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Purity: ≥98%
AP-III-a4 (also known as ENOblock) is a novel and potent small molecule inhibitor of enolase which is isolated by small molecule screening in a cancer cell assay to detect cytotoxic agents that function in hypoxic conditions, which has previously been shown to induce drug resistance. AP-III-a4 is the first, nonsubstrate analogue that directly binds to enolase and inhibits its activity (IC50=0.576 uM); inhibit cancer cell metastasis in vivo. In HCT116 cells, AP-III-a4 induces cell death under hypoxia, and inhibits cancer cell migration and invasion by down-regulation of AKT and Bcl-xL expression. In Huh7 hepatocytes and HEK kidney cells, AP-III-a4 induces glucose uptake and inhibits phosphoenolpyruvate carboxykinase (PEPCK) expression. Thus, ENOblock is the first reported enolase inhibitor that is suitable for biological assays. This new chemical tool may also be suitable for further study as a cancer and diabetes drug candidate.
| ln Vitro |
In a dose-dependent manner, AP-III-a4 (ENOblock) (0-10 μM; 24 h) decreases the viability of HCT116 cells[1]. Enolase is immediately bound by AP-III-a4, which therefore suppresses its activity[1]. III-a4 (0-10 μM; 24 or 48 h) causes apoptosis in cancer cells and inhibits their migration and invasion[1]. In hepatocytes and kidney cells, AP-III-a4 (10 μM; 24 h) can stimulate glucose uptake and decrease the production of phosphoenolpyruvate carboxykinase (PEPCK)[1].
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| ln Vivo |
In zebrafish, AP-III-a4 (ENOblock) (10 μM; 96 h) suppresses the gluconeogenesis regulator PEPCK and prevents cancer cells from metastasizing[1].
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: HCT116 Tested Concentrations: 1.25, 2.5, 5 and 10 μM Incubation Duration: 24 h Experimental Results: Induced higher levels of HCT116 colon cancer cell death in hypoxic conditions compared to normoxia. Western Blot Analysis[1] Cell Types: HCT116 Tested Concentrations: 1.25, 2.5, 5 and 10 μM Incubation Duration: 24 h for AKT, 48 h for Bcl-Xl Experimental Results: Bound to enolase in cell lysate and bound to purified enolase. diminished the expression of AKT and Bcl-Xl, which are negative regulators of apoptosis. Cell Invasion Assay[1] Cell Types: HCT116 Tested Concentrations: 0.156, 0.312, 0.625, 1.25 and 2.5 μM Incubation Duration: 24 h Experimental Results: Dramatically inhibits cancer cell invasion at a treatment concentration of 0.625 μM. Cell Migration Assay [1] Cell Types: HCT116 Tested Concentrations: 0.625, 1.25 and 2.5 μM Incubation Duration: 24 h Experimental Results: Inhibited cell migration dose-dependently. RT-PCR[1] Cell Types: Huh7 and HEK Tested Concentrations: 10 μM Incubation Duration: 24 h Experimental Results: Induced glucose uptake and inhibited PEPCK expression. |
| Animal Protocol |
Animal/Disease Models: The zebrafish cancer cell HCT116 xenograft model[1]
Doses: 10 μM Route of Administration: 96 h Experimental Results: decreased cancer cell dissemination. Inhibited PEPCK expression and induced glucose uptake. Inhibited adipogenesis and foam cell formation. |
| References |
[1]. Da-Woon Jung, et al. A Unique Small Molecule Inhibitor of Enolase Clarifies Its Role in Fundamental Biological Processes.ACS Chem. Biol., 2013, 8 (6), pp 1271–1282
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| Molecular Formula |
C31H44CLFN8O3
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| Molecular Weight |
594.72
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| CAS # |
1177827-73-4
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| Related CAS # |
AP-III-a4 hydrochloride;2070014-95-6
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| SMILES |
O=C(NCCOCCOCCN)CC1=CC=C(NC2=NC(NCC3CCCCC3)=NC(N(C4=CC=C(F)C=C4)C)=N2)C=C1
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| Chemical Name |
N-[2-[2-(2-Aminoethoxy)ethoxy]ethyl]-2-[4-[[4-(cyclohexylmethylamino)-6-[(4-fluorophenyl)methylamino]-1,3,5-triazin-2-yl]amino]phenyl]acetamide
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| Synonyms |
AP-III-a4; ENOblock
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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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.20 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 25.0 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. Solubility in Formulation 2: 2.5 mg/mL (4.20 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 ultrasonication. 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 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.20 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6815 mL | 8.4073 mL | 16.8146 mL | |
| 5 mM | 0.3363 mL | 1.6815 mL | 3.3629 mL | |
| 10 mM | 0.1681 mL | 0.8407 mL | 1.6815 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.
 ACS Chem. Biol.,2013,8(6), pp 1271–1282 |
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