GPX4/glutathione peroxidase 4
RSL3 targets glutathione peroxidase 4 (GPX4), a selenoprotein that plays a critical role in cellular redox homeostasis by reducing lipid hydroperoxides to their corresponding alcohols, thereby preventing iron-dependent lipid peroxidation and ferroptosis. RSL3 binds covalently to the active site of GPX4, inhibiting its enzymatic activity and rendering cells unable to detoxify lipid peroxides. This leads to the accumulation of lipid ROS and triggers ferroptotic cell death. RSL3 also inhibits system xc- (IC50 = 100 nM), which blocks GSH synthesis, although its primary mechanism is direct GPX4 inhibition. The compound exhibits selectivity for tumor cells bearing oncogenic RAS mutations.

With an IC50 of 5.8 μM in HN3-rslR cells and 0.48 μM in HN3 cells, RSL3 (0-8 μM, 72 hours) significantly lowers the viability of HN3 cells [1]. In HN3-rslR cells, RSL3 (0-8 μM, 24 hours) inactivates Keap1, raises the expression of p62 and Nrf2, and decreases the expression of GPX4 protein [1].
RSL3 or ML-162 induced the ferroptosis of HNC cells to varying degrees.[1]
Resistance to ferroptosis was associated with p62 and Nrf2 expression in RSL3-treated HNC cells.[1]
Inhibition of Nrf2 sensitized chemoresistant HNC cells to RSL3 treatment in vitro [1].
In vitro, RSL3 demonstrates potent activity as a ferroptosis inducer across a wide range of cancer cell lines. In human colorectal cancer cell lines (HCT116, HT29, LoVo), RSL3 treatment significantly reduces cell viability in a time- and dose-dependent manner, accompanied by increased intracellular ROS and lipid peroxidation levels, and impaired GPX4 protein function. RSL3 induces ferroptotic death in head and neck cancer cells by reducing GPX4 expression. The compound is active at nanomolar concentrations, with an IC50 of 100 nM for system xc- inhibition. RSL3-induced cell death can be blocked by the ferroptosis inhibitor ferrostatin-1 or by iron chelators, confirming the iron-dependent nature of the cell death pathway.

Factor HN3R cell tumor growth can be markedly inhibited by combining trigonelline with RSL3 (100 mg/kg, intratumoral injection, twice weekly for 20 days) [1].

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In mice xenograft models, all the mice survived well during and after cell implantation and treatment with vehicle, RSL3, trigonelline, or RSL3 plus trigonelline. They were euthanized 20 days after treatment. RSL3 or trigonelline alone did not significantly inhibit in vivo tumor growth compared with the vehicle control (P > 0.1) (Fig. 6A and B). However, tumor growth was significantly suppressed by RSL3 plus trigonelline (P < 0.01). Body weight and daily food intake did not change significantly in the control or treatment groups (P < 0.05) (Fig. 6C). The levels of ferrous iron, lipid ROS, and RPA measured in the in vivo tumors were significantly higher in the RSL3 plus trigonelline combination group than the control or other treatment groups (P < 0.01). A histological examination of vital organs did not reveal any significant differences between the groups [1].
In vivo, RSL3 has demonstrated antitumor efficacy in preclinical models. Cetuximab was found to enhance RSL3-induced ferroptosis by activating p38 MAPK and inhibiting the Nrf2/HO-1 axis, which further inhibited tumor growth. The compound's ability to selectively kill RAS-mutant cancer cells makes it a valuable tool for studying ferroptosis in the context of cancer therapy. However, detailed in vivo efficacy data in animal models are limited in the available literature. RSL3 is primarily used as a research tool for in vitro studies, and its in vivo pharmacokinetic and pharmacodynamic properties require further investigation.

ROS production measurement[1]
Cellular ROS generation in the supernatant of HNC cell lysates treated for 24 h was measured by adding, for 30 min at 37 °C, either 10 µM 2′,7′-dichlorofluorescein diacetate for cytosolic ROS or 2 µM C11-BODIPY C11 for lipid peroxidation. ROS levels were analyzed using a FACSCalibur flow cytometer equipped with CellQuest Pro.

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Labile iron pool (LIP) and iron assays[1]
Each sample was seeded in plates and treated with the indicated drugs. After 4 h, the supernatant was removed and the plates were washed twice with Hank's Balanced Salt Solution (HBSS). The cells were labeled by adding 8 μg/ML Calcein AM in HBSS and were incubated on plates for 30 min at 37 °C. After 30 min, the labeling solution was removed and the cells washed twice with PBS before trypsinization with Trypsin-EDTA. They were then neutralized by adding 4% FBS in HBSS and centrifuged for 3 min at 1500 rpm. The collected cells were washed once with HBSS while vortexing and were centrifuged for 3 min at 1500 rpm. They were then resuspended in 250 μL HBSS while vortexing, and go to FACS. Labile iron levels were analyzed using a FACSCalibur flow cytometer equipped with CellQuest Pro, and ferrous iron levels in the cells or tissue extracts were measured by using an iron assay kit.

