Cytoprotective Mechanism of Necrox-5 Against Toxicity Induced by Experimental Ferroptosis Instigators and the Pesticide Propargite

TL;DR

This study shows that Necrox-5 protects cells from ferroptosis and pesticide toxicity by acting as a powerful antioxidant.

Contribution

The paper is the first to demonstrate Necrox-5's protective effects against ferroptosis and propargite-induced toxicity.

Findings

Necrox-5 inhibits ferroptosis caused by multiple inducers like erastin and RSL3.

Necrox-5 protects against propargite toxicity but does not restore GSH or ATP levels.

Radical-scavenging antioxidant activity is the main mechanism of Necrox-5's cytoprotection.

Abstract

Necrox-5 is an indole-derived antioxidant that inhibits necrotic cell death, likely through prevention of mitochondrial stress, oxidative stress, inflammation, and hypoxia/reoxygenation. However, its protective role against ferroptotic toxicity has not yet been studied. In this study, we induced ferroptosis in HT-22 cells, an immortalized hippocampal neuronal cell line, using ferroptosis-inducing agents. We also tested Necrox-5 against toxicity induced by propargite, a pesticide known to inhibit complex V (mitochondrial adenosine triphosphate [ATP] synthase) and induce necrosis. We evaluated cytotoxicity using calcein AM and lactate dehydrogenase (LDH) release assays. Additionally, we conducted intracellular and cell-free C11-BODIPY assays to assess the efficacy of Necrox-5 in inhibiting lipid peroxidation. Intracellular glutathione (GSH) levels were measured using the mBCI-GSH assay, while ATP levels were determined through bioluminescence assays. Our findings show that Necrox-5 is a potent inhibitor of ferroptosis induced by erastin, RSL3, FINO2, and iron plus arachidonic acid. Furthermore, we demonstrated that Necrox-5 protects against ferroptosis-like propargite toxicity, although it did not prevent propargite-induced depletion of GSH and ATP. We identified radical-scavenging antioxidant activity as the primary mechanism by which Necrox-5 protects from ferroptosis and propargite toxicity. In conclusion, Necrox-5 is a potent cytoprotective compound that warrants further study for its potential role in ferroptosis-associated complications such as neurodegenerative diseases.

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1. Introduction

Necrox-5 (5-[(1,1-Dioxido-4-thiomorpholinyl)methyl]-2-phenyl-N-[(tetrahydro-2H-pyran-4-yl)methyl]-1H-indol-7-amine dimesylate) is a chemical compound from the Necrox series, developed by LG Life Sciences (now part of LG Chem Life Sciences) [1]. This compound has been shown to prevent a specific type of non-apoptotic necrotic cell death caused by oxidative stress [1,2,3]. Necrox-5 also provides protection from inflammation, hypoxia/reoxygenation, mitochondrial stress, and endoplasmic reticulum stress [4,5,6]. Research indicates that Necrox-5 functions as an inhibitor of mitochondrial calcium uniporter, thereby protecting the rat heart from harmful calcium overload [5]. It also helps maintain mitochondrial oxidative phosphorylation capacity and preserves peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α; a master regulator of cellular energy metabolism and important in mitochondrial biogenesis and function) during cardiac hypoxia-reperfusion injury [7]. Furthermore, the protective effects of Necrox-5 have been demonstrated in various models, including the attenuation of neomycin-induced hair cell loss in zebrafish [8], inhibition of bleomycin-induced pulmonary fibrosis [9], mitigation of IgE/Ag-stimulated anaphylaxis and mast cell activation [10], and reduction of lipopolysaccharide-induced acute respiratory distress [11]. Notably, Necrox-5 blocks activation of the NLR family pyrin domain containing 3 (NLRP3)-Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway, a major innate immune pathway [11,12]. Moreover, Necrox-5 shows neuroprotective effects against N-methyl-N-nitrosourea (MNU), a DNA-methylating agent, induced retinal degeneration in rats, and against 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced motor deficits in a zebrafish model of Parkinson’s disease (PD) [13,14]. In addition to causing motor deficits, MPTP decreased tyrosine hydroxylase expression (presumably through dopaminergic neuronal cell loss) in zebrafish, an effect attenuated by Necrox-5. While the neuroprotective mechanism of Necrox-5 remains unclear, its antioxidant activity is most likely the basis of its neuroprotective effects.

