PI3Kα (IC50 = 4 nM); PI3Kβ (IC50 = 0.5 nM); PI3Kγ (IC50 = 0.594 μM);PI3Kδ (IC50 = 0.1 nM); DNA-PK (IC50 = 8.6 nM)
KU-0060648 exhibits differential effects on growth inhibition, but is not profoundly cytotoxic in a panel of human cancer cell lines. When compared to SW620 cells, MCF7 cells exhibit more potent inhibition of DNA-PK and PI-3K. In MCF7 cells, exposure to 1 mM KU-0060648 for five days significantly reduces cell proliferation by more than 95%, but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increases etoposide and doxorubicin's cytotoxicity across a panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, demonstrating that the increased cytotoxicity of the topoisomerase II poisons is caused by DNA-PK inhibition. [1]

KU-0060648 exhibits differential effects on growth inhibition, but is not profoundly cytotoxic in a panel of human cancer cell lines. When compared to SW620 cells, MCF7 cells exhibit more potent inhibition of DNA-PK and PI-3K. In MCF7 cells, exposure to 1 mM KU-0060648 for five days significantly reduces cell proliferation by more than 95%, but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increases etoposide and doxorubicin's cytotoxicity across a panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, demonstrating that the increased cytotoxicity of the topoisomerase II poisons is caused by DNA-PK inhibition. [1]

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KU-0060648 inhibited cellular DNA-PK auto-phosphorylation at Ser2056 in a concentration-dependent manner, with IC₅₀ values of 0.02 µM in MCF7 breast cancer cells and 0.2 µM in SW620 colon cancer cells.[1]
It also inhibited PI-3K-mediated AKT phosphorylation at Ser473, with an IC₅₀ of 0.04 µM in MCF7 cells. However, its activity against PI-3K in SW620 cells was minimal (IC₅₀ >10 µM), suggesting cell line-dependent inhibition.[1]
Continuous exposure for 5 days inhibited cell proliferation across a panel of human breast (MCF7, T47D, MDA-MB-231) and colon (LoVo, SW620) cancer cell lines. The growth inhibition (GI₅₀) values ranged from 0.21 µM (LoVo) to 1 µM (MDA-MB-231 and SW620).[1]
KU-0060648 (1 µM, 16-hour exposure) alone showed low cytotoxicity (survival rates >80% in most lines, except MDA-MB-231 at 41%).[1]
It markedly enhanced the cytotoxicity of topoisomerase II poisons etoposide and doxorubicin. In DNA-PKcs-proficient cell lines (V3-YAC, M059-Fus-1), the compound enhanced etoposide cytotoxicity by 4- to 13-fold and doxorubicin cytotoxicity by up to 32-fold. This enhancement was minimal in DNA-PKcs-deficient cell lines (V3, M059J), confirming that chemosensitization is primarily due to DNA-PK inhibition.[1]
In a panel of human cancer cell lines, KU-0060648 (1 µM) enhanced etoposide cytotoxicity by up to 105-fold (in SW620 cells) and doxorubicin cytotoxicity by up to 107-fold (in MCF7 cells).[1]

KU-0060648 increases the anti-tumour activity of etoposide in both MCF7 and SW620 xenograft models, and has single-agent activity in the MCF7 xenograft model. [1]

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In vivo, intraperitoneal (i.p.) injection of KU-0060648 significantly inhibited HepG2 xenograft growth in nude mice. AKT-mTOR activation was also inhibited in xenografted tumors. Finally, we showed that DNA-PKcs expression was significantly upregulated in human HCC tissues.[2]

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In mice bearing MCF7 breast cancer xenografts, treatment with KU-0060648 alone (10 mg/kg i.p., twice daily for 14 days) caused a median tumor growth delay of 30 days compared to control. No significant single-agent activity was observed in SW620 colon cancer xenografts.[1]
Combination of KU-0060648 with etoposide phosphate significantly enhanced anti-tumor activity. In MCF7 xenografts, the combination caused a median growth delay of 55 days, compared to 38 days for etoposide alone and 30 days for KU-0060648 alone.[1]
In SW620 xenografts, a trend towards increased activity was observed when KU-0060648 was combined with etoposide, particularly with twice-daily dosing of the inhibitor.[1]

Determination of cellular activity of KU-0060648 against DNA-PK and PI-3K[1]
DNA-PK autophosphorylation was determined in cells exposed to a range of concentrations of KU-0060648 for 1 hr prior to X-irradiation (10 Gy). Cell lysates were prepared 30 minutes later using Phosphosafe Extraction Reagent according to the manufacturer’s instructions. Levels of DNA-PKcs auto-phosphorylation at Ser2056 relative to un-phosphorylated DNA-PKcs were determined by western blotting. To determine PI-3K activity, cells were exposed to a range of concentrations of KU-0060648 for 1 hr prior to a 30-minute treatment with 50 ng/ml insulin-like growth factor-1. The levels of PI-3K-dependent AKT phosphorylation (Ser473) relative to un-phosphorylated AKT were determined by western blotting.

