The ovarian tumor domain-containing protein 4 (OTUD4)-targeted ferroptosis inhibitor Maclekarpine E attenuates ulcerative colitis.

BACKGROUND Ulcerative colitis (UC) is a chronic inflammatory bowel disease driven by oxidative stress. Current therapies, including aminosalicylates and immunosuppressants, are limited by adverse effects. Natural product-derived compounds offer promising alternatives. Maclekarpine E (ME) is a dihydrobenzophenanthridine alkaloid with anti-inflammatory properties, but its efficacy and mechanism in UC are unclear.

OBJECTIVE This study aimed to evaluate the therapeutic effect of ME against UC and to elucidate its underlying molecular mechanism and potential target.

METHODS ME was prepared on a gram scale via a semi-synthetic and biomimetic route. Its biological activities were evaluated both in vitro using an LPS-induced RAW264.7 macrophage model, and in vivo using a DSS-induced murine model of ulcerative colitis. To elucidate the underlying mechanism, TMT-based proteomic profiling and an RSL3-induced ferroptosis model in RAW264.7 macrophages were employed. Potential targets associated with the biological activities of ME were identified by DIA-based SPIA proteomic analysis. The initial hit was subsequently validated using CETSA assay. Further validation was then conducted through CHX chase and ubiquitination assays in HEK-293T cells with overexpression or knockdown of the target protein, in order to confirm its functional relevance.

RESULTS ME demonstrated potent anti-inflammatory effects in both RAW264.7 cellular and C57BL/6 mice UC models. Proteomic and functional analyses identified ferroptosis inhibition as a key mechanism. ME up-regulated the NRF2/GPX4 antioxidant axis, down-regulated ACSL4, and mitigated lipid peroxidation, thereby counteracting RSL3-induced RAW264.7 cell ferroptosis. Furthermore, ME bounds to the deubiquitinase ovarian tumor domain-containing protein 4 (OTUD4) and reduced GPX4 ubiquitination and enhanced protein stability.

CONCLUSION ME could target OTUD4 to stabilize the core ferroptosis defense factor GPX4 and activate NRF2/GPX4 signaling pathway as a novel ferroptosis inhibitor to alleviate UC. These findings could suggest ME to be a promising lead compound for further modification and highlight OTUD4 as a potential new target for anti-inflammatory drug development.