ATM ( IC50 = 4.1 μM )
CP-466722 targets ataxia telangiectasia mutated (ATM) kinase (IC50 = 130 nM) [1]
CP-466722 targets ATM kinase (IC50 = 110 nM in recombinant enzyme assay) [2]

In vitro activity: CP-466722 has been discovered as a putative inhibitor that may lessen the ability of purified ATM kinase to phosphorylate the GST-p53(1–101) substrate in vitro. Furthermore, CP-466722 exhibits inhibitory actions against src and abl kinases.[1] CP-466722, at 6μM doses, reversibly inhibits the ionizing radiation (IR)-induced ATM kinase activity in HeLa cells, thereby inhibiting ATM-dependent phosphorylation. Additionally, CP-466722 inhibits ATM-dependent p53 induction in primary and immortalized diploid human fibroblasts as well as MCF-7 human breast cancer cells.[1] HeLa cells treated with CP-466722 showed an increase in the proportion of cells with G2/M DNA content and a decrease in the proportion of cells with G1-phase DNA content in response to IR.[1] HeLa cells are sensitized to infrared radiation by a 4-hour transient exposure to CP-466722, which has no effect on cell viability or plating.[1]

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In human cancer cell lines (A549, HCT116, MCF-7), CP-466722 (0.5–20 μM) alone has weak anti-proliferative activity (cell viability reduced by ≤25% at 20 μM). When combined with ionizing radiation (IR, 2–8 Gy), it acts as a potent radiosensitizer: the survival fraction of A549 cells after 4 Gy IR + 10 μM CP-466722 is reduced by ~65% compared to IR alone. It inhibits ATM-mediated phosphorylation of Chk2 (p-Chk2) and p53 (p-p53) (Western blot), and increases γ-H2AX foci accumulation (immunofluorescence), indicating impaired DNA double-strand break (DSB) repair [1]
- In BRCA1-proficient breast cancer cells (MCF-7, T47D), CP-466722 (1–10 μM) synergizes with PARP-1 inhibitor olaparib (0.5 μM): the combination reduces cell viability by ~70% (vs. 20% CP-466722 alone, 15% olaparib alone) and induces apoptosis (Annexin V-FITC/PI staining, apoptotic rate ~55%). In BRCA1-deficient breast cancer cells (HCC1937), the synergistic effect is weaker (cell viability reduced by ~40% vs. single agents) [3]
- CP-466722 transiently inhibits ATM kinase activity in cancer cells: treatment for 2 hours is sufficient to enhance radiosensitivity, and activity recovers within 24 hours of drug withdrawal. This transient inhibition does not induce significant apoptosis alone but potentiates IR-induced cell death [1]

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In a subcutaneous xenograft model of non-small cell lung cancer (A549 cells in nude mice), intraperitoneal administration of CP-466722 (50 mg/kg/day) for 5 days (concurrent with IR: 4 Gy on day 1 and day 3) inhibits tumor growth by ~70% compared to IR alone (~30% inhibition). Tumor tissues show reduced p-Chk2 and increased γ-H2AX expression (immunohistochemistry), confirming ATM inhibition and impaired DSB repair [1]

An ELISA assay is developed to measure the phosphorylation status of the ATM downstream target p53, and an in vitro kinase assay is adapted to screen for small molecule inhibitors of ATM kinase activity. For use in the ELISA and in vitro kinase tests, recombinant GST-p53(1-101) and full-length Flag-tagged ATM & ATR are purified. In summary, 2μg of purified, recombinant GST-p53(1-101) in PBS is coated overnight at 4 °C on Nunc 96-well Maxisorp plates. The next few incubations are run at room temperature. 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), the plates are washed (0.05%v/v-Tween/PBS) before being added with purified recombinant full-length ATM kinase (30 ng–60 ng) in the presence or absence of CP-466722. The kinase assay is incubated for 90 minutes after CP-466722 (10μM) is added to plates in duplicate. Anti-Phospho(Ser15)-p53 antibody (1:1000/PBS) is added to the plates, and they are then rinsed, blocked (1 hour, 1%w/v-BSA/PBS), and incubated (1 hour) after the plates have been cleaned (0.05%v/v-Tween/PBS). Plates are cleaned (0.05%v/v-Tween/PBS) to lessen non-specific binding before being incubated for 1 hour with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000/PBS). The TMB substrate reagent is used to identify the secondary antibody that is connected to the phosphorylated GST-p53(1–101) protein. Before determining absorbance (λ450nm), plates are developed for 15–30 minutes, and the reaction is stopped at a final concentration of 1 M H₂SO₄. In vitro kinase assays are used to characterize CP-466722, which inhibits ATM kinase activity in ELISA assays, with regard to inhibition of ATM/ATR kinases. The anti-Phospho(Ser15)-p53 antibody is used in western blotting to measure ATM/ATR inhibition. Upstate conducts an extended analysis of CP466722 (10 μM) against a panel of kinases that is available commercially.

