7-Dehydrocholesterol is an endogenous suppressor of ferroptosis.

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

Intrinsic and Extrinsic Limitations to the Design and Optimization of Inhibitors of Lipid Peroxidation and Associated Cell Death.

The archetype inhibitors of ferroptosis, ferrostatin-1 and liproxstatin-1, were identified via high-throughput screening of compound libraries for cytoprotective activity. These compounds have been shown to inhibit ferroptosis by suppressing propagation of lipid peroxidation, the radical chain reaction that drives cell death. Herein, we present the first rational design and optimization of ferroptosis inhibitors targeting this mechanism of action. Engaging the most potent radical-trapping antioxidant (RTA) scaffold known (phenoxazine, PNX), and its less reactive chalcogen cousin (phenothiazine, PTZ), we explored structure-reactivity-potency relationships to elucidate the intrinsic and extrinsic limitations of this approach. The results delineate the roles of inherent RTA activity, H-bonding interactions with phospholipid headgroups, and lipid solubility in determining activity/potency. We show that modifications which increase inherent RTA activity beyond that of the parent compounds do not substantially improve RTA kinetics in phospholipids or potency in cells, while modifications that decrease intrinsic RTA activity lead to corresponding erosions to both. The apparent "plateau" of RTA activity in phospholipid bilayers (kinh ∼ 2 × 105 M-1 s-1) and cell potency (EC50 ∼ 4 nM) may be the result of diffusion-controlled reactivity between the RTA and lipid-peroxyl radicals and/or the potential limitations on RTA turnover/regeneration by endogenous reductants. The metabolic stability of selected derivatives was assessed to identify a candidate for in vivo experimentation as a proof-of-concept. This PNX-derivative demonstrated stability in mouse liver microsomes comparable to liproxstatin-1 and was successfully used to suppress acute renal failure in mice brought on by tissue-specific inactivation of the ferroptosis regulator GPX4.

A non-canonical vitamin K cycle is a potent ferroptosis suppressor.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

Radical-Trapping Antioxidant Activity of Copper and Nickel Bis(Thiosemicarbazone) Complexes Underlies Their Potency as Inhibitors of Ferroptotic Cell Death.

Herein we demonstrate that copper(II)-diacetyl-bis(N4-methylthiosemicarbazone)(CuATSM), clinical candidate for the treatment of ALS and Parkinson's disease, is a highly potent radical-trapping antioxidant (RTA) and inhibitor of (phospho)lipid peroxidation. In THF autoxidations, CuATSM reacts with THF-derived peroxyl radicals with kinh = 2.2 × 106 M-1 s-1─roughly 10-fold greater than α-tocopherol (α-TOH), Nature's best RTA. Mechanistic studies reveal no H/D kinetic isotope effects and a lack of rate-suppressing effects from H-bonding interactions, implying a different mechanism from α-TOH and other canonical RTAs, which react by H-atom transfer (HAT). Similar reactivity was observed for the corresponding Ni2+ complex and complexes of both Cu2+ and Ni2+ with other bis(thiosemicarbazone) ligands. Computations corroborate the experimental finding that rate-limiting HAT cannot account for the observed RTA activity and instead suggest that the reversible addition of a peroxyl radical to the bis(thiosemicarbazone) ligand is responsible. Subsequent HAT or combination with another peroxyl radical drives the reaction forward, such that a maximum of four radicals are trapped per molecule of CuATSM. This sequence is supported by spectroscopic and mass spectrometric experiments on isolated intermediates. Importantly, the RTA activity of CuATSM (and its analogues) translates from organic solution to phospholipid bilayers, thereby accounting for its (their) ability to inhibit ferroptosis. Experiments in mouse embryonic fibroblasts and hippocampal cells reveal that lipophilicity as well as inherent RTA activity contribute to the potency of ferroptosis rescue, and that one compound (CuATSP) is almost 20-fold more potent than CuATSM and among the most potent ferroptosis inhibitors reported to date.

Metabolic determinants of cancer cell sensitivity to canonical ferroptosis inducers.

