KU-60019

ATM

547, 67

Dissolved in water, final concentrations ca.3 uM

Compared to KU-55933, KU-60019 is an improved more water-soluble inhibitor of the ATM kinase, while displaying similar target selectivity. KU-60019 has little activity against DNA-PKcs and ATR with IC50 values of 1.7 uM and >10 uM, respectively, as well as 229 other protein kinases such as PI3K, mTOR and mTOR/FKBP12. KU-60019 displays 3- to 10-fold more potency than KU-55933 at blocking radiation-induced phosphorylation of key ATM protein targets such as p53, gamma-H2AX, and CHK2, in human glioma U87 and U1242 cells, as 1 uM of KU-60019 significantly induces >70% decrease of p53 (S15) phosphorylation to which extent ca.10 uM of KU-55933 is required to achieve. KU-60019 effectively radiosensitizes human glioma cells with dose-enhancement ratio of 1.7 and 4.4 at 1 uM and 10 uM, respectively, and also radiosensitizes the normal fibroblasts but not the A-T fibroblasts. KU-60019 treatment (3 uM) blocks basal and insulin-induced AKT S473 phosphorylation by 70% and ca.50%, respectively, and completely reduces radiation-induced AKT phosphorylation below the level of control. The effect of KU-60019 on AKT S473 phosphorylation can be seen in glioma cell lines and normal fibroblasts but not in A-T (h-TERT) cells, and can be significantly blocked by phosphatase inhibitor okadaic acid, suggesting a critical role of ATM kinase in regulating AKT phosphorylation via unknown phosphatase. Consistent with the inhibition of prosurvival AKT signaling, KU-60019 at 3 uM significantly inhibits migration and invasion of human glioma U87 cells by >70% and ca.60%, respectively, as well as U1242 cells by >50% and ca.60% respectively. [1]

Cells are exposed to KU-60019 for 1, 3, and 5 days. Cell growth is determined by AlamarBlue. AlamarBlue is added to the medium to the recommended final concentration. Plates are incubated for 1 hour at 37 C, fluorescence is determined on a Fluoro-Count plate reader (excitation 530 nm, emission 590 nm), and values are taken as a measure of cell growth. Cell survival is determined by trypan blue/fluorescence activated cell sorting (FACS) assay.

KU-60019 is an improved analogue of KU-55933, with IC50 of 6.3 nM for ATM in cell-free assays, 270- and 1600-fold more selective for ATM than DNA-PK and ATR, and is a highly effective radiosensitizer.

An improved analog of KU-55933, more effective at blocking ATM-mediated DDR events.