2. Academic Validation
  3. Identification of RIPK3 as a target of flavonoids for anti-necroptosis in vitro

Identification of RIPK3 as a target of flavonoids for anti-necroptosis in vitro

  • Bioorg Chem. 2025 Jul 1:161:108503. doi: 10.1016/j.bioorg.2025.108503.
Hang Xie 1,  Wanchen Li 2,  Xiaoyu Han 3,  Minjun Li 4,  Qiang Zhao 5,  Yechun Xu 6,  Haixia Su 7,  Weihua Meng 8
Affiliations

Affiliations

  • 1 School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China.
  • 2 Nottingham Ningbo China Beacons of Excellence Research and Innovation Institute, University of Nottingham Ningbo China, Ningbo 315100, China.
  • 3 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China.
  • 4 Shanghai Synchrotron Radiation Facility, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China.
  • 5 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China; School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China.
  • 6 School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China; State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China; School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China. Electronic address: [email protected].
  • 7 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: [email protected].
  • 8 Nottingham Ningbo China Beacons of Excellence Research and Innovation Institute, University of Nottingham Ningbo China, Ningbo 315100, China; Division of Population Health and Genomics, Ninewells Hospital and Medical School, University of Dundee, Dundee DD2 4BF, UK; Center for Public Health, Faculty of Medicine, Health and Life Sciences, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast BT12 6BA, UK. Electronic address: [email protected].
PMID: 40328155 DOI: 10.1016/j.bioorg.2025.108503
Abstract

Receptor-interacting protein kinase 3 (RIPK3), a key regulator of Necroptosis, has emerged as an important target for therapeutic intervention. Flavonoids are natural compounds known for their anti-inflammatory and antioxidant properties, with recent studies highlighting their potential to modulate Necroptosis. In this study, we explored the potential of RIPK3 as a target for Flavonoids to achieve anti-necroptosis and anti-inflammatory effects. A library of 63 Flavonoids was tested for RIPK3 binding and kinase inhibition using fluorescence polarization (FP) competition assay and ADP-Glo kinase activity assay. Six Flavonoids, including scutellarein, robinetin, baicalin, myricetin, baicalein, and tricetin, showed significant inhibition of RIPK3, with IC50 values ranging from 2.5 to 13.7 μM. Structural studies of tricetin and robinetin through co-crystallization and molecular docking revealed distinct binding modes of these Flavonoids within the ATP-binding pocket of RIPK3. The anti-necroptosis effects of these Flavonoids were further evaluated in human HT-29 cells and mouse embryonic fibroblasts (MEFs) using a TSZ-induced cell death assay, resulting in EC50 values in the tens of micromolar range. Western blot analysis demonstrated that these Flavonoids inhibit the phosphorylation of RIPK3 and its downstream effector, Mixed Lineage Kinase domain-like protein (MLKL), and disrupt the formation of RIPK1 and RIPK3 aggregates in the Necroptosis pathway. These findings identify RIPK3 as a target of natural Flavonoids for the first time and elucidate the molecular mechanism underlying the anti-necroptotic activity of these Flavonoids.

Keywords

Anti-necroptosis effect; Co-crystal structure; Flavonoids; Inhibitors; RIPK3.

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