ATR kinase assay

ATR for use in the in vitro enzyme assay was obtained from HeLa nuclear extract by immunoprecipitation with rabbit polyclonal antiserum raised to amino acids 400 ~ 480 of ATR contained in the following buffer: 25 mM HEPES (pH 7.4), 2 mM MgCl2, 250 mM NaCl, 0.5 mM EDTA, 0.1 mM Na3VO4, 10% v/v glycerol, and 0.01% v/v Tween 20. ATR-antibody complexes were isolated from nuclear extract by incubating with protein A-Sepharose beads for 1 hr and then through centrifugation to recover the beads. In the well of a 96-well plate, 10 μL ATR-containing Sepharose beads were incubated with 1 μg of substrate glutathione S-transferase-p53N66 in ATR assay buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 6 mM MgCl2, 4 mM MnCl2, 0.1 mM Na3VO4, 0.1 mM DTT, and 10% (v/v) glycerol) at 37℃ in the presence or absence of inhibitor. After 10 mins with gentle shaking, ATP was added to a final concentration of 3 μM and the reaction continued at 37℃ for an additional 1 hr. The reaction was stopped by addition of 100 μL of PBS, and the reaction was transferred to a white opaque glutathione coated 96-well plate and incubated overnight at 4℃. This plate was then washed with PBS/0.05% (v/v) Tween 20, blotted dry, and analyzed by a standard ELISA technique with a phosphoserine 15 p53 antibody. The detection of phosphorylated glutathione S-transferase?p53N66 substrate was performed in combination with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody. Enhanced chemiluminescence solution was used to produce a signal, and chemiluminescent detection was carried out via a TopCount plate reader. The resulting calculated % enzyme activity was then used to determine the IC50 values for the compounds.

Cell lines

BT-474 cells and HT29 colorectal adenocarcinoma cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

60 mins

Applications

AZ20 inhibited pAKT T308 in BT-474 cells, and inhibited pATM Ser-1981 and pDNA-PK Ser-2056 in HT29 cells. In LoVo colorectal adenocarcinoma cells, AZ20 induced growth inhibition with the GI50 value of 0.20 μM.

Animal models

Female nude mice bearing LoVo tumors

Dosage form

5 mg/kg twice daily and 50 mg/kg once daily; p.o.

Applications

At the dose of 10 μM, AZ20 was found to inhibit the cytochrome 3A4-mediated metabolism of Midazolam by 50%. In a low dose rat PK study, it demonstrated that AZ20 has respectable bioavailability.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

AZ20 is a potent and selective inhibitor of ATR. AZ20 inhibits ATR immunoprecipitated from HeLa nuclear extracts with an IC50 value of 5 nM and ATR mediated phosphorylation of Chk1 in HT29 colorectal adenocarcinoma tumor cells with an IC50 value of 50 nM.
ATR is an attractive new anticancer drug target whose inhibitors have potential as chemo- or radiation sensitizers or as monotherapy in tumors addicted to particular DNA-repair pathways.
AZ20 is the first reported inhibitor of ATR protein kinase demonstrating tumor growth inhibition in vivo and is therefore a useful probe molecule to aid further investigation of ATR tumor biology. AZ20 potently inhibited the growth of LoVo colorectal adenocarcinoma tumor cells in vitro. In the mTOR (pAKT) cell assay, AZ20 weak inhibited recombinant mTOR enzyme activity. AZ20 shows good selectivity against all of the PI3K isoforms together with ATM and DNA-PK, and when tested in a large panel of kinases, AZ20 shows very high general kinase selectivity.
Female nude mice bearing LoVo tumors were treated with AZ20 orally at a dose of 25 mg/kg twice daily or 50 mg/kg once daily for 13 days. AZ20 showed monotherapy in vivo antitumor efficacy in LoVo colorectal xenografts in nude mice.
References:
[1]. Foote KM, Blades K, Cronin A et al. Discovery of 4-{4-[(3R)-3-Methylmorpholin-4-yl]-6- [1-(methylsulfonyl)cyclopropyl]pyrimidin-2-yl}-1H-indole (AZ20): a potent and selective inhibitor of ATR protein kinase with monotherapy in vivo antitumor activity. J Med Chem. 2013 Mar 14;56(5):2125-38.