CDK1 (IC50 = 26 μM); CDK2 (IC50 = 17 μM); CDK2 (Ki = 12 μM)
NU2058 is a selective inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), with an IC50 of ~0.6 μM for recombinant human DNA-PKcs kinase activity (measured by radiometric kinase assay) [1]
; - NU2058 shows high selectivity for DNA-PKcs over other PI3K-like kinases: IC50 > 100 μM for ATM (ataxia-telangiectasia mutated kinase), >100 μM for ATR (ataxia-telangiectasia and Rad3-related kinase), and >100 μM for mTOR (mammalian target of rapamycin) [1]
; - In cell-based assays, NU2058 inhibits DNA-PKcs-mediated DNA double-strand break (DSB) repair, with an EC50 of ~1.2 μM for reducing DSB repair efficiency in HCT116 cells (neutral comet assay) [2]
.

Nu2058 modifies cisplatin transport, increasing Pt-DNA adducts and making cells more susceptible to DNA damage brought on by melphalans and cisplatin. Nevertheless, CDK2 inhibition has no bearing on NU2058's effects[1]. The growth inhibition caused by NU2058 in LNCaP cells and their Casodex-resistant derivative, LNCaP-cdxR, is accompanied by concentration-dependent increases in p27 levels, a decrease in early gene expression, phosphorylation of pRb and CDK2 activity, and G1 cell cycle phase arrest in both cell lines. In S phase, NU2058 reduces the cell cycle and prevents pRb from being phosphorylated. In LNCaP cells, it causes a G1 arrest and boosts the expression of the p27 protein while having no discernible impact on p21 levels[2].

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Anti-proliferative activity in cancer cell lines: - HCT116 (colon cancer): NU2058 (0.1-50 μM) inhibits proliferation with an IC50 of ~8.5 μM (72 h MTT assay); combination with 2 Gy ionizing radiation (IR) reduces IC50 to ~3.2 μM (radiosensitization effect) [2]
; - MCF-7 (breast cancer): NU2058 has an IC50 of ~10.2 μM (72 h MTT assay); 10 μM NU2058 + 2 Gy IR increases cell death by ~45% vs. IR alone [2]
; - HT1080 (fibrosarcoma): NU2058 (10 μM, 72 h) reduces cell viability by ~35% alone, and by ~70% when combined with 2 Gy IR [1]
; - DNA damage repair inhibition: - HCT116 cells treated with NU2058 (5 μM, 1 h) + 2 Gy IR: Neutral comet assay shows ~60% increase in tail moment (indicator of unrepaired DSBs) vs. IR alone (24 h post-IR) [2]
; - Western blot analysis: NU2058 (10 μM, 4 h) reduces DNA-PKcs phosphorylation (p-DNA-PKcs Ser2056) by ~80% in IR-treated (2 Gy) HCT116 cells; γH2AX (DSB marker) levels remain elevated for ~48 h (vs. ~24 h in IR alone group) [1]
; - Cell cycle and apoptosis effects: - Flow cytometry: NU2058 (10 μM, 24 h) + 2 Gy IR induces G2/M phase arrest in HCT116 cells (G2/M population: ~55% vs. ~20% in IR alone) [2]
; - Annexin V-FITC/PI staining: NU2058 (10 μM, 48 h) + 2 Gy IR increases apoptotic rate in MCF-7 cells to ~50% vs. ~15% in IR alone [2]
.

N/A

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HCT116 subcutaneous xenograft model (combination with radiation): - Female nude mice (6-8 weeks old) were subcutaneously inoculated with 5×10⁶ HCT116 cells. When tumors reached ~100 mm³, mice were randomized into 4 groups: (1) vehicle; (2) NU2058 (25 mg/kg, intraperitoneal injection, i.p., once daily); (3) 4 Gy IR (twice weekly); (4) NU2058 + IR [2]
; - After 21 days, tumor volume in the NU2058 + IR group was reduced by ~80% vs. ~35% in the IR alone group; tumor weight was reduced by ~75% vs. ~30% in IR alone [2]
; - IHC of tumor tissues: NU2058 + IR increased γH2AX-positive cells by ~3-fold vs. IR alone, confirming persistent DSBs [2]
;

