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Purity: ≥98%
KU-55933 is a potent and specific ATM (Ataxia-telangiectasia mutated) kinase inhibitor with IC50/Ki of 12.9 nM/2.2 nM in cell-free assays, and is highly selective for ATM as compared to DNA-PK, PI3K/PI4K, ATR and mTOR. As an ATM inhibitor, KU-55933 dramatically reduced the rise in phospho-Akt at Ser473 in insulin- and IGF-I-treated MDA-MB-453 and PC-3 cells after serum starvation. In MDA-MB-453 and PC-3 cells, KU-55933 treatment reduced cell proliferation in the MTT assay by roughly 50% at a concentration of 10 μM. Treatment with KU-55933 inhibited cell proliferation in a panel of cell lines with varying Akt activities, and this effect was correlated with Akt phosphorylation. This new understanding of the mechanism governing ATM regulation may be helpful in developing more accurate plans for modulating ATM activity in cancer therapy, since it is thought that ATM inhibition makes cancer cells more susceptible to genotoxic substances.
| Targets |
ATM ( IC50 = 12.9 nM ); DNA-PK ( IC50 = 2500 nM ); mTOR ( IC50 = 9300 nM ); PI3K ( IC50 = 16600 nM )
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| ln Vitro |
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| ln Vivo |
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| Enzyme Assay |
In order to obtain ATM for the in vitro assay, rabbit polyclonal antiserum raised to the COOH-terminal 400 amino acids of ATM is immunoprecipitated from HeLa nuclear extract using a method that involves buffering the mixture with 25 mM HEPES (pH 7.4), 2 mM MgCl2, 250 mM KCl, 500 μM EDTA, 100 μM Na3VO4, 10% v/v glycerol, and 0.1% v/v Igepal. After an hour of incubation with protein A-Sepharose beads and subsequent centrifugation to recover the beads, ATM-antibody complexes are separated from nuclear extract. A 96-well plate's well is used to incubate ATM-containing Sepharose beads with 1 μg of glutathione S-transferase–p53N66 (p53's NH2-terminal 66 amino acids fused to glutathione S-transferase) in the ATM assay buffer [25 mM HEPES (pH 7.4), 75 mM NaCl, 3 mM MgCl2, 2 mM MnCl2, 50 μM Na3VO4, 500 μM DTT, and 5% v/v glycerol] at 37 °C with or without an inhibitor. The reaction is continued at 37 °C for an additional hour after adding ATP to a final concentration of 50 μM after 10 minutes of gentle shaking. Glutathione S-transferase-p53N66 binding is allowed to occur by centrifuging the plate at 250 × g for 10 minutes (4 °C) in order to remove the beads containing ATM. The supernatant is then taken out and put in a white opaque 96-well plate. This incubation process takes 1.5 hours at room temperature. The PBS wash, dry blotting, and standard ELISA analysis using a phospho-serine 15 p53 antibody are the next steps for this plate. When using a secondary antibody conjugated with horseradish peroxidase from goat antimouse, the substrate for phosphorylated glutathione S-transferase-p53N66 is detected. The process of creating a signal and chemiluminescent detection involves using an enhanced chemiluminescence solution. Chemiluminescent detection is done and a signal is generated using an enhanced chemiluminescence solution.
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| Cell Assay |
The ATM response is measured by Western blot analysis of p53 serine 15 phosphorylation and stabilization of wild-type p53 in U2OS cells that have been exposed to ionizing radiation (3, 5, or 15 Gy) or UV (5 or 50 J/m2). Each time point's whole cell extracts are extracted, proteins are separated using SDS-PAGE, and a p53 phospho-serine 15 specific antibody is used to measure the ATM-specific increase in phosphorylated serine 15. When using a p53-specific antibody (DO-1), overall p53 stabilization over time is also seen. Similarly, the following antibodies are used to study ATM-dependent phosphorylations on H2AX, CHK1, NBS1, and SMC1: NBS1 phospho-serine 343 and CHK1 phospho-serine 345 antibodies. SMC1 and SMC1 phospho-serine 966 antibodies are also used, along with antibodies against histone H2A (H-124) and CHK1. The peak response time of two hours for p53 serine 15 phosphorylation is used to track ATM inhibition in order to determine a cellular IC50 for KU-55933. Prior to applying ionizing radiation, KU-55933 is titrated onto cells and preincubated for one hour. The IC50 value is determined similarly to the in vitro determinations, and the percentage inhibition in relation to the vehicle control is computed using scanning densitometry.
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| Animal Protocol |
BALB/c nu/nu nude mice bearing LU1205 cells
10 μM |
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| References |
">[1]. Cancer Res
. 2004 Dec 15;64(24):9152-9.
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| Molecular Formula |
C21H17NO3S2
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| Molecular Weight |
395.49
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| Exact Mass |
395.06
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| Elemental Analysis |
C, 63.77; H, 4.33; N, 3.54; O, 12.14; S, 16.22
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| CAS # |
587871-26-9
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| Related CAS # |
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| Appearance |
White to off-white solid powder
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| SMILES |
C1COCCN1C2=CC(=O)C=C(O2)C3=C4C(=CC=C3)SC5=CC=CC=C5S4
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| InChi Key |
XRKYMMUGXMWDAO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H17NO3S2/c23-14-12-16(25-20(13-14)22-8-10-24-11-9-22)15-4-3-7-19-21(15)27-18-6-2-1-5-17(18)26-19/h1-7,12-13H,8-11H2
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| Chemical Name |
2-morpholin-4-yl-6-thianthren-1-ylpyran-4-one
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| Synonyms |
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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 |
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| 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) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5285 mL | 12.6425 mL | 25.2851 mL | |
| 5 mM | 0.5057 mL | 2.5285 mL | 5.0570 mL | |
| 10 mM | 0.2529 mL | 1.2643 mL | 2.5285 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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