Olaparib azd2281

Unless otherwise noted, all reagents were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. BODIPY FL and BODIPY 650/665 succinimidyl ester were purchased from Invitrogen (Carlsbad, CA). Olaparib (AZD2281) was purchased from Selleck Chemicals (Houston, TX). 4-[[4-Fluoro-3-(piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one and olaparib-BODIPY FL were synthesized as described elsewhere (40 –42 (link)). HPLC-ESI-MS analyses and HPLC purifications were performed on a Waters (Milford, MA) LC-MS system. For LC-ESI-MS analyses, a Waters XTerra^® C18 5 μm column was used. For preparative runs, an Atlantis^® Prep T3 OBDTM 5 μM column was used. High-resolution ESI mass spectra were obtained on a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform mass spectrometer (FT-ICR-MS) in the Department of Chemistry Instrumentation Facility at the Massachusetts Institute of Technology. Proton nuclear magnetic resonance (¹H NMR) spectra were recorded on a Varian AS-400 (400 MHz) spectrometer. Chemical shifts for protons are reported in parts per million (ppm) and are referenced against the dimethylsulfoxide lock signal (¹H, 2.50 ppm). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), coupling constants (Hz) and integration.

H23 and H522 lung adenocarcinoma cells, as well as SK-MES-1 squamous lung cancer cells were obtained from ATCC (Manassas, VA, USA). They were grown in RPMI 1640 supplemented with glutamine and 10% fetal bovine serum at 37°C in a humidified atmosphere consisting of 5% CO₂ and 95% air. Cell lines were validated by STR profiling at the MD Anderson Cancer Center core facilities and routinely tested for mycoplasma. The PARP inhibitors olaparib (AZD2281) and talazoparib (BMN673) were purchased from Selleckchem (Houston, TX, USA). BMN673 and AZD2281 were dissolved in dimethyl sulfoxide (DMSO) and kept as 10 mmol/L stock solutions in small aliquots at −20°C. The following primary antibodies were used: phosphorylated RAD17 (p-RAD17), p-CHK2, cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), RAD51, γH2AX antibody (Millipore), and p-RPA32 (Bethyl Laboratories, Montgomery, TX, USA). The transfection reagent Lipofectamine 3000 was purchased from Life Technologies (Carlsbad, CA, USA).