ATR ( IC50 = 19 nM ）
Berzosertib (VE-822; VX-970) targets ataxia telangiectasia mutated and Rad3 related (ATR) kinase [2]
Berzosertib (VE-822) is a highly potent and selective inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinase. It has a Ki of <0.0002 μM for ATR. It exhibits >100-fold cellular selectivity for ATR over the closely related phosphatidylinositol 3-kinase-related kinases (PIKKs) ATM and DNA-PK. Cellular IC50 values are 0.019 μM for ATR, 2.6 μM for ATM, and 18.1 μM for DNA-PK [1].

VE-822 (80 nM) attenuates the ATR signaling pathway and decreases tumor cell survival in response to XRT and gemcitabine. In normal cells, VE-822 (80 nM) attenuates ATR signaling without enhancing the killing effects of gemcitabine and radiation. Compared to XRT, VE-822 (80 nM) causes more γH2AX and 53BP1 foci to be induced by XRT in MiaPaCa-2 and PSN-1 cells. In MiaPaCa-2 and PSN-1 cells, VE-822 (80 nM) pre-treatment reduces Rad51 foci following XRT. In MiaPaCa-2 and PSN-1 cells, VE-822 (80 nM) by itself raises the G1-phase fraction. In MiaPaCa-2 and PSN-1 cells, VE-822 (80 nM) abolishes the XRT-enriched G2/M-phase-fraction. When combined with XRT and/or gemcitabine, VE-822 (80 nM) increases both early and late apoptosis in PSN-1 cells; this effect is greatest in the triple combination. In contrast, VE-822 has little effect when used alone.[1]
VE-822 enhances the tumor's reaction to agents that damage DNA and impede pChk1 Ser345 function.[2]

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Berzosertib (VE-822; VX-970) acts as a potent and selective ATR inhibitor, suppressing ATR-mediated signaling pathways in cancer cells. It enhances the cytotoxicity of DNA-damaging agents (e.g., chemotherapy, ionizing radiation) by impairing DNA damage repair, particularly in cancer cells with high replicative stress or defects in alternative DNA repair pathways [2]

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In pancreatic ductal adenocarcinoma (PDAC) cells (PSN-1, MiaPaCa-2), treatment with Berzosertib (80 nM) reduces phosphorylation of Chk1 at Ser345 (a marker of ATR activity) induced by gemcitabine (100 nM), radiation (6 Gy), or both [1].
Berzosertib decreases clonogenic survival of irradiated PDAC cells. Adding Berzosertib to gemcitabine reduces survival approximately 2-3 fold, and the effect is even more pronounced with the triple combination of Berzosertib, gemcitabine, and radiation [1].
In normal human fibroblasts (HFL-1, MRC5), Berzosertib reduces Chk1 phosphorylation but does not alter clonogenic survival after radiation or gemcitabine treatment. It also does not modify tube formation by human dermal microvascular endothelial cells after radiation or gemcitabine [1].
Berzosertib increases residual DNA damage in irradiated PDAC cells, as evidenced by increased γH2AX and 53BP1 foci at 24 h post-radiation. It also decreases Rad51 foci formation (a marker of homologous recombination repair) at 6 h post-radiation [1].
Berzosertib abrogates the radiation-induced G2/M phase arrest in PDAC cells. The combination of Berzosertib, radiation, and gemcitabine enhances early and late apoptosis in PSN-1 cells compared to single or double treatments [1].

