Design, synthesis, and biological evaluation of arctigenin derivatives as novel GPX4 inhibitors in colorectal cancer cells.

Glutathione peroxidase 4 (GPX4), a critical regulator of ferroptosis, has emerged as an attractive therapeutic target for cancer therapy. Leveraging the therapeutic potential of this target, we rationally designed and synthesized 25 novel arctigenin derivatives through structure-activity relationship (SAR) studies. Remarkably, compound W25 displayed the most potent antitumor efficacy against HCT-116 colorectal cancer cells, surpassing the activity of the parent compound arctigenin. Flow cytometry analysis revealed that W25 increased lipid reactive oxygen species (ROS) levels by 4.5 times in HCT-116 cells. Notably, ferroptosis inhibitors (ferrostatin-1 and deferoxamine mesylate) could reverse the increase of lipid ROS induced by W25, suggesting that W25 could selectively trigger ferroptosis in HCT-116 cells. Meanwhile, W25 suppressed the level of glutathione (GSH) and increased the level of malondialdehyde (MDA) in HCT-116 cells. More importantly, the Western blotting results displayed that W25 inhibited the protein expression of GPX4 in a dose-dependent manner, while it had no significant effect on other ferroptosis-related proteins (such as FSP1, FTH1, and SLC7A11). Enzyme activity data also showed that W25 could suppress the activity of GPX4. Furthermore, MG132 (a proteasome inhibitor) reversed the inhibition of GPX4 protein expression by W25. Correspondingly, the immunoprecipitation assay also showed that W25 promoted the ubiquitination level of GPX4, revealing that W25 induced ubiquitination-dependent proteasomal degradation of GPX4. These results suggest that W25 is a novel ferroptosis inducer against colorectal cancer through the inhibition of GPX4.