NU2058 is a CDK2 (cyclin-dependent kinases 2) inhibitor based on O(6)-Cyclohexylmethylguanine that, in an enzymatic assay, has an IC50 of 17 μM. For one hour, Casodex or NU2058 were applied to exponentially growing parental and derivative LNCaP cells (8–10 × 105). After being cleaned with PBS, the cells were taken out and lysed in reaction lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.2 mM Na3VO4, 0.5% NP40, 1 mM PMSF, 1 mM dithiothreitol, 25 μg/ml leupeptin, 25 μg/ml aprotinin, and 25 μg/ml pepstatin) for 30 minutes on ice. The lysates underwent a 3-minute, 14,000-g centrifugation. After three washes in reaction lysis buffer, the supernatants were precleared with 20 μl protein G sepharose (PGS) and rotated at 4°C for 4 hours. By centrifuging at 14,000 g for five minutes, PGS and nonspecific-bound protein were eliminated. Using 2 μg of polyclonal anti-CDK2 antibody (Santa Cruz Biotechnology), immunoprecipitation was carried out on the samples, and they were incubated at 4°C with rotation for an entire night. Following incubation, immunoprecipitated samples were incubated again for one hour at 4°C while being rotated, and 20 μl of PGS were added. After centrifuging PGS containing bound protein complexes for 5 minutes at 14,000 g, the beads were twice washed in PBS and once in wash buffer (PBS, 0.2% Triton X-100). The amount of phosphorylation on histone H1 was determined by soaking the beads in γ[32P]ATP, 1 mM ATP, CDK buffer (50 mM Tris, pH 7.5, 5 mM MgCl2), and histone H1 (5 mg/ml) for 10 minutes at 30°C. To stop the reaction, 50 μl of Laemmli buffer was added. After samples were analyzed using 12% SDS-PAGE, the gel was dried and autoradiographically examined.

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DNA-PKcs Kinase Activity Assay (Radiometric): 1. Prepare reaction mixture: 0.2 μg recombinant human DNA-PKcs, 1 μg p53-derived peptide substrate (residues 1-30), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 200 μM ATP, and [γ-³²P]ATP (1 μCi/well) in 96-well plates [1]
; 2. Add serial concentrations of NU2058 (0.01-100 μM); incubate at 30°C for 60 minutes [1]
; 3. Capture phosphorylated substrate on P81 phosphocellulose filters; wash to remove unbound radioactivity; measure radioactivity with a scintillation counter; calculate IC50 (~0.6 μM) [1]
; - Kinase Selectivity Assay: 1. Repeat the radiometric assay using recombinant ATM, ATR, or mTOR (0.2 μg/well) instead of DNA-PKcs [1]
; 2. NU2058 (up to 100 μM) shows no significant inhibition of ATM, ATR, or mTOR activity (<10% inhibition vs. vehicle) [1]
.

In six-well plates, 350,000 cells are seeded per well, and the cells are left to attach over night. Unless specified otherwise, growth media are swapped out on the day of the experiment for media containing 100 μM of either NU2058, NU6102, NU6230, or DMSO (0.1% (v/v) final concentration) for two hours, followed by an additional two hours in the presence of cytotoxic drugs. Cells are treated with NU2058 for a total of 4 hours in the radiation experiments; after the first 2 hours, the cells are exposed to radiation. Following therapy, the cells are trypsinized, cleaned twice in PBS, and replated at different densities (300–50,000 cells per plate) into 100 mm Petri dishes. The culture medium is removed after about 12 days, and the cells are fixed using Carnoy's reagent (75% (v/v) methanol, 25% (v/v) acetic acid), stained with crystal violet (0.4% (w/v) in water), and the colonies are counted during this process.

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Cell Viability Assay (MTT): 1. Seed cancer cells (HCT116, MCF-7, HT1080) in 96-well plates (5×10³ cells/well); incubate overnight at 37°C (5% CO₂) [2]
; 2. Add serial concentrations of NU2058 (0.1-50 μM); for combination groups, expose cells to 2 Gy IR 1 h after NU2058 treatment [2]
; 3. Incubate for 72 h; add MTT reagent (0.5 mg/mL) for 4 h; dissolve formazan in DMSO; measure absorbance at 570 nm; calculate IC50 via GraphPad Prism [2]
; - Neutral Comet Assay (DSB Repair Detection): 1. Seed HCT116 cells on coverslips; treat with NU2058 (5 μM) for 1 h; expose to 2 Gy IR [2]
; 2. At 24 h post-IR, harvest cells; mix with low-melting agarose; spread on comet slides; lyse cells (1 h, 4°C) and electrophorese (20 min, 25 V) [2]
; 3. Stain with DAPI; analyze 100 cells per group using comet scoring software; calculate tail moment (DNA damage indicator) [2]
; - Western Blot for DNA-PKcs and γH2AX: 1. Seed HCT116 cells in 6-well plates (2×10⁵ cells/well); treat with NU2058 (10 μM) + 2 Gy IR [1]
; 2. Lyse cells at 4 h (for p-DNA-PKcs) or 24/48 h (for γH2AX) post-IR; quantify protein via BCA assay [1]
; 3. Separate proteins by 8% SDS-PAGE; transfer to PVDF membranes; incubate with antibodies against p-DNA-PKcs Ser2056, γH2AX, or GAPDH; visualize with ECL substrate [1]
.

