Radical-trapping Antioxidants Research Articles

Suppression of ferroptosis by vitamin A or radical-trapping antioxidants is essential for neuronal development

The development of functional neurons is a complex orchestration of multiple signaling pathways controlling cell proliferation and differentiation. Because the balance of antioxidants is important for neuronal survival and development, we hypothesized that ferroptosis must be suppressed to gain neurons. We find that removal of antioxidants diminishes neuronal development and laminar organization of cortical organoids, which is fully restored when ferroptosis is inhibited by ferrostatin-1 or when neuronal differentiation occurs in the presence of vitamin A. Furthermore, iron-overload-induced developmental growth defects in C. elegans are ameliorated by vitamin E and A. We determine that all-trans retinoic acid activates the Retinoic Acid Receptor, which orchestrates the expression of anti-ferroptotic genes. In contrast, retinal and retinol show radical-trapping antioxidant activity. Together, our study reveals an unexpected function of vitamin A in coordinating the expression of essential cellular gatekeepers of ferroptosis, and demonstrates that suppression of ferroptosis by radical-trapping antioxidants or by vitamin A is required to obtain mature neurons and proper laminar organization in cortical organoids.

The hydrophobicity of the CARD8 N-terminus tunes inflammasome activation

Mounting evidence indicates that proteotoxic stress is a primary activator of the CARD8 inflammasome, but the complete array of signals that control this inflammasome have not yet been established. Notably, we recently discovered that several hydrophobic radical-trapping antioxidants (RTAs), including JSH-23, potentiate CARD8 inflammasome activation through an unknown mechanism. Here, we report that these RTAs directly alkylate several cysteine residues in the N-terminal disordered region of CARD8. These hydrophobic modifications destabilize the repressive CARD8 N-terminal fragment and accelerate its proteasome-mediated degradation, thereby releasing the inflammatory CARD8 C-terminal fragment from autoinhibition. Consistently, we also found that unrelated (non-RTA) hydrophobic electrophiles as well as genetic mutation of the CARD8 cysteine residues to isoleucines similarly potentiate inflammasome activation. Overall, our results not only provide further evidence that protein folding stress is a key CARD8 inflammasome-activating signal, but also indicate that the N-terminal cysteines can play key roles in tuning the response to this stress.

Lipid-derived electrophiles inhibit the function of membrane channels during ferroptosis

The therapeutic targeting of ferroptosis requires full understanding of the molecular mechanism of this regulated cell death pathway. While lipid-derived electrophiles (LDEs), including 4-hydroxy-2-nonenal (4-HNE), are important biomarkers of ferroptosis, a functional role for these highly reactive species in ferroptotic cell death execution has not been established. Here, through mechanistic characterization of LDE-detoxification impairment, we demonstrate that LDEs mediate altered protein function during ferroptosis. Applying live cell fluorescence imaging, we first identified that export of glutathione-LDE-adducts through multidrug resistance-associated protein (MRP) channels is inhibited following exposure to a panel of ferroptosis inducers (FINs) with different modes of action (type I-IV FINs erastin, RSL3, FIN56, and FINO2). This channel inhibition was recreated by both initiation of lipid peroxidation and treatment with 4-HNE. Importantly, treatment with radical-trapping antioxidants prevented impaired LDE-adduct export when working with both FINs and lipid peroxidation initiators but not 4-HNE, pinpointing LDEs as the cause of this inhibited MRP activity observed during ferroptosis. Our findings, when combined with reports of widespread LDE alkylation of key proteins following ferroptosis induction, including MRP1, set a precedent for LDEs as critical mediators of ferroptotic cell damage. Lipid hydroperoxide breakdown to form truncated phospholipids and LDEs may fully explain membrane permeabilization and modified protein function downstream of lipid peroxidation, offering a unified explanation of the molecular cell death mechanism of ferroptosis.

Construction of a screening system for lipid-derived radical inhibitors and validation of hit compounds to target retinal and cerebrovascular diseases

Recent studies have highlighted the indispensable role of oxidized lipids in inflammatory responses, cell death, and disease pathogenesis. Consequently, inhibitors targeting oxidized lipids, particularly lipid-derived radicals critical in lipid peroxidation, which are known as radical-trapping antioxidants (RTAs), have been actively pursued. We focused our investigation on nitroxide compounds that have rapid second-order reaction rate constants for reaction with lipid-derived radicals. A novel screening system was developed by employing competitive reactions between library compounds and a newly developed profluorescence nitroxide probe with lipid-derived radicals to identify RTA compounds. A PubMed search of the top hit compounds revealed their wide application as repositioned drugs. Notably, the inhibitory efficacy of methyldopa, selected from these compounds, against retinal damage and bilateral common carotid artery stenosis was confirmed in animal models. These findings underscore the efficacy of our screening system and suggest that it is an effective approach for the discovery of RTA compounds.

