DNA-PK (IC50: 0.019 μM in MCF7 cells; IC50: 0.17 μM in SW620 cells)
PI-3K (IC50: 0.039 μM in MCF7 cells; IC50: >10 μM in SW620 cells, with only 42% inhibition at 10 μM)
PI-3K isoforms: greatest potency against PI-3Kα and least potency against PI-3Kγ [1]

In vitro activity : KU-0060648 exhibits differential effects on growth inhibition, but is not profoundly cytotoxic in a panel of human cancer cell lines. It inhibits DNA-PK and PI-3K with greater potency in MCF7 than SW620 cell using cell-based assays. Five-day exposure to 1 mM KU-0060648 inhibits cell proliferation by more than 95% in MCF7 cells but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increases the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, confirming that enhanced cytotoxicity of the topoisomerase II poisons etoposide and doxorubicin is due to DNA-PK inhibition.

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KU-0060648 inhibited cellular DNA-PK autophosphorylation with IC50 of 0.019 μM in MCF7 cells and 0.17 μM in SW620 cells.
It inhibited PI-3K-mediated AKT phosphorylation with IC50 of 0.039 μM in MCF7 cells and >10 μM in SW620 cells (only 42% inhibition at 10 μM).
In a 5-day continuous exposure, 1 μM KU-0060648 inhibited cell proliferation by >95% in MCF7 cells, 55% in SW620 cells, and >50% in all tested cell lines (LoVo, T47D, MDA-MB-231).
GI50 values: SW620 0.95 μM, LoVo 0.21 μM, MCF7 0.27 μM, T47D 0.41 μM, MDA-MB-231 1 μM.
After 16 h exposure to 1 μM KU-0060648, cell survival was ~80% in all lines except MDA-MB-231 (41%).
In clonogenic assays, KU-0060648 (1 μM) enhanced etoposide cytotoxicity >100-fold in SW620, 9-fold in MCF7, and >100-fold in LoVo? (Table 1: SW620 etoposide fold enhancement 105±16; MCF7 8.9±4.9; LoVo 6.6±2.6; T47D 4.1±0.6; MDA-MB-231 3.6±1.2).
Enhanced doxorubicin cytotoxicity: >100-fold in MCF7, 12-fold in SW620, 73-fold in LoVo, 58-fold in T47D, 3.8-fold in MDA-MB-231.
In DNA-PKcs-deficient V3 cells vs proficient V3-YAC cells, chemosensitisation was largely DNA-PK-dependent: KU-0060648 enhanced etoposide cytotoxicity >13-fold in V3-YAC but only 2.5-fold in V3.
In M059J (deficient) vs M059-Fus-1 (proficient), KU-0060648 enhanced doxorubicin cytotoxicity up to 32-fold in M059-Fus-1 but only 1.4-fold in M059J.
KU-0060648 alone caused <15% reduction in viability in V3/V3-YAC and <5% in M059J/M059-Fus-1.
PI-3K activity (AKT phosphorylation) was comparable between M059J and M059-Fus-1 and similarly inhibited by KU-0060648 and ZSTK474 [1]

In mice bearing SW620 and MCF7 xenografts, KU-0060648 concentrations that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor at nontoxic doses for at least 4 hours. KU-0060648 alone delayed the MCF7 xenografts growth and increased etoposide-induced tumor growth delay in both in MCF7 and SW620 xenografts by up to 4.5 folds, without causing etoposide toxicity to unacceptable levels