Ferroptosis is an iron-mediated form of necrotic cell death characterized by oxidative stress and uncontrolled phospholipid peroxidation [15,16,17]. This process involves distinct mitochondrial shrinkage and disruption of membrane integrity, ultimately causing cell membrane rupture and cell death, which differ from apoptosis [18,19]. This type of cell death has been linked to degenerative conditions in various organ systems, including the brain [20,21,22]. Ferroptosis can be triggered by inactivating the key anti-ferroptotic checkpoint involving cyst(e)ine, glutathione (GSH), and glutathione peroxidase 4 (GPX4) [16]. Erastin, for example, blocks the Xc– channel, thereby reducing cystine uptake and suppressing glutathione synthesis, ultimately leading to ferroptosis. RSL3 is among the most potent inducers of ferroptosis; it blocks GPX4 enzymatic activity, thereby impairing its detoxification of lipid hydroperoxides and promoting ferroptosis. In contrast, FINO2 is a compound that contains an endoperoxide with a 1,2-dioxolane structure. The mechanism of ferroptosis induction by FINO2 differs from that of system Xc-inhibitors or RSL3. The presence of the endoperoxide group, together with a nearby hydroxyl group, is essential for FINO2’s ferroptotic effects. The mechanism of FINO2 involves indirect inhibition of GPX4 enzymatic activity and direct oxidation of iron, leading to significant lipid peroxidation and ferroptosis. In addition to these classical inducers, a combination of iron and arachidonic acid (AA) has been identified as a relevant inducer of ferroptosis in the brain [20]. Iron can catalyze the oxidation of AA, generating toxic lipid peroxides that can initiate ferroptosis. In experimental models, various pharmacological agents, including liproxstatin-1 (Lip-1), vitamin A, and certain indole compounds, can block ferroptosis [15,16,22,23]. The primary mechanism by which these compounds protect cells from ferroptosis is through their radical-trapping antioxidant (RTA) activity. Identification of ferroptosis blockers is a growing area of interest as a therapeutic for diverse incurable degenerative conditions linked to ferroptosis, such as neurodegenerative diseases.

Propargite is also known as a broad-spectrum sulfite ester acaricide and insecticide used in agriculture to control various mites on fruits, vegetables, nuts, and field crops. This chemical exerts its pesticidal effect mainly by inhibiting mitochondrial adenosine triphosphate (ATP) synthase [24,25]. This pesticide induces necrotic death in pancreatic β-like cells and dopaminergic neurons in an early study [26]. Recent evidence has also identified propargite as highly toxic to dopaminergic neurons [27] and as a factor that increases the risk of developing PD [28].

While Necrox-5 demonstrates regulatory and protective roles against various forms of toxicity and injury, the mechanism underlying its action in a ferroptosis model remains uncharacterized. Therefore, this study aims to explore the protective mechanism of Necrox-5 against ferroptosis. To understand the protective mechanism, we also aim to test Necrox-5 against propargite, an inducer of both necrosis and mitochondrial dysfunction [26,29].

2. Results

We tested the hypothesis that Necrox-5 provides protection against ferroptosis. To induce ferroptosis in HT-22 cells, we utilized classical inducers such as erastin, RSL3, and FINO2, as well as a combination of iron and AA (Figure 1A). Recent studies have demonstrated the ferroptosis-inducing effects of these agents [16,20]; therefore, we did not include these confirmatory data to avoid redundancy. We observed a dose-dependent cytoprotective effect of Necrox-5 against ferroptosis in the cell viability assay, with an estimated EC50 of 10–53 nM (Figure 1A and Table 1). We further confirmed anti-ferroptotic activity using a cell death assay, and a single dose of Necrox-5 (500 nM) significantly defends against cell death caused by erastin, RSL3, FIN02, and iron+AA (Figure 1B). Furthermore, a single dose of ferroptosis instigators increases lipid peroxidation, which was significantly blocked by Necrox-5 treatment (500 nM) (Figure 1C).

