CP-466722

ATM ( IC50 = 4.1 μM )

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.

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.

[1]. Cancer Res . 2008 Sep 15;68(18):7466-74.

[2]. J Biomol Screen . 2014 Apr;19(4):538-46.

C17H15N7O2

349.35

349.13

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

1080622-86-1

Solid powder

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

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)