Lipid Peroxidation and its Inhibition in Oxidative Cell Death: New Molecules, New Mechanisms and New Methods to Study Them

Abstract

The free radical chain reaction of lipid peroxidation (LPO) has long been implicated in various pathological conditions including neurodegenerative disorders, cardiovascular disease, diabetes and cancer. More recently, lipid peroxidation and the products derived therefrom have been associated with the execution of iron-dependent non-apoptotic cell death, coined ferroptosis in 2012. The elucidation of ferroptosis established a crucial connection between LPO and disease, facilitating advances in the design and evaluation of lipid peroxidation inhibitors as prospective therapeutics. The chain reaction can be suppressed by targeting initiation events with preventative antioxidants such as iron chelators or peroxide decomposers, or by quenching chain-propagating peroxyl radicals with radical-trapping antioxidants (RTAs); all of which have shown efficacy at inhibiting ferroptosis in cell models. Alternatively, sensitivity to ferroptosis can be modulated by altering the lipid composition of the cell towards less oxidizable lipids (i.e. monounsaturated fatty acids (MUFAs)), thereby sequestering autoxidizable lipid substrate (i.e. polyunsaturated fatty acids (PUFAs)) and lowering the rate of LPO.

Herein, we describe our efforts towards the development and validation of a new biologically relevant method to identify and characterise RTA and non-RTA inhibitors of LPO and subsequent oxidative cell death. Furthermore, we describe our investigation of new small molecule ferroptosis inhibitors and the structural requirements behind their remarkable potency, the importance of radical-trapping kinetics and stoichiometry in the inhibition of cell death and the mechanism of protection afforded by two distinct classes of lipids. In Chapter 2, we describe the development of a second-generation high-throughput fluorescence-based assay. A biomimetic initiation system comprised of the combination of an Fe2+-based initiation cocktail and liposome models of the phospholipid bilayer pre-loaded with phospholipid-derived hydroperoxides enables the identification of each of the various classes of LPO inhibitor which have been shown to rescue from cell death in ferroptosis. Further, we show that a limited dose-response profile of inhibitors enables the resolution of RTA and non-RTA inhibitors, providing not only relative efficacy but also mechanistic insight in the same microplate-based experiment. We have demonstrated the utility of this methodology by investigating various ferroptosis inhibitors with purported 'off-target' activity thought to be independent of LPO inhibition.

In Chapter 3, we systematically studied the RTA activity of unhindered phenolic ferroptosis inhibitors identified from high-throughput screens with remarkable potency at suppressing cell death. Our investigations in a variety of applications ranging from isotropic organic solution to mammalian cells uncovered the importance of modulating hydrogen-bond acidity of the reactive phenol O-H bond to maintain reactivity in the strong hydrogen-bonding environment of the phospholipid bilayer and thus potency at suppressing ferroptosis in the cell. Moreover, a structure-activity relationship study unveiled the structural motifs required for potent anti-ferroptotic activity. We show that the incorporation of a basic amine distal to the reactive RTA moiety dramatically improves anti-ferroptotic potency and highlights the importance of suppressing lysosomal lipid peroxidation to prevent the induction of cell death.

Previous work by our group demonstrated that the efficiency of the reaction between RTA and phospholipid-derived peroxyl radical, reflected by the magnitude of the inhibition rate constant, strongly correlates to the efficacy at suppressing ferroptosis in mouse embryonic fibroblasts. In Chapter 4, we evaluated the importance of radical-trapping kinetics and stoichiometry on anti-ferroptotic potency using isomeric hydroquinone RTAs, which display remarkably different inherent reactivity. We show that kinetics of radical-trapping is most important in the context of inhibition of ferroptotic cell death. Furthermore, we investigated the substrate specificity of ferroptosis suppressor protein-1 (FSP-1), a recently characterised enzyme responsible for recycling endogenous antioxidants and a critical component of the antioxidant defense system that contributes to ferroptosis resistance. We found that lipophilic para-quinones are privileged substrates for FSP-1 mediated regeneration and uncovered the most potent naturally derived RTAs identified to date, which benefit from endogenous recycling by FSP-1.

Considering the close relationship between LPO and the induction of ferroptosis, modulation of the lipid composition of the cell as well as the mechanisms involved in lipid metabolism can crucially dictate ferroptosis sensitivity. In Chapter 5, we investigated the modulation of ferroptosis by two distinct classes of lipids. Using oxidatively stable cyclopropanated fatty acids (C-PUFAs) we demonstrate the ferroptosis protection afforded by isotopically-reinforced polyunsaturated fatty acids (D-PUFAs) and MUFAs is a result of dilution of the pool of autoxidizable PUFA. In a second set of experiments, we focused on cis-vinyl ether lipids (i.e. plasmalogens) which have been implicated as both drivers and suppressors of ferroptosis. We show that the presence of the vinyl ether is indeed protective against LPO and subsequent cell death and propose a mechanism to account for the 'antioxidant' activity of vinyl ether lipids.