In vitro and in vivo activity of the aspartic protease inhibitor CWHM-117 against Toxoplasma gondii

Toxoplasmosis, caused by the protozoan Toxoplasma gondii , affects nearly one-third of the global population and may result in severe congenital, ocular, and neurological manifestations. Current therapies are limited by toxicity, poor efficacy against chronic infection, and lack of activity against tissue cysts, highlighting the need for new therapeutic strategies. Aspartic proteases represent promising but underexplored drug targets in T. gondii . In this study, we evaluated the anti- T . gondii potential of a panel of aspartic protease inhibitors initially developed for inhibition of Plasmodium . Eleven compounds were screened in vitro against intracellular tachyzoites (RH strain) in human foreskin fibroblasts (HFF), and their cytotoxicity was assessed to determine EC ₅₀ , CC ₅₀ , and selectivity indices. Most compounds displayed micromolar activity (EC ₅₀ range: 1.06–75.86 µM), with CWHM-032 (=TCMDC-134675), CWHM-033 (=TCMDC-136879), and CWHM-117 emerging as the most potent inhibitors. Based on its in vitro selective activity, predicted pharmacokinetic and safety profile, and previously reported efficacy against experimental malaria, CWHM-117 was selected for in vivo evaluation. In an acute murine model of toxoplasmosis, treatment with CWHM-117 delayed mortality compared to the vehicle-treated group. In a chronic infection model, CWHM-117 significantly reduced cerebral cyst burden ( P < 0.05), demonstrating activity against the bradyzoite stage, which remains a major therapeutic challenge. Overall, these findings indicate that aspartic protease inhibitors, particularly CWHM-117, represent promising lead compounds for the treatment of toxoplasmosis. This study supports T. gondii aspartic proteases as druggable targets and encourages further optimization and mechanistic studies to advance this class of compounds toward preclinical development.