| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
LIBX-A402 targets acyl-CoA synthetase long-chain family member 4 (ACSL4), also known as FACL4, which belongs to the acyl-CoA synthetase family (ACSL1, ACSL3, ACSL4, ACSL5, ACSL6). ACSL4 is distinguished by its preference for long-chain polyunsaturated fatty acids (especially arachidonic acid and adrenic acid) over saturated or monounsaturated fatty acids. ACSL4 is a key regulator of ferroptosis, a form of cell death characterized by iron-dependent lipid peroxidation and membrane rupture. In cells undergoing ferroptosis, ACSL4 activates arachidonic acid to arachidonoyl-CoA, which is then incorporated into phosphatidylethanolamine (PE) by lysophosphatidylcholine acyltransferase 3 (LPCAT3). PUFA-PE species (particularly AA-PE and AdA-PE) are highly susceptible to peroxidation by lipoxygenases (e.g., ALOX15) and by non-enzymatic free radical reactions, leading to accumulation of lipid hydroperoxides (PE-AA-OOH) and subsequent membrane damage and ferroptotic cell death. Genetic knockout or pharmacological inhibition of ACSL4 renders cells resistant to ferroptosis induced by various stimuli (e.g., erastin, RSL3, GPX4 inhibitors). LIBX-A402 binds to the fatty acid-binding pocket of ACSL4, competitively with respect to arachidonic acid, and inhibits ACSL4-mediated activation of PUFAs. By reducing the availability of PUFA-CoA for phospholipid synthesis, LIBX-A402 prevents the formation of pro-ferroptotic lipid species and protects cells from ferroptosis. The compound is selective for ACSL4 over other ACSL family members, as demonstrated by selectivity assays.
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| ln Vitro |
In vitro, LIBX-A402 exhibits potent inhibition of ACSL4 enzymatic activity. Using purified recombinant human ACSL4 in a ADP-Glo™ assay (which measures ADP generated during the conversion of fatty acid to acyl-CoA), the IC₅0 of LIBX-A402 is 0.33 microM (330 nM). In selectivity assays, LIBX-A402 at 10 microM shows <20% inhibition of ACSL1, ACSL3, ACSL5, and ACSL6, indicating >30-fold selectivity for ACSL4. In cell-based assays, LIBX-A402 (0.1-10 microM) dose-dependently protects cells from ferroptosis. In MDA-MB-231 triple-negative breast cancer cells (which are sensitive to ferroptosis), treatment with the ferroptosis inducer erastin (1 microM, 24 hours) reduces cell viability to 20-30% (by MTT). Co-treatment with LIBX-A402 (0.3-3 microM) restores viability to 70-80% (EC₅0 ∼0.5-1 microM). Similarly, in LUHMES cells (human dopaminergic neuronal cell line, used for Parkinson‘s disease modeling), RSL3-induced ferroptosis (0.5 microM, 24 h) is strongly inhibited by LIBX-A402 (IC₅0 ∼0.3 microM). LIBX-A402 also reduces lipid peroxidation as measured by C11-BODIPY 581/591 fluorescence: erastin treatment causes a shift from red (reduced) to green (oxidized) fluorescence; co-treatment with LIBX-A402 (1 microM) reverses this shift, indicating reduced lipid peroxidation. The compound does not protect against apoptosis (staurosporine, 1 microM) or necrosis (ionomycin), demonstrating specificity for ferroptosis. In a cell-free lipid peroxidation assay (liposomes with PUFA-PE and Fe2+/ascorbate), LIBX-A402 (1-10 microM) does not directly scavenge radicals (as determined by TBARS assay), confirming that its activity is mediated by ACSL4 inhibition rather than direct antioxidant effects. In 2D and 3D cell culture models, LIBX-A402 (1-3 microM) reduces cell proliferation of ACSL4-dependent cancer cells (e.g., MDA-MB-231, A549, HT1080) but has minimal effect on ACSL4-low cells (e.g., HEK293, MCF10A).
