CP-466722

Catalog No.S2245 Batch:S224501

Technical Data

Formula	C₁₇H₁₅N₇O₂
Molecular Weight	349.35	CAS No.	1080622-86-1
Solubility (25°C)*	In vitro	4-Methylpyridine	5 mg/mL warmed with 50ºC water bath (14.31 mM)
		DMSO	0.28 mg/mL (0.8 mM)
		Water	Insoluble
* <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. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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Biological Activity

Description

CP-466722 is a potent and reversible ATM inhibitor, does not affect ATR and inhibits PI3K or PIKK family members in cells.

Targets

ATM ^[2]

410 nM

In vitro

In vitro, CP-466722 is identified as a potential inhibitor to decrease the activity of purified ATM kinase to phosphorylate GST-p53(1–101) substrate. In addition, CP-466722 also shows the inhibitory activities against abl and src kinases. ^[1] In HeLa cells, CP-466722 at doses of 6μM, results in the inhibition in ATM-dependent phosphorylation by reversibly inhibiting ionizing radiation (IR)-induced ATM kinase activity. Besides, ATM-dependent p53 induction is also inhibited by CP-466722 in MCF-7 human breast cancer cells and primary and immortalized diploid human fibroblasts. ^[1] In response to IR, CP-466722 increased proportion of cells with G2/M DNA content and reduces proportion of cells with G1-phase DNA content in HeLa cells. ^[1] Transient exposure to CP-466722 for a period of 4 hours sensitizes HeLa cells to IR without affecting cell plating nor cell viability. ^[1]

Protocol (from reference)

Kinase Assay:^{^[1]}	In vitro kinase assays ^[1] To screen for small molecule inhibitors of ATM kinase activity, an in vitro kinase assay is adapted, and an ELISA assay develops which measured the phosphorylation status of the ATM downstream target p53. Recombinant GST-p53(1-101) and full-length Flag-tagged ATM & ATR are purified for use in the ELISA and in vitro kinase assays. Briefly, Nunc 96 well Maxisorp plates are coated overnight (4 °C) with 2μg of purified, recombinant GST-p53(1-101) in PBS. All subsequent incubations are performed at room temperature. The plates are washed (0.05%v/v-Tween/PBS) before addition of purified recombinant full-length ATM kinase (30 ng–60 ng) in a final volume of 80μL of reaction buffer (20 mM HEPES, 50 mM NaCl, 10 mM MgCl₂, 10 mM MnCl₂, 1 mM DTT and 1 μM ATP) in the presence or absence of CP-466722. CP-466722 (10μM) is added to plates in duplicate and the kinase assay is incubated (90 minutes). Plates are washed (0.05%v/v-Tween/PBS), blocked (1hour, 1%w/v-BSA/PBS) and rinsed before anti-Phospho(Ser15)-p53 antibody (1:1000/PBS) is added to the plates and incubated (1hour). To reduce non-specific binding plates are washed (0.05%v/v-Tween/PBS) prior to incubation (1hour) with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000/PBS). Secondary antibody that is linked to the phosphorylated GST-p53(1–101) protein is detected with TMB substrate reagent. Plates are developed (15 minutes–30 minutes) and the reaction is stopped (1 M H₂SO₄ final concentration) before absorbance is determined (λ450nm). CP-466722 that inhibits ATM kinase activity in ELISA assays, are characterized with respect to inhibition of ATM/ATR kinases using in vitro kinase assays. Western blotting using the anti-Phospho(Ser15)-p53 antibody is used as a readout of ATM/ATR inhibition. Extended analysis of CP466722 (10 μM) against a commercially available panel of kinases is performed by Upstate.
Cell Assay:^{^[1]}	Cell lines HeLa and A-T Concentrations 0-6 μM Incubation Time 4 hours Method HeLa or A-T (GM02052 expressing hTERT) cells are plated in triplicate and incubated for 24 hours. Cells are pre-treated: DMSO, CP466722 or KU55933 prior to IR (0-10Gy). Cells are incubated for 4 hours following IR before media is removed, cells washed (PBS), trypsined, counted and re-plated (2000 cells/plate, 10 cm plates) in the absence of drug and incubated for 10 days. Prior to colony counting, cells are washed (PBS), stained (PBS, 0.0037%v/v-formaldehyde, 0.1%w/v-crystal violet), rinsed (dH₂O) and dried. Defined populations (>50 cells) are counted as one surviving colony, data are calculated as percentage surviving colonies relative to control plates +/− SE.