VE-822 (60 mg/kg) inhibits phospho-Ser-345-Chk1 following DNA-damaging agents in mice with PSN-1 tumors. In mice with both PSN-1 and MiaPaCa-2 tumors, VE-822 (60 mg/kg) plus XTR doubles the time it takes for tumors to grow to 600 mm3 of XRT alone. In comparison to the Gem+XRT1 group of mice bearing both PSN-1 tumors, the addition of VE-822 (60 mg/kg) to the combination of gemcitabine and XRT significantly prolongs the tumor growth delay. The addition of VE-822 (60 mg/kg) to XRT1 increases uptake in tumors by 44% when compared to XRT1, indicating an increase in γH2AX phosphorylation and the persistence of DNA damage caused by XRT.[1]

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Berzosertib (VE-822; VX-970) exhibits antitumor activity in preclinical xenograft models when administered alone or in combination with DNA-damaging therapies. It inhibits tumor growth and improves the efficacy of chemotherapy/radiation, with no significant exacerbation of normal tissue toxicity [2]

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In mice bearing PSN-1 pancreatic tumor xenografts, oral administration of Berzosertib (60 mg/kg) inhibits radiation- and gemcitabine-induced Chk1 phosphorylation in tumor tissues, confirming ATR target engagement in vivo [1].
Berzosertib significantly enhances the antitumor efficacy of radiation in PSN-1 xenografts. Combining Berzosertib (60 mg/kg daily for 4-6 days) with a single 6 Gy radiation dose more than doubles the time for tumors to reach 600 mm³ compared to radiation alone. Similar radiosensitization is observed with fractionated radiation (5 × 2 Gy) [1].
In MiaPaCa-2 xenografts, Berzosertib combined with radiation results in complete tumor regression in 2 out of 4 mice, with the remaining two showing significantly delayed regrowth [1].
A clear dose-dependent radiosensitization effect is observed with intermittent Berzosertib dosing (15, 30, and 60 mg/kg on days 0, 2, and 4), with 60 mg/kg being the most efficacious [1].
Berzosertib substantially prolongs tumor growth delay when added to gemcitabine-based chemoradiation (gemcitabine 100 mg/kg + radiation 6 Gy + Berzosertib 60 mg/kg on days 1, 3, 5) in PSN-1 xenografts [1].
SPECT/CT imaging using an anti-γH2AX probe shows increased persistent DNA damage in PSN-1 tumors at 24 h post-radiation in mice treated with Berzosertib compared to radiation alone [1].

Broad kinase selectivity profiling and analysis of biochemical and cellular inhibition of ATR and related kinases were assessed. The specific methodology for the biochemical ATR kinase assay is not detailed in this paper, but reference is made to a previous publication (Reaper PM et al., Nat Chem Biol 2011) for the techniques used to determine Ki and IC50 values [1].
Berzosertib is an ATR inhibitor that has a Ki value of 0.2 nM or less. With a Ki of 34 nM, it also inhibits ATM.

Clonogenic survival assay: Cells are treated with Berzosertib (80 nM) 1 h before radiation and washed away 17 h after radiation. For chemotherapy, cells are exposed to gemcitabine (10, 20, or 50 nM) for 24 h before addition of Berzosertib (80 nM) for another 18 h. Gemcitabine is washed away immediately before Berzosertib addition. Colonies are stained and counted after 10-14 days [1].
Immunoblotting: Cells or tumor lysates are prepared using reducing Triton lysis buffer. Proteins are separated by SDS-PAGE and detected using antibodies against phospho-Chk1 (Ser345), phospho-Chk2 (T68), total Chk2, total Chk1, phospho-ATM (S1981), total ATM, phospho-DNA-PKcs (Ser2056), total DNA-PK, and β-actin [1].
Immunofluorescence for γH2AX, 53BP1, and Rad51 foci: Cells are fixed at indicated times post-treatment, stained with specific primary antibodies and fluorescent secondary antibodies. Nuclei are counterstained with DAPI. Foci are quantified using fluorescence microscopy [1].
Cell cycle analysis: Cells are harvested at 12 and 24 h post-treatment, fixed, stained with propidium iodide, and analyzed by flow cytometry [1].
Apoptosis assay: Cells are stained with Annexin V-FITC and propidium iodide 48 h post-treatment and analyzed by flow cytometry [1].
Capillary tube formation assay: Human dermal microvascular endothelial cells are treated, plated on Matrigel-coated plates, and tube formation is quantified 8 h post-treatment [1].
NSC 613327 (10 nM) is added twenty-four hours prior to XRT, and before adding Berzosertib (VE-822), it is replaced with new medium. For one hour prior to and eighteen hours following XRT (6 Gy), PSN-1 cells are treated with 80 nM Berzosertib (VE-822). Using an Annexin V-FITC kit with P, flow cytometry is used to analyze apoptosis 48 hours after XRT.

