In vitro activity: AZ32 is a novel, potent and orally bioavailable and blood-brain barrier-penetreable ATM inhibitor with IC50 of<6.2 nM for in cell free assays, and IC50 of 0.31 μM for ATM in cell 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.

Kinase Assay: AZ32 is a next-generation blood-brain barrier (BBB)-penetrating ATM inhibitor. AZ32 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.