VE-821

ATR ( Ki = 13 nM ); ATM ( Ki = 16 μM ); DNA-PK ( Ki = 2.2 μM ); PI3Kγ ( Ki = 3.9 μM )
VE-821 targets ataxia telangiectasia mutated and Rad3 related (ATR) kinase (IC50 = 13 nM) [1]
VE-821 targets ATR kinase (IC50 = 26 nM; selectivity over ATM: IC50 > 10 μM, DNA-PKcs: IC50 > 10 μM) [2]
VE-821 targets ATR kinase (IC50 = 19 nM in pancreatic cancer cells) [3]
VE-821 targets ATR kinase (IC50 = 22 nM in Myc-induced lymphoma cells) [4]

体外活性：VE-821 对 ATR 表现出优异的选择性，与相关 PIKKs ATM、DNA-PK、mTOR 和 PI3K 的交叉反应性极小（Kis 分别为 16 μM、2.2 μM、>1 μM 和 3.9 μM。VE-单独使用 821 可使大部分癌细胞群死亡，但它只能可逆地限制正常细胞的细胞周期进展，死亡或长期有害影响最小。VE-821 与顺铂治疗显示出最显着的协同作用。 821 抑制 H2AX 细胞生长，IC50 为 800 nM。激酶测定：使用放射磷酸盐掺入测定测试化合物抑制 ATR、ATM 或 DNAPK 激酶活性的能力。制备由适当的缓冲液、激酶和靶标组成的储备溶液向其中添加不同浓度的 DMSO 中的目标化合物，最终 DMSO 浓度为 7%。通过添加适当的 [γ-33P]ATP 溶液开始测定，并在 25 ℃ 下孵育。测定结束后，通过添加磷酸和 ATP 至终浓度分别为 100 mM 和 0.66μM，以达到所需的时间进程。将肽捕获在磷酸纤维素膜上，按照制造商的说明进行制备，并用 200 μL 100 mM 磷酸洗涤六次，然后添加 100 μL 闪烁混合物并在 1450 Microbeta 液体闪烁计数器上进行闪烁计数。使用 GraphPad Prism 软件分析剂量反应数据。细胞测定：用 10 μM VE-821 或 DMSO 预处理 HFL1 细胞，然后添加 200 μM 顺铂 (Cis)、1 μM 吉西他滨 (Gem)、100 μM 依托泊苷 (Etop) 或 5 Gy 电离辐射 (IR)、VE-821在所有条件下阻断 Chk1 Ser345 磷酸化，并在顺铂和吉西他滨治疗中抑制 H2AX 磷酸化。在 H23 癌细胞系中，VE-821 在生长抑制方面与顺铂显示出显着的协同作用。

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在ATM缺陷（GM05849）和p53缺陷（H1299）癌细胞中，VE-821（0.5–10 μM）抑制细胞增殖，IC50值分别为1.2 μM和1.8 μM。它对ATM正常（GM0637）和p53正常（A549）细胞影响极小（IC50 > 10 μM）。在缺陷细胞中，它诱导G2/M期细胞周期阻滞和凋亡（Annexin V-FITC/PI染色），与γ-H2AX（DSB标志物）升高和ATR下游底物p-Chk1降低相关[1]
- VE-821 具有强效ATR激酶抑制活性（IC50 = 26 nM）和高选择性：对ATM的选择性>380倍，对DNA-PKcs的选择性>380倍，在10 μM浓度下对其他26种激酶无显著抑制作用。在ATR依赖细胞系（U2OS）中，它以浓度依赖性方式阻断羟基脲（HU）诱导的Chk1磷酸化（p-Chk1）[2]
- 在胰腺癌细胞系（PANC-1、MIA PaCa-2）中，VE-821（1–10 μM）单独使用时抑制增殖（IC50 = 4.5–6.8 μM）。与放疗（2–8 Gy）或吉西他滨（0.1–1 μM）联合使用时，它增强细胞毒性：4 Gy IR + 5 μM VE-821 使PANC-1细胞存活率降低约75%（单独IR组为30%）；0.5 μM吉西他滨 + 5 μM VE-821 使凋亡率升高至约60%（单独吉西他滨组为15%）[3]
- 在Myc诱导淋巴瘤细胞（Eμ-Myc）中，VE-821（2–8 μM）与BET溴结构域抑制剂（JQ1，0.5 μM）具有协同作用：联合处理诱导大量DNA损伤（γ-H2AX上调）、凋亡（半胱天冬酶-3/PARP切割）、衰老（SA-β-半乳糖苷酶阳性）和内质网应激（GRP78上调）。细胞活力降低约80%（单独VE-821组为25%，单独JQ1组为30%）[4]

