In vitro activity: AZ31 is a novel, potent and orally bioavailable and blood-brain barrier-penetreable ATM inhibitor with IC50 in the nM range in cell free assays. It was identified after drug screening assays and refinements of lead compounds. Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. AZ31 was tested in vivo for efficacy and impact on tumor and healthy brain. AZ31 blocked the DNA damage response and radiosensitized GBM cells in vitro, with enhanced blood-brain barrier (BBB) penetration. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53.

Kinase Assay: AZ31 is a next-generation blood-brain barrier (BBB)-penetrating ATM inhibitor. AZ31 blocks the DNA damage response and radiosensitized GBM cells in vitro.

Cell Assay: Human glioma U1242, U87/luc-DsRed-p53(281G), and cell derivatives expressing reporter genes were previously described. Mouse glioma GL261 cells were infected with Fluc-DsRed2 lentivirus and sorted prior to cell injections. Similarly, certified NCI-H2228 non–small lung cancer cells were obtained from the ATCC. These cells were also modified to express luciferase (NCI-H2228-Luc) suitable for BLI. Cells were acquired and modified between 2009 and 2016. Cells were grown in complete DMEM (Gibco) supplemented with 10% FBS and penicillin–streptomycin at 37°C and 5% CO2. Cultures were maintained for no longer than 2 month and routinely tested negative for mycoplasma.