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Nrf2 transcriptional activity assay[1]
The transcriptional activity of Nrf2 was assayed using a Cignal Antioxidant Response Reporter kit, according to the manufacturer's instructions.
In vitro enzyme/receptor binding assays for RSL3 typically involve measuring its inhibitory activity against GPX4. GPX4 enzyme activity can be assessed using a cell-free system where purified GPX4 is incubated with a lipid hydroperoxide substrate (e.g., phosphatidylcholine hydroperoxide) and glutathione (GSH) in the presence of varying concentrations of RSL3. The inhibition of GPX4-mediated reduction of lipid hydroperoxides is measured, and the IC50 for GPX4 inhibition is determined from dose-response curves. Affinity purification and mass spectrometry studies have confirmed that RSL3 covalently binds to the active site of GPX4.

Cell viability assay[1]
Cell Types: HN3 cells, HN3-rslR Cell
Tested Concentrations: 0-8 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: IC50 values in HN3 and HN3-rslR cells were 0.48 µM and 5.8 µM respectively[1] .

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Western Blot Analysis [1]
Cell Types: HN3-rslR Cell
Tested Concentrations: 0-8 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibited GPX4 expression, increased p62 and Nrf2 levels, and diminished Keap1 levels.
In vitro cellular assays for RSL3 are performed using various cancer cell lines, including HT-1080 fibrosarcoma cells, colorectal cancer cells, and head and neck cancer cells. Cells are treated with varying concentrations of RSL3, and ferroptosis is assessed by measuring cell viability using MTT or resazurin reduction assays. Lipid peroxidation is measured using fluorescent probes such as C11-BODIPY581/591, and intracellular ROS levels are detected using DCFH-DA. GPX4 protein expression and activity are assessed by Western blotting. The specificity of ferroptosis is confirmed by co-treatment with ferrostatin-1 or iron chelators.

Animal/Disease Models: Tenweeks old athymic BALB/c male nude mice (nu/nu) carrying HN3R cells [1]
Doses: 100 mg/kg combined with trigonelline (50 mg/kg): intratumoral injection,
Experimental Results: Significant reduction in tumor volume in mice treated with trigonelline twice a week for 20 days.

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Tumor xenograft[1]
Ten-week-old athymic BALB/c male nude mice (nu/nu) were used and HN3R cells were injected subcutaneously into the flank of each. As soon as gross nodules from the tumor implants were detected, the mice were subjected to one of four treatments: vehicle; RSL3 (100 mg/kg intratumorally twice per week); trigonelline (50 mg/kg daily via oral administration); or RSL3 plus trigonelline. Each group included 10 mice. Tumor size and body weight were measured twice a week, and the tumor volume was calculated as (length × width2)/2. After scarification, the tumors were isolated and cellular lipid ROS and ferrous iron levels were measured.
In vivo animal experiments for RSL3 have been conducted in xenograft mouse models of cancer. Immunocompromised mice are implanted with human tumor cells (e.g., colorectal or head and neck cancer cells) and treated with RSL3 via intraperitoneal or intravenous administration. Tumor growth inhibition is monitored, and endpoints include tumor volume, tumor weight, and survival. Pharmacodynamic markers such as lipid peroxidation and GPX4 expression in tumor tissues are assessed by immunohistochemistry or biochemical assays. However, detailed protocols are not extensively documented in the available literature.

RSL3 has a molecular weight of 440.88 g/mol and a molecular formula of C23H21ClN2O5. It is soluble in DMSO at 40 mg/mL and is insoluble in water or ethanol. The compound should be stored as a powder at -20°C for up to 3 years. Detailed pharmacokinetic parameters such as half-life, Cmax, and bioavailability have not been extensively reported. As a research compound, RSL3 is primarily used in in vitro studies, and its in vivo pharmacokinetics require further investigation.

The toxicological profile of RSL3 has not been extensively characterized in publicly available literature. As a GPX4 inhibitor and ferroptosis inducer, RSL3 may have potential toxicity to normal cells, particularly those with high metabolic activity and iron content. The compound is classified as a hazardous substance and should be handled with appropriate safety precautions. No significant toxicity has been reported in the available research literature at the concentrations used for in vitro studies. Comprehensive toxicology studies would be necessary for therapeutic applications.

[1]. Nrf2 inhibition reverses resistance to GPX4 inhibitor-induced ferroptosis in head and neck cancer. Free Radic Biol Med. 2018 Dec;129:454-462.

[2]. Mitochondrial fission links ECM mechanotransduction to metabolic redox homeostasis and metastatic chemotherapy resistance. Nat Cell Biol. 2022 Feb;24(2):168-180.

[3]. CGI1746 targets σ1R to modulate ferroptosis through mitochondria-associated membranes. Nat Chem Biol. 2024 Jan 11.

[4]. Mitochondrial transplantation rescues neuronal cells from ferroptosis. Free Radic Biol Med. 2023 Nov 1;208:62-72.

[5]. Ibrutinib facilitates the sensitivity of colorectal cancer cells to ferroptosis through BTK/NRF2 pathway. Cell Death Dis. 2023 Feb 23;14(2):151.

