DNA-PK (IC50 = 14 nM); mTOR (IC50 = 1.7 μM); PI3K (IC50 = 5.0 μM); CRISPR/Cas9
NU7441 targets DNA-dependent protein kinase (DNA-PK), specifically the catalytic subunit DNA-PKcs. [2]

NU7441 increases the persistence of γH2AX foci after ionizing radiation–induced or etoposide-induced DNA damage. In both SW620 and LoVo cells, NU7441 (0.5 μM or 1 μM) significantly increases G2-M accumulation brought on by ionizing radiation, etoposide, and doxorubicin. [2] In addition to making DNA double-strand breaks more persistent, NU7441 also slightly reduces the activity of homologous recombination in DNA-PK-sufficient M059-Fus-1 and DNA-PK-sufficient M059 J human tumor cells.[3] In cells that express polymerase as well as those that lack it, NU7441 inhibits UV-induced RPA p34 hyperphosphorylation in a dose-dependent manner. [4] In chronic lymphocytic leukemia cells, NU7441 causes fludarabine-induced H2AX foci to accumulate and, in turn, reduces fludarabine-induced cell death. [5] In addition, DNA-PKcs autophosphorylation and repair brought on by mitoxantrone in cells of chronic lymphocytic leukemia are inhibited by NU7441.[6] In the aftermath of Cas9-mediated DNA cleavage, it decreases the frequency of NHEJ while increasing the rate of HDR[7].

---

NU7441 inhibited the proliferation of HepG2 liver cancer cells in a dose- and time-dependent manner as measured by CCK-8 assay; maximal inhibitory effect was observed at 10 μM after 72 h of treatment. [2]
Western blot analysis showed that NU7441 did not alter the total protein levels of DNA-PKcs, Ku70, or Ku80, but sharply decreased the amount of phosphorylated DNA-PKcs at serine 2056 (pDNA-PKcs (S2056)) in HepG2 cells. [2]
Compared with another DNA-PK inhibitor NU7026, NU7441 more potently reduced pDNA-PKcs (S2056) expression under the same conditions (4 Gy ⁶⁰Coγ radiation). [2]
Immunofluorescence staining revealed that NU7441 combined with ionizing radiation (IR) increased the number of γH2AX foci in HepG2 cells; the decrease in foci number over time was slower in the NU7441+IR group than in the IR-alone group (P<0.05). [2]
Neutral single-cell gel electrophoresis (comet assay) demonstrated that DNA damage (tail moment) was higher in HepG2 cells treated with NU7441+IR compared to IR alone, and the DNA repair rate was slower in the presence of NU7441 (P<0.05). [2]
Flow cytometry analysis showed that NU7441 alone did not significantly alter cell cycle distribution, but NU7441 combined with IR induced a dramatic increase in the percentage of cells arrested at G2/M phase compared to IR alone (P<0.05). [2]

In mice bearing SW620 xenografts, administration of NU7441 intraperitoneally at a dose of 10 mg/kg for at least 4 hours demonstrates nontoxicity and increases etoposide-induced tumor growth delay by a factor of two.[2]

---

NU7441 (also called KU-57788) is a highly potent, selective and ATP-competitive DNA-PK inhibitor with IC50 of 14 nM. It also inhibits PI3K with IC50 value of 5 μM in cell-free assays.

---

NU7441 was evaluated in cell-based assays without isolated enzyme experiments. No enzyme assay protocol is described in the paper. [2]

HepG2 cells (4000 per well) are cultured in a 96-well plate for 24 h. The culture media are then supplemented with concentrations of 0.1 µM, 1 µM, 5 µM, and 10 µM of KU-57788 once the cells have finished adhering. 10% CCK-8 solution is added to the culture media after KU-57788 treatment has been going on for 12 hours.Incubation is then continued for another 2 hours. A spectrometer is used to calculate the OD450 values, and the results are then analyzed to calculate the rate of cell growth.

