Implementation of the FENIX assay to screen for and assess inhibitors of lipid peroxidation and associated ferroptosis
Juliana A. Hüther, Melodie Mallais, Markus Rehm*, Derek A. Pratt*
Abstract: Ferroptosis is an iron-dependent form of regulated cell death characterized by the uncontrolled accumulation of lipid peroxides, primarily within phospholipid membranes. This oxidative process, driven by a radical-mediated chain reaction known as autoxidation, plays a central role in numerous pathological conditions including neurodegeneration, ischemia-reperfusion injury, and cancer. Pharmacological inhibition of lipid peroxidation by radical-trapping antioxidants (RTAs) has thus emerged as a promising strategy for therapeutic intervention. However, conventional ‘antioxidant assays’ such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) or oxygen radical absorbance capacity (ORAC) are inadequate for assessing reactivity toward lipid peroxyl radicals, limiting their utility in ferroptosis research. To address this gap, the Fluorescence-ENabled Inhibited autoXidation (FENIX) platform was developed as a physiologically relevant, high-throughput assay system for the mechanistic and kinetic characterization of lipid peroxidation inhibitors. FENIX-1 quantifies RTA activity by measuring rate constants for RTA-lipidperoxyl radical reactions initiated by a highly lipid-soluble radical initiator in liposomal systems wherein a fluorescent reporter is co-autoxidized along with the lipids. In FENIX-2 lipid peroxidation is initiated via iron-mediated decomposition of pre-formed phospholipid hydroperoxides, enabling the study of both RTA and non-RTA inhibitors, including iron chelators and peroxidase mimics. Together, these assays are complementary and mechanistically informative platforms for the identification and evaluation of inhibitors of lipid peroxidation and – by extension – ferroptosis. Here, we present a detailed overview and standardized protocols for both FENIX-1 and FENIX-2, with the aim of encouraging their application in lipid peroxidation research, ferroptosis modulation, and redox-targeted drug discovery.
Lay summary: Ferroptosis is a harmful form of cell death caused by damage in cell membranes, linked to diseases like stroke, neurodegeneration, and cancer. The FENIX assay is a new lab method that tests how compounds can block this damage, helping researchers find potential protective drugs. This tool makes it easier to study ferroptosis and develop therapies that keep cells healthy.
