell are higher in untreated controls than in cultures treated with 20 μM K11777. Standard deviations are shown as error bars. (B) K11777 does not induce cell toxicity, as evidenced by similar counts of BESM cell nuclei of untreated and treated controls. Standard deviations are shown as error bars.
FIG. 4.
FIG. 4.
Titration of fluphenazine and Iconix compound 130071 and EC50 values for T. cruzi and BESM cells. IC50, 50% inhibitory concentration.

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