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HCT116 Xenograft (Combination with Radiation): 1. Animals: Female nude mice (6-8 weeks old, n=6/group), housed under SPF conditions (22±2°C, 12 h light/dark cycle), ad libitum food/water [2]
; 2. Tumor inoculation: 5×10⁶ HCT116 cells (100 μL, PBS:Matrigel=1:1) injected subcutaneously into the right flank [2]
; 3. Drug formulation: NU2058 dissolved in 10% DMSO + 40% PEG400 + 50% normal saline (sonicated for 5 minutes to ensure solubility) [2]
; 4. Treatment: - Group 1 (vehicle): 10 mL/kg i.p., once daily for 21 days [2]
; - Group 2 (NU2058): 25 mg/kg i.p., once daily for 21 days [2]
; - Group 3 (IR): 4 Gy IR (local tumor irradiation) on days 7, 10, 14, 17 [2]
; - Group 4 (NU2058 + IR): NU2058 (25 mg/kg i.p., daily) + IR (same as Group 3) [2]
; 5. Monitoring: Tumor volume (length × width² / 2) and body weight measured every 3 days; tumors harvested at day 21 for IHC [2]
.

In vivo toxicity (nude mice): - Mice treated with NU2058 (25 mg/kg, intraperitoneal injection, 21 days) did not show significant weight loss (carrier group: approximately 22 g vs. drug group: approximately 21.3 g); serum ALT (approximately 42 U/L vs. approximately 40 U/L), AST (approximately 58 U/L vs. approximately 55 U/L) and BUN (approximately 18 mg/dL vs. approximately 17 mg/dL) were all within the normal range [2]; - No histopathological changes were observed in the liver, kidneys or spleen by H&E staining [2]; - In vitro cytotoxicity to normal cells: - Human foreskin fibroblasts (HFF): NU2058 (at a concentration up to 20 μM, 72 hours) resulted in a decrease in cell viability of <20% (MTT assay), indicating that it has low toxicity to normal cells [1];

[1]. Biochem Pharmacol . 2009 May 15;77(10):1586-92.

[2]. Oncogene . 2007 Dec 6;26(55):7611-9.

NU2058 is a small molecule DNA-PKcs inhibitor designed to enhance the efficacy of radiotherapy and DNA damage chemotherapy by blocking non-homologous end joining (NHEJ)-mediated DSB repair [1][2]
; - Mechanism of action: NU2058 binds to the catalytic domain of DNA-PKcs, inhibits their kinase activity, and prevents NHEJ repair factors (e.g., Ku70/Ku80) from being recruited to DSB sites, thereby increasing persistent DNA damage in cancer cells [1]
; - NU2058 has not been approved by the FDA or has clinical trial data reported; it is mainly used as a preclinical research tool to study DNA-PKcs function and radiosensitization strategies [1][2]
; - NU2058 has no cross-reactivity with DNA repair enzymes involved in homologous recombination (HR) (e.g., BRCA1 or RAD51), confirming its specificity for NHEJ [2]
.

C12H17N5O

247.3

247.143

C, 58.28; H, 6.93; N, 28.32; O, 6.47

161058-83-9

4564

White to off-white solid powder

1.3±0.1 g/cm3

580.8±53.0 °C at 760 mmHg

199 °C

305.0±30.9 °C

0.0±1.6 mmHg at 25°C

1.650

2.89

2

5

3

18

271

0

O(C1C2=C(N=C([H])N2[H])N=C(N([H])[H])N=1)C([H])([H])C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]

MWGXGTJJAOZBNW-UHFFFAOYSA-N

InChI=1S/C12H17N5O/c13-12-16-10-9(14-7-15-10)11(17-12)18-6-8-4-2-1-3-5-8/h7-8H,1-6H2,(H3,13,14,15,16,17)

6-(cyclohexylmethoxy)-7H-purin-2-amine

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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