Temperature-Dependent Effects of Alkyl Substitution on Diarylamine Antioxidant Reactivity.

Alkylated diphenylamines are among the most efficacious radical-trapping antioxidants (RTAs) for applications at elevated temperatures since they are able to trap multiple radical equivalents due to catalytic cycles involving persistent diphenylnitroxide and diphenylaminyl radical intermediates. We have previously shown that some heterocyclic diarylamine RTAs possess markedly greater efficacy than typical alkylated diphenylamines, and herein, report on our efforts to identify optimal alkyl substitution of the scaffold, which we had found to be the ideal compromise between reactivity and stability. Interestingly, the structure-activity relationships differ dramatically with temperature: para-alkyl substitution slightly increased reactivity and stoichiometry at 37 and 100 °C due to more favorable (stereo)electronic effects and corresponding diarylaminyl/diarylnitroxide formation, while ortho-alkyl substitution slightly decreased both reactivity and stoichiometry. No such trends were evident at 160 °C; instead, the compounds were segregated into two groups based on the presence/absence of benzylic C-H bonds. Electron spin resonance spectroscopy indicates that increased efficacy was associated with lesser diarylnitroxide formation, and deuterium-labeling suggests that this is due to abstraction of the benzylic H atom, precluding nitroxide formation. Computations predict that this reaction path is competitive with established fates of the diarylaminyl radical, thereby minimizing the formation of off-cycle products and leading to significant gains in high-temperature RTA efficacy.

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.

FSP1 is a glutathione-independent ferroptosis suppressor.

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

Resolving the Role of Lipoxygenases in the Initiation and Execution of Ferroptosis.

Lipoxygenases (LOXs) have been implicated as central players in ferroptosis, a recently characterized cell death modality associated with the accumulation of lipid hydroperoxides: the products of LOX catalysis. To provide insight on their role, human embryonic kidney cells were transfected to overexpress each of the human isoforms associated with disease, 5-LOX, p12-LOX, and 15-LOX-1, which yielded stable cell lines that were demonstrably sensitized to ferroptosis. Interestingly, the cells could be rescued by less than half of a diverse collection of known LOX inhibitors. Furthermore, the cytoprotective compounds were similarly potent in each of the cell lines even though some were clearly isoform-selective LOX inhibitors. The cytoprotective compounds were subsequently demonstrated to be effective radical-trapping antioxidants, which protect lipids from autoxidation, the autocatalytic radical chain reaction that produces lipid hydroperoxides. From these data (and others reported herein), a picture emerges wherein LOX activity may contribute to the cellular pool of lipid hydroperoxides that initiate ferroptosis, but lipid autoxidation drives the cell death process.

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.

The Potency of Diarylamine Radical-Trapping Antioxidants as Inhibitors of Ferroptosis Underscores the Role of Autoxidation in the Mechanism of Cell Death.

Two aromatic amines (ferrostatin-1 and liproxstatin-1) were recently identified from high-throughput screening efforts to uncover potent inhibitors of ferroptosis, the necrotic-like cell death induced by inhibition of glutathione peroxidase 4 (GPX4), deletion of the corresponding gpx4 gene, or starvation of GPX4 of its reducing cosubstrate, glutathione (GSH). We have since demonstrated that these two aromatic amines are highly effective radical-trapping antioxidants (RTAs) in lipid bilayers, suggesting that they subvert ferroptosis by inhibiting lipid peroxidation (autoxidation) and, thus, that this process drives the execution of ferroptosis. Herein, we show that diarylamine RTAs used to protect petroleum-derived products from autoxidation can be potent inhibitors of ferroptosis. The diarylamines investigated include representative examples of additives to engine oils, greases and rubber (4,4'-dialkyldiphenylamines), core structures of dyes and pharmaceuticals (phenoxazines and phenothiazines), and aza-analogues of these three classes of compounds that we have recently shown can be modified to achieve much greater reactivity. We find that regardless of how ferroptosis is induced (GPX4 inhibition, gpx4 deletion or GSH depletion), compounds which possess good RTA activity in organic solution (kinh > 105 M-1 s-1) and lipid bilayers (kinh > 104 M-1 s-1) are generally potent inhibitors of ferroptosis (in mouse embryonic fibroblasts). Likewise, structural analogs that do not possess RTA activity are devoid of antiferroptotic activity. These results further support the argument that lipid peroxidation (autoxidation) plays a major role in the mechanism of cell death induced by either GPX4 inhibition, gpx4 deletion, or GSH depletion. Moreover, it offers clear direction that ongoing medicinal chemistry efforts on liproxstatin and ferrostatin derivatives, which have been proposed as lead compounds for the treatment and/or prevention of ischemia/reperfusion injury, renal failure, and neurodegeneration, can be widened to include other aminic RTAs. To aid in these efforts, some relevant structure-reactivity relationships are discussed.

