Development of Trypanosoma cruzi in vitro assays to identify compounds suitable for progression in Chagas’ disease drug discovery

Chagas’ diseaseis responsible for significant mortality and morbidity in Latin America. Current treatments display variable efficacy and have adverse side effects, hence more effective, better tolerated drugs are needed. However, recent efforts have proved unsuccessful with failure of the ergosterolbiosynthesis inhibitor posaconazolein phase II clinical trials despite promising in vitro and in vivo studies. The lack of translation between laboratory experiments and clinical outcome is a major issue for further drug discovery efforts. Our goal was to identify cell-based assays that could differentiate current nitro-aromatic drugs nifurtimoxand benznidazolefrom posaconazole. Using a panel of T. cruzi strains including the six major lineages (TcI-VI), we found that strain PAH179 (TcV) was markedly less susceptible to posaconazolein vitro. Determination of parasite doubling and cycling times as well as EdU labelling experiments all indicate that this lack of sensitivity is due to the slow doubling and cycling time of strain PAH179. This is in accordance with ergosterolbiosynthesis inhibition by posaconazoleleading to critically low ergosterollevels only after multiple rounds of division, and is further supported by the lack of effect of posaconazoleon the non-replicative trypomastigote form. A washoutexperiment with prolonged posaconazoletreatment showed that, even for more rapidly replicating strains, this compound cannot clear all parasites, indicative of a heterogeneous parasite population in vitro and potentially the presence of quiescent parasites. Benznidazolein contrast was able to kill all parasites. The work presented here shows clear differentiation between the nitro-aromatic drugs and posaconazolein several assays, and suggests that in vitro there may be clinically relevant heterogeneity in the parasite population that can be revealed in long-term washoutexperiments. Based on these findings we have adjusted our in vitro screening cascade so that only the most promising compounds are progressed to in vivo experiments.