Kinase assays

DNA-PK activity was measured at 30℃, 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 in polypropylene 96-well plates. Varying concentrations of inhibitor were added. After 10 min of incubation, ATP was added to give a final concentration of 50 μm, along with a 30-mer double-stranded DNA oligonucleotide to initiate the reaction. After 1 h with shaking, 150 μl of PBS were added to the reaction, and 5 μl were then transferred to a 96-well opaque white plate containing 45 μl of PBS per well, where the GSTp53N66 substrate was 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 was used in a basic ELISA procedure.

Cell lines

CHO cell lines V3 transfected with human DNA-PKcs gene

Preparation method

The solubility of this compound in DMSO is<2.81mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

10 μM

Applications

NU7026 potentiated ionizing radiation cytotoxicity in exponentially growing DNA-PK proficient cells, which can act as a potent radiosensitizer and shows potential as tools for anticancer therapeutic intervention.

Animal models

Female BALB/c mice

Dosage form

four times per day at 100 mg/kg, i.p.

Application

NU7026 shows a significant radiosensitisation effect in BALB/c mice.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

NU 7026 is a selective inhibitor of  DNA-PK with IC50 value of 0.23 μM [1].
DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase and involves in a variety of cellular processes, like, DNA double-strand break (DSB) repair, V(D)J recombination apparatus, chromatin structure and telomere maintenance. It has been shown that increased DNA-PK expression resulted in tumor cells resistance to radio- or chemo- therapy [2].
NU 7026 is a potent DNA-PK inhibitor and often is used combined with PARP-1 inhibitor AG14361 to sensitize tumor cells to radio- or chemo-therapy. When tested with primary PARP-1-/- and cells PARP-1+/+ cells, NU 7026 treatment (<50 μM) sensitized cells to IR-induced cytotoxicity and reduced clonogenic survival by inhibiting DNA-PK [1]. In N87 gastric cancer cells, administration NU 7026 combined with radiation increased DNA double-srand break, cell apoptosis and reduced cell survival through inhibiting DNA-PK [2]. When tested with CH1 human ovarian cancer cells, administration of NU 7026 combined with radiation reduced cell survival rate and clonogenic ability [3].
In female BALB/c mice model with CH1 cells subcutaneous xenograft, administration of NU 7026 orally or intraperitoneally sensitized mice to the radiotherapy with the dose of 100 mg/kg [3].
It has also been reported that NU 7026 is a potent inhibitor of PI-3K, ATM and ATR with IC50 value of 13 μM, >100μM and >100μM, respectively [1].
References:
[1].    Veuger, S.J., et al., Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1. Cancer Res, 2003. 63(18): p. 6008-15.
[2].    Niazi, M.T., et al., Effects of dna-dependent protein kinase inhibition by NU7026 on dna repair and cell survival in irradiated gastric cancer cell line N87. Curr Oncol, 2014. 21(2): p. 91-6.
[3].    Nutley, B.P., et al., Preclinical pharmacokinetics and metabolism of a novel prototype DNA-PK inhibitor NU7026. Br J Cancer, 2005. 93(9): p. 1011-8.