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

Abstract

Chagas' disease is 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 ergosterol biosynthesis inhibitor posaconazole in 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 nifurtimox and benznidazole from 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 posaconazole in 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 ergosterol biosynthesis inhibition by posaconazole leading to critically low ergosterol levels only after multiple rounds of division, and is further supported by the lack of effect of posaconazole on the non-replicative trypomastigote form. A washout experiment with prolonged posaconazole treatment 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. Benznidazole in contrast was able to kill all parasites. The work presented here shows clear differentiation between the nitro-aromatic drugs and posaconazole in several assays, and suggests that in vitro there may be clinically relevant heterogeneity in the parasite population that can be revealed in long-term washout experiments. 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.

Fig 1. Drug efficacy against T. cruzi intracellular amastigotes.

Strains Silvio X10/7, Y and PAH1790 assessed after 5 days treatment with nifurtimox (A) benznidazole (B) and posaconazole (C). (Representative dose-response curves of at least 3 biological replicates normalised to 0% effect DMSO and 100% effect 50 μM nifurtimox controls. Average±SD). The x axis is the Log of the compound concentration in Molar.

Fig 2. Raw dose-response data for T. cruzi intracellular amastigotes after treatment with posaconazole at 72 h, 96 h and 120 h for strains Silvio X10/7 (A-C) and PAH179 (D-F) (3 biological replicates shown).

The x axis is the Log of the compound concentration in Molar.

Fig 3. Drug efficacy against T. cruzi trypomastigotes.

Silvio X10/7 (A-C) and Tulahuen βgal (D-F) trypomastigotes after 24, 48 and 72 h treatment with nifurtimox (A & D), benznidazole (B & E) and posaconazole (C & F). (Dose-response curves of three biological replicates. Average±SD). The x axis is the Log of the concentration of the drug in Molar.

Fig 4. Detection of cells in S phase growth by EdU labelling of intracellular T. cruzi strains Silvio X10/7 and PAH179.

Number of amastigotes per well (average ±SD) determined by Hoechst staining after 5 days treatment with DMSO, 5 μM nifurtimox, 50 μM benznidazole and 1 μM posaconazole (A). Percent EdU positive amastigotes (B) after treatments described above using the automated image analysis algorithm. Imaging by HTS microscopy of Silvio X10/7 (C) and PAH179 (D) infected Vero labelled with EdU AlexaFluor 488 and Hoechst, treated for 5 days with DMSO, 5 μM nifurtimox, 50 μM benznidazole and 1 μM posaconazole. Parasites remaining after treatment are highlighted by white arrows (Hoechst), replicating parasites remaining after treatment are highlighted by red arrows (EdU AlexaFluor488). Bar 10μm.

Fig 5. Updated in vitro screening cascade for Chagas’ disease with addition of the newly developed T. cruzi strain panel and washout assays.

(Cpd, compound; TA, turnaround time).