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The ovarian tumor domain-containing protein 4 (OTUD4)-targeted ferroptosis inhibitor Maclekarpine E attenuates
Xinye Lv1, Zhehao He1, Jiaojiao Xue1
1Hunan Key Laboratory of Traditional Chinese Veterinary Medicine, Hunan Agricultural University, Changsha 410128, Hunan, China.
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.
Insights
Maclekarpine E (ME) effectively treats ulcerative colitis (UC) by inhibiting ferroptosis and stabilizing GPX4. This natural compound targets OTUD4, offering a promising new therapeutic strategy for UC.
Area of Science:
- Pharmacology
- Molecular Biology
- Immunology
Background:
- Ulcerative colitis (UC) is a chronic inflammatory condition.
- Current UC therapies have limitations and adverse effects.
- Natural compounds like Maclekarpine E (ME) show therapeutic potential.
Purpose of the Study:
- Evaluate the therapeutic efficacy of Maclekarpine E (ME) in ulcerative colitis (UC).
- Elucidate the molecular mechanisms underlying ME's anti-inflammatory effects.
- Identify potential molecular targets of ME.
Main Methods:
- ME was synthesized and tested in vitro (macrophage model) and in vivo (murine UC model).
- Proteomic profiling (TMT, DIA-SPIA) identified mechanisms and targets.
- Ferroptosis inhibition was assessed using RSL3-induced models.
- Target validation involved CETSA, CHX chase, and ubiquitination assays.
Main Results:
- ME exhibited significant anti-inflammatory effects in UC models.
- ME acts as a ferroptosis inhibitor by up-regulating NRF2/GPX4 and down-regulating ACSL4.
- ME targets OTUD4, stabilizing GPX4 by reducing its ubiquitination.
Conclusions:
- ME stabilizes GPX4 via OTUD4 interaction, activating the NRF2/GPX4 pathway.
- ME functions as a novel ferroptosis inhibitor for UC treatment.
- ME is a potential lead compound, and OTUD4 is a novel anti-inflammatory drug target.
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