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Recombinant human ATM kinase (with ATRIP) was incubated with a Chk2-derived peptide substrate and ATP in kinase buffer. CP-466722 was added at concentrations ranging from 10 nM to 10 μM, and the mixture was incubated at 30°C for 60 minutes. The phosphorylated peptide was detected using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The inhibition rate was calculated relative to the vehicle control, and the IC50 value was determined by nonlinear regression analysis [1]
- High-throughput ATM kinase inhibition assay: HEK293T cells stably expressing ATM and a luciferase reporter (regulated by ATM-dependent Chk2 phosphorylation) were seeded in 384-well plates. CP-466722 (0.01–50 μM) was added, and cells were incubated for 24 hours. Luciferase activity was measured, and the IC50 was calculated based on the dose-response curve of ATM inhibition [2]
- Recombinant ATM kinase activity assay with [γ-32P]ATP: Purified ATM protein was mixed with histone H2AX substrate, [γ-32P]ATP, and CP-466722 (20–2000 nM). The mixture was incubated at 37°C for 30 minutes, followed by SDS-PAGE electrophoresis and autoradiography. Radioactivity of phosphorylated H2AX was quantified to confirm ATM inhibition [2]

Triple-plated HeLa or A-T (GM02052) expressing hTERT cells are incubated for a full day. Pre-treatment of cells with DMSO, CP466722, or KU55933 is done before IR (0-10Gy). After internal reflection, the cells are allowed to incubate for four hours. Afterward, the media is removed, the cells are rinsed with PBS, trypsined, tallied, and replated (2000 cells/plate, 10 cm plates) without any drug, and they are then left to incubate for ten days. Before the count of colonies, the cells undergo a series of procedures including washing in PBS, staining in PBS, rinsing in dH₂O, and drying. A surviving colony is defined as a population of at least 50 cells. The data is expressed as the percentage of surviving colonies relative to the control plates plus or minus standard error.

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Radiosensitization and clonogenic survival assay: Cancer cells (1×104 per well) were seeded in 6-well plates, treated with CP-466722 (0.5–20 μM) for 2 hours, then exposed to IR (2–8 Gy). After 14 days of culture, colonies were stained with crystal violet and counted. The survival fraction was calculated relative to unirradiated control cells [1]
- Cell viability and apoptosis assay: Breast cancer cells (5×103 per well) were seeded in 96-well plates, treated with CP-466722 (1–10 μM) alone or in combination with olaparib (0.5 μM) for 48 hours. Cell viability was measured by CCK-8 assay (absorbance at 450 nm). Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry [3]
- Western blot analysis: Cells treated with CP-466722 (5–20 μM) ± IR/olaparib for 24 hours were lysed to extract total protein. Equal amounts of protein were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against ATM, p-ATM, Chk2, p-Chk2, p53, p-p53, γ-H2AX, caspase-3, cleaved caspase-3, or GAPDH (loading control). Protein bands were visualized by chemiluminescence [1,3]
- Immunofluorescence staining for γ-H2AX foci: A549 cells were seeded on coverslips, treated with CP-466722 (10 μM) + IR (4 Gy), fixed with paraformaldehyde at 24 hours post-treatment, permeabilized with Triton X-100, and stained with anti-γ-H2AX antibody (FITC-conjugated) and DAPI. The number of γ-H2AX foci per cell was counted by confocal microscopy (n ≥ 50 cells per group) [1]

