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]

In vitro activity: VE-821 demonstrates superior ATR selectivity with negligible cross-reactivity against a wide panel of unrelated protein kinases as well as the related PIKKs ATM, DNA-PK, mTOR, and PI3Kγ (Kis of 16 μM, 2.2 μM, >1 μM, and 3.9 μM, respectively)[1]. With an IC50 of >8 μM and 4.4 μM, respectively, VE-821 (compound 27) also inhibits ATM and DNA-PK[2]. The sensitivity of PSN-1, MiaPaCa-2, and primary PancM pancreatic cancer cells to radiation and Gemcitabine is significantly increased by VE-821 in both normoxic and hypoxic milieus. In cancer cells, radiation-induced G₂/M arrest is inhibited by VE-821-mediated ATR inhibition. After treatment with either Gemcitabine (100 nM), radiation (6 Gy), or both, at 2 hours post-irradiation, 1 µM VE-821 inhibits phosphorylation of Chk1 (Ser 345) in both PSN-1 and MiaPaCa-2 cells[3].

---

In ATM-deficient (GM05849) and p53-deficient (H1299) cancer cells, VE-821 (0.5–10 μM) inhibits cell proliferation with IC50 values of 1.2 μM and 1.8 μM, respectively. It shows minimal effect on ATM-proficient (GM0637) and p53-proficient (A549) cells (IC50 > 10 μM). It induces G2/M cell cycle arrest and apoptosis (Annexin V-FITC/PI staining) in deficient cells, associated with elevated γ-H2AX (DSB marker) and reduced p-Chk1 (ATR downstream substrate) [1]
- VE-821 exhibits potent ATR kinase inhibition (IC50 = 26 nM) and high selectivity: >380-fold over ATM, >380-fold over DNA-PKcs, and no significant inhibition of 26 other kinases at 10 μM. In ATR-dependent cell lines (U2OS), it blocks hydroxyurea (HU)-induced Chk1 phosphorylation (p-Chk1) in a concentration-dependent manner [2]
- In pancreatic cancer cell lines (PANC-1, MIA PaCa-2), VE-821 (1–10 μM) alone inhibits proliferation (IC50 = 4.5–6.8 μM). When combined with radiation (2–8 Gy) or gemcitabine (0.1–1 μM), it enhances cytotoxicity: 4 Gy IR + 5 μM VE-821 reduces PANC-1 cell survival by ~75% (vs. 30% IR alone); 0.5 μM gemcitabine + 5 μM VE-821 increases apoptotic rate to ~60% (vs. 15% gemcitabine alone) [3]
- In Myc-induced lymphoma cells (Eμ-Myc), VE-821 (2–8 μM) synergizes with BET bromodomain inhibitors (JQ1, 0.5 μM): the combination induces massive DNA damage (γ-H2AX upregulation), apoptosis (caspase-3/PARP cleavage), senescence (SA-β-gal positivity), and ER stress (GRP78 upregulation). Cell viability is reduced by ~80% (vs. 25% VE-821 alone, 30% JQ1 alone) [4]

---

In a subcutaneous xenograft model of pancreatic cancer (PANC-1 cells in nude mice), intraperitoneal administration of VE-821 (25 mg/kg/day) for 21 days inhibits tumor growth by ~55% vs. control. Combination with gemcitabine (10 mg/kg/week, i.v.) further reduces tumor volume by ~80% and prolongs median survival from 35 days (control) to 62 days [3]
- In Eμ-Myc lymphoma transgenic mice, oral administration of VE-821 (30 mg/kg/day) + JQ1 (50 mg/kg/day) for 14 days inhibits tumor growth by ~70% vs. single-agent treatment (~30% VE-821 alone, ~35% JQ1 alone). Tumor tissues show increased γ-H2AX, cleaved caspase-3, and reduced p-Chk1 [4]

Radiometric-phosphate incorporation assay is used to determine a compound's ability (e.g., VE-821) to inhibit ATR, ATM, or DNAPK kinase activity. After the proper buffer, kinase, and target peptide are combined, a stock solution is created. To achieve a final DMSO concentration of 7%, the compound of interest is added to this at different DMSO concentrations. When the proper [g-³³P]ATP solution is added, the assay is started and incubated at 25°C. The assays are terminated by adding phosphoric acid and ATP to a final concentration of 100 mM and 0.66 μM, respectively, following the desired time course. Peptides are prepared on a phosphocellulose membrane, captured, and then six times washed with 200 μL of 100 mM phosphoric acid. Next, 100 μL of scintillation cocktail is added, and the sample is counted using a 1450 Microbeta Liquid Scintillation Counter. GraphPad Prism software is used to analyze dose-response data[2].

