Inhibition of Ferroptotic Toxicity by 4-Hydroxyindole

Hydroxyindoles are organic compounds characterized by the presence of a hydroxy group attached to an indole ring (six-membered benzene ring fused to a five-membered pyrrole ring). These compounds are naturally occurring and play a role in the synthesis of various medicinal drugs. One notable example is 4-Hydroxyindole (4-HI), which contains a hydroxy group at the fourth position of the indole ring. In a recent study, we tested various hydroxyindole compounds for their antiferroptotic activity, including 3-hydroxyindole, which demonstrated strong resistance to ferroptosis. Ferroptosis is a regulated form of cell death that occurs due to uncontrolled phospholipid peroxidation and is associated with the development of degenerative conditions, such as neurodegenerative diseases. Here, we tested the hypothesis that 4-HI could protect against ferroptosis, similar to other hydroxyindole compounds. To induce ferroptosis, we utilized established modulators, including erastin, RSL3, and FINO2. We assessed cytotoxicity using the calcein AM assay and measured lipid peroxidation caused by ferroptosis inducers with the C11-BODIPY assay. Our results indicated that 4-HI protects various brain-related cell types, including HT-22, N27, and RBE4 cells, from ferroptosis. We also utilized our newly developed cell-free assay, in which combined iron and arachidonic acid were used to oxidize C11-BODIPY, allowing us to investigate the radical scavenging activity of 4-HI. We discovered that 4-HI exhibits antioxidant effects in cell-free assays, suggesting that its protective action against ferroptosis is likely due to its radical-scavenging capabilities. Interestingly, we found that 4-hydroxyindole-3-carbaldehyde, a structural analog of 4-HI, did not effectively prevent ferroptosis. This suggests that the carbaldehyde group, which is an electron-withdrawing group, may reduce the antiferroptotic activity of 4-HI. In summary, 4-HI appears to be a promising inhibitor of ferroptosis, warranting further research to explore its potential in protecting against neurotoxicity and neurodegeneration associated with this type of cell death.