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In MCF7 xenograft-bearing mice, KU-0060648 alone (10 mg/kg i.p. twice daily for 14 days) caused a median tumour growth delay of 30 days (time to RTV4 = 40 days, mean 43.3±3.6 days, P=0.0467).
Etoposide phosphate alone (11.35 mg/kg i.p. daily x5) caused a median growth delay of 38 days (time to RTV4=48 days, mean 31.7±8.6 days, P=0.0001).
Combination of KU-0060648 (once daily? Actually twice daily? Figure 4A: KU-0060648 twice daily x14? But text says "once daily for 14 days" for MCF7 combination? Clarify: In MCF7, combination used KU-0060648 once daily? Text: "For combinations, KU-0060648 was administered i.p. once or twice daily for 5 days (SW620) or once daily for 14 days (MCF7)". So once daily for MCF7 combination. However Figure 4A legend says KU-0060648 alone twice daily x14, but combination? Need to be precise. Extract from text: "combination of treatments caused a median growth delay of 55 days (time to RTV4=65 days, mean 52±6.8 days, P=0.006)". Toxicity: max body weight loss 3% for single agents, 7% for combination.
In SW620 xenograft model, KU-0060648 alone (10 mg/kg i.p. twice daily? Actually once daily x5? Text: "single agent KU-0060648 10 mg/kg i.p. twice daily for either 5 days" but also "once daily for 5 days" for combination? Figure 4B shows once daily and twice daily dosing. Text: "When KU-0060648 was administered twice daily, a growth delay of a further 2 days". However no statistically significant delays due to rapid tumour growth.
Ex vivo pharmacodynamic assay: In SW620 tumours, KU-0060648 (2.5 or 25 mg/kg i.v.) caused dose-dependent reduction of DNA-PK activity (p53 Ser15 phosphorylation). At 25 mg/kg, nearly 100% inhibition of DNA-PK activity for at least 4 hours, with tumour concentrations sufficient for biological effect [1]

Not described in the provided paper (no cell-free enzyme assay details). [1]

But there are cell-based assays described. The "Cell Assay" field should include detailed procedures for cell-based experiments. Let me extract from Materials and Methods.
Cell culture: All human cells (LoVo, SW620, T47D, MCF7, MDA-MB-231, V3, V3-YAC, M059J, M059-Fus-1) cultured in RPMI 1640 or DMEM with 10% FBS, penicillin (50 U/ml), streptomycin (50 μg/ml), at 37°C, 5% CO2.
Determination of cellular activity against DNA-PK and PI-3K: Cells exposed to KU-0060648 for 1 h then X-irradiated (10 Gy) for DNA-PK autophosphorylation; lysates prepared 30 min later using Phosphosafe reagent. Western blot with anti-pSer2056 DNA-PKcs and total DNA-PKcs. For PI-3K activity: cells exposed to KU-0060648 for 1 h then treated with IGF-1 (50 ng/ml) for 30 min; western blot for pSer473 AKT and total AKT. Densitometry performed; IC50 calculated from sigmoidal dose-response curve.
Cytotoxicity and growth inhibition: Clonogenic assays – cells exposed to etoposide or doxorubicin with/without KU-0060648 (1 μM) for 16 h, then seeded in drug-free medium, colonies stained after 10-14 days and counted. Growth inhibition (5-day continuous exposure) determined by SRB assay; GI50 calculated.
Western blot details: Tris-Acetate 3-8% gradient gels, transferred to nitrocellulose, probed with primary antibodies (DNA-PKcs, pSer2056 DNA-PKcs, AKT, pSer473 AKT, actin), HRP-linked secondary, ECL detection, LAS-3000 imager [1]

Mice bearing SW620 and MCF7 xenografts

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Animal protocols described:
Plasma pharmacokinetics: Female Balb C mice received KU-0060648 at 10 mg/kg i.v., p.o., or i.p. Formulation: equimolar phosphoric acid in saline, final pH 5. Mice killed at intervals up to 360 min; plasma concentrations by LC-MS/MS.
Tissue distribution: Female athymic CD1-nu/nu mice bearing MCF7 or SW620 xenografts (~650 mm³) received KU-0060648 12.5 mg/kg i.v. Killed at 60 or 240 min; tumours excised, homogenized in PBS (1:3 w/v); plasma and tumour concentrations by LC-MS/MS.
Ex vivo pharmacodynamic assay: SW620 tumour-bearing mice received KU-0060648 2.5 or 25 mg/kg i.v. or vehicle. After 1 or 4 h, tumours excised and homogenized; DNA-PK activity measured by ELISA using p53 peptide substrate (Ser15 phosphorylation).
Anti-tumour efficacy: Mice bearing SW620 or MCF7 xenografts (palpable, ~5x5 mm) treated. For SW620: KU-0060648 10 mg/kg i.p. twice daily for 5 days (doses 8 h apart) or once daily for 5 days; etoposide phosphate 11.35 mg/kg i.p. daily x5 (equivalent to 10 mg/kg free etoposide). Combinations: KU-0060648 given immediately prior to etoposide. For MCF7: KU-0060648 10 mg/kg i.p. twice daily for 14 days (single agent) or once daily for 14 days (combination); etoposide same. Tumour volumes measured by caliper, calculated as a²×b/2; relative tumour volume (RTV) used [1]