Cytotoxicity and Growth inhibition Studies[1]
Cytotoxicity was measure by clonogenic assays. Cells grown in 6-well plates were exposed to etoposide or doxorubicin, with or without KU-0060648 (1 μM) for 16 hours, prior to harvesting and seeding into 10 cm diameter Petri dishes, in drug-free medium. Colonies were stained with crystal violet after 10 to 14 days and counted with an automated colony counter. Cell growth inhibition following 5-day continuous exposure to KU-0060648 was determined by SRB assay, as described previously. The GI50 is the concentration causing 50% cell growth inhibition.

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DNA-PK and PI-3K Cellular Activity Assay: Cells were exposed to a range of concentrations of KU-0060648 for 1 hour. For DNA-PK inhibition assessment, cells were then irradiated (10 Gy X-rays) and lysed 30 minutes later. For PI-3K inhibition assessment, cells were treated with insulin-like growth factor-1 (50 ng/ml) for 30 minutes after the 1-hour pre-incubation. Cell lysates were prepared, and levels of phosphorylated DNA-PKcs (Ser2056) or phosphorylated AKT (Ser473) relative to total protein were determined by Western blotting using specific antibodies. Densitometry analysis was performed to calculate the percentage inhibition and IC₅₀ values.[1]
Clonogenic Survival Assay: To assess cytotoxicity and chemosensitization, cells grown in plates were exposed to etoposide or doxorubicin, with or without KU-0060648 (1 µM), for 16 hours. Cells were then harvested, seeded into Petri dishes in drug-free medium, and allowed to form colonies for 10-14 days. Colonies were stained and counted.[1]
Growth Inhibition (SRB) Assay: Cells were continuously exposed to KU-0060648 for 5 days. Cell density was then measured using the Sulforhodamine B (SRB) colorimetric assay to determine growth inhibition (GI₅₀).[1]

human-tumor SW620 or MCF7 xenograft models
\n10 mg/kg, twice daily
\ni.p.

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\nKU-0060648 plasma pharmacokinetics following different routes of administration[1]
\nAll in vivo experiments were reviewed and approved by the relevant institutional animal welfare committees and performed according to national law. We determined the plasma pharmacokinetics of KU-0060648 following administration intravenously (i.v.), intra-peritoneally (i.p.) or orally (p.o.) at 10 mg/kg to female Balb C mice. KU-0060648 was formulated in a vehicle of equimolar phosphoric acid, made up to volume with sterile saline and at final pH 5. Mice were killed at intervals up to 360 minutes after KU-0060648 administration and plasma concentrations of KU-0060648 were determined by LC-MS/MS analysis, as previously described.\nKU-0060648 distribution to tumour xenografts[1]
\nFemale athymic mice were maintained and handled in isolators under specific pathogen free conditions for tissue distribution and efficacy studies. KU-0060648 (12.5 mg/kg i.v.) was administered to MCF7 or SW620 tumour-bearing mice (650 mm3), which were killed 60 or 240 minutes later. Tumours were excised and homogenised in PBS (1:3 w/v), using a stirrer mascercarator homogenizer , in 10 second bursts, on ice. Plasma and tumour KU-0060648 concentrations were determined by LC-MS/MS analysis, as previously described.\n

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\nDNA-PK ex vivo pharmacodynamic assay[1]
\nKU-0060648 at 2.5 or 25 mg/kg or vehicle alone was administered to SW620 tumour-bearing mice i.v.. After 1 or 4 hours, animals were killed and tumours were excised and homogenised. DNA-PK activity within tumour homogenates was determined by measuring the DNA-PK-dependent phosphorylation of a p53 peptide substrate (Ser15), using an ELISA assay, as described previously.\n

