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

Abstract

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.