| In Vitro | In vitro activity: AZ20 shows good selectivity against all of the PI3K isoforms together with ATM and DNA-PK. In vitro, AZ20 decreases pChk1 Ser345, pChk1 Ser317 and pChk1 Ser296 levels in a concentration-dependent manner. Prolonged exposure with AZ20 increases γH2AX pan-nuclear staining, indicative of replication stress. This is associated with S-phase arrest and increase in phospho-histone H3. AZ20 induces growth inhibition and cell death in vitro and its profile of activity is distinct from other cytotoxic agents. The cytotoxic effect of AZ20 can be increased in combination with the selective ATM inhibitor KU-60019.
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°C 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°C 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°C. 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 Assay: 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. |
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