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| Targets |
AGPAT4-IN-1 targets the enzyme 1-acyl-sn-glycerol-3-phosphate acyltransferase 4 (AGPAT4), also known as lysophosphatidic acid acyltransferase delta (LPAATdelta). AGPAT4 catalyzes the conversion of LPA to PA, a key step in the synthesis of phosphatidylcholine and phosphatidylethanolamine, and a critical node in lipid metabolism and cell signaling. AGPAT4-IN-1 covalently binds to the active site cysteine residue Cys228 of AGPAT4, irreversibly inhibiting its enzymatic activity. This leads to reduced PA production, which in turn inhibits the downstream mTOR/S6K signaling pathway, a key regulator of cell growth, proliferation, and survival. The compound operates within the lipid metabolism and PI3K/AKT/mTOR pathways. It is selective for AGPAT4 over other AGPAT isoforms.
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| ln Vitro |
In vitro, AGPAT4-IN-1 inhibits AGPAT4 enzymatic activity with an IC50 of 795 nM in a cell-free assay using purified enzyme and radiolabeled LPA substrate. In HCC cell lines (e.g., HepG2, Huh7, PLC/PRF/5), treatment with AGPAT4-IN-1 at 1-10 uM for 24-72 hours reduces PA levels, decreases phosphorylation of S6K and S6 (Western blot), and inhibits cell proliferation (MTT assay) with IC50 values around 2-5 uM. The compound induces apoptosis at concentrations ≥5 uM after 48 h (Annexin V/PI staining, increased cleaved PARP). It also reduces the stem cell characteristics of HCC cells, as shown by decreased spheroid formation and reduced expression of stemness markers (CD133, EpCAM, Nanog). AGPAT4-IN-1 enhances the sensitivity of HCC cells to sorafenib, reducing the IC50 of sorafenib by 3-5 fold. No significant cytotoxicity is observed in normal hepatocytes (THLE-2) up to 10 uM.
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| ln Vivo |
In vivo, AGPAT4-IN-1 exhibits antitumor activity in a hepatocellular carcinoma (HCC) xenograft mouse model. Female BALB/c nude mice (6-8 weeks, n=8/group) are implanted subcutaneously with HepG2 or Huh7 cells (5×10⁶ in Matrigel). When tumors reach ~150 mm3, mice are treated with AGPAT4-IN-1 intraperitoneally at 10, 20, or 40 mg/kg every other day for 3-4 weeks. Formulation: 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline. The compound significantly inhibits tumor growth (TGI ~40-60%) at 20-40 mg/kg. It also reduces tumor PA levels and p-S6K expression by immunohistochemistry. In combination with sorafenib (10 mg/kg oral daily), AGPAT4-IN-1 enhances tumor growth inhibition (TGI ~70-80%) and induces tumor regression in some animals. The combination also reduces the percentage of CD133+ cancer stem cells in tumors. No significant body weight loss is observed at these doses. For research use only.
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| Enzyme Assay |
For non-cellular AGPAT4 enzyme activity assay, express and purify recombinant human AGPAT4 from insect cells. Prepare assay buffer: 50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.05% Triton X-100, 1 mM DTT. Incubate AGPAT4 (5-10 nM) with AGPAT4-IN-1 (0.1-10000 nM in DMSO, final DMSO 1%) for 15 min at 25degC. Add substrates: 25 uM LPA (1-oleoyl-lysophosphatidic acid, sonicated) and 10 uM [14C]oleoyl-CoA (55 mCi/mmol) in a final volume of 50 uL. Incubate at 37degC for 30 min. Terminate reaction by adding 200 uL of chloroform:methanol (1:1). Extract lipids by vortexing and centrifugation, collect organic phase, dry under nitrogen, and resuspend in 20 uL chloroform. Spot onto silica gel TLC plate, develop with petroleum ether:ethyl ether:formic acid (80:20:1.5), and visualize by autoradiography. Scrape the PA spot and quantify by liquid scintillation. Calculate IC50 from dose-response curve. Positive control: AGPAT4 inhibitor (e.g., compound from literature). Negative control: DMSO only.
