Ferroptosis (IC50 = 22 nM)

In mouse embryonic fibroblasts, lipostatin-1 has anti-ferroptosis action with an IC50 of about 38 nM[2].Fer-1 and Lip-1 Are Inherently Good, but Not Great, Radical-Trapping Antioxidants; Fer-1 and Lip-1 Are Excellent Radical-Trapping Antioxidants in Phospholipid Bilayers; Fer-1 and Lip-1 Are Poor Inhibitors of 15-LOX-1 at Best, As Is α-TOH. [2]
Supporting the involvement of ferroptosis, treatment with Liproxstatin-1 was able to protect HRPTEpiCs from RSL3-induced cell death (Fig. 7a). Similar findings were obtained in the immortalized human renal proximal tubule epithelial cell line, HK-2 (Supplementary Fig. 7b). Next, we knocked down Gpx4 in HK-2 cells using a pool of siRNAs, revealing a small yet significant decrease in cell viability sensitive to αToc treatment (Supplementary Fig. 7c). Inducing cell death through Gpx4 knockdown, however, turned out to be challenging for the high expression levels of Gpx4 in kidney tubular epithelial cells (Supplementary Fig. 7d). Nonetheless, the Gpx4 knockdown rendered cells more sensitive to ferroptosis-inducing agents (Supplementary Fig. 7e), indicating a Gpx4-regulated ferroptotic machinery in human proximal tubular epithelial cells. Moreover, RSL3-induced BODIPY 581/591 C11 oxidation could be blocked by Liproxstatin-1 (Fig. 7b), demonstrating that Liproxstatin-1 prevents ferroptotic cell death also in humans.[1]

In human cells, Gpx4/kidney, and ischemia/reperfusion-induced tissue injury models, liprostatin-1 (10 mg/kg, i.p.) reduces ferroptosis [1].

---

Next, researchers assessed the in vivo potential of liprostatin-1 to prevent the consequences of inducible Gpx4 disruption in animals. On TAM treatment of CreERT2;Gpx4fl/fl mice, mice were injected daily with liprostatin-1 intraperitoneally (i.p.) until the mice showed signs of acute renal failure (ARF), at which point they were euthanized (Fig. 7c). Notably, Liproxstatin-1 remarkably extended survival compared with the vehicle-treated group. TUNEL staining at day 9 after TAM treatment showed a strongly reduced number of TUNEL+ cells in Liproxstatin-1 compared with the vehicle-treated group (Fig. 7d), suggesting that Liproxstatin-1 delays ferroptosis in tubular cells. The discrepancy between death of mice due to ARF in Fig. 1c and vehicle-treated animals in Fig. 7c is explained by the mode of TAM administration, feeding versus i.p. injection. As an independent proof-of-concept, we analysed the in vivo efficacy of Liproxstatin-1 in a bona fide model of hepatic ischaemia/reperfusion injury, providing evidence that liprostatin-1 mitigated tissue injury in ischaemia/reperfusion-induced liver injury (Fig. 7e). Hence, these data implicate ferroptosis as a contributor in ischaemia/reperfusion-induced tissue injury and hold great promise for the development of therapeutics to treat related pathologies.[1]

Inhibited Autoxidation of Styrene[2]
These experiments were carried out in a manner similar to that described in our previous work.36 In brief, styrene was washed thrice with 1 M aqueous NaOH, dried over MgSO4, filtered, distilled under vacuum, and purified by percolating through silica, then basic alumina. To a cuvette of 1.25 mL of styrene was added 1.18 mL of chlorobenzene, and the solution equilibrated for 5 min at 37 °C. The cuvette was blanked, 12.5 μL of 2 mM PBD-BODIPY in 1,2,4-trichlorobenzene was added followed by 50 μL of 0.3 M AIBN in chlorobenzene, and the solution was thoroughly mixed. After 20 min, an aliquot of liprostatin-1, Fer-1, C15-THN, PMHC, or α-TOH stock solution (1 mM) in chlorobenzene was added and the loss of absorbance at 591 nm followed. The inhibition rate constant (kinh) and stoichiometry (n) were determined for each experiment according to Figure 1B (see the Supporting Information for complete details). Autoxidations were carried out with three technical replicates at each concentration, and kinetics are reported as the mean ± standard deviation.