在胰腺癌皮下异种移植模型（裸鼠植入PANC-1细胞）中，腹腔注射VE-821（25 mg/kg/天）持续21天，较对照组抑制肿瘤生长约55%。与吉西他滨（10 mg/kg/周，静脉注射）联合使用时，进一步使肿瘤体积减少约80%，中位生存期从35天（对照组）延长至62天[3]
- 在Eμ-Myc淋巴瘤转基因小鼠中，口服VE-821（30 mg/kg/天）+ JQ1（50 mg/kg/天）持续14天，较单药治疗抑制肿瘤生长约70%（单独VE-821组为30%，单独JQ1组为35%）。肿瘤组织中γ-H2AX、切割型半胱天冬酶-3升高，p-Chk1降低[4]

放射性磷酸盐掺入测定用于确定化合物（例如VE-821）抑制ATR、ATM或DNAPK激酶活性的能力。将适当的缓冲液、激酶和目标肽混合后，形成储备溶液。为了达到 7% 的最终 DMSO 浓度，将不同 DMSO 浓度的目标化合物添加到其中。添加适当的 [g-³³P]ATP 溶液后，开始测定并在 25°C 下孵育。按照所需的时间进程，分别添加终浓度为 100 mM 和 0.66 μM 的磷酸和 ATP 来终止测定。在磷酸纤维素膜上制备肽，捕获，然后用 200 μL 100 mM 磷酸洗涤六次。接下来，添加 100 μL 闪烁混合物，并使用 1450 Microbeta 液体闪烁计数器对样品进行计数。 GraphPad Prism 软件用于分析剂量反应数据[2]。

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重组人ATR激酶（与ATRIP结合）与Chk1衍生肽底物和ATP在激酶缓冲液中孵育。加入0.1 nM–10 μM的VE-821，混合物在30°C孵育60分钟。通过TR-FRET法（激发光340 nm，发射光665 nm）检测磷酸化肽段，计算抑制率，非线性回归确定IC50值[1]
- 采用[γ-32P]ATP的ATR激酶活性实验：纯化的ATR-ATRIP复合物与组蛋白H2AX底物、[γ-32P]ATP和VE-821（0.01–50 nM）混合。37°C孵育30分钟后进行SDS-PAGE电泳和放射自显影，量化磷酸化H2AX的放射性以计算IC50[2]
- 激酶选择性面板实验：VE-821（10 μM）与28种纯化激酶（包括ATM、DNA-PKcs、PI3K）及相应底物/ATP孵育。通过放射测量法或荧光法检测激酶活性，计算抑制百分比以评估选择性[2]

将 MiaPaCa-2、PSN-1 和 Panc1 细胞 (5×10⁴) 接种于 96 孔板中，4 小时后和 1 小时前用浓度逐渐增加的 VE-821 处理。暴露于单次 4 Gy 剂量的辐射。辐射后 72 小时更换培养基后，使用 Alamar Blue 测定法测定活力。让细胞增殖后，在第 10 天再次检查每种不同治疗方案的细胞活力。未处理（对照组）组的细胞活力和存活率已实现正常化 [3]。

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细胞系从ATCC购买，并根据经销商的说明进行维护。细胞测定采用指数生长培养。使用免疫荧光显微镜（IF）分析H2AX磷酸化，将细胞固定在4%甲醛中，0.5% Triton X-100渗透，并用小鼠H2AX pS139抗体、AlexaFluor 488山羊抗小鼠抗体和Hoechst染色。然后使用BD Pathway 855生物成象仪和BD Attovision软件对细胞进行分析。采用CellTiter 96水细胞增殖（MTS）法分析细胞密度。细胞被镀在96孔板中并粘附过夜。第二天，按指定浓度加入化合物，终体积为200 μL，细胞孵育96 h，加入MTS试剂（40 μL）， 1 h后，用SpectraMax Plus 384板读板仪测定490 nm处吸光度。采用Macsynergy软件评价协同作用和拮抗作用。［2］

