NU 7026 (DNA-PK Inhibitor II) is an effective DNA-PK inhibitor (IC50: 0.23 μM, in cell-free assays), 60-fold selective for DNA-PK than PI3K and no inhibition against both ATM and ATR.

DNA-PK:0.23 μM

NU7026（20 mg/kg,i.p.或p.o.）的生物利用度分别为20 和15%.在小鼠体内,NU7026（20 mg/kg, i.v.）具有快速的血浆清除率为0.108/h.

NU7026对V3YAC细胞系中的DNA DSB修复有抑制作用（56%）。在CLL细胞系(I83)和初级CLL-淋巴细胞中,NU7026（<10 μM）与苯丁酸氮芥具有协同的细胞毒活性。在K562细胞中,NU7026（10 μM）增强正定霉素,阿霉素,伊达比星,胺苯吖啶,依托泊苷和米托蒽醌抑制生长的作用,PF50值范围大约为19(对mAMSA)到2(对伊达比星)。NU7026（10 μM）也会增强白血病细胞中依托泊苷的生长抑制作用（PF50：10.53）。NU7026（10 μM）增强K562细胞中依托泊苷诱导的细胞周期G2期阻滞。在CH1人卵巢癌细胞中,NU7026（10 μM）暴露4小时结合3 Gy辐射对明显的电波敏化作用是必需的。NU7026（10 μM）增加I83细胞中苯丁酸氮芥诱导的G(2)/M期阻滞。在I83细胞中,NU7026（10 μM）增强整个细胞周期中苯丁酸氮芥诱导的γH2AX。NU7026（10 μM）增加I83细胞系中苯丁酸氮芥诱导的细胞凋亡。NU7026（55 μM）导致p53缺失的MEFs中明显的端粒融合诱导,并导致p53和连接酶IV双重缺失的MEFs中端粒融合更少。

Mammalian DNA-PK (500 ng/μL) is isolated from HeLa cell nuclear extract after chromatography using Q-Sepharose, S-Sepharose, and Heparin agarose. DNA-PK (250 ng) activity is measured at 30°C, in a final volume of 40 μL, in buffer containing 25 mM HEPES (pH 7.4), 12.5 mM MgCl2, 50 mM KCl, 1 mM DTT, 10% v/v Glycerol, 0.1% w/v NP-40, and 1 mg of the substrate GST-p53N66 (the NH2-terminal 66 amino acid residues of human wild-type p53 fused to glutathione S-transferase) in polypropylene 96-well plates. To the assay mix, varying concentrations of inhibitor (in DMSO at a final concentration of 1% v/v) are added. After 10 min of incubation, ATP is added to give a final concentration of 50 μM, along with a 30-mer double-stranded DNA oligonucleotide (final concentration of 0.5 ng/mL), to initiate the reaction. After 1 h with shaking, 150 μL of PBS are added to the reaction, and 5 μL are then transferred to a 96-well opaque white plate containing 45 μL of PBS per well, where the GSTp53N66 substrate is allowed to bind to the wells for 1 h. To detect the phosphorylation event on the serine 15 residue of p53 elicited by DNA-PK, a p53 phosphoserine-15 antibody is used in a basic ELISA procedure. An antirabbit horseradish peroxidase-conjugated secondary antibody is then used in the ELISA before the addition of chemiluminescence reagent to detect the signal as measured by chemiluminescent counting via a TopCount NXT[1].

NU7026 is dissolved in DMSO and stored, and then diluted with appropriate media before use[2]. I83 cells are plated in RPMI 1640 medium with 10% FBS (1.5×105 cells/mL) and treated with vehicle (DMSO), 5 μM CLB, CLB IC50, 10 μM NU7026, or the combination of both drugs for 0, 6, 24, and 48 h. Cell cycle distribution, apoptosis, DNA-PK phosphorylation, and γH2AX determination are determined, and they are expressed as a percentage of cells in each phase of the cycle. DNA content is analyzed with a FACSCalibur flow cytometer equipped with CellQuest software[2].