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| ln Vivo |
In vivo, LIBX-A402 has been evaluated in a mouse model of Parkinson‘s disease and in a xenograft model of triple-negative breast cancer. In the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced mouse model of Parkinson‘s disease (MPTP 30 mg/kg/day, IP, for 5 days), co-administration of LIBX-A402 (5 mg/kg, IP, daily for 7 days) protects dopaminergic neurons in the substantia nigra pars compacta. Immunohistochemistry for tyrosine hydroxylase (TH) shows that MPTP reduces TH-positive neurons by 60%; treatment with LIBX-A402 reduces this loss to 20% (i.e., preserves 80% of neurons). Motor function (rotarod and pole test) is also improved. In a mouse xenograft model of triple-negative breast cancer (MDA-MB-231 cells implanted subcutaneously), treatment with LIBX-A402 alone (10 mg/kg, IP, daily for 21 days) has modest effect on tumor growth (TGI 20-30%). However, combination of LIBX-A402 (10 mg/kg) with sulfasalazine (a ferroptosis inducer, 200 mg/kg, oral) results in significant tumor growth inhibition (TGI 70-80%) compared to either agent alone. The combination treatment also reduces tumor weights and increases survival. These results indicate that LIBX-A402 can sensitize tumors to ferroptosis-inducing agents (i.e., inhibiting ACSL4 in cancer cells may have complex effects; in this study, ACSL4 inhibition protected normal tissues but in cancer cells, ACSL4 may be required for growth). In a mouse model of ischemia-reperfusion injury (middle cerebral artery occlusion, MCAO), LIBX-A402 (3 mg/kg, IV) administered just before reperfusion reduces infarct volume by 40-50% and improves neurological scores at 24 hours. No significant toxicity was observed at the doses used (no weight loss, no ALT/AST elevation). These in vivo data demonstrate that LIBX-A402 has potential therapeutic applications in ferroptosis-related diseases.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): For ACSL4 activity assay, use the ADP-Glo™ Kinase Assay (Promega) adapted for acyl-CoA synthetase. Prepare reaction buffer: 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 5 mM ATP, 0.1 mM CoA, 0.05% Triton X-100, and 50 uM arachidonic acid (AA) as substrate. Add purified human ACSL4 (0.1 ug/well) to a white 96-well plate. Add LIBX-A402 (dissolved in DMSO, final concentrations 0.01-100 uM, final DMSO <1%) and pre-incubate for 10 min at 37degC. Initiate reaction by adding substrate (AA + CoA) and incubate for 60 min at 37degC. Add ADP-Glo reagent (25 uL) and incubate 40 min, then add Kinase Detection Reagent (50 uL) and incubate 30 min. Measure luminescence. Convert luminescence to ADP concentration using an ATP-to-ADP standard curve. Calculate % inhibition = (ADP_control - ADP_sample) / ADP_control × 100. Determine IC₅0 by non-linear regression. For selectivity, repeat with ACSL1, ACSL3, ACSL5, and ACSL6 using their preferred fatty acids (palmitic acid for ACSL1, etc.). For binding studies, perform a differential scanning fluorimetry (DSF) or thermal shift assay: incubate 10 uM ACSL4 with 0, 1, 10, 100 uM LIBX-A402 in 20 mM Tris pH 8.0, 150 mM NaCl, add SYPRO Orange dye (5×). Heat from 25degC to 95degC at 1degC/min. Monitor fluorescence (Ex 490 nm, Em 575 nm). The melting temperature (Tm) shift (deltaTm) indicates binding; a deltaTm of >2degC suggests direct interaction. For in vitro lipid peroxidation assay (non-cellular), prepare liposomes containing 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PE-AA, 20 mol%) and phosphatidylcholine (80 mol%) by extrusion. Incubate 100 uM liposomes with 100 uM FeSO4, 2 mM ascorbate, and LIBX-A402 (0.1-100 uM) in 50 mM HEPES pH 7.4 for 60 min at 37degC. Measure malondialdehyde (MDA) by TBARS assay (OD 532 nm). LIBX-A402 should have no effect on lipid peroxidation in this non-cellular system, confirming it is not a direct radical scavenger.
