Beyond DPPH: Use of Fluorescence-Enabled Inhibited Autoxidation to Predict Oxidative Cell Death Rescue

Highlights

• FENIX enables screening or counter-screening of lipid peroxidation inhibitors

• Equally simple to the DPPH assay, but relays information on kinetics and stoichiometry

• FENIX in liposomes predicts the anti-ferroptotic potency of antioxidants in cells

• H-bonding to the phospholipid head group attenuates RTA activity in lipid bilayers

Summary

“Antioxidant activity” is an often invoked, but generally poorly characterized, molecular property. Several assays are available to determine antioxidant activity, the most popular of which is based upon the ability of a putative antioxidant to reduce 2,2-diphenyl-1-picrylhydrazyl. Here, we show that the results of this assay do not correlate with the potency of putative antioxidants as inhibitors of ferroptosis, the oxidative cell death modality associated with (phospho)lipid peroxidation. We subsequently describe our efforts to develop an approach that quantifies the reactivity of putative antioxidants with the (phospho)lipid peroxyl radicals that propagate (phospho)lipid peroxidation (dubbed FENIX [fluorescence-enabled inhibited autoxidation]). The results obtained with FENIX afford an excellent correlation with anti-ferroptotic potency, which facilitates mechanistic characterization of ferroptosis inhibitors, and reveals the importance of H-bonding interactions between antioxidant and phospholipid that underlie both the lackluster antioxidant activity of phenols under physiologically relevant conditions and the emergence of arylamines as inhibitors of choice.