Suppression of Lipid Peroxidation by Necrostatins and Their Potential for Dual Targeting of Ferroptosis and Necroptosis

Necrostatin-1 (Nec-1), a widely used RIPK1 inhibitor and necroptosis probe, also suppresses ferroptosis, complicating distinction between these cell death pathways. Here, we show that the thiohydantoin moiety of Nec-1 reacts with hydroperoxides to form a sulfenic acid intermediate capable of trapping peroxyl radicals and inhibiting the lipid peroxidation (LPO) that drives ferroptosis. To investigate this mechanism, several analogs were synthesized and evaluated in the FENIX cell-free LPO assay. Anti-LPO activity was enhanced by increasing steric bulk or nucleophilicity around the thiocarbonyl, relocating the thiohydantoin methyl group, or introducing a fully conjugated linker between the thiohydantoin and indole rings. The fully conjugated analog of Nec-1 (dsaturated Nec-1 or dsNec-1) was isolated as a stable species from preparative reactions of Nec-1 and a hydroperoxide, suggesting it mediates Nec-1's antiferroptotic effects. Desaturated derivatives were, however, inactive against necroptosis, indicating that the Nec-1 scaffold is unsuitable for dual-pathway inhibitor development.