Non-Tertiary Alkyl Substituents Enhance High-Temperature Radical Trapping by Phenothiazine and Phenoxazine Antioxidants.

Radical-trapping antioxidants (RTAs) are an indispensable class of additive used to preserve hydrocarbon materials from oxidative degradation. Materials that are regularly subjected to elevated temperatures where autoxidation is self-initiated (i.e., >120 °C) require high concentrations of RTA for protection. Not only is this costly, but it can negatively impact material performance. Herein we show that inhibition of the autoxidation of a model hydrocarbon (n-hexadecane) by phenothiazine (PTZ) at ≥160 °C can be greatly enhanced by the incorporation of either 1° or 2° alkyl substituents in the 3- and/or 7-positions of the scaffold. Structure-reactivity studies, product analyses and computations suggest that this results from hydrogen atom transfer (HAT) from the benzylic carbon of these alkyl substituents in the PTZ-derived aminyl radical intermediate. The resultant iminoquinone methide can then undergo further radical-trapping reactions, depending on the nature of the alkyl substituent. Similar structure-reactivity relationships are observed for the phenoxazine (PNX) scaffold. These results not only have significant implications for the design and development of new high-temperature RTA technology, but also for understanding aminic RTA activity at elevated temperatures. Specifically, they suggest that a stoichiometric model better accounts for the RTA activity of aromatic amines in saturated hydrocarbons than the widely accepted catalytic model.

Interrupted Homolytic Substitution Enables Organoboron Compounds to Inhibit Radical Chain Reactions Rather than Initiate Them.

The reactions of organoboranes with peroxyl radicals are key to their use as radical initiators for a vast array of radical chain reactions, particularly at low temperatures where high stereoselectivity or regioselectivity is desired. Whereas these reactions generally proceed via concerted homolytic substitution (SH2) mechanisms, organoboranes that bear groups that can stabilize tetracoordinate boron radical "ate" complexes (e.g., catecholboranes) undergo this reaction via a stepwise addition/fragmentation sequence and serve as useful stoichiometric alkyl radical precursors. Here we show that arylboronic esters and amides derived from catecholborane and diaminonaphthaleneborane, respectively, are potent radical-trapping antioxidants (RTAs). Mechanistic studies reveal that this is because the radical "ate" complexes derived from peroxyl radical addition to boron are sufficiently persistent to trap another radical in an interrupted SH2 reaction. Remarkably, the reactivity of these organoboranes as inhibitors of autoxidation was shown to translate from simple hydrocarbons to the phospholipids of biological membranes such that they can inhibit ferroptosis, the cell death modality driven by lipid autoxidation and relevant in neurodegeneration and other major pathologies. The unique mechanism of these organoboranes is one of only a handful of RTA mechanisms that are not based on H-atom transfer processes and provide a new dimension to boron chemistry and its applications.

Enhancement of Diarylamine Antioxidant Activity by Molybdenum Dithiocarbamates.

Molybdenum dithiocarbamates (MDTCs) are indispensable lubricant additives. Although their role as antiwear agents is well established, they have also been attributed antioxidant properties that are not understood. MDTCs do not inhibit autoxidation, but they markedly enhance the capacity of diphenylamines (DPAs)─ubiquitous radical-trapping antioxidants (RTAs)─to do so. We find this synergy to be evident not only at elevated temperatures (160 °C in n-hexadecane) but also at moderate temperatures, where autoxidations can be continuously monitored and kinetics more easily interpreted (100 °C in squalane). Interestingly, the synergy disappeared in an unsaturated hydrocarbon (n-hexadec-1-ene), where the RTA activity of the DPA is known to result from the diarylnitroxide derived therefrom. Autoxidations of squalane carried out in the presence of the diarylnitroxide─wherein it is a poor inhibitor─were much better inhibited in the presence of MDTC, suggesting that it converts the nitroxide to (a) more competent RTA(s). Indeed, preparative experiments revealed two species: DPA and a DPA dimer into which a single oxygen atom had been incorporated. This conversion is accelerated by the oxidation of MDTC to a dioxo molybdenum species. A mechanism is proposed to account for these observations, and the implications of our findings and their interpretation are discussed.

