Innovative Assay Development and Targeted Screening Unveil Compounds Against Toxoplasma gondii Bradyzoite and Tachyzoite Forms

Toxoplasma gondii, a pathogen of global concern, and its ability to interconvert between bradyzoite and tachyzoite forms necessitates innovative approaches for therapeutic discovery. This study centers on the pioneering development of a high-throughput assay and subsequent screening of the Library of Pharmacologically Active Compounds (LOPAC) against both tachyzoite and bradyzoite forms of T. gondii.

We used a unique isolate from a patient with congenital toxoplasmosis, called Tg68, which exhibits robust differentiation into mature bradyzoites under stress conditions in vitro. Comparative analyses highlighted Tg68's slower replication and enhanced bradyzoite differentiation relative to ME49. Notably, Tg68 maintains high rates of bradyzoite formation at high MOI under stress conditions, presenting a valuable model for identifying compounds effective against the bradyzoite stage.

In this study, Tg68 parasites were engineered to express nano luciferase under pBAG1 for investigating bradyzoite stage inhibitors. Additionally, Tg68 parasites expressing firefly luciferase constitutively under the pTub1 promoter were used to study tachyzoite stage inhibitors. A 384-well format assay was developed using automated liquid handlers. Screening the LOPAC library revealed 44 compounds with >50% inhibitory effects against Tg68 bradyzoites. Subsequent characterization included TgEC50, HepG2 CC50, and in vitro EC50 assessment against bradyzoites. Now we are expanding the screen to include the ReFRAME (Repurposing, Focused Rescue, and Accelerated Medchem) library comprising ~15,000 FDA-approved and late clinical leads in collaboration with Scripps. Our approach prioritizes compounds based on efficacy against Tg68, specifically targeting bradyzoites, while ensuring safety profiles. These findings not only unveil a promising set of compounds for further preclinical development against T. gondii including bradyzoites that are responsible for chronic infection.