We next tested the possible mechanism by which Necrox-5 protects cells from ferroptosis. Since the loss of glutathione (GSH) can sensitize cells to ferroptosis, we tested whether Necrox-5 protects against GSH depletion induced by erastin and buthionine sulfoximine (BSO); however, we found that Necrox-5 does not prevent GSH depletion (Figure 2A). Given the antioxidant function of Necrox-5 and the anti-ferroptotic functions of indole-containing molecules due to their inherent RTA properties [1,16,30,31], we tested the hypothesis that Necrox-5 protects against ferroptosis due to its RTA property. We found a potent RTA activity of Necrox-5 in a cell-free lipid peroxidation assay. For this experiment, we tested higher concentrations of Necrox-5 against iron+AA-induced BODIPY oxidation for a 30 min treatment. In contrast, our cytotoxicity assessments utilized lower concentrations (~500 nM) over a longer 24 h period. Notably, we found that the lowest concentration tested (1.562 μM) inhibited iron+AA-induced BODIPY oxidation by more than 50% for 30 min of treatment (Figure 2C).

Given Necrox-5’s role in protecting mitochondrial oxidative capacity and providing bioenergetic support [7], we extended the study to assess its effect on toxicity induced by a mitochondrial respiration inhibitor, propargite. Similar to its anti-ferroptotic effect, Necrox-5 exhibits potent cytoprotective activity against propargite-induced toxicity (Figure 3A,B).

Since propargite can cause necrotic cell death [26], it is important to identify whether Necrox-5 can protect against propargite toxicity by preventing necrosis and/or mitochondrial dysfunction. We next assessed the mechanism by which Necrox-5 protects against propargite toxicity. Since propargite inhibits mitochondrial complex V [24], it can deplete ATP, as we confirmed (Figure 4A). ATP depletion can affect GSH levels, and we confirmed that propargite treatments (2.5 and 5 μM) deplete GSH (Figure 4B). Notably, propargite depletes ATP and GSH, a finding that is not attributable to oxidative stress, as we supplemented with Lip-1 (a ferroptosis inhibitor). We first tested whether low doses of Necrox-5 (up to 500 nM) could protect against propargite-induced GSH depletion, and subsequently whether a higher dose of Necrox-5 (2.5 μM) would have a similar effect. Our findings revealed that Necrox-5 did not protect against propargite-induced depletion of GSH and ATP (Figure 4C,D). This indicates that the protective effect of Necrox-5 is related to preventing necrosis via the RTA mechanism rather than addressing mitochondrial dysfunction.

3. Discussion

While the antioxidant effects of Necrox-5 were published in 2010 (prior to the introduction of the term “ferroptosis”) [1], the protective role of this compound in a specific ferroptosis model had not been tested until now. In this study, we report the potent anti-ferroptotic activity of Necrox-5, which is comparable to that of the standard ferroptosis inhibitor, Lip-1 [16]. We also elucidate the cytoprotective mechanism of Necrox-5 against ferroptosis, which we attribute to its RTA activity. In addition to its antioxidant functions, Necrox-5 has been reported to have other effects, including mitochondria-specific effects. Since we detected a potent ferroptosis inhibitory effect at nanomolar concentration, caution should be applied before interpreting Necrox-5 other effects. Overall, our findings support previous studies indicating that Necrox-5 protects against oxidative stress through its antioxidant properties.