[6]. A potent GPX4 degrader to induce ferroptosis in HT1080 cells. Eur J Med Chem. 2024 Feb 5;265:116110.

[7]. Sorafenib fails to trigger ferroptosis across a wide range of cancer cell lines. Cell Death Dis. 2021 Jul 13;12(7):698.

[8]. The ferroptosis inducing compounds RSL3 and ML162 are not direct inhibitors of GPX4 but of TXNRD1. Redox Biol. 2023 Jun;62:102703.

[9]. Inhibition of cannabinoid receptor type 1 sensitizes triple-negative breast cancer cells to ferroptosis via regulating fatty acid metabolism. Cell Death Dis. 2022 Sep 21;13(9):808.

[10]. Prominin2 Drives Ferroptosis Resistance by Stimulating Iron Export. Dev Cell. 2019 Dec 2;51(5):575-586.e4.

Glutathione peroxidase 4 (GPX4) is a regulator of ferroptosis (an iron-dependent, non-apoptotic cell death process); inhibition of GPX4 sensitizes drug-resistant cancer cells to ferroptosis. However, some cancer cells develop protective mechanisms against ferroptosis; understanding these mechanisms could help overcome chemotherapeutic resistance. This study investigated the molecular mechanisms by which GPX4 inhibition induces ferroptosis resistance in head and neck cancer (HNC) cells. We tested the effects of two GPX4 inhibitors (1S, 3R)-RSL3 and ML-162, as well as trigonelline, in HNC cell lines, including the cisplatin-resistant cell line (HN3R) and the acquired RSL3-resistant cell line (HN3-rslR). The effects of the inhibitors and trigonelline, as well as the inhibition of p62, Keap1, or Nrf2 genes, were evaluated by cell viability, cell death, lipid reactive oxygen species (ROS) production, and protein expression, and in a mouse tumor xenograft model. Treatment with RSL3 or ML-162 can induce ferroptosis in head and neck cancer (HNC) cells to varying degrees. Treatment with RSL3 or ML-162 can increase the expression of p62 and Nrf2 in chemotherapeutic resistant HN3R and HN3-rslR cells, inactivate Keap1, and increase the expression of the phosphorylated PERK-ATF4-SESN2 pathway. Transcriptional activation of Nrf2 is associated with ferroptosis resistance. Transfection with overexpression of Nrf2 by inhibiting Keap1 or Nrf2 genes can induce resistance to RSL3 in chemotherapeutic HN3 cells. However, Nrf2 inhibition or p62 silencing can sensitize HN3R cells to RSL3. In a mouse model transplanted with HN3R cells, trigonelline can sensitize chemotherapeutic head and neck cancer cells to RSL3 treatment. Therefore, activation of the Nrf2-ARE pathway leads to resistance of head and neck cancer cells to GPX4 inhibitors, while inhibition of this pathway can reverse the resistance of head and neck cancer cells to ferroptosis. [1]
RSL3 is a research compound widely used as a pharmacological tool to study ferroptosis, a form of non-apoptotic cell death characterized by iron-dependent lipid peroxidation. It is also known as (1S,3R)-RSL3 and RAS-selective lethal compound 3. RSL3 is not an approved drug and is intended for research use only. It has been instrumental in elucidating the role of GPX4 in ferroptosis and has implications in cancer research, neurodegeneration, and other diseases where oxidative stress is a key factor. RSL3 is supplied by various research chemical suppliers for biochemical and cell-based assays.

C23H21CLN2O5

440.8762

440.113

C, 62.66; H, 4.80; Cl, 8.04; N, 6.35; O, 18.14

1219810-16-8

1750826

White to off-white solid powder

1.4±0.1 g/cm3

641.3±55.0 °C at 760 mmHg

341.6±31.5 °C

0.0±1.9 mmHg at 25°C

1.636

3.07

1

5

6

31

696

2

COC(=O)[C@H]1CC2=C([C@@H](N1C(=O)CCl)C3=CC=C(C=C3)C(=O)OC)NC4=CC=CC=C24

TXJZRSRTYPUYRW-NQIIRXRSSA-N

InChI=1S/C23H21ClN2O5/c1-30-22(28)14-9-7-13(8-10-14)21-20-16(15-5-3-4-6-17(15)25-20)11-18(23(29)31-2)26(21)19(27)12-24/h3-10,18,21,25H,11-12H2,1-2H3/t18-,21+/m1/s1

methyl (1S,3R)-2-(2-chloroacetyl)-1-(4-methoxycarbonylphenyl)-1,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylate

(1S,3R)-RSL3; 1S,3R-RSL3; RSL3 (1S,3R-); CHEMBL4747331; (1S,3R)-Methyl 2-(2-chloroacetyl)-1-(4-(methoxycarbonyl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate; RSL3 1S,3R-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~100 mg/mL (~226.82 mM)

Solubility in Formulation 1: 5 mg/mL (11.34 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.67 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.72 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 0.56 mg/mL (1.27 mM) (saturation unknown) in 10% DMF 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.

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Solubility in Formulation 5: 20 mg/mL (45.36 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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