Home DNA Damage/DNA Repair ATM/ATR inhibitor CP-466722

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CP-466722 ATM/ATR inhibitor

Cat.No.S2245

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

Chemical Structure

Molecular Weight: 349.35

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Quality Control

Batch: S224501 4-Methylpyridine]5 mg/mL]true]DMSO]0.28 mg/mL]false]Water]Insoluble]false Purity: 99.99%
99.99

Solubility

In vitro
Batch:

4-Methylpyridine : 5 mg/mL

DMSO : 0.28 mg/mL (0.8 mM)
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Water : Insoluble

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In vivo
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Chemical Information, Storage & Stability

Molecular Weight 349.35 Formula

C17H15N7O2

 Storage (From the date of receipt)
CAS No. 1080622-86-1 Download SDF Storage of Stock Solutions

Synonyms N/A Smiles COC1=C(C=C2C(=C1)C(=NC=N2)N3C(=NC(=N3)C4=CC=CC=N4)N)OC

Mechanism of Action

Targets/IC50/Ki
ATM
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, this compound also shows the inhibitory activities against abl and src kinases. In HeLa cells, this compound 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 this chemical in MCF-7 human breast cancer cells and primary and immortalized diploid human fibroblasts. In response to IR, this compound increased proportion of cells with G2/M DNA content and reduces proportion of cells with G1-phase DNA content in HeLa cells. Transient exposure to this chemical for a period of 4 hours sensitizes HeLa cells to IR without affecting cell plating nor cell viability.
Kinase Assay
In vitro kinase assays
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. This compound (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). This compound 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 this chemical (10 μM) against a commercially available panel of kinases is performed by Upstate.
References

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