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

  • ACS Cent Sci. 2017 Mar 22;3(3):232-243. doi: 10.1021/acscentsci.7b00028.
Omkar Zilka  1 ,  Ron Shah  1 ,  Bo Li  1 ,  José Pedro Friedmann Angeli  2 ,  Markus Griesser  1 ,  Marcus Conrad  2 ,  Derek A Pratt  1
Affiliations
  • 1. Department of Chemistry and Biomolecular Sciences, University of Ottawa , Ottawa, Ontario K1N 6N5, Canada.
  • 2. Institute of Developmental Genetics, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) , 85764 Neuherberg, München, Germany.
PMID: 28386601 DOI: 10.1021/acscentsci.7b00028
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.

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