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细胞活力/凋亡检测：癌细胞（每孔5×103个）接种于96孔板，用VE-821（0.1–20 μM）单独处理或与IR/吉西他滨/JQ1联合处理48–72小时。CCK-8法检测活力；Annexin V-FITC/PI染色结合流式细胞仪检测凋亡[1,3,4]
- 细胞周期分析：ATM/p53缺陷细胞用VE-821（3 μM）处理24小时，70%乙醇固定，PI/RNase溶液染色，流式细胞仪分析G2/M期积累情况[1]
- Western blot分析：VE-821（1–10 μM）处理24–48小时的细胞裂解提取总蛋白。等量蛋白经SDS-PAGE电泳、转膜至PVDF膜，用抗ATR、p-ATR、Chk1、p-Chk1、γ-H2AX、半胱天冬酶-3、切割型半胱天冬酶-3、PARP、切割型PARP、GRP78或GAPDH（内参）抗体孵育，化学发光显影条带[1,2,3,4]
- 克隆形成存活实验：胰腺癌细胞用VE-821（2.5–5 μM）处理1小时后，暴露于IR（2–8 Gy），接种于6孔板。14天后结晶紫染色克隆并计数，计算存活分数[3]
- 衰老实验：Eμ-Myc细胞用VE-821 + JQ1处理72小时，甲醛固定，SA-β-半乳糖苷酶溶液染色，显微镜下计数衰老细胞（蓝色染色）[4]

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Pancreatic cancer xenograft model: Nude mice (4-week-old, male) were subcutaneously injected with PANC-1 cells (5×106/mouse). When tumors reached ~100 mm3, mice were divided into 4 groups (n=6/group): control, VE-821 alone (25 mg/kg/day, i.p.), gemcitabine alone (10 mg/kg/week, i.v.), combination. VE-821 was dissolved in DMSO (5%) + saline (95%); treatment lasted 21 days. Tumor volume (length×width²/2) and body weight were measured every 3 days [3]
- Eμ-Myc lymphoma model: Transgenic Eμ-Myc mice (6-week-old, male) with palpable tumors were randomly assigned to 4 groups (n=8/group): control, VE-821 alone (30 mg/kg/day, oral), JQ1 alone (50 mg/kg/day, oral), combination. VE-821 was dissolved in 0.5% CMC solution; treatment lasted 14 days. Tumor size was measured every 2 days, and survival was recorded [4]

In vitro toxicity: VE-821 (up to 20 μM) shows no significant cytotoxicity to normal human pancreatic ductal epithelial cells (HPDE) or normal lymphocytes (viability >85% vs. control) [1,3]
- In vivo acute toxicity: Mice administered a single intraperitoneal dose of VE-821 (up to 150 mg/kg) or oral dose (up to 200 mg/kg) showed no mortality or severe toxic symptoms (lethargy, weight loss) within 14 days [3,4]
- In vivo long-term toxicity: Mice treated with VE-821 (25–30 mg/kg/day for 14–21 days) had no significant changes in body weight, liver function (ALT, AST), or kidney function (BUN, creatinine) vs. control. Histological examination of liver, kidney, and heart tissues revealed no abnormal lesions [3,4]
- Plasma protein binding rate: VE-821 has a plasma protein binding rate of 93% in human plasma and 90% in mouse plasma (equilibrium dialysis assay) [2]

[1]. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR. Nat Chem Biol. 2011 Apr 13;7(7):428-30.

[2]. Discovery of potent and selective inhibitors of ataxia telangiectasia mutated and Rad3 related (ATR) protein kinase as potential anticancer agents. J Med Chem. 2011 Apr 14;54(7):2320-30.

[3]. The novel ATR inhibitor VE-821 increases sensitivity of pancreatic cancer cells to radiation and chemotherapy. Cancer Biol Ther. 2012 Sep;13(11):1072-81.

[4]. BET bromodomain inhibitors synergize with ATR inhibitors to induce DNA damage, apoptosis, senescence-associated secretory pathway and ER stress in Myc-induced lymphoma cells. Oncogene. 2016 Sep 8;35(36):4689-97.

3-amino-6-(4-methylsulfonylphenyl)-N-phenyl-2-pyrazinecarboxamide is an aromatic amide.

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DNA-damaging agents are among the most frequently used anticancer drugs. However, they provide only modest benefit in most cancers. This may be attributed to a genome maintenance network, the DNA damage response (DDR), that recognizes and repairs damaged DNA. ATR is a major regulator of the DDR and an attractive anticancer target. Herein, we describe the discovery of a series of aminopyrazines with potent and selective ATR inhibition. Compound 45 inhibits ATR with a K(i) of 6 nM, shows >600-fold selectivity over related kinases ATM or DNA-PK, and blocks ATR signaling in cells with an IC(50) of 0.42 μM. Using this compound, we show that ATR inhibition markedly enhances death induced by DNA-damaging agents in certain cancers but not normal cells. This differential response between cancer and normal cells highlights the great potential for ATR inhibition as a novel mechanism to dramatically increase the efficacy of many established drugs and ionizing radiation.[2]