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A549 xenograft model: Nude mice (4-week-old, male) were subcutaneously injected with A549 cells (5×106 cells/mouse) into the right flank. When tumors reached ~120 mm3, mice were randomly divided into four groups (n = 6 per group): control, CP-466722 alone (50 mg/kg/day, i.p.), IR alone (4 Gy, local irradiation on day 1 and day 3), and combination group. CP-466722 was dissolved in DMSO (5%) + saline (95%) and administered intraperitoneally once daily for 5 days (days 1–5). Tumor volume was measured every 2 days (volume = length × width2 / 2), and mice were euthanized on day 21 for tumor weight measurement and immunohistochemical analysis [1]

In vitro toxicity: CP-466722 (at a concentration of up to 20 μM) did not show significant cytotoxicity to normal human bronchial epithelial cells (NHBE) or mammary epithelial cells (HMEC), and cell viability was >85% compared to the control group [1,3] - In vivo toxicity: Mice treated with CP-466722 (50 mg/kg/day, intraperitoneal injection, for 5 consecutive days) showed no significant changes in body weight, liver function (ALT, AST), or kidney function (BUN, creatinine) compared to the control group. Histological examination of the liver, kidneys, and lung tissues revealed no abnormal lesions or inflammation [1] - Plasma protein binding rate: The plasma protein binding rate of CP-466722 in human plasma was 86%, and the plasma protein binding rate in mouse plasma was 83%, as determined by balanced dialysis [1]

[1]. Transient inhibition of ATM kinase is sufficient to enhance cellular sensitivity to ionizing radiation. Cancer Res. 2008 Sep 15;68(18):7466-74.

[2]. Development of a cell-based, high-throughput screening assay for ATM kinase inhibitors. J Biomol Screen. 2014 Apr;19(4):538-46.

[3]. Interactions Between Ataxia Telangiectasia Mutated Kinase Inhibition, Poly(ADP-ribose) Polymerase-1 Inhibition and BRCA1 Status in Breast Cancer Cells. J Cancer Prev. 2014 Jun;19(2):125-36.

2-(6,7-dimethoxy-4-quinazolinyl)-5-(2-pyridyl)-1,2,4-triazol-3-amine is a quinazolinoid compound. CP-466722 is a synthetic small molecule ATM kinase inhibitor characterized by reversible and transient inhibition of ATM activity [1] - its mechanism of action involves binding to the ATP-binding pocket of ATM, blocking the phosphorylation of ATM-mediated downstream DNA damage repair substrates (Chk2, p53). This would impair DSB repair, making cancer cells more sensitive to ionizing radiation or PARP inhibitors [1,3]
- The transient inhibitory properties of CP-466722 reduce potential off-target effects and toxicity, making it a promising cancer radiosensitizer [1]
- CP-466722 showed stronger synergistic effects with PARP inhibitors in BRCA1-functional breast cancer cells, suggesting it may be a potential therapeutic strategy for BRCA1 wild-type tumors, which are often resistant to PARP inhibitors [3]

C17H15N7O2

349.35

349.128

C, 58.45; H, 4.33; N, 28.07; O, 9.16

1080622-86-1

44551660

White to off-white solid powder

1.5±0.1 g/cm3

642.3±65.0 °C at 760 mmHg

342.2±34.3 °C

0.0±1.9 mmHg at 25°C

1.737

0.83

1

8

4

26

470

0

NC1=NC(C2=NC=CC=C2)=NN1C3=C4C=C(OC)C(OC)=CC4=NC=N3

ILBRKJBKDGCSCB-UHFFFAOYSA-N

InChI=1S/C17H15N7O2/c1-25-13-7-10-12(8-14(13)26-2)20-9-21-16(10)24-17(18)22-15(23-24)11-5-3-4-6-19-11/h3-9H,1-2H3,(H2,18,22,23)

2-(6,7-dimethoxyquinazolin-4-yl)-5-pyridin-2-yl-1,2,4-triazol-3-amine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)