Targeted Degradation\nof Receptor-Interacting Protein\nKinase 1 to Modulate the Necroptosis Pathway

Necroptosis\nis a highly regulated form of necrotic cell death that\nplays an essential role in pathogen defense and tissue homeostasis.\nAbnormal regulation of the necroptotic pathway has been implicated\nin the pathogenesis of various human diseases, including cancer, inflammatory,\nand neurodegenerative diseases. Receptor-interacting protein kinase\n1 (RIPK1) serves as a crucial regulator of the necroptotic signaling\npathway and has been identified as a potential therapeutic target.\nMechanistically, RIPK1 serves as both a protein kinase and a scaffolding\nprotein, fulfilling its dual function through a combination of kinase\nactivity-dependent and kinase activity-independent mechanisms. Thus,\nemploying a targeted RIPK1 knockdown strategy is a highly effective\nmeans of inhibiting RIPK1 functions. To achieve a targeted RIPK1 knockdown,\nwe generated a RIPK1-PROTAC, MS2031, by connecting the ZB-R-55 RIPK1\nbinder to the VHL ligand, thereby recruiting the CUL2-RING-VHL (CRL2<sup>VHL</sup>) E3 ubiquitin ligase complex for targeted degradation of\nRIPK1 through the 26S proteasome. Notably, MS2031 treatment effectively\nreduced the abundance of RIPK1 protein in the nanomolar range in various\ncell lines we examined, including HT-29 and T47D cells, and modulated\nthe necroptosis signaling pathway. These results suggest that MS2031\nmay hold potential for the treatment of human diseases resulting from\naberrant regulation of RIPK1.