---

NU7441 was tested in the following cell-based assays:
Cell proliferation assay (CCK-8): HepG2 cells (4000 per well) were cultured in 96-well plates for 24 h, then treated with 0.1, 1, 5, or 10 μM NU7441 for 12–72 h. After treatment, 10% CCK-8 solution was added and incubated for 2 h, and OD450 was measured to determine cell growth. [2]
Western blot: Total protein (100 μg) isolated from cells was separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked with 5% milk, incubated with primary antibodies (anti-DNA-PKcs, anti-pDNA-PKcs (S2056), anti-Ku70, anti-Ku80, anti-β-actin, 1:1000 dilution) overnight at 4 °C, then with secondary antibody (1:4000) for 1 h at room temperature. Signals were developed using enhanced chemiluminescence. [2]
Immunofluorescence staining for γH2AX: HepG2 cells grown on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100 for 20 min, blocked with 1% BSA for 30 min, incubated with anti-γH2AX (S139) antibody (1:500) overnight at 4 °C, washed, incubated with fluorescence-conjugated secondary antibody for 1 h at room temperature, and counterstained with DAPI (1 μg/mL). Images were captured under a confocal microscope. [2]
Neutral single-cell gel electrophoresis (comet assay): Slides were coated with 1% normal melting agarose, then a mixture of ~1000 cells and 0.65% low-melting agarose was added. After lysis in pre-chilled neutral lysis buffer for 2 h at 4 °C, slides were washed, denatured in 1% TBE buffer for 40 min at 4 °C, subjected to electrophoresis at 20 V for 20 min, stained with propidium iodide (2 μg/mL) for 15 min, and imaged under a fluorescence microscope. Tail moment was analyzed by CASP software (≥60 cells per group). [2]
Cell cycle analysis by flow cytometry: HepG2 cells at log phase were treated with 5 μM NU7441 for 12 h, with or without 4 Gy ⁶⁰Coγ radiation (NU7441 added 1 h before radiation). Cells were collected, fixed with 70% ethanol at -20 °C for ≥12 h, digested with RNase (50 μg/mL) for 30 min at 37 °C, stained with propidium iodide, and analyzed by flow cytometry. [2]

Female rude mice bearing SW620 xenografts
10 mg/kg
Intraperitoneally administrated

---

[1]. Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. Bioorg Med Chem Lett. 2004 Dec 20;14(24):6083-7.

[2]. NU7441 Enhances the Radiosensitivity of Liver Cancer Cells. Cell Physiol Biochem. 2016;38(5):1897-905.

[3]. Discovery of potent chromen-4-one inhibitors of the DNA-dependent protein kinase (DNA-PK) using a small-molecule library approach. J Med Chem. 2005 Dec 1;48(24):7829-46.

[4]. The acetyl-lysine binding site of bromodomain-containing protein 4 (BRD4) interacts with diverse kinase inhibitors. ACS Chem Biol. 2014 Feb 25.

[5]. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med. 2015 Aug 27;7:93.

8-(4-Dibenzothiophene)-2-(4-morpholino)-1-benzopyran-4-one is a member of the dibenzothiophene class of compounds.

---

NU7441 (also known as KU57788) is a specific DNA-PK inhibitor that blocks non-homologous end joining (NHEJ), a major DNA double-strand break repair pathway. [2]
Liver cancer cells have a strong capacity to repair radiation-induced DNA damage, which contributes to recurrence after radiotherapy; targeting DNA-PK with NU7441 enhances radiosensitization. [2]
In this study, NU7441 increased radiation-induced DNA damage, prolonged γH2AX foci persistence, delayed DNA repair, induced G2/M cell cycle arrest, and promoted apoptosis in HepG2 cells, suggesting its potential as a radiotherapy sensitizer for liver cancer treatment. [2]

C25H19NO3S

413.49

413.108

C, 72.62; H, 4.63; N, 3.39; O, 11.61; S, 7.75

503468-95-9

11327430

Off-white to beige solid powder

1.4±0.1 g/cm3

646.9±55.0 °C at 760 mmHg

246.28° C

345.0±31.5 °C

0.0±1.9 mmHg at 25°C

1.732

5.98

0

5

2

30

690

0

S1C2=C([H])C([H])=C([H])C([H])=C2C2C([H])=C([H])C([H])=C(C1=2)C1=C([H])C([H])=C([H])C2C(C([H])=C(N3C([H])([H])C([H])([H])OC([H])([H])C3([H])[H])OC1=2)=O

JAMULYFATHSZJM-UHFFFAOYSA-N

InChI=1S/C25H19NO3S/c27-21-15-23(26-11-13-28-14-12-26)29-24-17(6-3-9-20(21)24)19-8-4-7-18-16-5-1-2-10-22(16)30-25(18)19/h1-10,15H,11-14H2

8-dibenzothiophen-4-yl-2-morpholin-4-ylchromen-4-one

NU7441; NU 7441; NU-7441; KU 57788; KU57788; KU-57788

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~4 mg/mL warming (~9.7 mM)
Water: <1 mg/mL (slightly soluble or insoluble)
Ethanol: <1 mg/mL (slightly soluble or insoluble)

Solubility in Formulation 1: ≥ 1.43 mg/mL (3.46 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

AO:AO 4%DMSO+30%PEG 300+5%Tween 80+ddH2O: 1.0mg/mL

---

 (Please use freshly prepared in vivo formulations for optimal results.)