RSL3 GPX4 Inhibitor: Properties, Uses & FAQs

RSL3 is a potent, covalent small-molecule inhibitor of glutathione peroxidase 4 (GPX4) widely used in research to induce ferroptotic cell death and study oxidative lipid-damage pathways.

Target

Glutathione peroxidase 4 (GPX4)

Mechanism

Covalent binding to the selenocysteine residue in the active site of GPX4, irreversibly inhibiting its lipid hydroperoxide-reducing activity

Cell Death Type Induced

Ferroptosis (iron-dependent, non-apoptotic regulated cell death driven by lipid peroxidation)

Solubility

Soluble in DMSO; prepare stock solutions in DMSO and dilute into aqueous media immediately before use

Storage

Store at -20 °C, protected from light and moisture; avoid repeated freeze-thaw cycles

Research Applications

Ferroptosis induction, cancer vulnerability studies, GPX4 pathway dissection, oxidative stress modelling

What Is RSL3 and How Does It Work?

RSL3 (1S,3R-RSL3) is a small-molecule compound originally identified in a phenotypic screen for molecules that selectively kill RAS-mutant cancer cells. It acts as a direct, covalent inhibitor of glutathione peroxidase 4 (GPX4), the only mammalian enzyme capable of reducing phospholipid hydroperoxides within membranes.

GPX4 uses glutathione (GSH) as a cofactor and relies on a catalytic selenocysteine residue. RSL3 alkylates this selenocysteine, permanently blocking enzymatic activity. Without functional GPX4, toxic lipid peroxides accumulate in cellular membranes, ultimately triggering ferroptosis—a regulated, iron-dependent form of cell death distinct from apoptosis, necrosis, and autophagy.

Because RSL3 acts downstream of GSH synthesis (unlike erastin, which depletes GSH by inhibiting system Xc⁻), it can induce ferroptosis even in cells with elevated GSH levels, making it a uniquely valuable tool for mechanistic studies.

Key Properties and Handling of RSL3

Chemical identity: RSL3 is a chloroacetamide-containing compound. The active stereoisomer is (1S,3R)-RSL3; the inactive enantiomer (1R,3S)-RSL3 is commonly used as a negative control in ferroptosis experiments. Researchers should confirm which stereoisomer is supplied before designing controls.

Solubility and stock preparation: RSL3 is poorly soluble in water but dissolves readily in DMSO. Typical stock concentrations are 10–50 mM in DMSO. Working concentrations in cell culture media are in the nanomolar-to-low-micromolar range (commonly 0.1–10 µM depending on cell line sensitivity), with final DMSO concentrations kept ≤0.1% v/v to avoid solvent cytotoxicity.

Stability: RSL3 should be stored at −20 °C in a desiccated, light-protected environment. Aliquot stock solutions to minimise freeze-thaw cycles. Once diluted in aqueous media, use promptly as stability decreases.

Safety: RSL3 is a research reagent and should be handled with standard laboratory precautions—gloves, eye protection, and a well-ventilated area. Consult the Safety Data Sheet (SDS) supplied with the product for full hazard information.

RSL3 in Ferroptosis Research: Applications and Experimental Considerations

Inducing ferroptosis: RSL3 is among the most widely used tools to induce ferroptosis experimentally. It is used to validate ferroptosis in a given cell context, assess the ferroptotic vulnerability of cancer cell lines, and investigate rescue pathways (e.g., supplementation with ferrostatin-1, liproxstatin-1, or vitamin E analogues).

Cancer research: GPX4 inhibition by RSL3 is particularly lethal to cancer cells that are mesenchymal, therapy-resistant, or have undergone epithelial-to-mesenchymal transition (EMT), because such cells often exhibit high lipid peroxidation vulnerability. RSL3 is therefore used to probe ferroptosis as a potential anti-cancer strategy.

Important controls: Always include (1R,3S)-RSL3 (inactive enantiomer) as a negative control; include ferrostatin-1 or liproxstatin-1 as ferroptosis rescue agents to confirm on-target cell death mechanism. Iron chelation with deferoxamine (DFO) can further confirm iron dependence.

Readouts: Cell viability assays (e.g., CellTiter-Glo), lipid peroxidation probes (C11-BODIPY, DPPP), malondialdehyde (MDA) assays, and GPX4 activity assays are standard downstream readouts used alongside RSL3 treatment.

