Structure-based design of potent and selective inhibitors targeting RIPK3 for eliminating on-target toxicity in vitro

• Published in: Nature communications Vol. 16; no. 1; pp. 4288 - 15
• Main Authors: Su, Haixia, Chen, Guofeng, Xie, Hang, Li, Wanchen, Xiong, Muya, He, Jian, Hu, Hangchen, Zhao, Wenfeng, Shao, Qiang, Li, Minjun, Zhao, Qiang, Xu, Yechun
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 08.05.2025; Nature Publishing Group; Nature Portfolio
• Subjects: 13/1; 13/44; 631/154/309/2420; 631/250/256; 631/535/1266; 82/16; 82/80; 82/81; 82/83; Animals; Apoptosis; Apoptosis - drug effects; Cell Line; Conformation; Design; Dimerization; Drug Design; HT29 Cells; Humanities and Social Sciences; Humans; Hydrophobicity; Inhibitors; Kinases; Locking; Mice; multidisciplinary; Necroptosis; Necroptosis - drug effects; Protein Kinase Inhibitors - chemistry; Protein Kinase Inhibitors - pharmacology; Receptor-Interacting Protein Serine-Threonine Kinases - antagonists & inhibitors; Receptor-Interacting Protein Serine-Threonine Kinases - chemistry; Receptor-Interacting Protein Serine-Threonine Kinases - genetics; Receptor-Interacting Protein Serine-Threonine Kinases - metabolism; Science; Science (multidisciplinary); Selectivity; Structure-Activity Relationship; Toxicity; Tumor Necrosis Factor-alpha - pharmacology; Tumor necrosis factor-α
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-025-59432-8

The essential role of RIPK3 in necroptosis makes its inhibition a promising therapeutic strategy. However, the development of RIPK3 inhibitors has been hampered by on-target apoptosis and limited kinase selectivity. Inspired by the R69H mutation, which prevents on-target apoptosis by disrupting RIPK3 dimerization, we design LK-series inhibitors that effectively inhibit RIPK3 in biochemical assays and block TNF-α-induced necroptosis in both mouse L929 and human HT29 cells without inducing apoptosis. The representative compound, LK01003, shows high selectivity across a panel of 379 kinases. Our structural studies reveal that LK compounds act as Type I 1/2 inhibitors, engaging a unique hydrophobic site and stabilizing an inactive conformation of RIPK3. Moreover, several type II inhibitors are also revealed to maintain RIPK3 in the inactive conformation and do not induce on-target apoptosis. These findings suggest a promising strategy for rational design of safe and selective inhibitors by locking the inactive conformation of RIPK3. Here the authors described the structure-based design of novel, selective RIPK3 inhibitors that do not induce on-target apoptosis by occupying a unique hydrophobic site and locking RIPK3 into an inactive conformation.