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\nAnti-tumour efficacy study[1]
\nMice bearing SW620 or MCF7 xenografts s.c. (n = 5 per group) were treated when tumours were palpable (approx 5 mm × 5 mm, 8-10 days post implantation). Animals received normal saline i.p. once daily (control), single agent KU-0060648 10 mg/kg i.p. twice daily for either 5 days (2 × d × 5) in SW620 tumour-bearing, or 14 days (2 × d × 14) in MCF7 tumour bearing mice with doses on each day 8 hours apart, or etoposide phosphate once daily i.p. (11.35 mg/kg in saline, equivalent to 10 mg/kg free etoposide, i.p., d × 5). For combinations, KU-0060648 was administered i.p. once or twice daily for 5 days (SW620) or once daily for 14 days (MCF7), with the first dose immediately prior to etoposide phosphate.\n

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\nIn vivo anti-tumor efficiency assay[2]
\nA significant amount of HepG2 cells (5 millions/mice) were injected subcutaneously into the right flanks of female nude mice (6-8 weeks old). When tumors reached around 100 mm3, mice were randomized into three groups with 12 mice per group: vehicle control (saline), 10 mg/kg of KU-0060648 (intraperitoneal injection or i.p., daily, for 21 days), and 50 mg/kg of KU-0060648 (i.p., daily, for 21 days). The injection was started when the tumors were established (volumes around 100 mm3). Tumor volumes, recorded every week, were calculated through the established formula: Volume (mm3) = (d2 × D)/2, in which d and D were the shortest and the longest diameter, respectively. Two weeks after initial KU-0060648 administration, xenografted tumors of two mice per group were isolated, and were subjected to Western blotting and immunohistochemistry (IHC) staining assays. Humane endpoints were applied to minimize suffering. Five weeks after initial KU-0060648 administration, HepG2 xenografts were separated through surgery and weighted.

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\nPharmacokinetic Study: KU-0060648 was formulated in a vehicle of equimolar phosphoric acid, adjusted to pH 5 with sterile saline. Female Balb C mice received a single dose of 10 mg/kg via intravenous (i.v.), intraperitoneal (i.p.), or oral (p.o.) routes. Blood was collected at various time points up to 360 minutes for plasma concentration analysis.[1]
\nTumor Distribution Study: Female athymic mice bearing MCF7 or SW620 subcutaneous xenografts (~650 mm³) received a single i.v. dose of KU-0060648 (12.5 mg/kg). Mice were sacrificed at 60 or 240 minutes post-dose. Tumors were excised, homogenized in PBS, and analyzed for drug concentration alongside plasma.[1]
\nEx vivo DNA-PK Activity Assay: SW620 tumor-bearing mice received a single i.v. dose of KU-0060648 (2.5 or 25 mg/kg) or vehicle. Mice were sacrificed at 1 or 4 hours post-dose. Tumors were excised and homogenized. DNA-PK activity in the homogenate was determined by measuring the DNA-PK-dependent phosphorylation of a p53 peptide substrate (Ser15) using an ELISA-based assay.[1]
\nEfficacy Study in Xenografts: Mice bearing established SW620 or MCF7 subcutaneous xenografts were treated when tumors reached approximately 5x5 mm.
\nFor SW620 model: Animals received vehicle (control), KU-0060648 alone (10 mg/kg i.p., once or twice daily for 5 days), etoposide phosphate alone (11.35 mg/kg i.p., once daily for 5 days), or the combination.
\nFor MCF7 model: Animals received vehicle, KU-0060648 alone (10 mg/kg i.p., twice daily for 14 days), etoposide phosphate alone (11.35 mg/kg i.p., once daily for 5 days), or the combination, with the first dose of KU-0060648 given immediately prior to etoposide.[1]

Table 2 lists the plasma pharmacokinetic parameters determined after administration of 10 mg/kg KU-0060648 to Balb/C mice via different routes. The bioavailability of KU-0060648 after oral administration was ≥100%. The pharmacokinetic parameters of KU-0060648 after intraperitoneal administration were similar to those after intravenous administration, with a bioavailability of 78%. [1]