Female Balb/c nude mice are subcutaneously inoculated with 10⁶ PDAC cells (PSN-1 or MiaPaCa-2) in 50 μL serum-free medium mixed with 50 μL Matrigel. When tumors reach ~80 mm³, mice are randomized [1].
Berzosertib is dissolved in 10% Vitamin E d-alpha tocopheryl polyethylene glycol 1000 succinate and administered by oral gavage in a volume of 200 μL. Dosing schedules vary: daily on days 0-5 (6 days total), daily on days 0-3 (4 days total), or on days 1, 3, and 5. The dose used is 60 mg/kg unless otherwise specified [1].
Gemcitabine (100 mg/kg) is administered by intraperitoneal injection on day 0 [1].
Tumor irradiation: Mice are anesthetized and tumors are irradiated using a biological irradiator. Radiation doses are either single 6 Gy or fractionated 5 × 2 Gy daily from day 1 to day 5. XRT is given 2 h after Berzosertib administration [1].
Tumor volume is measured three times weekly using calipers and calculated as (π/6) × length × width². Tumor growth delay is assessed as time to reach a specified volume (400 or 600 mm³) [1].
For intestinal toxicity assessment, mice bearing PSN-1 tumors are treated with vehicle or Berzosertib (60 mg/kg p.o. on days 0, 2, and 4) and abdominal XRT (6 Gy) on day 0, 2 h after Berzosertib. On day 5, small intestine is harvested, fixed, sectioned, and stained with H&E and TUNEL to assess villus length, villus tip loss, and apoptosis [1].
For γH2AX SPECT imaging, mice are administered the radioimmunoconjugate ¹¹¹In-DTPA-anti-γH2AX-Tat 2 h before radiation (6 Gy). SPECT/CT is performed 24 h post-radiation. Berzosertib (60 mg/kg) is given on days 0 and 1, with radiation on day 1 [1].

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Dissolved in saline; 60 mg/kg; Oral administration
Mice bearing PSN-1 or MiaPaCa-2 tumors

Berzosertib is a close analogue of VE-821, possessing stronger anti-ATR activity and favorable pharmacokinetic properties, supporting in vivo studies. This compound has oral bioavailability, and all in vivo studies were conducted via gavage. Specific parameters such as half-life, percentage of bioavailability, Cmax, or AUC are not detailed in this article [1].

In vitro studies have shown that Berzosertib does not enhance the cytotoxicity of radiation- or gemcitabine-induced normal human fibroblasts (HFL-1, MRC5) or affect the formation of tubular structures in human dermal microvascular endothelial cells [1]. In vivo studies have shown that Berzosertib is well-tolerated, either alone or in combination with radiation therapy. In all xenograft studies, no weight loss was observed, whether Berzosertib (60 mg/kg) was used alone or in combination with radiation therapy or chemoradiotherapy [1]. Importantly, Berzosertib does not exacerbate radiation-induced gastrointestinal damage. Compared with radiation therapy alone, the addition of Berzosertib to abdominal radiation therapy did not significantly increase jejunal cell apoptosis, villus tip loss, or villus shortening. Weight loss was comparable in the radiation therapy-only group and the combination therapy group [1].

[1]. Cell Death Dis . 2012 Dec 6;3(12):e441.

[2]. Cancer Treat Rev . 2014 Feb;40(1):109-17.

