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

Abstract

New assay designs are needed to improve the predictive value of the Trypanosoma cruzi in vitro tests 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 of T. cruzi amastigotes 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 point immediately before the fold change becomes negative, independent of the drug concentration and exposure time. This time-to-kill over drug concentration revealed specific pharmacodynamic profiles of the benchmark drugs benznidazole and posaconazole. Author SummaryChagas disease, caused by Trypanosoma 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 predictive in vitro assays. To address this, we have developed an assay design that enables the growth of T. cruzi intracellular 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 parasites stop multiplying and start to die. The resulting pharmacodynamics profiles can provide robust and informative details on anti-chagasic candidates, as demonstrated for the benchmark drugs benznidazole and posaconazole.