DNA-PK; DDR/DNA damage; DSBs (DNA double-strand breaks)

In U251 and NSC11 cells, VX-984 (0-500 nM, 30 minutes) suppresses the phosphorylation of DNA-PKcs caused by radiation [1]. In U251 and NSC11 cells, VX-984 (0-500 nM) increases DNA-PKcs phosphorylation in a concentration-dependent manner [1]. Alternative mechanisms for DSB repair, including as homologous recombination (HR) and radiogravity, are enhanced by VX-984 (0-1 μM) [1]. and mNHEJ, or mutagenic NHEJ[2]. Drug compounds contain stable heavy isotopes of hydrogen, carbon, and other elements. These isotopes are mostly employed as quantitative tracers in drug development. have drawn interest because of their pharmacokinetic properties and derived traits [4]. Crepuscular substances may have the following benefits: (1) Extended half-life in vivo. Compounds that cause death may be able to extend the compound's half-life in vivo, or its pharmacokinetic properties. This may raise the drug's convenience while also enhancing the compound's safety, effectiveness, and tolerability. Enhancement of intestinal bioavailability (2). In the intestinal wall and umbilicus, deuterated substances can decrease desired metabolism (first-pass metabolism), which increases the amount of the undesired medication that reaches its intended site of action. Better tolerance and activity at low doses are determined by high bioavailability. (3) Enhanced performance. Deuterated substances enhance death safety and lessen toxicity or reactivity (4). Death generation substances are harmless and have the ability to lessen or remove the negative effects of medicinal compounds. (5) Preserve its therapeutic qualities. predicted death in a prior investigation

In orthotopic brain tumor xenografts, VX-984 (0-100 mg/kg, oral gavage, daily) suppresses phosphorylation of DNA-PKcs caused by radiation [1]. The oral administration of VX-984 (0-50 mg/kg) twice daily for two days improves the radiosensitivity of xenografts of brain tumors [1].

---

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].

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.

Western Blot Analysis [1]
Cell Types: U251 and NSC11 Cell
Tested Concentrations: 0, 100, 250 and 500 nM
Incubation Duration: 30 minutes
Experimental Results: Concentrations shown. When VX-984 was administered 1 hour before irradiation, radiation-induced DNA-PKcs phosphorylation-dependent reduction was observed in each glioma line. Treatment with VX-984 alone was ineffective.

---

Class switch recombination assay [2]
B cells were isolated by lysing red blood cells with ACK lysing buffer, followed by reverse selection using anti-CD43 MACS micro-beads and a Mini-MACS column. Collected B cells were stained with 5 μM CFSE for 10 minutes at 37°C and then cultured in RPMI-1640 containing 10% FBS, 1x penicillin/streptomycin, 1% L-glutamine, 1x MEM non-essential amino acids, 1% sodium pyruvate, 1% HEPES, and 53 mM 2-mercaptoethanol. Media was supplemented with 25 μg/ml LPS from E.coli, 50 U/ml Interleukin-4, and 1:1000 rat anti-mouse anti-CD180. Cells were cultured for 72 hours with VX-984 (0.2, 0.4, and 0.8 μM) at 37°C with 5% CO2. B cells were blocked with 5 μl of anti-mouse CD16/CD32. Primary antibodies Biotin-rat anti-mouse IgG1 and FITC-rat anti-mouse CD45R/B220 were added at 10 μl of a 1:100 dilution, followed by 10 μl of a 1:100 dilution of Streptavidin-A647. Cell proliferation and IgG+ cells were measured using a Becton Dickinson FACSCalibur and data were analyzed by FlowJo software.

---

End-joining dual reporter (EJ-DR) assay [2]
U2OS EJ-DR cells were plated in a 6 well plate in DMEM containing 10% charcoal stripped FBS and 1% antibiotic-antimycotic. After 72 hours, the cells were washed and the medium was replaced with DMEM containing 10% tet-free FBS and 1% antibiotic-antimycotic and VX-984 (0.5, 0.7, and 1.0 μM) was added. To induce double-strand breaks at the I-Sce1 sites, 1 μM Shield1 and 100 nM Triamcinolone Acetonide were added 30 minutes after drug addition. After 24 hours, cells were washed and fresh EJ-DR Tet-free medium and drug was added. After 72 hours, cells were collected and the levels of HR and NHEJ were measured using a Becton Dickinson FACSCalibur and data were analyzed by FlowJo software.

---

Immunofluorescence assay [2]
This assay was used to visualize the presence and ensuing resolution of H2AX foci following DNA damage with and without VX-984 over time. The level of phosphorylated histone variant H2AX (γ-H2AX) is a surrogate marker for levels of DNA damage. Immediately after double strand breakage (DSB), γH2AX forms bright nuclear foci that can be stained and visualized by immunoflourescent microscopy. The presence of γ-H2AX foci is widely regarded as marker for DSBs in cells and has been shown to be specific for DSB [38]. We used the antibody to γ-H2AX to visualize cells with DNA damage.

