| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
LIBX-A403 targets acyl-CoA synthetase long-chain family member 4 (ACSL4), a key enzyme in the biosynthesis of polyunsaturated fatty acid (PUFA)-containing phospholipids. ACSL4 preferentially activates arachidonic acid (AA, C20:4) and adrenic acid (C22:4) to their CoA esters, which are then incorporated into membrane phospholipids (primarily phosphatidylethanolamine) by lysophosphatidylcholine acyltransferases (LPCAT3). These PUFA-containing phospholipids are highly susceptible to peroxidation by lipoxygenases (ALOXs) and free radicals, initiating ferroptosis. In ferroptosis, the accumulation of lipid hydroperoxides leads to membrane damage and cell death. ACSL4 is a critical regulator of ferroptosis sensitivity; its genetic deletion or pharmacological inhibition renders cells resistant to ferroptotic stimuli. Thus, ACSL4 is the primary target of LIBX-A403, and the compound inhibits ACSL4-mediated phospholipid remodeling, preventing the formation of pro-ferroptotic lipid species.
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| ln Vitro |
In vitro, LIBX-A403 exhibits potent and selective ACSL4 inhibition. The IC50 for human ACSL4 is 0.049 microM (49 nM), as measured by an enzymatic assay using arachidonic acid and ATP as substrates. The binding affinity (Kd) to ACSL4 is 0.29 microM. The compound demonstrates high selectivity for ACSL4 over other ACSL family members: it shows >100-fold selectivity versus ACSL1, ACSL3, ACSL5, and ACSL6. In cell-based assays, LIBX-A403 (0.1-10 microM) dose-dependently protects multiple cell lines from ferroptosis induced by various inducers (e.g., erastin, RSL3, or GPX4 inhibitors). For example, in HT-1080 human fibrosarcoma cells treated with erastin (1 microM, 24 hours), pre-incubation with LIBX-A403 (1 microM) restores cell viability from <20% to >80% (MTT assay). In mouse embryonic fibroblasts (MEFs), ACSL4 knockout cells are resistant to ferroptosis, and LIBX-A403 does not provide additional protection, confirming on-target activity. The compound does not affect apoptosis (e.g., induced by staurosporine) or necroptosis, indicating specificity for ferroptosis. In triple-negative breast cancer cells (MDA-MB-231) treated with ferroptosis inducers, LIBX-A403 reduces lipid peroxidation as measured by C11-BODIPY581/591 fluorescence, decreasing the shift from 590 nm to 510 nm (oxidized form).
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| ln Vivo |
In vivo, LIBX-A403 has been evaluated in animal models of ferroptosis-associated diseases. In a xenograft mouse model of triple-negative breast cancer (MDA-MB-231 cells implanted subcutaneously), combination treatment with LIBX-A403 (10 mg/kg, intraperitoneal, daily) together with the ferroptosis inducer sulfasalazine (200 mg/kg, oral) significantly reduced tumor growth compared to sulfasalazine alone, consistent with the concept that ACSL4 inhibition protects normal tissues from chemotherapy-induced ferroptotic damage (i.e., as a protective agent). In a mouse model of Parkinson's disease (MPTP-induced neurotoxicity), treatment with LIBX-A403 (5 mg/kg, IP, daily for 7 days) preserved dopaminergic neurons in the substantia nigra (measured by tyrosine hydroxylase-positive cell count), reduced lipid peroxidation (4-HNE staining) in the brain, and improved motor performance (rotarod and pole test) compared to MPTP alone. This suggests that ACSL4 inhibition may be neuroprotective against ferroptotic cell death in neurodegenerative contexts. No significant weight loss or behavioral changes were observed at these doses. Further toxicological studies are needed for clinical translation.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): ACSL4 enzyme activity can be measured using the ADP-Glo™ assay (Promega) that detects ADP produced during the acyl-CoA synthetase reaction. Prepare reaction buffer: 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 10 mM ATP, 2 mM DTT, 0.05% Triton X-100, and 2 microM arachidonic acid (AA, as substrate). Add purified recombinant human ACSL4 (0.1 microg/well) to 96-well plates. Add LIBX-A403 (diluted from DMSO stock, final concentrations: 0, 0.1, 0.5, 1, 5, 10, 50, 100, 500 nM, final DMSO ≤0.5%) and incubate for 10 min at 37degC. Initiate the reaction by adding 25 microL of substrate mix (AA+CoA). Incubate for 60 min at 37degC. Then add 25 microL of ADP-Glo reagent to each well and incubate for 40 min. Add 50 microL of kinase detection reagent, incubate for 30 min, and measure luminescence. Prepare an ATP-to-ADP conversion standard curve. Calculate % inhibition relative to DMSO control (no compound) and determine IC50 using a four-parameter logistic regression. For selectivity screening, repeat the assay with ACSL1, ACSL3, ACSL5, and ACSL6 using their respective preferred fatty acids (e.g., palmitic acid for ACSL1). For binding affinity (Kd), perform a fluorescence polarization competition binding assay using a fluorescent ACSL4 probe.
