| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
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| Other Sizes |
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| Targets |
DNA-PK; DDR/DNA damage; DSBs (DNA double-strand breaks)
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|---|---|
| ln Vitro |
Radiotherapy is a primary treatment modality for glioblastomas (GBM). Because DNA-PKcs is a critical factor in the repair of radiation-induced double strand breaks (DSB), this study evaluated the potential of VX-984, a new DNA-PKcs inhibitor, to enhance the radiosensitivity of GBM cells. Treatment of the established GBM cell line U251 and the GBM stem-like cell (GSC) line NSC11 with VX-984 under in vitro conditions resulted in a concentration-dependent inhibition of radiation-induced DNA-PKcs phosphorylation. In a similar concentration-dependent manner, VX-984 treatment enhanced the radiosensitivity of each GBM cell line as defined by clonogenic analysis. As determined by γH2AX expression and neutral comet analyses, VX-984 inhibited the repair of radiation-induced DNA double-strand break in U251 and NSC11 GBM cells, suggesting that the VX-984-induced radiosensitization is mediated by an inhibition of DNA repair[1].
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| ln Vivo |
Extending these results to an in vivo model, treatment of mice with VX-984 inhibited radiation-induced DNA-PKcs phosphorylation in orthotopic brain tumor xenografts, indicating that this compound crosses the blood-brain tumor barrier at sufficient concentrations. For mice bearing U251 or NSC11 brain tumors, VX-984 treatment alone had no significant effect on overall survival; radiation alone increased survival. The survival of mice receiving the combination protocol was significantly increased as compared with control and as compared with radiation alone. These results indicate that VX-984 enhances the radiosensitivity of brain tumor xenografts and suggest that it may be of benefit in the therapeutic management of GBM [1].
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| Enzyme Assay |
Neutral comet assay [1]
The neutral comet assay was performed using a commercially available kit according to the recommendations from the manufacturer with slight modifications. Briefly, mono-layers were irradiated (10 Gy) and returned to the incubator. At specified times, single-cell suspensions were generated, washed with PBS, mixed with low melting agarose (1:10), and transferred to the provided slides. Cells were lysed at 4°C for 1 hour on wet ice, subjected to electrophoresis for 20 minutes at room temperature and fixed with 70% EtOH. DNA was stained with SYBR Green, and digital fluorescent images were analyzed with TriTek CometScore as described. Data are expressed as % damage remaining in which the Olive tail moment from cultures irradiated on ice and collected immediately after irradiation was set to 100% damage, with the remaining times after irradiation normalized accordingly. All time points were corrected for VX-984 or vehicle treatment alone by subtracting the Olive tail moment of sham irradiated vehicle or VX-984–treated samples. At least 50 cells per condition were measured. Data presented are the mean ± SEM of 3 independent experiments.
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| Animal Protocol |
Orthotopic xenografts [1]
U251 (2.5 × 105) cells or CD133+ NSC11 cells (1.0 × 105) transduced to express luciferase and GFP with the lentivirus LVpFUGQ-UbC-ffLuc2-eGFP2 were intracranially implanted into the right striatum of 6- to 8-week-old athymic female nude mice (Ncr nu/nu; NCI Animal Production Program) at 1.0 mm anterior and 2.0 mm lateral to the bregma to a depth of 3.0 mm as previously described. Bioluminescent imaging (BLI) and local irradiation were all performed as described previously. VX-984 was dissolved in freshly made 5% methylcellulose and delivered by oral gavage. On day 6 (U251) or day 20 (NSC11) after implantation, consistent BLI was detected in all mice, which were then randomized according to the signal obtained from BLI into four groups: vehicle, VX-984, radiation (3 × 3 Gy), and VX-984 plus radiation (7–8 mice/group), and the treatments initiated the next day. Three Gy was delivered on 3 consecutive days with VX-984 (dissolved in 5% methylcellulose) delivered by oral gavage each day 0.5 hour before and 4 hours after irradiation. For irradiation, mice were anesthetized using a cocktail of keta-mine/xylazine/acepromazine and placed in well-ventilated Plexi glass jigs with shielding for the entire torso of the mouse along with critical normal structures of the head (ears, eyes, and neck). Radiation was delivered using an X-Rad 320 X-irradiator with a 2.0 mm aluminum filtration (300 kV peak; 10 mA) X-ray at a dose rate of 2.9 Gy/minute. All in vivo irradiation experiments were performed using the same instrument located within the animal facility; output and quality assurance are performed annually. Mice were monitored every day until the onset of neurologic symptoms (morbidity). BLI and weights were measured biweekly (U251) or weekly (NSC11) after irradiation until the first mouse of the group was lost.