Novel 3-hydroxypyridin-4(1H)-One derivatives as ferroptosis inhibitors with iron-chelating and reactive oxygen species scavenging activities and therapeutic effect in cisplatin-induced cytotoxicity

Recent advances in understanding the role of iron and ROS in cell death suggest new therapeutic avenues to treat organ damage including acute kidney injury (AKI). Inhibiting ferroptosis was expected to have great potential for the treatment of this disease. Ferroptosis is characterized by iron-dependent lipid peroxidation and currently, a majority of reported ferroptosis inhibitors belong to either radical-trapping antioxidants or iron chelators. However, clinically used iron chelators such as deferoxamine and deferiprone have limited efficacy against ferroptosis (generally with EC50 > 100 μM), despite their proven safety. Herein, we present the rational design of novel ferroptosis inhibitors by incorporating the naturally occurring cinnamic acid scaffold and the 3-hydroxypyridin-4(1H)-one iron-chelating pharmacophore. Through ABTS˙+ radical-scavenging assay, oxygen radical absorbance capacity (ORAC) measurement, Fe3+ affinity evaluation, and anti-erastin-induced HT22 cell ferroptosis assays, we identified compound 9c as the most prospective ferroptosis inhibitor (ABTS˙+, IC50 = 4.35 ± 0.05 μM; ORCA = 23.79 ± 0.56 TE; pFe3+ = 18.59; EC50 = 14.89 ± 0.08 μM, respectively). Notably, 9c dose-dependently alleviated cell death in cisplatin-induced AKI model. Our results provide insight into the development of new ferroptosis inhibitors through rational incorporation of pharmacophores from existing ferroptosis inhibitors, and compound 9c could be a promising lead compound worth further investigation.

General Approach to Identify, Assess, and Characterize Inhibitors of Lipid Peroxidation and Associated Cell Death.

Lipid peroxidation (LPO) is associated with a variety of pathologies and drives a form of regulated necrosis called ferroptosis. There is much interest in small-molecule inhibitors of LPO as potential leads for therapeutic development for neurodegeneration, stroke, and acute organ failure, but this has been hampered by the lack of a universal high-throughput assay that can identify and assess candidates. Herein, we describe the development and validation of such an approach. Phosphatidylcholine liposomes loaded with ∼10% phospholipid hydroperoxide and STY-BODIPY, a fluorescent signal carrier that co-autoxidizes with polyunsaturated phospholipids, are shown to autoxidize at convenient and constant rates when subjected to an optimized Fe2+-based initiation cocktail. The use of this initiation system enables the identification of each of the various classes of LPO inhibitors which have been shown to rescue from cell death in ferroptosis: radical-trapping antioxidants (RTAs), peroxidase mimics, and iron chelators. Furthermore, a limited dose-response profile of inhibitors enables the resolution of RTA and non-RTA inhibitors─thereby providing not only relative efficacy but mechanistic information in the same microplate-based experiment. Despite this versatility, the approach can still be used to estimate rate constants for the reaction of RTAs with chain-propagating peroxyl radicals, as demonstrated for a representative panel of RTAs. To illustrate the utility of this assay, we carried out a preliminary investigation of the 'off-target' activity of several ferroptosis suppressors that have been proposed to act independently of inhibition of LPO, including lipoxygenase inhibitors, cannabinoids, and necrostatins, the archetype inhibitors of necroptosis.

Tryptophan Metabolism Acts as a New Anti-Ferroptotic Pathway to Mediate Tumor Growth.