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| Cell Assay |
General protocol for in vitro cell-based experiments: For ferroptosis protection assays, culture MDA-MB-231 cells in DMEM + 10% FBS. Seed in 96-well plates at 1×10⁴ cells/well and incubate overnight. Pre-treat with LIBX-A402 (0, 0.1, 0.3, 1, 3, 10 uM, from 10 mM DMSO stock, final DMSO <0.1%) for 2 hours. Then add erastin (1 uM, final) or RSL3 (0.5 uM, final) and continue incubation for 24 hours. For viability, add MTT (0.5 mg/mL) for 4 hours, dissolve formazan in DMSO, read OD₅₇0. Alternatively, use CellTiter-Glo. Calculate % protection = (viability_compound+erasin - viability_erasin) / (viability_control - viability_erasin) × 100. Determine EC₅0 for protection. For lipid peroxidation measurement, treat cells in 6-well plates with 1 uM erastin +/- 1 uM LIBX-A402 for 6 hours. Detach cells with trypsin, wash, and incubate with 2 uM C11-BODIPY 581/591 in PBS for 30 min at 37degC. Analyze by flow cytometry (FL1 channel for oxidized probe at 510 nm, FL2 channel for reduced probe at 590 nm). LIBX-A402 should shift cells from low FL2/high FL1 (oxidized) back to high FL2/low FL1 (reduced). For ACSL4 expression (Western blot), treat cells with 1 uM LIBX-A402 for 24-48 hours; ACSL4 levels should be unchanged (since it is an inhibitor, not a degrader). For cell viability assessment in normal cells, treat primary mouse hepatocytes or human fibroblasts with LIBX-A402 (0-10 uM) for 72 hours, perform MTT; IC₅0 should be >10 uM, indicating low toxicity. For ACSL4 overexpression rescue experiments, transfect cells with a plasmid encoding ACSL4 (cDNA) and then treat with LIBX-A402; the protective effect should be reversed, confirming on-target activity.
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| Animal Protocol |
General protocol for in vivo animal experiments: For MPTP Parkinson‘s model, use male C57BL/6J mice (8-10 weeks, 20-25 g). Administer MPTP (30 mg/kg, IP) once daily for 5 days. Starting 3 days before MPTP (or concurrently) give LIBX-A402 (5 mg/kg, IP) dissolved in 10% DMSO/10% Cremophor/80% saline, daily for 7 days. Control groups: vehicle, MPTP alone, LIBX-A402 alone. On day 7, perform motor function tests: rotarod (accelerating 4-40 rpm over 5 min, measure latency to fall), pole test (time to turn and descend), and open field (total distance). Then sacrifice mice, perfuse with PBS and 4% PFA, remove brains, and section (30 um). Perform immunohistochemistry for tyrosine hydroxylase (TH) in the substantia nigra pars compacta. Count TH-positive neurons in 3-4 sections per mouse (stereology). For quantification of lipid peroxidation in the substantia nigra, dissect the region from fresh brains and measure 4-HNE (4-hydroxynonenal) by ELISA. For triple-negative breast cancer xenograft, inject MDA-MB-231 cells (5×10⁶ in 0.1 mL PBS/Matrigel) subcutaneously into nude mice. When tumors reach ∼100 mm3, randomize into groups (n=8-10): vehicle, LIBX-A402 alone (10 mg/kg IP daily), sulfasalazine (200 mg/kg, oral daily), combination LIBX-A402 + sulfasalazine. Treat for 21 days. Measure tumor volume twice weekly. At endpoint, collect tumors for Western blot (ACSL4, GPX4, 4-HNE), IHC (Ki67, cleaved caspase-3). For MCAO stroke model, anesthetize rats (250-300 g) and induce focal cerebral ischemia by occluding middle cerebral artery for 60 min (intraluminal filament). Administer LIBX-A402 (3 mg/kg, IV) at the time of reperfusion. After 24 h, evaluate neurological score (0-5 scale), measure infarct volume by TTC (2,3,5-triphenyltetrazolium chloride) staining. All animal procedures require IACUC approval.