Every parasite counts?! Improving in vitro assay design for Chagas’ disease drug discovery

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. Symptoms of the chronic disease occur decades after infection in about 30% of the infected people. The major symptoms of chronic disease are cardiomyopathies and mega-syndromes of the digestive tract. Antiparasitic treatment is important to prevent and reduce chronic symptoms. However, the only two licensed antichagasic drugs, benznidazole and nifurtimox, are compromised by limited efficacy and side effects. Therefore, new drugs are urgently needed. Surprisingly high numbers of relapses in the clinical trials of ergosterol inhibitors questioned the preclinical drug discovery pipeline for Chagas' disease. In recent years, progress has been made in in vitro drug discovery towards higher throughput in screening, testing against strain panels, and testing against reversibility of drug action. However, inhibition of T. cruzi amastigote replication is still a dominant feature in the in vitro assays in contrast to cidality.
The aim of this PhD project was to develop an in vitro assay that contributes new read-outs to the preclinical drug discovery pipeline for Chagas' disease.
As a first step, methods for T. cruzi molecular genetics were established in our laboratory: genotyping, a transfection protocol including the prioritization of selection antibiotics, and the sequence of the genome of our T. cruzi strain STIB980. Next, we established an eGFP-expressing parasite line and characterized it phenotypically. This eGFP expressing parasite line was employed in a novel assay design that allows to monitor parasite replication and drug action in four-hour intervals over 6 days with a high-content microscope occupancy of 24 h only. The results of this assay revealed high levels of variability. Statistical modelling of the development of parasite numbers over 24 h led to the fold-change in parasite numbers as a robust read-out. For drug exposed samples, the fold-change in parasite numbers is dependent on the drug concentration and the time of drug exposure. Using the fold-change, we can determine the timepoint when parasite numbers start to drop with statistical confidence. This time-to-kill is a novel pharmacodynamic parameter that enhances the characterization of drug candidates.
The established methods and the novel assay design will hopefully contribute to Chagas' disease drug discovery in our laboratory and promote the discovery of the much needed effective drugs.