Emerging evidence reveals that amino acid metabolism plays an important role in ferroptotic cell death. The conversion of methionine to cysteine is well known to protect tumour cells from ferroptosis upon cysteine starvation through transamination. However, whether amino acids-produced metabolites participate in ferroptosis independent of the cysteine pathway is largely unknown. Here, the authors show that the tryptophan metabolites serotonin (5-HT) and 3-hydroxyanthranilic acid (3-HA) remarkably facilitate tumour cells to escape from ferroptosis distinct from cysteine-mediated ferroptosis inhibition. Mechanistically, both 5-HT and 3-HA act as potent radical trapping antioxidants (RTA) to eliminate lipid peroxidation, thereby inhibiting ferroptotic cell death. Monoamine oxidase A (MAOA) markedly abrogates the protective effect of 5-HT via degrading 5-HT. Deficiency of MAOA renders cancer cells resistant to ferroptosis upon 5-HT treatment. Kynureninase (KYNU), which is essential for 3-HA production, confers cells resistant to ferroptotic cell death, whereas 3-hydroxyanthranilate 3,4-dioxygenase (HAAO) significantly blocks 3-HA mediated ferroptosis inhibition by consuming 3-HA. In addition, the expression level of HAAO is positively correlated with lipid peroxidation and clinical outcome. Together, the findings demonstrate that tryptophan metabolism works as a new anti-ferroptotic pathway to promote tumour growth, and targeting this pathway will be a promising therapeutic approach for cancer treatment.

The NLRP1 and CARD8 inflammasomes detect reductive stress

The danger signals that activate the related nucleotide-binding domain leucine-rich repeat pyrin domain-containing 1 (NLRP1) and caspase activation and recruitment domain-containing 8 (CARD8) inflammasomes have not been fully established. We recently reported that the oxidized form of TRX1 binds to NLRP1 and represses inflammasome activation. These findings suggested that intracellular reductive stress, which would reduce oxidized TRX1 and thereby abrogate the NLRP1-TRX1 interaction, is an NLRP1 inflammasome-activating danger signal. However, no agents that induce reductive stress were known to test this premise. Here, we identify and characterize several radical-trapping antioxidants, including JSH-23, that induce reductive stress. We show that these compounds accelerate the proteasome-mediated degradation of the repressive N-terminal fragments of both NLRP1 and CARD8, releasing the inflammasome-forming C-terminal fragments from autoinhibition. Overall, this work validates chemical probes that induce reductive stress and establishes reductive stress as a danger signal sensed by both the NLRP1 and CARD8 inflammasomes.

Ferroptosis Inhibitors as Potential New Therapeutic Targets for Cardiovascular Disease.

Ferroptosis is a novel form of programmed cell death that occurs due to an increase in iron levels. Ferroptosis is implicated in a number of cardiovascular diseases, including myocardial infarction (MI), reperfusion damage, and heart failure (HF). As cardiomyocyte depletion is the leading cause of patient morbidity and mortality, it is critical to thoroughly comprehend the regulatory mechanisms of ferroptosis activation. In fact, inhibiting cardiac ferroptosis can be a useful therapeutic method for cardiovascular disorders. The iron, lipid, amino acid, and glutathione metabolisms strictly govern the beginning and execution of ferroptosis. Therefore, ferroptosis can be inhibited by iron chelators, free radical-trapping antioxidants, GPX4 (Glutathione Peroxidase 4) activators, and lipid peroxidation (LPO) inhibitors. However, the search for new molecular targets for ferroptosis is becoming increasingly important in cardiovascular disease research. In this review, we address the importance of ferroptosis in various cardiovascular illnesses, provide an update on current information regarding the molecular mechanisms that drive ferroptosis, and discuss the role of ferroptosis inhibitors in cardiovascular disease.

Chalcogen Atoms as Electron Donors in Proton-Coupled Electron Transfer Reactions.

Proton-coupled electron transfer (PCET) reactions are crucial for the optimal functioning of a broad scope of chemical and biological processes. In this report, we present an unprecedented type of concerted PCET (cPCET), in which a chalcogen atom acts as the electron donor. The nature of this mechanism is key for understanding the reactivity of different radical-trapping antioxidants having heavy chalcogens (S, Se, or Te) in their structures. Moreover, this chalcogen-assisted cPCET is likely to be occurring in multiple systems of biological interest.

Synthesis and antioxidant activities of N-thiophenyl ebselenamines: a 77Se{1H} NMR mechanistic study

Synthesis of N-thiophenyl ebselenamines and selenenyl sulphides as efficient radical-trapping and hydroperoxide-decomposing antioxidants, respectively has been described.

Antiradical Activity of Dopamine, L-DOPA, Adrenaline, and Noradrenaline in Water/Methanol and in Liposomal Systems.

