Kinase experiment:

Compounds (e.g., ETP-46464) and control inhibitors are delivered directly to cell media (100 μL per well) with a multi-well pipette at a final concentration of 10 μM. Media content is homogenized by carefully vortexing plates at 500 rpm. Prior to 4-hydroxy-tamoxifen (4-OHT) addition, Compounds (e.g., ETP-46464 ) are incubated at 37oC for 15 minutes. Next, to induce ATR activity, 4-OHT is added to all wells and incubated for 60 minutes at 37oC. Finally, cells are fixed with paraformaldehyde and processed for IF. Every compound (e.g., ETP-46464) is analyzed at least in three independent experiments[1].

Cell experiment:

Cells are trypsinized with 0.25% Trypsin-EDTA and counted with 0.4% Trypan Blue using an automated cell counter and plated in 96-well plates at 5000 cells per well for KLE, HEC1B and HELA and 10,000 cells per well for OVCAR3, A2780, A2780-CP20 and SIHA. After cells attach and reach approximately 60% confluency (24-48 h post seeding), media is removed and replaced with fresh media containing Cisplatin (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 μM) or Carboplatin (0, 1.56, 3.13, 6.25, 12.5, 25, 50 or 100 μM) in 0.15% DMSO, 5 μM ETP-46464, 10 μM KU55933, or a combination of 5 μM ETP-46464 and 10 μM KU55933 and incubated for 72 h. Final concentrations of ETP-46464 and KU55933 utilized are based on prior evidence indicating inhibition of ATR and ATM signaling, respectively. Single-agent dose response analyses of ETP-46464 and KU55933 in a subset of cell lines revealed a wide LD50 range (10.0±8.7 and 38.3±7.6 μM, respectively). Similarly, cells are treated with fresh media containing Cisplatin (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 μM) in 0.08% DMSO and 5 μM VE-821. Cell viability is assessed using the MTS CellTiter 96 Aqueous One Solution Cell Proliferation Assay. After a 2 h incubation at 37℃, absorbance is measured at 490 nm using a microplate spectrophotometer. Three biological replicates are performed for each cell line where each inhibitor(s)/Cisplatin concentration is assayed in triplicate for each experiment[2].

ETP-46464 is a potent and selective inhibitor for ATR (IC50 = 25 nM).

ATR (ATM- and Rad3-related) is a member of PIKK (phosphatidylinositol 3-kinase-like kinases) that regulates the DNA damage response pathways. It is a DNA damage sensor that is activated upon genotoxic stresses (e.g. ionizing radiation, UV radiation and DNA replication stalling) and phosphorylates its downstream substrates (e.g. p53, BRCA1 and CHEK1).

ETP-46464 abolished the G2/M checkpoint. It caused the presence of micronuclei or completely fragmented nuclei in cells under ionizing radiation. Cells treated simultaneously with hydroxyurea and ETP-46464 exhibited elevated ATM and Chk2 phosphorylation. In U2OS cells, ETP-46464 promoted the breakage of stalked replication forks. [1]

Reference:
1. Toledo LI, Murga M, Zur R et al.  A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations. Nat Struct Mol Biol. 2011 Jun;18(6):721-7.