Animal/Disease Models: Athymic female nude mice (6-8 weeks old, 7-8 mice/group, U251 intracerebral xenograft) [1]
Doses: 0, 50 and 100 mg/kg
Route of Administration: Oral tube Feed, daily, 1 or 4 hrs (hrs (hours)) before irradiation (10 Gy)
Experimental Results: DNA-PKcs phosphorylation levels diminished after irradiation.

---

Animal/Disease Models: Athymic female nude mice (6-8 weeks old, 7/group, U251 intracerebral xenograft) [1]
Doses: 0, 50 mg/kg
Route of Administration: po (oral gavage), twice a day, before Local tumor irradiation (3Gy) for 30 minutes, followed by 4 hrs (hrs (hours)) for 3 days (3 × 3Gy)
Experimental Results: VX-984 alone in U251 tumors had no significant impact on overall survival compared with vehicle; radiation alone improved survival Rate. The combination of VX-984 and radiotherapy increased tumor radiosensitivity and Dramatically improved mouse survival compared with radiotherapy alone.

---

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.

[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.

[2]. VX-984 is a selective inhibitor of non-homologous end joining, with possible preferential activity in transformed cells. Oncotarget. 2018 May 25;9(40):25833-25841.

[3]. Abstract 3716: Potent radiation enhancement with VX-984, a selective DNA-PKcs inhibitor for the treatment of NSCLC. Cancer Res (2016) 76 (14_Supplement): 3716.

[4]. Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals. Ann Pharmacother. 2019 Feb;53(2):211-216.

DNA-dependent Protein Kinase Inhibitor VX-984 is an ATP-competitive inhibitor of the catalytic subunit of DNA-dependent protein kinase (DNA-PK), with potential sensitizing and enhancing activities for both chemo- and radiotherapies. Upon administration, DNA-PK inhibitor VX-984 binds to and inhibits the catalytic subunit of DNA-PK, thereby interfering with the non-homologous end joining (NHEJ) process and preventing repair of DNA double strand breaks (DSBs) caused by ionizing radiation or chemotherapeutic treatment. This increases chemo- and radiotherapy cytotoxicity and leads to enhanced tumor cell death. The enhanced ability of tumor cells to repair DSBs plays a major role in the resistance of tumor cells to chemo- and radiotherapy; DNA-PK plays a key role in the NHEJ pathway and DSB repair.

---

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]

---

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.[2]

---

Ionizing radiation (IR), which is widely used for the treatment of cancer, causes double-strand breaks (DSBs) in DNA. If left unrepaired, these DSBs are lethal to the cell. DNA-dependent protein kinase (DNA-PK) is a key enzyme in the non-homologous end joining (NHEJ) pathway that repairs DSBs caused by IR, or chemotherapeutic agents that cause DSBs such as doxorubicin. The goal of these studies was to characterize the radiation enhancing effects of VX-984, a selective and potent ATP-competitive inhibitor of the catalytic subunit of DNA-PK (DNA-PKcs), with a focus on non-small cell lung cancer (NSCLC) cells and tumor xenografts. VX-984 enhances the cytotoxicity of IR in a panel of cancer cell lines including NSCLC cell lines in vitro with dose enhancement factors (DEF) greater than 3. Notably, VX-984 combined with IR in normal human lung fibroblasts minimally enhanced the cytotoxicity compared to IR alone. Additionally, VX-984 decreased DNA-PKcs autophosphorylation on S2056 both in vitro and in vivo in NSCLC cells and attenuated the decay of the DNA damage markers γH2AX and pKAP1 in response to IR. In NSCLC PDX models VX-984, in combination with IR (2 Gy x 3), caused durable complete responses while IR alone only led to a delay in tumor growth, consistent with delayed DNA damage repair. In these models, the combination of VX-984 and IR was well tolerated. These data demonstrate that VX-984 is a potent radiation-enhancing agent and provide a strong rationale for the use of VX-984 in combination with IR for the treatment of NSCLC.[3]

C23H23N7O

415.487346887589

415.209

C, 66.49; H, 6.06; N, 23.60; O, 3.85

1476074-39-1

1476071-49-4 (normal);1562396-65-9 (demethyl);1476074-39-1 (deuterium);

72188357

White to off-white solid powder

2.7

2

7

6

31

582

1

O=C(C1C=CN=C2C=1C=CC=C2[C@H](C)CNC1C=C(C2=C([2H])N=C(C)N=C2[2H])N=CN=1)NC

PEACIOGDEQRHFA-KIYKJNLWSA-N

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-/m1/s1/i11D,12D

(S)-N-methyl-8-(1-((2'-methyl-[4,5'-bipyrimidin]-6-yl-4',6'-d2)amino)propan-2-yl)quinoline-4-carboxamide

VX-984; VX 984; 1476074-39-1; UNII-0C33IBK195; 0C33IBK195; VX984; 8-[(2S)-1-[[6-(4,6-dideuterio-2-methylpyrimidin-5-yl)pyrimidin-4-yl]amino]propan-2-yl]-N-methylquinoline-4-carboxamide; VX984; M9831; M-9831; M 9831

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 mg/mL (~24.07 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (2.41 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 10.0 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 mg/mL (2.41 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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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