Untargeted metabolomics for triaging of cytochrome b inhibitors during Chagas’ disease drug discovery

Chagas' disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a potentially fatal condition for which new treatments are urgently needed. Due to the lack of validated drug targets, phenotypic screening followed by target deconvolution is the dominant approach in Chagas' disease drug discovery. However, as most phenotypic screening hits act through a small number of promiscuous targets, implementation of counter-screening methodology for these targets as early as possible in the workflow is essential to enable prioritisation of compounds with novel Modes of Action (MoA). Here, we demonstrate that untargeted metabolomic profiling using liquid chromatography mass spectrometry (LC-MS) can reliably identify compounds that act through one of the most common targets, cytochrome b. Treatment of epimastigote form T. cruzi in culture with cytochrome b inhibitors resulted in rapid and pronounced perturbation of the metabolome. We identified a signature of 79 metabolites that were differentially expressed by at least 2-fold (p < 0.05). Unsupervised multivariate analysis using these features allowed clear separation of cytochrome b inhibitors from compounds acting through other MoA, and through disruption of oxidative phosphorylation by other mechanisms. Flexibility was observed in this cytochrome b signature between experiments, and depending on the compounds used, suggesting that this approach could be readily implemented in other laboratories. Triage of cytochrome b inhibitors early in the Chagas' disease drug discovery workflow using untargeted metabolomics will aid in prioritisation of medicinal chemistry resources towards compounds acting through novel mechanisms.