---

Inhibited Autoxidation of PC Liposomes[2]
To a cuvette of 2.34 mL of 10 mM PBS at pH 7.4 were added liposomes (125 μL of 20 mM stock in PBS at pH 7.4),32 and the solution was equilibrated for 5 min at 37 °C. The cuvette was blanked, 10 μL of 2 mM STY-BODIPY in DMSO was added followed by 10 μL of 0.05 M MeOAMVN in acetonitrile, and the solution was thoroughly mixed. After 5 min, an aliquot of liprostatin-1, Fer-1, C15-THN, PMHC, or α-TOH stock solution (1 mM) in DMSO was added and the loss of absorbance at 565 nm followed. The inhibition rate constant (kinh) and stoichiometry (n) were determined for each experiment according to Figure 3B (see the Supporting (see the Supporting Information for complete details). Autoxidations were carried out with three technical replicates at each concentration, and kinetics are reported as the mean ± standard deviation. Indistinguishable results were obtained in select control experiments where the antioxidant was added prior to liposome extrusion.

Phenotypic screening for ferroptosis inhibitors[1]
In brief, compound seeding onto 96-well plates (1,000 cells per well) was carried out simultaneously with 1 μM TAM administration (leading to Gpx4 inactivation) obviating multiple medium changes, followed by incubation for 72 h. Cell viability was assessed subsequently using the live/dead assay dye AquaBluer. In the primary screening round, all compounds were tested at a single concentration of 10 μM and positive hits were selected from wells with >80% cell viability. To confirm primary hits, compounds were re-screened in the same assay and dose-dependent survival as well as toxicity curves were obtained using concentrations of 0–100 μM. IC50 and TC50 values were calculated using the GraphPad Prism software. Validated hits were then evaluated based on efficacy, selectivity for ferroptosis, therapeutic range and physicochemical properties. In addition, an in silico ADME-Tox screening was implemented to exclude compounds with potential in vivo side effects. To further validate liprostatin-1, SAR studies were performed using commercially available derivatives.

Animals included in the treatment study of inducible Gpx4−/− mice were equally distributed between sex and weight, with typically 8–10 weeks of age. The average weight within the groups was between 22 and 24 g. Groups were formed to have comparable numbers of females/males of the same age. Animal weight was arranged to have a similar distribution between females and males. For the pharmacological inhibitor experiments, CreERT2;Gpx4fl/fl mice were injected on day 1 and 3 with 0.5 mg TAM dissolved in Miglyol. On day 4, compound treatment was started (liprostatin-1: 10 mg kg−1) along with vehicle control (1% dimethylsulphoxide (DMSO) in PBS). Liproxstatin-1 and vehicle control were administered once daily by i.p. injection. Survival analysis was performed using the GraphPad Prism software and statistical analysis was done according to the log-rank (Mantel–Cox) test. The compounds, vehicle and liprostatin-1, were both odourless and colourless ensuring no detectable bias. Injections and daily animal assessment were performed in a blinded fashion. When animals showed terminal signs, they were euthanized. No statistical method was used to predetermine sample size for the treatment of the Gpx4−/− mice. Mice were kept under standard conditions with food and water ad libitum (ssniff). All experiments were performed in compliance with the German Animal Welfare Law and have been approved by the institutional committee on animal experimentation and the government of Upper Bavaria.[1]

---

Dissolved in % DMSO in PBS; 10 mg/kg; i.p. injection

GreERT2; Gpx4fI/fI mice

[1]. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat Cell Biol. 2014 Dec;16(12):1180-91.

[2]. On the Mechanism of Cytoprotection by Ferrostatin-1 and Liproxstatin-1 and the Role of Lipid Peroxidation in Ferroptotic Cell Death. ACS Cent Sci. 2017 Mar 22;3(3):232-243.

Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4(-/-) mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4(-/-) mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.[1]

---

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.[2]

C19H22CL2N4

377.310781955719

376.122

2250025-95-5

Liproxstatin-1;950455-15-9;Liproxstatin-1-13C6;Liproxstatin-1-15N

136590563

Typically exists as solid at room temperature

4

3

2

25

460

0

HEHOHTKMIOBTKC-UHFFFAOYSA-N

InChI=1S/C19H21ClN4.ClH/c20-15-5-3-4-14(12-15)13-22-18-19(8-10-21-11-9-19)24-17-7-2-1-6-16(17)23-18;/h1-7,12,21,24H,8-11,13H2,(H,22,23);1H

N-[(3-chlorophenyl)methyl]spiro[1,4-dihydroquinoxaline-3,4'-piperidine]-2-imine;hydrochloride

Liproxstatin-1 (hydrochloride); 2250025-95-5; Liproxstatin-1 hydrochloride; HY-12726A; AKOS034834095; CS-0120787; Liproxstatin-1 HCl (950455-15-9 free base); N-(3-Chlorobenzyl)-1'H-spiro[piperidine-4,2'-quinoxalin]-3'-amine hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)