Ferroptosis-mediated anticancer activity of endoperoxide-containing steroids derived from <i>Daedaleopsis confragosa</i> via targeting NOS2

Mushrooms belonging to the genus <i>Daedaleopsis</i> are known to contain various biologically active constituents, including steroids. However, they have not been studied as extensively as other medicinal mushrooms. In this study, chemical analysis was performed on the methanolic extract of the fruiting bodies of <i>D. confragosa</i>, a species that has not been thoroughly explored. Seven steroidal compounds were identified, and among these compounds, <b>1</b>, <b>2</b>, <b>3</b> and <b>5</b>, which contain an endoperoxide bridge, exhibited strong cytotoxicity against HCT116, A549, MDA-MB-231, and A375SM cancer cell lines. Computational target prediction identified inducible nitric oxide synthase (NOS2) as their most probable molecular target. Based on this, compound <b>2</b>, with the most potent cytotoxicity, along with its intercellular byproducts, was subjected to molecular docking with inducible nitric oxide synthase (NOS2). The byproduct <b>2-1</b> demonstrated the highest binding affinity, forming hydrogen bonds with key residues of the NOS2 heme-binding site. Furthermore, compounds <b>1</b>, <b>2</b>, <b>3</b> and <b>5</b> significantly increased the expression of various ferroptosis markers, PTGS2, SLC7A11, CHAC1, and HMOX1, in a dose-dependent manner. Finally, NOS2 knockdown using siRNA also showed enhanced expression of ferroptosis markers which is the same response observed with the treatment of the compounds. In conclusion, this study suggests that fungal endoperoxide-containing steroids from <i>D. confragosa</i> represent new ferroptosis inducers <i>via</i> targeting NOS2, supporting their potential as anticancer drug candidates.