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.

Ferroptosis Inhibition: Mechanisms and Opportunities.

The past decade has yielded tremendous insights into how cells die. This has come with our understanding that several distinct forms of cell death are encompassed under the umbrella term necrosis. Among these distinct forms of regulated necrotic cell death, ferroptosis has attracted considerable attention owing to its putative involvement in diverse pathophysiological processes. A key feature of the ferroptosis process is the requirement of phospholipid peroxidation, a process that has been linked with several human pathologies. Now with the establishment of a connection between lipid peroxidation and a distinctive cell death pathway, the search for new small molecules able to suppress lipid peroxidation has gained momentum and may yield novel cytoprotective strategies. We review here advances in our understanding of the ferroptotic process and summarize the development of lipid peroxidation inhibitors with the ultimate goal of suppressing ferroptosis-relevant cell death and related pathologies.

Determination of Key Hydrocarbon Autoxidation Products by Fluorescence.

Hydroperoxides and carboxylic acids are key primary products that arise in the autoxidation of hydrocarbons. We have developed a simple approach to rapidly and simultaneously determine both types of products using hydroperoxide- and acid-sensitive moieties conjugated to nonpolar coumarin- and BODIPY-based fluorophores. The coumarin- and BODIPY-conjugated amine probes described here undergo 38- and 8-fold enhancement, respectively, upon protonation in a solvent system compatible with heavy hydrocarbons. The latter can be used directly with our previously described hydroperoxide-sensitive coumarin-conjugated phosphine probe to enable rapid quantification of both carboxylic acids and hydroperoxides in hydrocarbon samples. The utility of the approach is illustrated by the ready determination of the differing relative rates of hydroperoxide and acid formation with changes in hydrocarbon structure (e.g., n-hexadecane vs 1-hexadecene vs a lubricant base stock). The method offers significant versatility and automation compared with common but laborious titration approaches, and greatly improves screening efficiency and accuracy for the identification of novel radical-trapping antioxidants for high temperature applications. This application was demonstrated by the automated analysis of hydroperoxides and carboxylic acids (by microplate reader) in samples from 24 inhibited autoxidations of a lubricating oil, which were carried out on a parallel synthesizer at 160 °C in triplicate in a single day.

Unprecedented inhibition of hydrocarbon autoxidation by diarylamine radical-trapping antioxidants.

The reactivities of novel heterocyclic diarylamine radical-trapping antioxidants (RTAs) are profiled in a heavy hydrocarbon at 160 °C, conditions representative of those at which diphenylamine RTAs are used industrially. While carboxylic acids produced during the autoxidation are shown to deactivate these more basic RTAs, the addition of a sacrificial base leads to efficacies that are unprecedented in the decades of academic and industrial research in this area.

The catalytic mechanism of diarylamine radical-trapping antioxidants.

Diarylamine radical-trapping antioxidants are important industrial additives, finding widespread use in petroleum-derived products. They are uniquely effective at elevated temperatures due to their ability to trap multiple radicals per molecule of diarylamine. Herein we report the results of computational and experimental studies designed to elucidate the mechanism of this remarkable activity. We find that the key step in the proposed catalytic cycle-decomposition of the alkoxyamine derived from capture of a substrate-derived alkyl radical with a diarylamine-derived nitroxide-proceeds by different mechanisms depending on the structure of both the substrate and the diarylamine. N,N-Diarylalkoxyamines derived from saturated substrates and diphenylamines decompose by N-O homolysis followed by disproportionation. Alternatively, those derived from unsaturated substrates and diphenylamines, or saturated substrates and N-phenyl-ß-naphthylamine, decompose by an unprecedented concerted retro-carbonyl-ene reaction. The alkoxyamines that decompose by the concerted process inhibit hexadecane autoxidations at 160 °C to the same extent as the corresponding diarylamine, whereas those alkoxyamines that decompose by the N-O homolysis/disproportionation pathway are much less effective. This suggests that the competing cage escape of the alkoxyl radicals following N-O homolysis leads to significantly less effective regeneration of diarylamines and implies that the catalytic efficiency of diarylamine antioxidants is substrate-dependent. The results presented here have significant implications in the future design of antioxidant additives: diarylamines designed to yield intermediate alkoxyamines that undergo the retro-carbonyl-ene reaction are likely to be much more effective than existing compounds and will display catalytic radical-trapping activities at lower temperatures due to lower barriers to regeneration.