Necrox-5 contains several important structural components. It includes a phenyl group, which is a six-carbon aromatic ring (C_6_H_5_), attached to the 2-position of the indole ring. Additionally, an amine group connects the indole core to a tetrahydropyran ring structure and a 1,1-dioxido-4-thiomorpholinyl group. This latter group is a six-membered ring containing a sulfur atom double-bonded to two oxygen atoms, forming a sulfone group, and is also part of an amine ring. Compounds with phenyl, amine, or indole moieties, such as N-acetylcysteine and ferrostatin-1, are recognized for their antioxidant and anti-ferroptotic activities [30,32,33]. Therefore, the presence of these groups perhaps explains how Necrox-5 protects cells from ferroptosis. In earlier research, we demonstrated that indole-containing compounds, such as 3- and 4-hydroxyindoles, can prevent ferroptosis-mediated cytotoxicity through their inherent RTA activity [16,31]. Because Necrox-5 is also an indole-containing compound, this study further underscores the potential of the indole scaffold for discovering ferroptosis inhibitors. Notably, Necrox-5 exhibits potent anti-ferroptotic activity compared to previously reported hydroxyindole compounds [16,31].

Although we identified RTA as the primary mechanism by which Necrox-5 inhibits ferroptosis, we do not rule out the possibility of other mechanisms. The standard ferroptosis inhibitor Lip-1 was found to accumulate in lysosomes, where it interacts with iron. In the acidic environment of lysosomes, ferrous iron (Fe^2+^) can be released and catalyze the formation of harmful lipid peroxides, thereby inducing ferroptosis. Lip-1 disrupts this process, halting the cycle of lipid damage [34]. Since Necrox-5 is a similarly effective molecule to Lip-1, it is important to determine whether Necrox-5 also accumulates in lysosomes and interacts with iron to block ferroptosis.

Among the various oxidative stress conditions, Necrox-5 shows promise as a neuroprotective therapy [13,14]. However, its ability to penetrate the blood–brain barrier (BBB) remains unclear. If Necrox-5 cannot penetrate the BBB, structural modifications could yield improved analogs that could then be further studied for neuroprotective therapy against neurodegenerative diseases associated with oxidative stress and ferroptosis.

Propargite has been shown to induce necrotic cell death and is strongly linked to the pathogenesis of PD [26,27,28]. However, the specific mechanisms by which it causes neurotoxicity and the strategies to mitigate this toxicity remain largely unclear. In addition to the ferroptosis model, we found that Necrox-5 offers protection against propargite-induced toxicity. Notably, our report is the first to demonstrate that the lipid peroxidation inhibitor Necrox-5 can counteract the toxic effects of propargite. This finding suggests that propargite induces a specific type of necrotic cell death—ferroptosis, and that Necrox-5 prevents the toxic effect (necrosis) by inhibiting ferroptosis. While we propose that propargite induces ferroptosis-like toxicity, further studies are needed to determine whether and how it triggers lipid peroxidation to mediate this process.

Consistent with existing literature on propargite as a mitochondrial ATP synthase inhibitor [24,25], we found that propargite also depletes ATP. Given that ATP depletion can lead to GSH insufficiency [20], we tested and confirmed that propargite also depletes GSH. However, the relationship between GSH sufficiency and ATP depletion requires additional investigation. We also evaluated the ability of Necrox-5 to rescue ATP and GSH depletion caused by propargite. Although Necrox-5 did not rescue depletion under our experimental conditions (glucose-supplemented media), we recommend conducting further experiments using low-glucose or galactose-supplemented media. This experimental setup would enhance oxidative phosphorylation (OXPHOS) in cell cultures, thereby forcing cells to rely more heavily on mitochondria, increasing oxygen consumption (OCR), and improving metabolic fitness. Additionally, identifying strategies to protect against propargite-induced ATP and GSH depletion would be an exciting area of research for developing neuroprotective therapies for PD.