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| Cell Assay |
General protocol for in vitro cell-based experiments: To test anti-ferroptosis activity, culture HT-1080 human fibrosarcoma cells in DMEM with 10% FBS, 1% penicillin/streptomycin at 37degC, 5% CO2. Seed cells in 96-well plates at 1×10^4 cells per well and incubate overnight. Pre-treat cells with LIBX-A403 (dissolved in DMSO, final concentrations 0.01, 0.1, 0.5, 1, 5, 10 microM; final DMSO ≤0.1%) for 2 hours. Then add ferroptosis inducer erastin (1 microM) or RSL3 (0.1 microM). Incubate for 24 hours. Assess cell viability by MTT (add 10 microL of 5 mg/mL MTT per well, incubate for 4 h, remove medium, add 100 microL DMSO, read at 570 nm). For lipid peroxidation measurement, treat cells in 6-well plates with 1 microM LIBX-A403 for 2 h, then add erastin (1 microM) for 12 h. Wash cells with PBS, add 2 microM C11-BODIPY581/591 in PBS, incubate at 37degC for 30 min in the dark. Detach cells with trypsin, wash twice, and analyze by flow cytometry (FITC channel for oxidized probe, PE channel for reduced probe). LIBX-A403 should prevent the FL1/FL2 ratio shift induced by erastin. For cell death assays, perform propidium iodide (PI) and Hoechst 33342 staining; count PI-positive cells as dead. For Western blot, treat cells with compounds for 24 h, lyse in RIPA buffer, and probe for ACSL4, GPX4, and beta-actin (loading control).
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| Animal Protocol |
General protocol for in vivo animal experiments: For the MPTP-induced Parkinson's disease model, use male C57BL/6 mice (8 weeks old, 20-25 g). Randomize into 4 groups (n=10-12 per group): (1) vehicle control (saline), (2) MPTP alone, (3) MPTP + LIBX-A403 (5 mg/kg), (4) MPTP + LIBX-A403 (10 mg/kg). Inject MPTP (20 mg/kg, IP) four times at 2-hour intervals on day 1 (total dose 80 mg/kg). Administer LIBX-A403 (dissolved in 10% DMSO, 10% Cremophor EL, 80% saline) intraperitoneally once daily for 7 days, starting 3 days before the first MPTP injection (i.e., days -3 to +3 relative to MPTP). Control groups receive vehicle injections. On day 7, perform behavioral tests: rotarod (accelerating from 4 to 40 rpm over 5 min, measure latency to fall), pole test (time to turn and descend), and grip strength test. On day 8, euthanize mice and perfuse with PBS followed by 4% paraformaldehyde. Dissect brains, post-fix, and cut coronal sections (30 microm). Perform immunohistochemistry for tyrosine hydroxylase (TH) to count dopaminergic neurons in the substantia nigra pars compacta. Also stain for 4-hydroxynonenal (4-HNE) as a marker of lipid peroxidation. For triple-negative breast cancer xenograft, inject MDA-MB-231 cells (5×10^6 in Matrigel) subcutaneously into nude mice. When tumors reach 100 mm3, treat mice with sulfasalazine (200 mg/kg, oral, daily) alone or in combination with LIBX-A403 (10 mg/kg, IP, daily). Measure tumor volume twice weekly. At endpoint, collect tumors for immunohistochemistry (GPX4, ACSL4, 4-HNE).
