In vitro profiling of Trypanosoma cruzi inhibitors identified from High throughput Screening and application to...

Chagas disease, a neglected tropical disease caused by Trypanosoma cruzi, urgently requires next-generation therapeutics due to the limitations of use of benznidazole and nifurtimox, including adverse effects, long treatment period and still unproven efficacy in chronic cases. With limited knowledge of T. cruzi biology regarding validated targets that could be addressed from a drug discovery perspective, most drug discovery efforts associated to the identification of new T. cruzi active chemotypes has been relying upon cell-based assays. The failure of repurposing the CYP51 acting antifungal drug posaconazole through clinical trials has highlighted the need for a more sophisticated characterization of phenotypically identified T. cruzi inhibitors via more stringent triage compounds before engaging with downstream drug development. In this study, we have evaluated 2422 compounds against the intracellular amastigotes of T. cruzi to identify novel starting points for drug development. The 30 identified inhibitors of T. cruzi growth including 5 serotonin-dopamine receptor antagonists and 7 TGF-β receptor inhibitors were then used as chemical tools to profile the drug action effects in terms of T. cruzi amastigote quantification measurements, kinetics of action, susceptibility in different strains as well as forms of the parasite. In terms of kinetics of action, fast-acting compounds inhibited the proliferation of intracellular amastigotes within 48 h of incubation, whereas slow-acting compounds required prolonged exposure to achieve comparable growth inhibition. Although most compounds were active against both Y and Dm28c strains, the correlation between their IC₅₀ values was only moderate. Among the 30 identified inhibitors, 6 displayed potent inhibitory activity against all three parasite life-cycle stages-amastigotes, trypomastigotes, and epimastigotes. Lastly, the "parasite painting" methodology was applied to trypomastigotes of the parasite to classify compounds based on morphological perturbations. The series of assays applied in this study offer tools to characterize and prioritize inhibitors for the downstream discovery process.