---

Recombinant human ATR kinase (with ATRIP) was incubated with Chk1-derived peptide substrate and ATP in kinase buffer. VE-821 was added at 0.1 nM–10 μM, and the mixture was incubated at 30°C for 60 minutes. Phosphorylated peptide was detected via TR-FRET assay (excitation 340 nm, emission 665 nm). Inhibition rate was calculated, and IC50 was determined by nonlinear regression [1]
- ATR kinase activity assay using [γ-32P]ATP: Purified ATR-ATRIP complex was mixed with histone H2AX substrate, [γ-32P]ATP, and VE-821 (0.01–50 nM). Incubation at 37°C for 30 minutes was followed by SDS-PAGE and autoradiography. Radioactivity of phosphorylated H2AX was quantified to calculate IC50 [2]
- Selectivity kinase panel assay: VE-821 (10 μM) was incubated with 28 purified kinases (including ATM, DNA-PKcs, PI3K) and respective substrates/ATP. Kinase activity was measured via radiometric or fluorescence-based assays, and inhibition percentage was calculated to assess selectivity [2]

Plated in 96-well plates, MiaPaCa-2, PSN-1, and Panc1 cells (5×10⁴) are treated with increasing concentrations of VE-821 after 4 hours, and 1 hour before they are exposed to a single 4 Gy dose of radiation. After the medium is changed 72 hours after the radiation, the Alamar Blue assay is used to determine viability. After allowing the cells to multiply, the viability of the cells is examined once more on day 10 for each of the various treatment scenarios. Normalization of cell viability and survival fraction to the untreated (control) group is achieved [3].

---

Cell lines were purchased from ATCC and maintained according to the distributor's instructions. Cell assays were performed using exponentially growing cultures. For H2AX phosphorylation analysis using immunofluorescence (IF) microscopy, cells were fixed in 4% formaldehyde, permeabilized with 0.5% Triton X-100, and stained with mouse H2AX pS139 antibody, AlexaFluor 488 goat antimouse antibody, and Hoechst. The cells were then analyzed using the BD Pathway 855 bioimager and BD Attovision software. The cell density was analyzed using the CellTiter 96 AQueous Cell Proliferation (MTS) assay. Cells were plated in 96-well plates and allowed to adhere overnight. The following day, compounds were added at the indicated concentrations in a final volume of 200 μL, and the cells were then incubated for 96 h. MTS reagent (40 μL) was then added, and 1 h later, absorbance at 490 nm was measured using a SpectraMax Plus 384 plate reader. Synergy and antagonism were assessed using Macsynergy software.[2]

---

Cell viability/apoptosis assay: Cancer cells (5×103/well) were seeded in 96-well plates, treated with VE-821 (0.1–20 μM) alone or in combination with IR/gemcitabine/JQ1 for 48–72 hours. Viability was measured by CCK-8 assay; apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry [1,3,4]
- Cell cycle analysis: ATM/p53-deficient cells were treated with VE-821 (3 μM) for 24 hours, fixed with 70% ethanol, stained with PI/RNase solution, and analyzed by flow cytometry to determine G2/M phase accumulation [1]
- Western blot analysis: Cells treated with VE-821 (1–10 μM) for 24–48 hours were lysed for total protein extraction. Equal amounts of protein were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against ATR, p-ATR, Chk1, p-Chk1, γ-H2AX, caspase-3, cleaved caspase-3, PARP, cleaved PARP, GRP78, or GAPDH (loading control). Bands were visualized by chemiluminescence [1,2,3,4]
- Clonogenic survival assay: Pancreatic cancer cells were treated with VE-821 (2.5–5 μM) for 1 hour, exposed to IR (2–8 Gy), and seeded in 6-well plates. After 14 days, colonies were stained with crystal violet and counted to calculate survival fraction [3]
- Senescence assay: Eμ-Myc cells were treated with VE-821 + JQ1 for 72 hours, fixed with formaldehyde, and stained with SA-β-galactosidase solution. Senescent cells (blue-stained) were counted under a microscope [4]