Kinase Assay:^{^[1]}

In vitro kinase assays ^[1]
To screen for small molecule inhibitors of ATM kinase activity, an in vitro kinase assay is adapted, and an ELISA assay develops which measured the phosphorylation status of the ATM downstream target p53. Recombinant GST-p53(1-101) and full-length Flag-tagged ATM & ATR are purified for use in the ELISA and in vitro kinase assays. Briefly, Nunc 96 well Maxisorp plates are coated overnight (4 °C) with 2μg of purified, recombinant GST-p53(1-101) in PBS. All subsequent incubations are performed at room temperature. The plates are washed (0.05%v/v-Tween/PBS) before addition of purified recombinant full-length ATM kinase (30 ng–60 ng) in a final volume of 80μL of reaction buffer (20 mM HEPES, 50 mM NaCl, 10 mM MgCl₂, 10 mM MnCl₂, 1 mM DTT and 1 μM ATP) in the presence or absence of CP-466722. CP-466722 (10μM) is added to plates in duplicate and the kinase assay is incubated (90 minutes). Plates are washed (0.05%v/v-Tween/PBS), blocked (1hour, 1%w/v-BSA/PBS) and rinsed before anti-Phospho(Ser15)-p53 antibody (1:1000/PBS) is added to the plates and incubated (1hour). To reduce non-specific binding plates are washed (0.05%v/v-Tween/PBS) prior to incubation (1hour) with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000/PBS). Secondary antibody that is linked to the phosphorylated GST-p53(1–101) protein is detected with TMB substrate reagent. Plates are developed (15 minutes–30 minutes) and the reaction is stopped (1 M H₂SO₄ final concentration) before absorbance is determined (λ450nm). CP-466722 that inhibits ATM kinase activity in ELISA assays, are characterized with respect to inhibition of ATM/ATR kinases using in vitro kinase assays. Western blotting using the anti-Phospho(Ser15)-p53 antibody is used as a readout of ATM/ATR inhibition. Extended analysis of CP466722 (10 μM) against a commercially available panel of kinases is performed by Upstate.

Cell Assay:^{^[1]}

Cell lines
HeLa and A-T
Concentrations
0-6 μM
Incubation Time
4 hours
Method
HeLa or A-T (GM02052 expressing hTERT) cells are plated in triplicate and incubated for 24 hours. Cells are pre-treated: DMSO, CP466722 or KU55933 prior to IR (0-10Gy). Cells are incubated for 4 hours following IR before media is removed, cells washed (PBS), trypsined, counted and re-plated (2000 cells/plate, 10 cm plates) in the absence of drug and incubated for 10 days. Prior to colony counting, cells are washed (PBS), stained (PBS, 0.0037%v/v-formaldehyde, 0.1%w/v-crystal violet), rinsed (dH₂O) and dried. Defined populations (>50 cells) are counted as one surviving colony, data are calculated as percentage surviving colonies relative to control plates +/− SE.

References

Customer Product Validation

: Data from [PLoS One, 2012, 7(11), e50423]

: Data from [PLoS One, 2012, 7(11), e50423]

: Data from [J Neurooncol, 2012, 110(3), 349-57]

: Data from [J Neurooncol, 2012, 110(3), 349-57]

Selleck's CP-466722 has been cited by 11 publications

Loss of N-Glycanase 1 Alters Transcriptional and Translational Regulation in K562 Cell Lines [ G3 (Bethesda), 2020, 4;10(5):1585-1597]	PubMed: 32265286
Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras. [ Cell, 2019, 36(2):179-193]	PubMed: 28162770
A Pharmacogenomic Landscape in Human Liver Cancers. [ Cancer Cell, 2019, 36(2):179-193]	PubMed: 31378681
An Anticancer Drug Cocktail of Three Kinase Inhibitors Improved Response to a Dendritic Cell-Based Cancer Vaccine [ Cancer Immunol Res, 2019, 7(9):1523-1534]	PubMed: 31266784
Upregulation of long noncoding RNA SNHG20 promotes cell growth and metastasis in esophageal squamous cell carcinoma via modulating ATM-JAK-PD-L1 pathway [ J Cell Biochem, 2019, 10.1002/jcb.28444]	PubMed: 30767270
Alleviation of Senescence via ATM Inhibition in Accelerated Aging Models. [ Mol Cells, 2019, 42(3):210-217]	PubMed: 30726661
ATM‑JAK‑PD‑L1 signaling pathway inhibition decreases EMT and metastasis of androgen‑independent prostate cancer. [ Mol Med Rep, 2018, 17(5):7045-7054]	PubMed: 29568923
Simultaneous targeting of ATM and Mcl-1 increases cisplatin sensitivity of cisplatin-resistant non-small cell lung cancer [ Cancer Biol Ther, 2017, 18(8):606-615]	PubMed: 28686074
Monitoring the Activation of the DNA Damage Response Pathway in a 3D Spheroid Model. [Mondesert O, et al. PLoS One, 2015, 10(7):e0134411]	PubMed: 26225756
ATM inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM cells. [Nadkarni A, et al. J Neurooncol, 2012, 110(3):349-57]	PubMed: 23054561

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