Discovery and Optimization of Ergosterol Peroxide Derivatives as Novel Glutaminase 1 Inhibitors for the Treatment of Triple-Negative Breast Cancer

In this study, novel ergosterol peroxide (EP) derivatives were synthesized and evaluated to assess their antiproliferative activity against four human cancer cell lines (A549, HepG2, MCF-7, and MDA-MB-231). Compound <b>3g</b> exhibited the most potent antiproliferative activity, with an IC₅₀ value of 3.20 µM against MDA-MB-231. This value was 5.4-fold higher than that of the parental EP. Bioassay optimization further identified <b>3g</b> as a novel glutaminase 1 (GLS1) inhibitor (IC₅₀ = 3.77 µM). In MDA-MB-231 cells, <b>3g</b> reduced the cellular glutamate levels by blocking the glutamine hydrolysis pathway, which triggered reactive oxygen species production and induced caspase-dependent apoptosis. Molecular docking indicated that <b>3g</b> interacts with the reaction site of the variable binding pocket by forming multiple interactions with GLS1. In a mouse model of breast cancer, <b>3g</b> showed remarkable therapeutic effects at a dose of 50 mg/kg, with no apparent toxicity. Based on these results, <b>3g</b> could be further evaluated as a novel GLS1 inhibitor for triple-negative breast cancer (TNBC) therapy.