Plasma pharmacokinetics of KU-0060648 in Balb C mice after 10 mg/kg:
i.v.: Cmax 1.9 μg/ml, Tmax 5 min, AUCinf 242 μg/mlmin, T1/2 102 min, CL 41 ml/kg/min.
p.o.: Cmax 0.9 μg/ml, Tmax 120 min, AUCinf 287 μg/mlmin, T1/2 142 min, CL 35 ml/kg/min, bioavailability 119%.
i.p.: Cmax 0.9 μg/ml, Tmax 30 min, AUCinf 189 μg/mlmin, T1/2 106 min, CL 54 ml/kg/min, bioavailability 78%.
Tissue distribution in tumour-bearing mice after 12.5 mg/kg i.v.:
In MCF7 xenografts: at 60 min – plasma 1.0±0.6 μM, tumour 7.0±3.8 μM; at 120 min – plasma 0.8±0.5 μM, tumour 4.7±1.1 μM.
In SW620 xenografts: at 60 min – plasma 2.1±0.5 μM, tumour 4.9±0.3 μM; at 120 min – plasma 0.7±0.2 μM, tumour 2.9±0.4 μM.
Concentrations >1 μM (sufficient for in vitro chemosensitisation) maintained in tumour for at least 4 hours [1]

Toxicity observed in efficacy studies:
In MCF7 xenograft model, KU-0060648 alone and etoposide alone caused negligible toxicity (max body weight loss 3%). Combination did not cause unacceptable toxicity (max body weight loss 7%).
In SW620 xenograft model, KU-0060648 or etoposide alone caused no significant toxicity (no body weight loss). Combination caused max body weight loss 11% (not unacceptable). [1]

Mol Cancer Ther.2012 Aug;11(8):1789-98.

KU-0060648 is a dual inhibitor of DNA-PK and PI-3K, developed from LY294002 pharmacophore, with >500-fold greater solubility, greater potency against DNA-PK, and selectivity over other PIKK family members (ATM, ATR, mTOR).
It is an ATP-competitive inhibitor.
The compound showed cell-line dependent PI-3K inhibition; lack of PI-3K inhibition in SW620 cells may be due to differential isoform expression (KU-0060648 has greatest potency against PI-3Kα and least against PI-3Kγ).
MCF7 and T47D cells (harboring PIK3CA mutations) were more sensitive to KU-0060648 single-agent growth inhibition than MDA-MB-231 (no mutation).
In vivo, KU-0060648 single agent had antitumour activity in MCF7 xenografts (PIK3CA mutant) but not in SW620 xenografts.
Chemosensitisation by KU-0060648 is primarily due to DNA-PK inhibition, confirmed using isogenically paired DNA-PKcs-proficient and deficient cells [1]

C₂₇H₂₉CL₂N₇O₂S

586.54

881375-00-4

Typically exists as solid at room temperature

O=C(NCC1=CC=C(S(C2=CC(F)=CC(F)=C2)(=O)=O)C=C1)C3=CN4C(C=C3)=NC=C4.Cl

KU-0060648 HCl

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: ~10 mM
Water: N/A
Ethanol: N/A

30% propylene glycol, 5% Tween 80, 65% D5W: 20mg/mL (Please use freshly prepared in vivo formulations for optimal results.)