KU-55933 (ATM Kinase Inhibitor)

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S1092

5~200mg

Brand

Selleckchem

Description

KU-55933 (ATM Kinase Inhibitor)

Catalog No.S1092

4 Reviews 15 Product Citations

KU-55933 is a potent and specific ATM inhibitor with IC50/K_i of 12.9 nM/2.2 nM, and is highly selective for ATM as compared to DNA-PK, PI3K/PI4K, ATR and mTOR.

Distributor Tel: +886-2-2827-1197jimmy@new.abscience.com.tw

Biological Activity

Description	KU-55933 is a potent and specific ATM inhibitor with IC50/K_i of 12.9 nM/2.2 nM, and is highly selective for ATM as compared to DNA-PK, PI3K/PI4K, ATR and mTOR.
Targets	ATM	ATM
IC50	13 nM	2.2 nM (K_i) ^[1]
In vitro	KU-55933 inhibits DNA-PK and PI3K with IC50 of 2.5 μM and 16.6 μM, respectively. Besides, KU-55933 also prevents the activity of mTOR with IC50 of 9.3 μM. KU-55933 is active at the cellular level in ablating a well-characterized ATM-dependent phosphorylation event. KU-55933 has a dose-dependent effect in inhibiting this ATM-dependent phosphorylation event with IC50 of 300 nM. KU-58050 does not prevent the ATM-dependent phosphorylation of p53 serine 15 until a dose of 30 μM. Addition of KU-55933 has no appreciable effects on UV-induced phosphorylation of H2AX on serine 139, NBS1 on serine 343, CHK1 on serine 345, and SMC1 on serine 966. In stark contrast to the UV responses, KU-55933 ablates the ionizing radiation-induced phosphorylation of these ATM substrates. KU-55933 sensitizes HeLa cells to a range of ionizing radiation doses. ^[1] KU-55933 inhibits the phosphorylation of Akt induced by growth factors in cancer cells. KU-55933 suppresses the proliferation of cancer cells. Furthermore, suppression of ATM by KU-55933 improves survival, probably via prevention of downstream activation of TAp63α. ^[2]
In vivo	Suppression of ATM-dependent STAT3 activation by KU-55933 enhances TRAIL-mediated apoptosis through up-regulation of surface DR5 expression, whereas suppression of both STAT3 and NF-κB appeares to be involved in down-regulation of cFLIP accompanied by an additional increase in apoptotic levels. The ATM inhibitor KU-55933 affectes TRAIL-mediated apoptosis more strongly than the JAK2 inhibitor, AG490, or overexpression of STAT3β. ^[3]
Features

Protocol(Only for Reference)

Kinase Assay: ^[1]

Purified enzyme assays	ATM for use in the in vitro assay is obtained from HeLa nuclear extract by immunoprecipitation with rabbit polyclonal antiserum raised to the COOH-terminal 400 amino acids of ATM in buffer containing 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. ATM-antibody complexes are isolated from nuclear extract by incubating with protein A-Sepharose beads for 1 hour and then through centrifugation to recover the beads. In the well of a 96-well plate, ATM-containing Sepharose beads are incubated with 1 μg of substrate glutathione S-transferase–p53N66 (NH₂-terminal 66 amino acids of p53 fused to glutathione S-transferase) in 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 in the presence or absence of inhibitor. After 10 minutes with gentle shaking, ATP is added to a final concentration of 50 μM and the reaction continued at 37 °C for an additional 1 hour. The plate is centrifuged at 250 × g for 10 minutes (4 °C) to remove the ATM-containing beads, and the supernatant is removed and transferred to a white opaque 96-well plate and incubated at room temperature for 1.5 hours to allow glutathione S-transferase-p53N66 binding. This plate is then washed with PBS, blotted dry, and analyzed by a standard ELISA technique with a phospho-serine 15 p53 antibody. The detection of phosphorylated glutathione S-transferase-p53N66 substrate is performed in combination with a goat antimouse horseradish peroxidase-conjugated secondary antibody. Enhanced chemiluminescence solution is used to produce a signal and chemiluminescent detection is carried out.

