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Non-invasive monitoring of drug action: A new live in vitro assay design for Chagas' disease drug discovery

Fesser, Anna F. and Braissant, Olivier and Olmo, Francisco and Kelly, John M. and Maeser, Pascal and Kaiser, Marcel. (2020) Non-invasive monitoring of drug action: A new live in vitro assay design for Chagas' disease drug discovery. PLOS Neglected tropical diseases, 14 (7). e0008487.

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Abstract

Author summary Chagas' disease, caused byTrypanosoma cruzi, is a chronic debilitating infection occurring mostly in Latin America. There is an urgent need for new, well tolerated drugs. However, the latest therapeutic candidates have yielded disappointing outcomes in clinical trials, despite promising preclinical results. This demands new and more predictivein vitroassays. To address this, we have developed an assay design that enables the growth ofT.cruziintracellular forms to be monitored in real time, under drug pressure, for 6 days post-infection. This allowed us to establish the tipping point of drug action, when the death rate of the parasites exceeded the growth rate. The resulting pharmacodynamics profiles can provide robust and informative details on anti-chagasic candidates, as demonstrated for the benchmark drugs benznidazole and posaconazole.
New assay designs are needed to improve the predictive value of the Trypanosoma cruzi in vitrotests used as part of the Chagas' disease drug development pipeline. Here, we employed a green fluorescent protein (eGFP)-expressing parasite line and live high-content imaging to monitor the growth ofT.cruziamastigotes in mouse embryonic fibroblasts. A novel assay design allowed us to follow parasite numbers over 6 days, in four-hour intervals, while occupying the microscope for only 24 hours per biological replicate. Dose-response curves were calculated for each time point after addition of test compounds, revealing how EC50 values first decreased over the time of drug exposure, and then leveled off. However, we observed that parasite numbers could vary, even in the untreated controls, and at different sites in the same well, which caused variability in the EC50 values. To overcome this, we established that fold change in parasite number per hour is a more robust and informative measure of drug activity. This was calculated based on an exponential growth model for every biological sample. The net fold change per hour is the result of parasite replication, differentiation, and death. The calculation of this fold change enabled us to determine the tipping point of drug action, i.e. the time point when the death rate of the parasites exceeded the growth rate and the fold change dropped below 1, depending on the drug concentration and exposure time. This revealed specific pharmacodynamic profiles of the benchmark drugs benznidazole and posaconazole.
Faculties and Departments:03 Faculty of Medicine > Departement Biomedical Engineering
UniBasel Contributors:Braissant, Olivier
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Public library of Science
ISSN:1935-2735
Note:Publication type according to Uni Basel Research Database: Journal article
Language:English
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edoc DOI:
Last Modified:29 Oct 2020 14:42
Deposited On:29 Oct 2020 14:42

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