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| Cell Assay |
For in vitro cell assays, culture HCC cells (HepG2, Huh7) in DMEM with 10% FBS, 1% P/S at 37degC, 5% CO2. For viability, seed cells in 96-well plates (5×103 cells/well), treat with AGPAT4-IN-1 (0.1-100 uM) for 72 h. Add MTT (0.5 mg/mL, 4 h), solubilize in DMSO, read OD570. For apoptosis, treat with 5 or 10 uM compound for 48 h, stain with Annexin V-FITC and PI, analyze by flow cytometry. For Western blot, treat cells with 1-10 uM compound for 24 h, lyse in RIPA buffer with protease/phosphatase inhibitors, and blot for p-S6K (Thr389), total S6K, p-S6 (Ser235/236), total S6, and beta-actin. For combination studies, treat with AGPAT4-IN-1 (2-5 uM) plus sorafenib (0.5-5 uM) for 48 h, then measure viability (MTT) and calculate combination index (CompuSyn). For cancer stem cell assays, treat with 5 uM compound for 48 h, then culture in serum-free stem cell medium in ultra-low attachment plates for 7 days; count spheroids (>50 um). DMSO vehicle control (≤0.1%). All experiments in triplicate.
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| Animal Protocol |
For in vivo xenograft study, use female BALB/c nude mice (6-8 weeks, 18-22 g, n=8-10/group). Inject HepG2 cells (5×10⁶ in 100 uL PBS + 100 uL Matrigel) subcutaneously into right flank. When tumors reach ~150 mm3 (day 10-14), randomize mice into groups: vehicle control, AGPAT4-IN-1 20 mg/kg, AGPAT4-IN-1 40 mg/kg, sorafenib 10 mg/kg, combination (AGPAT4-IN-1 20 mg/kg + sorafenib 10 mg/kg). AGPAT4-IN-1 is formulated in 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline to 2-4 mg/mL for 20-40 mg/kg (dosing volume 10 mL/kg). Administer intraperitoneally (IP) every other day for 21 days. Sorafenib is formulated in 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline or in 0.5% methylcellulose and given orally daily. Tumor volume measured twice weekly (calipers, V=length×width2/2). Body weight recorded. At endpoint (day 21), tumors are excised, weighed, and processed for immunohistochemistry (p-S6, CD133) and Western blot (p-S6K, PA levels by lipidomics). For stem cell analysis, tumor cells are dissociated and cultured in serum-free stem cell medium for spheroid formation. Statistical analysis by two-way ANOVA.
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| ADME/Pharmacokinetics |
Specific PK parameters for AGPAT4-IN-1 are not publicly available. The compound has a molecular weight of 306.77 and is formulated for intraperitoneal administration at 20-40 mg/kg every other day, suggesting that it is not orally bioavailable or that IP is the preferred route. Half-life (t½) is not reported, but the every-other-day dosing schedule suggests a sufficiently long duration of action (>24 h). Volume of distribution (Vd) is not known. Clearance (CL) is likely hepatic. Protein binding not reported. Solubility: DMSO (≥100 mg/mL). For in vivo formulation, the compound is soluble in 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline at concentrations up to 5 mg/mL. Storage: powder at -20degC for up to 3 years; in DMSO at -80degC for 1 year. For research use only.
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| Toxicity/Toxicokinetics |
No formal toxicity data are available for AGPAT4-IN-1. In xenograft studies, doses up to 40 mg/kg IP every other day for 3 weeks are well-tolerated, with no significant body weight loss or gross signs of toxicity observed. No histopathological analysis of major organs has been reported. As a covalent inhibitor of AGPAT4, potential on-target adverse effects may include disruption of lipid metabolism in normal tissues (e.g., liver, adipose). However, no such studies have been conducted. Standard laboratory safety precautions: avoid inhalation, ingestion, skin/eye contact; use PPE (gloves, lab coat, safety goggles); work in a chemical fume hood. For research use only-not for human use. Dispose of waste according to local hazardous waste regulations.
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| References | |
| Additional Infomation |
CAS: 2411250-59-2. Molecular formula: C14H11ClN2O2S, molecular weight: 306.77. Also known as Compound CL26. Purity typically >98% by HPLC. Appearance: light yellow to yellow solid powder. Solubility: DMSO (≥100 mg/mL). Storage: powder -20degC for 3 years, 4degC for 2 years; in solvent -80degC for 6 months. Target: AGPAT4 (Cys228 covalent inhibitor). IC50: 795 nM. Research areas: hepatocellular carcinoma (HCC), lipid metabolism, cancer stem cells. Pathway: Lipid metabolism, mTOR/S6K signaling. Not for human use. For research only.
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| Molecular Formula |
C14H11CLN2O2S
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| Molecular Weight |
306.77
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| CAS # |
2411250-59-2
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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 (~325.98 mM; with sonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (16.30 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 | 3.2598 mL | 16.2989 mL | 32.5977 mL | |
| 5 mM | 0.6520 mL | 3.2598 mL | 6.5195 mL | |
| 10 mM | 0.3260 mL | 1.6299 mL | 3.2598 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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