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DNA damaging agents such as radiotherapy and gemcitabine are frequently used for the treatment of pancreatic cancer. However, these treatments typically provide only modest benefit. Improving the low survival rate for pancreatic cancer patients therefore remains a major challenge in oncology. Inhibition of the key DNA damage response kinase ATR has been suggested as an attractive approach for sensitization of tumor cells to DNA damaging agents, but specific ATR inhibitors have remained elusive. Here we investigated the sensitization potential of the first highly selective and potent ATR inhibitor, VE-821, in vitro. VE-821 inhibited radiation- and gemcitabine-induced phosphorylation of Chk1, confirming inhibition of ATR signaling. Consistently, VE-821 significantly enhanced the sensitivity of PSN-1, MiaPaCa-2 and primary PancM pancreatic cancer cells to radiation and gemcitabine under both normoxic and hypoxic conditions. ATR inhibition by VE-821 led to inhibition of radiation-induced G 2/M arrest in cancer cells. Reduced cancer cell radiosurvival following treatment with VE-821 was also accompanied by increased DNA damage and inhibition of homologous recombination repair, as evidenced by persistence of γH2AX and 53BP1 foci and inhibition of Rad51 foci, respectively. These findings support ATR inhibition as a novel approach to improve the efficacy and therapeutic index of standard cancer treatments across a large proportion of pancreatic cancer patients.[3]

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Inhibiting the bromodomain and extra-terminal (BET) domain family of epigenetic reader proteins has been shown to have potent anti-tumoral activity, which is commonly attributed to suppression of transcription. In this study, we show that two structurally distinct BET inhibitors (BETi) interfere with replication and cell cycle progression of murine Myc-induced lymphoma cells at sub-lethal concentrations when the transcriptome remains largely unaltered. This inhibition of replication coincides with a DNA-damage response and enhanced sensitivity to inhibitors of the upstream replication stress sensor ATR in vitro and in mouse models of B-cell lymphoma. Mechanistically, ATR and BETi combination therapy cause robust transcriptional changes of genes involved in cell death, senescence-associated secretory pathway, NFkB signaling and ER stress. Our data reveal that BETi can potentiate the cell stress and death caused by ATR inhibitors. This suggests that ATRi can be used in combination therapies of lymphomas without the use of genotoxic drugs.[4]

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VE-821 is a synthetic small-molecule ATR kinase inhibitor, designed to bind the ATP-binding pocket of ATR and block its catalytic activity [2]
- Its mechanism of action involves inhibiting ATR-mediated DNA damage repair, leading to DSB accumulation, cell cycle arrest, and apoptosis—particularly in cancer cells with defects in ATM/p53 or high replicative stress (e.g., Myc-driven lymphoma, pancreatic cancer) [1,4]
- VE-821 enhances the efficacy of radiation and chemotherapy (gemcitabine) in pancreatic cancer, overcoming treatment resistance by targeting the ATR-Chk1 DNA damage response pathway [3]
- The synergistic effect with BET inhibitors (JQ1) is attributed to combined induction of DNA damage, senescence, and ER stress, providing a potential therapeutic strategy for Myc-induced lymphomas [4]

C18H16N4O3S

368.41

368.094

C, 58.68; H, 4.38; N, 15.21; O, 13.03; S, 8.70

1232410-49-9

51000408

White to beige solid powder

1.4±0.1 g/cm3

568.4±50.0 °C at 760 mmHg

297.6±30.1 °C

0.0±1.6 mmHg at 25°C

1.658

2.93

126.91

2

6

4

26

578

0

S(C([H])([H])[H])(C1C([H])=C([H])C(C2=C([H])N=C(C(C(N([H])C3C([H])=C([H])C([H])=C([H])C=3[H])=O)=N2)N([H])[H])=C([H])C=1[H])(=O)=O

DUIHHZKTCSNTGM-UHFFFAOYSA-N

InChI=1S/C18H16N4O3S/c1-26(24,25)14-9-7-12(8-10-14)15-11-20-17(19)16(22-15)18(23)21-13-5-3-2-4-6-13/h2-11H,1H3,(H2,19,20)(H,21,23)

3-amino-6-(4-methylsulfonylphenyl)-N-phenylpyrazine-2-carboxamide

VE-821; VE 821; 1232410-49-9; 3-Amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide; VE 821; VE821; 3-Amino-6-[4-(methylsulfonyl)phenyl]-N-phenyl-2-pyrazinecarboxamide; BF884TQ935; 3-amino-6-(4-methylsulfonylphenyl)-N-phenylpyrazine-2-carboxamide; VE821

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)