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| References |
[1]. The DNA-PK Inhibitor VX-984 Enhances the Radiosensitivity of Glioblastoma Cells Grown In Vitro and as Orthotopic Xenografts. Mol Cancer Ther. 2018 Jun;17(6):1207-1216.
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| Additional Infomation |
Purpose: DNA double-strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). We demonstrate the selectivity of VX-984, a DNA-PK inhibitor, using assays not previously reported. Experimental design: The class switch recombination assay (CSR) in primary B cells was used to measure efficiency of NHEJ. A cellular reporter assay (U2OS EJ-DR) was used to assess the efficiency of HR and NHEJ in cells treated with VX-984. Immunofluorescence assays (IF) evaluated γ-H2AX foci for DSB repair kinetics in human astrocytes and T98G glioma cells. Western blotting was used to evaluate phosphorylation of DNA-PKcs substrates. Results: We found a dose-dependent reduction in CSR efficiency with VX-984, and through the EJ-DR assay, dramatic dose-dependent increases in HR and mNHEJ. Immunofluorescence assays showed an inability of malignant cells to resolve γ-H2AX foci in the presence of VX-984. Radiation-induced phosphorylation of DNA-PK substrates was further reduced by treatment with VX-984. Conclusions: VX-984 efficiently inhibits NHEJ, resulting in compensatory increases in alternative repair pathways, increases DSBs, and appears to affect transformed cells preferentially.[Oncotarget. 2018 May 25;9(40):25833-25841.]
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| Molecular Formula |
C23H23N7O
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|---|---|
| Molecular Weight |
413.475023508072
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| Exact Mass |
415.209
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| Elemental Analysis |
C, 66.49; H, 6.06; N, 23.60; O, 3.85
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| CAS # |
2448475-19-0
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| Related CAS # |
1476071-49-4 (normal);1562396-65-9 (demethyl);1476074-39-1 (deuterium);
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| PubChem CID |
139035013
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| Appearance |
White to off-white solid powder
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| LogP |
2.7
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
31
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| Complexity |
582
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| Defined Atom Stereocenter Count |
1
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| SMILES |
[C@H](C1=CC=CC2C(=CC=NC1=2)C(=O)NC)(C)CNC1=NC=NC(C2=C([H])N=C(C)N=C2[H])=C1
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| InChi Key |
PEACIOGDEQRHFA-WXFXTCFHSA-N
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| InChi Code |
InChI=1S/C23H23N7O/c1-14(17-5-4-6-18-19(23(31)24-3)7-8-25-22(17)18)10-28-21-9-20(29-13-30-21)16-11-26-15(2)27-12-16/h4-9,11-14H,10H2,1-3H3,(H,24,31)(H,28,29,30)/t14-/m0/s1/i11D,12D
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| Chemical Name |
8-[(2R)-1-[[6-(4,6-dideuterio-2-methylpyrimidin-5-yl)pyrimidin-4-yl]amino]propan-2-yl]-N-methylquinoline-4-carboxamide
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| Synonyms |
(R)-VX-984; 2448475-19-0; (R)-N-Methyl-8-(1-((2'-methyl-[4,5'-bipyrimidin]-6-yl-4',6'-d2)amino)propan-2-yl)quinoline-4-carboxamide;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : 11.36 mg/mL (27.34 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.14 mg/mL (2.74 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 11.4 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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. Solubility in Formulation 2: ≥ 1.14 mg/mL (2.74 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 11.4 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. View More
Solubility in Formulation 3: ≥ 1.14 mg/mL (2.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4185 mL | 12.0925 mL | 24.1850 mL | |
| 5 mM | 0.4837 mL | 2.4185 mL | 4.8370 mL | |
| 10 mM | 0.2418 mL | 1.2092 mL | 2.4185 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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