Identification of novel neuroprotective N,N-dimethylaniline derivatives that prevent oxytosis/ferroptosis and localize to late endosomes and lysosomes

Oxidative stress has been implicated in the aging process and the progression of many neurodegenerative disorders. We previously reported that a novel oxindole compound, GIF-0726-r, effectively prevents endogenous oxidative stress, such as oxytosis/ferroptosis, an iron-dependent form of non-apoptotic cell death, in mouse hippocampal cells. In this study, using two hundred compounds that were developed based on the structure-activity relationship of GIF-0726-r, we screened for the most potent compounds that prevent glutamate- and erastin-induced oxytosis and ferroptosis. Using submicromolar concentrations, we identified nine neuroprotective compounds that have N,N-dimethylaniline as a common structure but no longer contain an oxindole ring. The most potent derivatives, GIF-2114 and GIF-2197-r (the racemate of GIF-2115 and GIF-2196), did not affect glutathione levels, had no antioxidant activity in vitro, or ability to activate the Nrf2 pathway, but prevented oxytosis/ferroptosis via reducing reactive oxygen production and decreasing ferrous ions. Furthermore, we developed fluorescent probes of GIF-2114 and GIF-2197-r to image their distribution in live cells and found that they preferentially accumulated in late endosomes/lysosomes, which play a central role in iron metabolism. These results suggest that GIF-2114 and GIF-2197-r protect hippocampal cells from oxytosis/ferroptosis by targeting late endosomes and lysosomes, as well as decreasing ferrous ions.