Improving antichagasic drug discovery through stage-specific monitoring of Trypanosoma cruzi
About 6-7 million people are infected with Trypanosoma cruzi, a protozoan parasite causing Chagas’ disease. Most of the symptoms appear in the chronic phase of Chagas’ disease: cardiomyopathies and mega-syndromes of the digestive tract. There is a high need for new drug, as the only two available drugs are not very effective and often display side effects. However, the most advanced new drug candidates, the CYP51 inhibitors, were found to lead to a high number of relapses in clinical trials. The fact that this failure had not been predicted by the current preclinical drug testing systems reflects a lack of understanding of T. cruzi biology.
For antichagasic drug discovery, it will be crucial to understand the processes involved in replication and differentiation of the T. cruzi life-cycle forms in the mammalian host and to translate this understanding into the drug discovery pipeline. We are exploring novel in vitro read-outs such as growth rates, differentiation and stage-specific markers to understand replication, differentiation and the effect of drugs on these processes.
Such read-outs require transgenic parasites with reporter genes. For this purpose, we have phenotypically characterized a clonal T. cruzi line, genotyped it, and sequenced the genome. This T. cruzi line will be transfected with reporter genes and characterized to use it for forward and reverse genetics of drug action and resistance. We already established an eGFP-expressing clone for use in a real-time assay for non-invasive monitoring of growth and drug action. With this model, we can now determine pharmacodynamics parameter of in vitro drug action. To obtain stage-specific reporter expression, we have selected stage-specific proteins from published data sets. Constructs for transfection will be made, and the stage-specific expression of the transgenic reporter proteins in T. cruzi will be tested phenotypically and with quantitative RT-PCR.
This approach will result in T. cruzi in vitro systems that allow to monitor cell growth, differentiation, and drug action in real-time and in a non-invasive way. This, in turn, will enhance our understanding of T. cruzi biology and promote drug discovery for Chagas' disease.