On the Mechanism of Cytoprotection by Ferrostatin-1 and Liproxstatin-1 and the Role of Lipid Peroxidation in Ferroptotic Cell Death

TLDR

Ferroptosis is an iron‑dependent, lipid‑hydroperoxide–driven form of regulated necrosis implicated in degenerative diseases, and high‑throughput screens have identified ferrostatin‑1 and liproxstatin‑1 as potent inhibitors that slow lipid peroxidation. The study aims to demonstrate that ferrostatin‑1 and liproxstatin‑1 inhibit ferroptosis by acting as radical‑trapping antioxidants rather than lipoxygenase inhibitors, and to evaluate similarly designed, highly reactive THN antioxidants for antiferroptotic activity. Using kinetic assays, the authors show that ferrostatin‑1 and liproxstatin‑1 react more slowly with peroxyl radicals than α‑tocopherol yet are more reactive in phosphatidylcholine bilayers, do not inhibit 15‑lipoxygenase‑1, and that THNs, as radical‑trapping antioxidants, effectively block ferroptosis in cell culture. These experiments reveal that ferrostatin‑1 and liproxstatin‑1, as radical‑trapping antioxidants, subvert ferroptosis without inhibiting 15‑LOX‑1, and that the highly reactive THNs are equally effective in preventing ferroptosis induced by Gpx4 inhibition or glutamate toxicity, supporting a central role for lipid peroxidation in ferroptotic cell death.

Abstract

Ferroptosis is a form of regulated necrosis associated with the iron-dependent accumulation of lipid hydroperoxides that may play a key role in the pathogenesis of degenerative diseases in which lipid peroxidation has been implicated. High-throughput screening efforts have identified ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as potent inhibitors of ferroptosis − an activity that has been ascribed to their ability to slow the accumulation of lipid hydroperoxides. Herein we demonstrate that this activity likely derives from their reactivity as radical-trapping antioxidants (RTAs) rather than their potency as inhibitors of lipoxygenases. Although inhibited autoxidations of styrene revealed that Fer-1 and Lip-1 react roughly 10-fold more slowly with peroxyl radicals than reactions of α-tocopherol (α-TOH), they were significantly more reactive than α-TOH in phosphatidylcholine lipid bilayers − consistent with the greater potency of Fer-1 and Lip-1 relative to α-TOH as inhibitors of ferroptosis. None of Fer-1, Lip-1, and α-TOH inhibited human 15-lipoxygenase-1 (15-LOX-1) overexpressed in HEK-293 cells when assayed at concentrations where they inhibited ferroptosis. These results stand in stark contrast to those obtained with a known 15-LOX-1 inhibitor (PD146176), which was able to inhibit the enzyme at concentrations where it was effective in inhibiting ferroptosis. Given the likelihood that Fer-1 and Lip-1 subvert ferroptosis by inhibiting lipid peroxidation as RTAs, we evaluated the antiferroptotic potential of 1,8-tetrahydronaphthyridinols (hereafter THNs): rationally designed radical-trapping antioxidants of unparalleled reactivity. We show for the first time that the inherent reactivity of the THNs translates to cell culture, where lipophilic THNs were similarly effective to Fer-1 and Lip-1 at subverting ferroptosis induced by either pharmacological or genetic inhibition of the hydroperoxide-detoxifying enzyme Gpx4 in mouse fibroblasts, and glutamate-induced death of mouse hippocampal cells. These results demonstrate that potent RTAs subvert ferroptosis and suggest that lipid peroxidation (autoxidation) may play a central role in the process.