4. Materials and Methods

4.1. Reagents and Chemicals

RPMI 1640 (Cat#MT10041CM) was obtained from Corning Inc., Corning, NY, USA; penicillin and streptomycin (Cat# 15140122), amphotericin B (Cat#15290026), fetal bovine serum (FBS; Cat#A5256801), phosphate-buffered saline (PBS; Cat#10010023), and Hank’s balanced salt solution (HBSS; Cat# 14–175–079) were obtained from Gibco, part of Thermo Fisher Scientific, Waltham, MA, USA; calcein AM (Cat# C1430) was from Invitrogen (Carlsbad, CA, USA), also part of Thermo Fisher Scientific; ammonium iron(II) sulfate hexahydrate (Iron; Cat#201370250) was from Thermo Fisher Scientific. BSO (Catalog #309475000) was obtained from Acros Organics (Geel, Belgium), which is now part of Thermo Scientific Chemicals. Ferroptosis blocker (Lip-1; Cat#S7699) and RSL3 (Cat# S8155) were purchased from Selleck Chemicals LLC, Houston, TX, USA; erastin (Cat#329600), monochlorobimane (mBCI; Cat#69899), BODIPY 581/591 C11 (Cat#SML3717), and IGEPAL CA-630 (Cat#I8896) were obtained from MilliporeSigma (Burlington, MA), an affiliate of Merck KGaA, Darmstadt, Germany. NecroX-5 (Cat#HY-104015) was obtained from MedChemExpress (MCE), Monmouth Junction, NJ, USA; Arachidonic acid (AA, Cat#ICN19462510) was obtained from MP Biomedicals, CA, USA. The Cytotoxicity Detection Kit/KitPLUS (LDH; Cat#11644793001 and 4744926001) was purchased from Roche via MilliporeSigma.

4.2. Cell Culture and Cytotoxicity Assays

In this study, we used an immortalized HT-22 cell line derived from HT-4. This cell line was kindly provided by Dr. Val J. Watts from Purdue University and has been utilized in several prior studies [16,31,35,36]. HT-22 cells were cultured in RPMI 1640 media supplemented with 10% FBS, 1% penicillin-streptomycin, and amphotericin B (an antifungal) added at 0.5–1 μg/mL. Cell-based assays were conducted in the same media as the cell culture.

Cytotoxicity was evaluated using two assays: the calcein AM assay and the LDH release assay. In the calcein AM assay, the cell viability was assessed after treatment. Calcein AM is a nonfluorescent compound that can penetrate the membranes of living cells. Once inside the cell, calcein AM is converted by esterases to calcein, a fluorescent compound that remains in the cytoplasm. The fluorescence intensity is directly proportional to the number of viable cells [16,35]. In the experiments, cells were seeded at 1.5 × 10^4^ per well in 96-well plates and treated/cotreated with various compound(s) for 24 h. After the treatment, the cells were washed with HBSS and then incubated for 30 min to 1 h with a 1 μM solution of calcein AM in HBSS. Following incubation, cell viability was measured fluorometrically using a Molecular Devices SpectraMax M2e reader (San Jose, CA, USA), with excitation at 494 nm and emission at 517 nm. The percentages of viable cells were calculated relative to the control group.

The lactate dehydrogenase (LDH) release assay was performed using the Cytotoxicity Detection Kit to measure cell death, as described previously [15,16,35]. This assay evaluated LDH levels in culture supernatants, indicating changes in cell permeability. Cells were seeded and treated according to the cell viability protocol. After the treatment, 100 μL of supernatant was transferred to a 96-well plate and mixed with 100 μL of assay reagent. This mixture was incubated in the dark for 30 min, and then the absorbance was measured at 492 and 600 nm using a Molecular Devices SpectraMax M2e reader (San Jose, CA, USA). DMSO-treated cells released LDH spontaneously, whereas Triton X-100 induced maximal LDH release. The percentage of LDH release was calculated using the following formula: % of LDH release = (experimental value − spontaneous release)/(maximum release − spontaneous release) × 100.

4.3. Lipid Peroxidation Measurement

We conducted an intracellular lipid peroxidation assay using the fluorescent probe C11-BODIPY 581/591, as previously described [15,30]. This probe displays an increase in green fluorescence and a decrease in red fluorescence in response to lipid peroxidation. In this assay, cells were seeded as in the cell viability experiment and treated simultaneously with the tested compounds and a ferroptosis inducer in medium containing 2.5 μM C11-BODIPY 581/591. After washing the cells with PBS, the cells were supplemented with additional PBS, and fluorescence was measured using a Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA). Red fluorescence was recorded at 565/600 nm, while green fluorescence was recorded at 477/525 nm. The level of lipid peroxidation was quantified as the ratio of green to red fluorescence.

