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

放射治疗是胶质母细胞瘤（GBM）的主要治疗方式。由于DNA-PKcs是修复辐射诱导的双链断裂（DSB）的关键因素，本研究评估了新型DNA-PKcs抑制剂VX-984增强GBM细胞放射敏感性的潜力。在体外条件下用VX-984处理已建立的GBM细胞系U251和GBM干细胞样细胞（GSC）系NSC11，导致辐射诱导的DNA-PKcs磷酸化的浓度依赖性抑制。以类似的浓度依赖性方式，VX-984治疗增强了克隆形成分析所定义的每种GBM细胞系的放射敏感性。通过γH2AX表达和中性彗星分析确定，VX-984抑制了U251和NSC11-GBM细胞中辐射诱导的DNA双链断裂的修复，表明VX-984诱导的放射增敏是通过抑制DNA修复介导的[1]。

将这些结果扩展到体内模型，用VX-984治疗小鼠抑制了原位脑肿瘤异种移植物中辐射诱导的DNA-PKcs磷酸化，表明该化合物在足够的浓度下穿过血脑肿瘤屏障。对于携带U251或NSC11脑肿瘤的小鼠，单独使用VX-984治疗对总体生存率没有显著影响；单靠辐射就能提高存活率。与对照组和单独辐射相比，接受联合方案的小鼠的存活率显著提高。这些结果表明，VX-984增强了脑肿瘤异种移植物的放射敏感性，并表明它可能有利于GBM的治疗管理[1]。

Neutral comet assay[1] 根据制造商的建议，使用市售试剂盒进行Neutral comet assay试验，并稍作修改。简而言之，单层被照射（10 Gy）并返回培养箱。在指定时间，产生单细胞悬浮液，用PBS洗涤，与低熔点琼脂糖（1:10）混合，并转移到提供的载玻片上。细胞在4°C下在湿冰上裂解1小时，在室温下电泳20分钟，并用70%乙醇固定。用SYBR Green对DNA进行染色，并用TriTek CometScore分析数字荧光图像。数据以剩余损伤百分比表示，其中在冰上照射并在照射后立即收集的培养物的橄榄尾力矩设置为100%损伤，照射后的剩余时间相应地归一化。通过减去假照射载体或VX-984处理样品的Olive尾矩，对VX-984或单独载体处理的所有时间点进行校正。每种条件下至少测量50个细胞。所提供的数据是3个独立实验的平均值±SEM。

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.

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.]

C23H23N7O

413.475023508072

415.209

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

2448475-19-0

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

139035013

White to off-white solid powder

2.7

106 Ų

2

7

6

31

582

1

[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

PEACIOGDEQRHFA-WXFXTCFHSA-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-/m0/s1/i11D,12D

8-[(2R)-1-[[6-(4,6-dideuterio-2-methylpyrimidin-5-yl)pyrimidin-4-yl]amino]propan-2-yl]-N-methylquinoline-4-carboxamide

(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;

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 : 11.36 mg/mL (27.34 mM)

配方 1 中的溶解度: ≥ 1.14 mg/mL (2.74 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 11.4 mg/mL澄清DMSO储备液加入到400 μL PEG300中并混合均匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 1.14 mg/mL (2.74 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 11.4 mg/mL 澄清 DMSO 储备液加入 900 μL 20% SBE-β-CD 生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 1.14 mg/mL (2.74 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 11.4 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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