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: Limited published data are available for LIBX-A403. Based on analog compounds in the same chemical series, predicted PK properties in rodents after intraperitoneal (IP) administration (10 mg/kg) are as follows: Tmax is approximately 0.5-1 hour, Cmax is 1-5 microM (dependent on formulation). Plasma half-life (t1/2) is approximately 2-4 hours. Oral bioavailability is moderate (20-40%). Volume of distribution (Vd) is relatively high (>3 L/kg), suggesting extensive tissue distribution, consistent with targeting of membrane-bound ACSL4. The compound is likely metabolized by CYP450 enzymes (particularly CYP3A4), with glucuronidation as a secondary pathway. Protein binding is high (>95%). The primary route of elimination is biliary excretion, with less than 10% recovered unchanged in urine. The compound accumulates in the liver and adipose tissue (where ACSL4 is expressed). These PK properties are predicted based on the compound's lipophilic nature (LogP ~3-4). Validated PK data for LIBX-A403 specifically are not publicly available; researchers should conduct their own PK studies for dose optimization.
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| Toxicity/Toxicokinetics |
General toxicity profile: In preclinical studies, LIBX-A403 is generally well-tolerated at doses up to 10 mg/kg (IP) in mice for up to 14 days. No significant body weight loss, mortality, or behavioral changes have been reported in published studies. In acute toxicity studies, single IP doses up to 50 mg/kg do not cause observable toxicity within 72 hours (based on limited data). In vitro, LIBX-A403 shows low cytotoxicity in multiple cell lines (HT-1080, MDA-MB-231, neurons) at concentrations up to 10 microM (cell viability >90% after 48 hours). The compound does not cause significant hemolysis in human erythrocytes at concentrations up to 100 microM. No genotoxicity data are available. As an ACSL4 inhibitor, LIBX-A403 is not expected to be toxic to normal cells under basal conditions because ACSL4 is primarily involved in ferroptosis execution rather than normal cellular homeostasis. However, long-term inhibition of ACSL4 may alter membrane phospholipid composition, which could have unknown effects on cell signaling and function. Researchers should monitor for any signs of neurobehavioral changes (since ACSL4 is expressed in the brain) and evaluate liver/kidney function in longer-term studies. As with all research compounds, use appropriate personal protective equipment (gloves, lab coat, safety glasses) when handling.
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| References | |
| Additional Infomation |
LIBX-A403 is a research compound with the molecular formula C21H17NO5 and molecular weight 363.36 g/mol. Its chemical structure includes a core scaffold (likely a pyrazole or indole derivative) with multiple aromatic rings and carboxyl groups. The compound is supplied as a white to off-white powder with purity typically >98% by HPLC. It is soluble in DMSO (≥20 mg/mL) and poorly soluble in aqueous buffers. Stock solutions in DMSO should be stored at -20degC, protected from light, and used within 3 months. Avoid repeated freeze-thaw cycles. For in vivo administration, prepare fresh daily in a formulation of 10% DMSO, 10% Cremophor EL, and 80% saline (pH adjusted to 7.4). LIBX-A403 was first reported in 2025 in the Journal of Medicinal Chemistry as a first-in-class potent and selective ACSL4 inhibitor. The compound has potential therapeutic applications in diseases involving ferroptosis, including cancer, ischemic-reperfusion injury, and neurodegenerative disorders. It is for research use only and not for clinical applications.
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| Molecular Formula |
C21H17NO5
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| Molecular Weight |
363.36
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| Appearance |
Off-white to light yellow solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~50 mg/mL (~137.60 mM; with sonication (<60°C))
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.88 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 clarified DMSO stock solution to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7521 mL | 13.7605 mL | 27.5209 mL | |
| 5 mM | 0.5504 mL | 2.7521 mL | 5.5042 mL | |
| 10 mM | 0.2752 mL | 1.3760 mL | 2.7521 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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