Catecholamines play a crucial role in signal transduction and are also expected to act as endogeneous antioxidants, but the mechanism of their antioxidant action is not fully understood. Here, we describe the impact of pH on the kinetics of reaction of four catecholamines (L-DOPA, dopamine, adrenaline, and noradrenaline) with model 2,2-diphenyl-1-picrylhydrazyl radical (dpph•) in methanol/water. The increase in pH from 5.5 to 7.4 is followed by a 2 order of magnitude increase in the rate constant, e.g., for dopamine (DA) kpH5.5 = 1,200 M–1 s–1 versus kpH7.4 = 170,000 M–1 s–1, and such rate acceleration is attributed to a fast electron transfer from the DA anion to dpph•. We also proved that at pH 7.0 DA breaks the peroxidation chain of methyl linoleate in liposomes assembled from neutral and negatively charged phospholipids. In contrast to no inhibitory effect during peroxidation in non-ionic emulsions, in bilayers one molecule of DA traps approximately four peroxyl radicals, with a rate constant kinh >103 M–1 s–1. Our results from a homogeneous system and bilayers prove that catecholamines act as effective, radical trapping antioxidants with activity depending on the ionization status of the catechol moiety, as well as microenvironment: organization of the lipid system (emulsions vs bilayers) and interactions of catecholamines with the biomembrane.

Nitroxides as Building Blocks for Nanoantioxidants.

Nitroxides are an important class of radical trapping antioxidants whose promising biological activities are connected to their ability to scavenge peroxyl (ROO•) radicals. We have measured the rate constants of the reaction with ROO• (kinh) for a series of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) derivatives as 5.1 × 106, 1.1 × 106, 5.4 × 105, 3.7 × 105, 1.1 × 105, 1.9 × 105, and 5.6 × 104 M–1 s–1 for −H, −OH, −NH2, −COOH, −NHCOCH3, −CONH(CH2)3CH3, and =O substituents in the 4 position, with a good Marcus relationship between log (kinh) and E° for the R2NO•/R2NO+ couple. Newly synthesized Pluronic-silica nanoparticles (PluS) having nitroxide moieties covalently bound to the silica surface (PluS–NO) through a TEMPO–CONH–R link and coumarin dyes embedded in the silica core, has kinh = 1.5 × 105 M–1 s–1. Each PluS-bound nitroxide displays an inhibition duration nearly double that of a structurally related "free" nitroxide. As each PluS–NO particle bears an average of 30 nitroxide units, this yields an overall ≈60-fold larger inhibition of the PluS–NO nanoantioxidant compared to the molecular analogue. The implications of these results for the development of novel nanoantioxidants based on nitroxide derivatives are discussed, such as the choice of the best linkage group and the importance of the regeneration cycle in determining the duration of inhibition.

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.

Targeting Ferroptosis against Ischemia/Reperfusion Cardiac Injury

Ischemic heart disease is a leading cause of death worldwide. Primarily, ischemia causes decreased oxygen supply, resulting in damage of the cardiac tissue. Naturally, reoxygenation has been recognized as the treatment of choice to recover blood flow through primary percutaneous coronary intervention. This treatment is the gold standard therapy to restore blood flow, but paradoxically it can also induce tissue injury. A number of different studies in animal models of acute myocardial infarction (AMI) suggest that ischemia-reperfusion injury (IRI) accounts for up to 50% of the final myocardial infarct size. Oxidative stress plays a critical role in the pathological process. Iron is an essential mineral required for a variety of vital biological functions but also has potentially toxic effects. A detrimental process induced by free iron is ferroptosis, a non-apoptotic type of programmed cell death. Accordingly, efforts to prevent ferroptosis in pathological settings have focused on the use of radical trapping antioxidants (RTAs), such as liproxstatin-1 (Lip-1). Hence, it is necessary to develop novel strategies to prevent cardiac IRI, thus improving the clinical outcome in patients with ischemic heart disease. The present review analyses the role of ferroptosis inhibition to prevent heart IRI, with special reference to Lip-1 as a promising drug in this clinicopathological context.

Autoxidation vs. antioxidants - the fight for forever.

Autoxidation limits the longevity of essentially all hydrocarbons and materials made therefrom - including us. The radical chain reaction responsible often leads to complex mixtures of hydroperoxides, alkyl peroxides, alcohols, carbonyls and carboxylic acids, which change the physical properties of the material - be it a lubricating oil or biological membrane. Autoxidation is inhibited by addtitives such as radical-trapping antioxidants, which intervene directly in the chain reaction. Herein we review the most salient features of autoxidation and its inhibition, emphasizing concepts and mechanistic considerations important in understanding this chemistry across the wide range of contexts in which it is relevant.

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.