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Tissue distribution of KU-0060648 in MCF7 and SW620 xenograft mice after intravenous administration [1]
After intravenous administration of KU-0060648 (12.5 mg/kg) to mice carrying MCF7 or SW620 xenograft tumors, KU-0060648 was widely distributed in tumor tissue and remained in tumor tissue after plasma clearance (Table 2). The concentration of KU-0060648 in the tumor was maintained above 1 μM (which produces chemosensitizing effect in vitro) for at least 4 hours. Following a single intravenous injection of 10 mg/kg in mice, the peak plasma concentration (Cmax) of KU-0060648 was 19 µg/ml (5 min), the elimination half-life (T₁/₂) was 102 min, and the clearance (CL) was 41 ml/kg/min. [1] The bioavailability of oral administration (10 mg/kg po) was close to 100% (119%), with a Tmax of 120 min and a T₁/₂ of 142 min. [1] The bioavailability of intraperitoneal administration (10 mg/kg ip) was 78%, with a Tmax of 30 min and a half-life of 106 min. [1] Following intravenous administration of 12.5 mg/kg in tumor-bearing mice, KU-0060648 was widely distributed in MCF7 and SW620 xenograft tumors. After plasma clearance, the tumor concentration was maintained at at least 1 µM (the effective concentration for in vitro chemosensitization). [1]

In the MCF7 xenograft efficacy study, the toxicity of KU-0060648 monotherapy was negligible (maximum weight loss of 3%). When used in combination with etoposide phosphate, the maximum weight loss was 7%, which was considered acceptable. [1]
In the SW620 xenograft study, neither KU-0060648 monotherapy nor etoposide phosphate monotherapy caused significant weight loss. When used in combination, the maximum weight loss was 11%. [1]

[1]. Chemosensitization of cancer cells by KU-0060648, a dual inhibitor of DNA-PK and PI-3K. Mol Cancer Ther. 2012 Aug;11(8):1789-98.

[2]. KU-0060648 inhibits hepatocellular carcinoma cells through DNA-PKcs-dependent and DNA-PKcs-independent mechanisms. Oncotarget. 2016 Mar 29;7(13):17047-59.

2-(4-ethyl-1-piperazinyl)-N-[4-[2-(4-morpholinyl)-4-oxo-1-benzopyran-8-yl]-1-dibenzothiophene]acetamide is a dibenzothiophene compound. KU-0060648 is a dual ATP-competitive DNA-PK and PI-3K inhibitor developed from the pharmacophore LY294002. It is selective for other PIKK family members such as ATM, ATR and mTOR. [1] Its chemosensitizing effect on topoisomerase II inhibitors (etoposide, doxorubicin) is mainly attributed to the inhibition of DNA-PK, thereby weakening the non-homologous end joining (NHEJ) repair pathway of DNA double-strand breaks. [1]
Single-agent antitumor activity was observed in MCF7 xenografts (carrying PIK3CA mutations), but not in SW620 xenografts, suggesting that tumors with PI-3K pathway activation may be sensitive to it. [1]
This study provides a validation of the principle for a treatment strategy of dual inhibition of DNA-PK and PI-3K. [1]

C33H34N4O4S

582.7125

582.23

C, 68.02; H, 5.88; N, 9.61; O, 10.98; S, 5.50

881375-00-4

881375-00-4

11964036

Off-white to brown solid powder

1.3±0.1 g/cm3

819.9±65.0 °C at 760 mmHg

449.7±34.3 °C

0.0±3.0 mmHg at 25°C

1.694

5.56

1

8

6

42

1010

0

O=C1C2C=CC=C(C=2OC(N2CCOCC2)=C1)C1C2SC3C(C=2C(NC(CN2CCN(CC)CC2)=O)=CC=1)=CC=CC=3

AATCBLYHOUOCTO-UHFFFAOYSA-N

InChI=1S/C33H34N4O4S/c1-2-35-12-14-36(15-13-35)21-29(39)34-26-11-10-23(33-31(26)25-6-3-4-9-28(25)42-33)22-7-5-8-24-27(38)20-30(41-32(22)24)37-16-18-40-19-17-37/h3-11,20H,2,12-19,21H2,1H3,(H,34,39)

2-(4-ethylpiperazin-1-yl)-N-[4-(2-morpholin-4-yl-4-oxochromen-8-yl)dibenzothiophen-1-yl]acetamide

KU0060648; KU 0060648; LM6DZS6PYA; 2-(4-ethylpiperazin-1-yl)-N-[4-(2-morpholin-4-yl-4-oxochromen-8-yl)dibenzothiophen-1-yl]acetamide; CHEMBL1086377; 2-(4-ethylpiperazin-1-yl)-N-(4-(2-morpholino-4-oxo-4H-chromen-8-yl)dibenzo[b,d]thiophen-1-yl)acetamide; KU-0060648

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: 2~2.8 mg/mL (3.4~4.8 mM)

Solubility in Formulation 1: ≥ 0.28 mg/mL (0.48 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 2.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.28 mg/mL (0.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 2.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 0.28 mg/mL (0.48 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 2.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 20 mg/mL

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