Purified enzyme assays

ATM for use in the in vitro assay is obtained from HeLa nuclear extract by immunoprecipitation with rabbit polyclonal antiserum raised to the COOH-terminal 400 amino acids of ATM in buffer containing 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. ATM-antibody complexes are isolated from nuclear extract by incubating with protein A-Sepharose beads for 1 hour and then through centrifugation to recover the beads. In the well of a 96-well plate, ATM-containing Sepharose beads are incubated with 1 μg of substrate glutathione S-transferase–p53N66 (NH₂-terminal 66 amino acids of p53 fused to glutathione S-transferase) in 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 in the presence or absence of inhibitor. After 10 minutes with gentle shaking, ATP is added to a final concentration of 50 μM and the reaction continued at 37 °C for an additional 1 hour. The plate is centrifuged at 250 × g for 10 minutes (4 °C) to remove the ATM-containing beads, and the supernatant is removed and transferred to a white opaque 96-well plate and incubated at room temperature for 1.5 hours to allow glutathione S-transferase-p53N66 binding. This plate is then washed with PBS, blotted dry, and analyzed by a standard ELISA technique with a phospho-serine 15 p53 antibody. The detection of phosphorylated glutathione S-transferase-p53N66 substrate is performed in combination with a goat antimouse horseradish peroxidase-conjugated secondary antibody. Enhanced chemiluminescence solution is used to produce a signal and chemiluminescent detection is carried out.

Cell Assay: ^[1]

Cell lines	U2OS cells
Concentrations	10 μM
Incubation Time	2 hours
Method	U2OS cells are exposed to ionizing radiation (3, 5, or 15 Gy) or UV (5 or 50 J/m²) and the ATM response determined by Western blot analysis of p53 serine 15 phosphorylation and stabilization of wild-type p53. Whole cell extracts are obtained from each time point, proteins separated by SDS-PAGE, and the ATM-specific increase in phosphorylated serine 15 measured with a p53 phospho-serine 15 specific antibody. Overall p53 stabilization with time is also observed with a p53-specific antibody (DO-1). Similarly, for studying ATM-dependent phosphorylations on H2AX, CHK1, NBS1, and SMC1, the following antibodies are used: CHK1 phospho-serine 345 and NBS1 phospho-serine 343 antibodies. Histone H2A (H-124) and CHK1 antibodies are also used, as well as SMC1 and SMC1 phospho-serine 966 antibodies. For determination of a cellular IC50 for KU-55933, the peak response time for p53 serine 15 phosphorylation of 2 hours is used to monitor inhibition of ATM. KU-55933 is titrated onto cells and preincubated for 1 hour before ionizing radiation. Using scanning densitometry, the percentage inhibition relative to vehicle control is calculated, and the IC50 value is calculated as for the in vitro determinations.

Animal Study: ^[3]

Animal Models	BALB/c nu/nu nude mice bearing LU1205 cells
Dosages	10 μM
Administration
Solubility	30% PEG400/0.5% Tween80/5% propylene glycol, 30 mg/mL

References

Chemical Information

Download KU-55933 (ATM Kinase Inhibitor) SDF

Molecular Weight (MW)	395.49
Formula	C₂₁H₁₇NO₃S₂
CAS No.	587871-26-9

Storage	3 years -20â„ƒPowder
Storage	6 months-80â„ƒin DMSO
Syonnyms	N/A

Solubility (25°C) *	In vitro	DMSO	33 mg/mL (83 mM)
		Water	<1 mg/mL (<1 mM)
		Ethanol	<1 mg/mL (<1 mM)
	In vivo	30% PEG400/0.5% Tween80/5% propylene glycol,	30 mg/mL
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Name	2-morpholino-6-(thianthren-1-yl)-4H-pyran-4-one

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.39549	3.9549	7.9098	11.8647

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Customer Reviews (4)

Click to enlarge	Rating
	Source	Cancer Discovery, 2012, 2, 1048-1063. KU-55933 (ATM Kinase Inhibitor) purchased from Selleck
	Method	Western Blot
	Cell Lines	HCC1937 cells
	Concentrations	10 uM
	Incubation Time	24 h
	Results	As expected, KU-55933 led to a decrease in autophosphorylation of ATM ( Fig. 5A , third and fourth lane of each panel), and prevented the increase in H2AX phosphorylation seen in response to ionizing radiation. However, KU-55933 did not prevent the NVP-BKM120–induced induction of γ-H2AX, which was robust both at baseline and in response to ionizing radiation ( Fig. 5A , last lane of each panel), suggesting that an alternative kinase, such as DNA-PK, phosphorylates H2AX in response to PI3K inhibition.

Product Citations (15)

Combining a PI3K inhibitor with a PARP inhibitor provides an effective therapy for a mouse model of BRCA1-related breast cancer. [Juvekar A, et al. Cancer Discov 2012;2(11):1048-63]

PubMed: 22915751
Defective chromatin recruitment and retention of NHEJ core components in human tumor cells expressing a Cyclin E fragment [Chatterjee P, et al. Nucleic Acids Res 2013;41(22):10157-69]

PubMed: 24021630
Systems Biology Approach Identifies the Kinase Csnk1a1 as a Regulator of the DNA Damage Response in Embryonic Stem Cells. [Jordi Carreras Puigvert, et al. Sci Signal 2013;6(259):ra5]

PubMed: 23354688

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KU-55933 (ATM Kinase Inhibitor)