Unveiling p65 as the target of diphyllin in ameliorating metabolic dysfunction-associated steatotic liver disease via targeted protein degradation technology

Xuejing Zhu ¹ Lei Zhang ² Wenqian Cui ² Liangjie Wang ² Fengjing Xu ² Mengyuan Liu ² Shuangcheng Chen ³ Haowen Jiang ⁴ Zhiying He ^1* Chang Peng ^5* Jinlong Li ^2*

• ¹ Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, Shanghai Municipality, China
• ² Nantong University, Nantong, Jiangsu Province, China
• ³ Nantong Health College of Jiangsu Province, Nantong, Jiangsu Province, China
• ⁴ Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai, China
• ⁵ Hangzhou Institute for Advanced Study, University of Chinese Academy of Science, Hangzhou, Jiangsu Province, China

Metabolic dysfunction-associated steatotic liver disease (MASLD), characterized by hepatic steatosis, inflammation and fibrosis, is becoming a global epidemic. However, the currently available effective clinical strategies remain limited. Herein, we determine that diphyllin, a natural arylnaphthalene lignan lactone, is effective on MASLD, evidenced by the inhibition of hepatic lipid accumulation through promoting fatty acid oxidation in vivo and in vitro. To uncover the underlying mechanisms, we employ the targeted protein degradation technology (protac) applied for the discovery of compound d/protein-protein interaction. We design and synthesize diphyllin-based protac and identify p65 as a potential target protein. Under p65 deficiency, the effects of diphyllin on lipid metabolism are blocked in vitro. As p65 acts as a repressor of NRF2, diphyllin interacts with p65, leading to the induction of the NRF2 transcriptional activity and the enhancement of antioxidant capacity. When NFR2 is inhibited, the lowering effects of diphyllin on lipid are abolished. In conclusion, our study not only presents diphyllin as a potential lead compound for MASLD therapy, but also offers a novel approach for elucidating the mechanisms of action of natural products.