High-Throughput Screen of Advanced Repurposing Library Identifies Compounds Active Against Acute and Chronic Stages of Toxoplasma gondii

Abstract

Toxoplasma gondii, a pathogen with a global burden impacting millions, has the capability to interconvert between the actively replicating tachyzoite form into a semidormant bradyzoite for, complicating treatment. Here, we utilized an advanced high throughput screen (HTS) to identify repurposing candidates from the ReFRAME library, which consolidates 14,095 molecules from three major databases, including FDA approved and late-stage clinical leads. To facilitate screening, the permissive cyst-forming Tg68 strain was genetically engineered to express nano luciferase under the pBAG1 promoter to track bradyzoite growth, and firefly luciferase under the pTub1 promoter to monitor tachyzoite growth. HTS in 384 well format identified 160 compounds that exhibited > 80% inhibition at 10 micromolar of both tachyzoites and bradyzoites. By cross referencing against a publicly available HEK293T cytotoxicity dataset, we filtered out 61 compounds based on half-maximal cytotoxicity (CC50) less < 5 micromolar, leaving 99 for further evaluation. From this set, 15 top hits were identified based on potency and the selectivity index (CC50:EC50; SI > 20) against HepG2. The top hits were profiled using in vitro growth assays for tachyzoites, bradyzoites, and ex vivo liberated and intact cysts. Marizomib, monensin, narasin, and LY2090314 showed the strongest inhibition following 24 h treatment of intact cysts. When tested in combination with clinical compounds atovaquone and pyrimethamine, the top hits were predominantly additive. Additionally, they were evaluated for their potential for developing resistance using a minimum inoculum of resistance (MIR) assay. Six of nine compounds tested in MIR assays yielded no detectable parasites over 30 days. In contrast, LY2090314 resistant populations exhibited 26 to 29-fold resistance and carried a TgGSK3 L53V mutation, identifying a candidate resistance determinant. Collectively, our findings identify compounds with activity against tachyzoites and bradyzoites and with low propensity for resistance, as starting points for mechanistic investigation and further evaluation of in vivo efficacy and host-toxicity liabilities.