Cancer cells rewire their metabolism and rely on endogenous antioxidants to mitigate lethal oxidative damage to lipids. However, the metabolic processes that modulate the response to lipid peroxidation are poorly defined. Using genetic screens, we compared metabolic genes essential for proliferation upon inhibition of cystine uptake or glutathione peroxidase-4 (GPX4). Interestingly, very few genes were commonly required under both conditions, suggesting that cystine limitation and GPX4 inhibition may impair proliferation via distinct mechanisms. Our screens also identify tetrahydrobiopterin (BH4) biosynthesis as an essential metabolic pathway upon GPX4 inhibition. Mechanistically, BH4 is a potent radical-trapping antioxidant that protects lipid membranes from autoxidation, alone and in synergy with vitamin E. Dihydrofolate reductase catalyzes the regeneration of BH4, and its inhibition by methotrexate synergizes with GPX4 inhibition. Altogether, our work identifies the mechanism by which BH4 acts as an endogenous antioxidant and provides a compendium of metabolic modifiers of lipid peroxidation.

Potent Ferroptosis Inhibitors Can Catalyze the Cross-Dismutation of Phospholipid-Derived Peroxyl Radicals and Hydroperoxyl Radicals.

Nitroxides were recently shown to catalyze the cross-dismutation of alkylperoxyl and hydroperoxyl radicals, making them uniquely effective radical-trapping antioxidants (RTAs) in unsaturated hydrocarbons where both species are formed. Given the abundance of unsaturated lipids in biological membranes, the continuous generation of hydroperoxyl (superoxide) as a byproduct of aerobic respiration, and the demonstrated cytoprotective properties of some nitroxides, we probed if cross-dismutation operates in phospholipid bilayers and cell culture. Interestingly, only nitroxides that were efficiently converted to amines in situ were effective, with their activity paralleling the stability of the incipient aminyl radicals. The ether-linked diarylamine phenoxazine, one of the most potent RTAs known, was particularly effective as a cross-dismutation catalyst. In contrast, phenolic RTAs such as α-tocopherol (α-TOH), the most potent form of vitamin E, were found to be inefficient due to the preference for the combination of hydroperoxyl and phenoxyl radicals over H-atom transfer between them. Experiments carried out in mouse embryonic fibroblasts corroborated these findings. Cells cotreated with phenoxazine (or its nitroxide) and a superoxide source were better protected from ferroptosis than those treated with phenoxazine (or its nitroxide) alone. No such synergy was observed for cells treated with α-TOH. Live cell imaging established that cytoprotection was associated with suppression of (phospho)lipid peroxidation. These results highlight the remarkable capacity for select amines to act as effective phase-transfer catalysts for a reducing equivalent (an H atom), such that a water-soluble "reactive oxygen species" can be used to quench a lipid-soluble one.

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

"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.

The Catalytic Reaction of Nitroxides with Peroxyl Radicals and Its Relevance to Their Cytoprotective Properties.

Sterically-hindered nitroxides such as 2,2,6,6-tetramethylpiperidin- N-oxyl (TEMPO) have long been ascribed antioxidant activity that is thought to underlie their chemopreventive and anti-aging properties. However, the most commonly invoked reactions in this context-combination with an alkyl radical to give a redox inactive alkoxyamine or catalysis of superoxide dismutation-are unlikely to be relevant under (most) physiological conditions. Herein, we characterize the kinetics and mechanisms of the reactions of TEMPO, as well as an N-arylnitroxide and an N, N-diarylnitroxide, with alkylperoxyl radicals, the propagating species in lipid peroxidation. In each of aqueous solution and lipid bilayers, they are found to be significantly more reactive than Vitamin E, Nature's premier radical-trapping antioxidant (RTA). Inhibited autoxidations of THF in aqueous buffers reveal that nitroxides reduce peroxyl radicals by electron transfer with rate constants ( k ≈ 106 to >107 M-1 s-1) that correlate with the standard potentials of the nitroxides ( E° ≈ 0.75-0.95 V vs NHE) and that this activity is catalytic in nitroxide. Regeneration of the nitroxide occurs by a two-step process involving hydride transfer from the substrate to the nitroxide-derived oxoammonium ion followed by H-atom transfer from the resultant hydroxylamine to a peroxyl radical. This reactivity extends from aqueous solution to phosphatidylcholine liposomes, where added NADPH can be used as a hydride donor to promote nitroxide recycling, as well as to cell culture, where the nitroxides are shown to be potent inhibitors of lipid peroxidation-associated cell death (ferroptosis). These insights have enabled the identification of the most potent nitroxide RTA and anti-ferroptotic agent yet described: phenoxazine- N-oxyl.

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

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.