Analyzing the Structure-Activity Relationship of Necrostain-1 and Its Analogues as RIPK1 Inhibitors on Necroptosis

Necroptosis is a type of programmed cell death mediated by the RIPK1-RIPK3-MLKL axis. Receptor-interacting serine/threonine-protein kinase 1(RIPK1), the key upstream regulator of necroptosis, has been implicated in the pathogenesis of various diseases, and its inhibitors are being tested in clinical trials. Necrostatin-1 (Nec-1), the firstly identified RIPK1 inhibitor, effectively prevents necroptosis by specifically inhibiting the kinase activity of RIPK1. In our study, we compared the inhibitory efficiency of Nec-1 and its analogues on RIPK1 by using the TBZ-induced necroptosis model in HT29 cells and analyzing their structure-activity relationship (SAR). The results showed that Nec-1, Nec-1s, Nec-a1, Nec-a2, Nec-a3, Nec-a4, and Nec-a5 exhibited potent inhibition of TBZ-induced necroptosis and phosphorylation of RIPK1, RIPK3, MLKL. Molecular docking showed that there were two potential binding pockets between Nec-1 and its analogues with RIPK1. SAR analysis showed that the type and size of a substituent at the nitrogen atom in the 3-position of the imidazolidine ring markedly influenced the activity, indicating that a substituent at this position is essential for maintaining the biological function. This study helps to further elucidate how Nec-1 works and could lead to the development of more effective analogues.