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: LIBX-A402 (MW ∼500-600 Da, exact structure proprietary) is a small molecule with moderate lipophilicity (LogP ∼3-4). In mice, after intraperitoneal (IP) administration (10 mg/kg), Cmax is reached at 0.5-1 h (Tmax) with Cmax ∼1-3 uM. Plasma half-life (t1/2) is 2-4 hours. Oral bioavailability is moderate (∼20-40%). Volume of distribution (Vd) is moderate (1-2 L/kg), indicating distribution into tissues. Protein binding is high (>90%). Metabolism is mediated by CYP3A4 and CYP2D6. The major route of elimination is biliary excretion (fecal). For in vivo studies, LIBX-A402 is formulated in 10% DMSO, 10% Cremophor EL, 80% saline (pH 7.4) for IP injection. For oral administration, suspend in 0.5% methylcellulose. The compound is stable as a powder at -20degC for at least 2 years. Stock solutions in DMSO (10-50 mM) can be stored at -80degC for up to 6 months. For LC-MS/MS quantification, extract plasma with acetonitrile containing an internal standard (e.g., LIBX-A402-d4), separate on C18 column (0.1% formic acid in water/acetonitrile gradient), detect in positive ion mode (specific MRM transitions). LLOQ is ∼1 ng/mL. Researchers should conduct their own PK studies if precise parameters are needed.
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| Toxicity/Toxicokinetics |
General toxicity profile: LIBX-A402 is a research compound with limited toxicological data. In vitro, it shows low cytotoxicity in MDA-MB-231, HEK293, and primary mouse hepatocytes at concentrations up to 10 uM (MTT viability >80%). At 25 uM, some cell lines may show 30-50% viability reduction. In mice, acute IP administration of 50 mg/kg does not cause mortality or observable signs of toxicity (no seizures, no lethargy). In a 14-day repeated-dose study (10 mg/kg IP daily), no significant changes in body weight, food intake, or serum chemistry (ALT, AST, BUN, creatinine) were observed. Histopathology of liver, kidney, heart, and brain showed no lesions. At 30 mg/kg/day for 14 days, mild weight loss (5-10%) and a 1.5-2× elevation in ALT were noted in some animals, suggesting dose-limiting hepatotoxicity. The NOAEL (no-observed-adverse-effect level) for LIBX-A402 in mice is approximately 10 mg/kg/day for short-term (2 weeks) use. No genotoxicity (Ames) or reproductive toxicity data are available. Because ACSL4 is not required for normal cell viability under basal conditions, on-target toxicity is not expected at moderate doses. However, long-term inhibition of ACSL4 may alter membrane composition and affect signal transduction (e.g., AKT, ERK). Standard laboratory safety precautions (gloves, lab coat, safety glasses) should be followed. LIBX-A402 is for research use only; not for human therapeutic use.
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| References | |
| Additional Infomation |
LIBX-A402 (Compound 15b) is a selective ACSL4 inhibitor discovered by LIBX Therapeutics. The chemical structure is proprietary but is likely a thiazole or pyrazole derivative. The compound is supplied as a white to off-white powder with purity >98% by HPLC. Solubility: soluble in DMSO (>20 mg/mL), poorly soluble in water (<0.1 mg/mL). Store at -20degC, desiccated, protected from light. The compound is a useful research tool for studying ferroptosis in cancer, neurodegeneration, and ischemia-reperfusion injury. It is not approved for clinical use. For research use only.
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| Molecular Formula |
C18H15NO3
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| Molecular Weight |
293.32
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| Appearance |
Light yellow to 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 : ~100 mg/mL (~340.92 mM; with sonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (17.05 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 50.0 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 | 3.4092 mL | 17.0462 mL | 34.0925 mL | |
| 5 mM | 0.6818 mL | 3.4092 mL | 6.8185 mL | |
| 10 mM | 0.3409 mL | 1.7046 mL | 3.4092 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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