Distinguishing RSL3 from Other Ferroptosis Inducers

Multiple classes of ferroptosis inducers exist, and understanding how RSL3 differs from others is critical for experimental design:

• RSL3 vs. Erastin: Erastin inhibits system Xc⁻ (cystine/glutamate antiporter), depleting intracellular cysteine and thereby reducing GSH levels. RSL3 bypasses this and directly inhibits GPX4 regardless of GSH status. RSL3 is therefore effective in contexts where erastin may not be.
• RSL3 vs. FIN56: FIN56 promotes GPX4 protein degradation via a mechanism distinct from direct active-site inhibition. RSL3 covalently blocks GPX4 enzymatic activity without necessarily depleting the protein.
• RSL3 vs. FINO2: FINO2 induces ferroptosis partly through iron oxidation and indirect GPX4 inactivation, whereas RSL3 acts directly and covalently on GPX4.

Researchers often combine or compare these inducers to dissect pathway dependencies or to identify biomarkers of ferroptosis sensitivity in different cell models.

Ordering, Specifications and Quality Assurance

Aladdin Scientific product: RSL3 is available from Aladdin Scientific under SKU R302648. Please refer to the product page for current specifications including purity (typically ≥98% by HPLC), molecular weight, CAS number, and available pack sizes.

Purity and analytical data: Each lot is accompanied by a Certificate of Analysis (CoA) documenting purity, identity confirmation (e.g., NMR, HPLC, MS), and physical appearance. Customers can request the CoA for any specific lot through Aladdin Scientific customer service.

Regulatory note: RSL3 is supplied for research use only and is not intended for diagnostic, therapeutic, or veterinary use.

For volume pricing, custom pack sizes, or bulk orders, please contact Aladdin Scientific sales or submit an inquiry through the website.

Frequently asked questions

What is RSL3 used for in research?

RSL3 is primarily used as a tool compound to induce ferroptosis by covalently inhibiting GPX4. It is applied in cancer biology to study ferroptotic vulnerabilities, in oxidative stress research to model lipid peroxidation-driven cell death, and in drug discovery efforts aimed at exploiting ferroptosis for therapeutic benefit.

How does RSL3 differ from erastin as a ferroptosis inducer?

Erastin induces ferroptosis indirectly by inhibiting the system Xc⁻ transporter, thereby depleting intracellular cysteine and reducing GSH levels. RSL3 acts downstream by directly and covalently inhibiting GPX4, bypassing GSH status entirely. This means RSL3 can induce ferroptosis in cells with high GSH levels that may be resistant to erastin.

What negative control should I use in RSL3 experiments?

The standard negative control for RSL3 experiments is the inactive enantiomer (1R,3S)-RSL3, which lacks GPX4-inhibitory activity but is otherwise structurally identical. This controls for non-specific effects of the chemical scaffold. Ferroptosis rescue agents such as ferrostatin-1 or liproxstatin-1 should also be included to confirm that observed cell death is ferroptosis-specific.

At what concentration should RSL3 be used in cell culture experiments?

Working concentrations of RSL3 typically range from 0.1 µM to 10 µM, but the optimal concentration varies significantly between cell lines due to differences in GPX4 expression and lipid peroxidation susceptibility. We recommend performing a dose-response experiment for each new cell model. Final DMSO concentrations should be kept at or below 0.1% v/v to avoid solvent-related cytotoxicity.

How should RSL3 stock solutions be prepared and stored?

Dissolve RSL3 in DMSO to prepare a concentrated stock solution (typically 10–50 mM). Aliquot into single-use volumes to avoid repeated freeze-thaw cycles, and store at −20 °C in a sealed, desiccated container protected from light. Once thawed, keep on ice and use promptly. Aqueous dilutions should be prepared fresh immediately before each experiment.

Can RSL3 be used in vivo?

RSL3 has been used in some in vivo studies to investigate ferroptosis in animal models, but its pharmacokinetic properties—including metabolic stability, bioavailability, and tissue distribution—require careful evaluation for each application. In vivo use may require formulation optimization. Researchers should consult the relevant primary literature and conduct appropriate pilot studies before proceeding with in vivo experiments.

References

1. Yang WS, Stockwell BR. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem Biol. 2008.
2. Yang WS et al. Regulation of ferroptotic cancer cell death by GPX4. Cell. 2014.
3. Dixon SJ et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012.
4. Stockwell BR et al. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell. 2017.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.