Toxoplasma gondii forms enduring bradyzoites within cysts in brain and muscle tissues that are crucial for the transmission and remission. Current medical treatments effectively target the pathogenic tachyzoites, however they fail to eradicate the chronic T. gondii infections. It is currently not well understood why treatments fail and how drugs impact bradyzoites metabolism.

To address these shortcomings, we screened 400 compounds from the MMV Pathogen Box against in vitro tachyzoites and bradyzoites. Our screening identified 16 compounds that simultaneously inhibit tachyzoite growth and act cidal against bradyzoites. Notably, the known bc₁-complex inhibitors ELQ-400 and buparvaquone were among the identified hits.

To characterize parasite responses to inhibitors and delineate their modes of actions we used HILIC-UHPLC-MS to profile and trace stable-isotope incorporation into metabolites of treated parasites. The impact of a non-metabolic control inhibitor (BKI 1294) revealed a general stress response marked by elevated oxidized glutathione and AMP and a depletion of mitochondrial and glycolytic metabolites. We also identified a bc₁-complex inhibitor by comparing its metabolic fingerprint to known inhibitors. The metabolic impact of MMV1028806 mirrored that of buparvaquone and atovaquone in both tachyzoites and bradyzoites. While treated tachyzoites exhibited many metabolic changes including dysregulation of the TCA cycle and pyrimidine synthesis pathway, bradyzoites showed a more conservative response consistent with an energy shortage. Direct measurements of mitochondrially produced ATP demonstrated very low ATP levels in bradyzoites that can be further depleted by atovaquone but not by a non-bradyzocidal coenzyme Q analog. Our findings further challenge the notion of metabolically inactive bradyzoites. Our data also suggest that a functional bc₁-complex plays a crucial role in ATP production and bradyzoite survival, both of which can be disrupted by approved bc₁-complex targeting drugs in vitro.