3-[3-[4-(methylaminomethyl)phenyl]-5-isoxazolyl]-5-(4-propyl-2-ylsulfonylphenyl)-2-pyrazinamide is a sulfonamide compound. Berzosertib (VX-970) has been used in clinical trials for the treatment of various tumors, including ovarian tumors, ovarian serous tumors, adult solid tumors, and advanced solid tumors. Berzosertib is an ataxia-telangiectasia and Rad3-related (ATR) kinase inhibitor. ATR kinase is a DNA damage-responsive kinase with potential antitumor activity. After administration, berzosertib selectively binds to and inhibits ATR kinase activity, thereby blocking ATR-mediated signaling in the ATR checkpoint kinase 1 (Chk1) signaling pathway. This prevents activation of DNA damage checkpoints, disrupts DNA damage repair, and induces tumor cell apoptosis. ATR is a serine/threonine protein kinase that is upregulated in various cancer cell types and plays a key role in DNA repair, cell cycle progression, and survival; it is activated by DNA damage caused by DNA replication-related stress. Berzosertib (VE-822; VX-970) is a second-generation, orally bioavailable ATR kinase inhibitor with improved potency and pharmacokinetic properties compared to earlier ATR inhibitors [2]. Its mechanism of action includes binding to the ATP-binding pocket of ATR, blocking the ATR-Chk1 signaling pathway, and inducing cell cycle arrest or apoptosis in cancer cells with unrepaired DNA damage [2]. Its efficacy in treating various solid tumors is currently under investigation, particularly those tumors that are resistant to conventional chemotherapy or radiotherapy due to defects in the DNA damage response pathway [2]. Berzosertib (VE-822) is a novel, highly potent and selective ATR inhibitor, an analogue of VE-821 with greater potency and pharmacokinetic properties more suitable for in vivo studies. The drug is designed to treat cancers with defective DNA damage responses, such as ATM or p53 mutations, which are common in pancreatic ductal adenocarcinoma (up to 70% of patients have such mutations) by utilizing synthetic lethal effects. The compound inhibits ATR-mediated Chk1 phosphorylation, blocks cell cycle checkpoints, impairs homologous recombination repair, and specifically increases persistent DNA damage in cancer cells. Preclinical studies have shown that Berzosertib significantly enhances the sensitivity of pancreatic tumors to radiation and gemcitabine-based chemoradiotherapy without increasing toxicity to normal tissues, supporting its clinical development as a promising radiosensitizer for locally advanced pancreatic cancer [1].

C24H25N5O3S

463.55

463.167

C, 62.18; H, 5.44; N, 15.11; O, 10.35; S, 6.92

1232416-25-9

59472121

Yellow solid powder

1.3±0.1 g/cm3

674.4±55.0 °C at 760 mmHg

361.7±31.5 °C

0.0±2.1 mmHg at 25°C

1.603

3.36

2

8

7

33

714

0

S(C1C([H])=C([H])C(C2=C([H])N=C(C(C3=C([H])C(C4C([H])=C([H])C(=C([H])C=4[H])C([H])([H])N([H])C([H])([H])[H])=NO3)=N2)N([H])[H])=C([H])C=1[H])(C([H])(C([H])([H])[H])C([H])([H])[H])(=O)=O

JZCWLJDSIRUGIN-UHFFFAOYSA-N

InChI=1S/C24H25N5O3S/c1-15(2)33(30,31)19-10-8-18(9-11-19)21-14-27-24(25)23(28-21)22-12-20(29-32-22)17-6-4-16(5-7-17)13-26-3/h4-12,14-15,26H,13H2,1-3H3,(H2,25,27)

3-[3-[4-(methylaminomethyl)phenyl]-1,2-oxazol-5-yl]-5-(4-propan-2-ylsulfonylphenyl)pyrazin-2-amine

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)

5% DMSO+ 40%PEG300+ 5%Tween 80+ 50% ddH2O: 1.0mg/ml (2.16mM) (Please use freshly prepared in vivo formulations for optimal results.)