We conducted a cell-free iron + AA-Induced C11-BODIPY oxidation assay to evaluate the potential RTA activity of Necrox-5. Considering the limitations of common assays for the identification of antioxidant activity, such as the ABTS assay, which do not use lipids, we recently developed this new assay to address this issue [31,35], following previously described protocols [30,37]. The basic principle of this assay is that iron reacts with AA to produce lipid-derived ROS that oxidize C11-BODIPY. To conduct this assay, we first prepared solutions of the tested compound at various concentrations, C11-BODIPY581/591 (2 μM), and iron (20 μM) + AA (100 μM), all dissolved in PBS. Next, 50 μL of the C11-BODIPY solution, 50 μL of the Iron + AA solution, and 50 μL of the tested compound (Necrox-5) at the desired concentration were mixed in a 96-well plate. The mixture was then incubated for 30 min at 37 °C, protected from light. Following incubation, fluorescence was measured at 477 nm (excitation) and 525 nm (emission) using a Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA). DMSO was utilized as a vehicle control because the tested drug and other chemicals were dissolved in it. The inhibition of C11-BODIPY oxidation by the tested compound was assessed using the following formula: Iron + AA-induced C11-BODIPY oxidation (%) = [(Control value − Sample value)/Control value] × 100.

4.4. ATP-Glo™ Bioluminometric Cell Viability Assay

Intracellular levels of ATP (the primary energy source for cellular metabolic functions) were measured by an ATP-Glo™ Bioluminometric Cell Viability Assay [16]. This kit contains firefly luciferase, which catalyzes the oxidation of D-luciferin by ATP to produce light. The ATP content of cells correlates with the amount of light generated, indicating that metabolically active cells are present. Therefore, this kit can determine viable cell numbers based on the ATP levels detected. We seeded cells at 1.5 × 10^4^ cells per well in 96-well plates and treated them with the test compounds for 24 h. The treated cells were then washed with HBSS, treated with ATP-Glo™ detection cocktail, and luminescence was measured using a Molecular Devices SpectraMax M2e Multi-Mode Microplate Reader (San Jose, CA, USA) with an emission wavelength of 560 nm. ATP levels were calculated relative to control cells and expressed as a percentage.

4.5. Glutathione (GSH) Measurement

A previously described modified mBCI method [38] was used to assess total intracellular GSH. The mBCI dye is inherently nonfluorescent until conjugated, at which point it exhibits strong reactivity with several low-molecular-weight thiols, such as GSH. We seeded cells at the concentrations specified by the cell viability assay and washed them with HBSS after treatment. Next, 100 μL of the mBCI test solution was added, which consisted of IGEPAL (0.2%), glutathione S-transferase (0.5 units/mL), and mBCI (30 μM) in HBSS. The plate was then shaken and incubated for 60 min (at room temperature) before measuring GSH fluorometrically using a Molecular Devices SpectraMax M2e Multi-Mode Microplate Reader (San Jose, CA, USA). The reading was conducted with an excitation wavelength of 394 nm and an emission wavelength of 490 nm. GSH percentage was expressed relative to control cells.

4.6. Statistical Analysis

Statistical analysis was performed using Microsoft Excel 365 and GraphPad Prism 10. We used nonlinear regression with a variable-slope model to fit a logistic curve to the dose–response data, thereby determining EC50 values and 95% confidence intervals. EC50 values were reported when effectiveness exceeded 50%. A two-way ANOVA followed by Šídák’s multiple comparisons test was used to assess statistical significance between inducers and treatments.

5. Conclusions

Necrox-5 has been identified as a potent inhibitor of ferroptosis in a neuronal culture model (Figure 5). Our research also demonstrated that propargite, a PD-relevant pesticide, induces ferroptosis-like toxicity, which can be effectively blocked by Necrox-5 treatment (Figure 5). Further studies could clarify the rationale for using Necrox-5 as a protective agent against ferroptosis-related complications.