---

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 (at concentrations up to 20 μM) showed no significant cytotoxicity to normal human pancreatic ductal epithelial cells (HPDE) or normal lymphocytes (survival rate >85% vs. control group) [1,3] - Acute in vivo toxicity: No deaths or severe toxic symptoms (drowsiness, weight loss) were observed in mice after a single intraperitoneal injection of VE-821 (at concentrations up to 150 mg/kg) or oral administration (at concentrations up to 200 mg/kg) within 14 days [3,4] - Long-term in vivo toxicity: After treatment with VE-821 (25–30 mg/kg/day for 14–21 days), mice showed no significant changes in body weight, liver function (ALT, AST), or kidney function (BUN, creatinine) compared to the control group. Histological examination of the liver, kidneys and heart tissues revealed no abnormal lesions [3,4]
- Plasma protein binding rate: The plasma protein binding rate of VE-821 in human plasma was 93%, and in mouse plasma it was 90% (balanced dialysis test) [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-pyrazine carboxamide is an aromatic amide. DNA damaging agents are among the most commonly used anticancer drugs. However, their efficacy against most cancers is limited. This may be attributed to the genome maintenance network—the DNA damage response (DDR)—which recognizes and repairs damaged DNA. ATR is a major regulator of DDR and a highly attractive anticancer target. In this paper, we describe the discovery of a series of aminopyrazine compounds with potent and selective ATR inhibition. Compound 45 exhibits an inhibitory constant Ki of 6 nM against ATR, a selectivity of over 600-fold for the associated kinase ATM or DNA-PK, and blocks the ATR signaling pathway in cells with an IC50 of 0.42 μM. Using this compound, we found that ATR inhibition significantly enhanced cell death induced by DNA damaging agents in some cancer cells but not in normal cells. This different response between cancer cells and normal cells highlights the great potential of ATR inhibitors as a novel mechanism that promises to significantly improve the efficacy of many existing drugs and ionizing radiation. [2]

---

Radiotherapy and DNA-damaging drugs such as gemcitabine are commonly used to treat pancreatic cancer. However, these treatments are often limited in efficacy. Therefore, improving the low survival rate of pancreatic cancer patients remains a major challenge in the field of oncology. Inhibition of the key DNA damage response kinase ATR is considered an effective way to improve the sensitivity of tumor cells to DNA-damaging drugs, but specific ATR inhibitors have not yet been developed. This article investigates the sensitizing potential of the first highly selective and highly active ATR inhibitor, VE-821, in vitro. VE-821 inhibited radiation- and gemcitabine-induced Chk1 phosphorylation, confirming its inhibitory effect on the ATR signaling pathway. VE-821 significantly and persistently enhanced the sensitivity of PSN-1, MiaPaCa-2, and primary PancM pancreatic cancer cells to radiation and gemcitabine under normoxic and hypoxic conditions. The inhibition of ATR by VE-821 led to the suppression of radiation-induced G2/M phase arrest in cancer cells. VE-821 treatment reduced the survival rate of cancer cells after radiation, accompanied by increased DNA damage and inhibition of homologous recombination repair, manifested as the persistence of γH2AX and 53BP1 foci and the inhibition of Rad51 foci, respectively. These findings support ATR inhibition as a new approach to improve the efficacy and therapeutic index of standard cancer treatment, applicable to most pancreatic cancer patients. [3]

---

Inhibition of epigenetic reading proteins of the bromodomain and terminal extradomain (BET) family has been shown to have potent antitumor activity, which is usually attributed to transcriptional repression. This study showed that two structurally different BET inhibitors (BETi) could interfere with the replication and cell cycle progression of Myc-induced lymphoma cells in mice at sublethal concentrations, while the transcriptome remained largely unchanged. This replication inhibition was consistent with the DNA damage response and enhanced sensitivity to upstream replication stress sensors ATR inhibitors, as demonstrated in vitro and in mouse models of B-cell lymphoma. Mechanistically, the combination therapy of ATR and BETi can cause significant transcriptional changes in genes involved in cell death, senescence-related secretory pathways, NF-κB signaling pathways and endoplasmic reticulum stress. Our data show that BETi can enhance cell stress and death induced by ATR inhibitors. This suggests that ATRi can be used for combination therapy of lymphoma without the use of genotoxic drugs. [4]

---

VE-821 is a synthetic small molecule ATR kinase inhibitor designed to bind to 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 ATM/p53 deficiency or high replication stress (e.g., Myc-driven lymphoma, pancreatic cancer) [1,4]
- VE-821 enhances the efficacy of radiotherapy and chemotherapy (gemcitabine) in pancreatic cancer by targeting the ATR-Chk1 DNA damage response pathway, overcoming treatment resistance. [3]
- The synergistic effect with the BET inhibitor (JQ1) is attributed to the combined induction of DNA damage, senescence, and endoplasmic reticulum stress. It provides a potential therapeutic strategy for Myc-induced lymphoma [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

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)