VE-821

VE-821 is a potent, highly-selective, and ATP-competitive DNA damage response (DDR) kinase ATR inhibitor with Ki value of 13nM. VE-821 specifically inhibits ATR, revealing low cross-reactivity against the mammalian target of rapamycin (mTOR), DNA-dependent protein kinase (DNA-PK), phosphoinositol 3-kinase-γ (PI3K) and the related PIKKs ATM [1].

HL-60 cells treated with VE-821 (10μM) showed reduction of phosphorylatin of Chk1 (Ser 345), inhibition of cell growth, and a radiosensitizing effect after Gamma-ray irradiation [2].

VE-821 has also been demonstrated to down-regulate the phosphorylated Chk1 (Ser 345) but it does not inhibit the phosphorylation of Chk2 (Thr68) and ATM (Ser1981) in pancreatic cancer cell lines, including PSN-1 and MiaPaCa-2 cells that are treated with gemcitabine or radiation. VE-821 combined with gemcitabine (a nucleoside analog) has caused a remarkable increase of cytotoxic effect of gemcitabine against hypoxia [3].

References:
[1] Reaper PM1, Griffiths MR, Long JM, Charrier JD, Maccormick S, Charlton PA, Golec JM, Pollard JR. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR. Nat Chem Biol. 2011 Apr 13;7(7):428-30.
[2] Vávrová J1, Zárybnická L, Lukášová E, Řezáčová M, Novotná E, Sinkorová Z, Tichý A, Pejchal J, Durišová K. Inhibition of ATR kinase with the selective inhibitor VE-821 results in radiosensitization of cells of promyelocytic leukaemia (HL-60). Radiat Environ Biophys. 2013 Nov;52(4):471-9.
[3] Prevo R1, Fokas E, Reaper PM, Charlton PA, Pollard JR, McKenna WG, Muschel RJ, Brunner TB.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.

Radiosensitization of human leukemic HL-60 cells by ATR kinase inhibitor (VE-821): phosphoproteomic analysis.[Pubmed:25003641]

Int J Mol Sci. 2014 Jul 7;15(7):12007-26.

DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)-triggered by radiation-induced double strand breaks-is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

Synergistic potentiation of (-)-lomaiviticin A cytotoxicity by the ATR inhibitor VE-821.[Pubmed:27177826]

Bioorg Med Chem Lett. 2016 Jul 1;26(13):3122-3126.

(-)-Lomaiviticin A (1) is a cytotoxic bacterial metabolite that induces double-strand breaks in DNA. Here we show that the cytotoxicity of (-)-lomaiviticin A (1) is synergistically potentiated in the presence of VE-821 (7), an inhibitor of ataxia telangiectasia and Rad3-related protein (ATR). While 0.5nM 1 or 10muM 7 alone are non-lethal to K562 cells, co-incubation of the two leads to high levels of cell kill (81% and 94% after 24 and 48h, respectively). Mechanistic data indicate that cells treated with 1 and 7 suffer extensive DNA double-strand breaks and apoptosis. These data suggest combinations of 1 and 7 may be a valuable chemotherapeutic strategy.

VE-821, an ATR inhibitor, causes radiosensitization in human tumor cells irradiated with high LET radiation.[Pubmed:26286029]

Radiat Oncol. 2015 Aug 19;10:175.

BACKGROUND: High linear energy transfer (LET) radiation such as carbon ion particles is successfully used for treatment of solid tumors. The reason why high LET radiation accomplishes greater tumor-killing than X-rays is still not completely understood. One factor would be the clustered or complex-type DNA damages. We previously reported that complex DNA double-strand breaks produced by high LET radiation enhanced DNA end resection, and this could lead to higher kinase activity of ATR protein recruited to RPA-coated single-stranded DNA. Although the effect of ATR inhibition on cells exposed to low LET gamma-rays has recently been reported, little is known regarding the effect of ATR inhibitor on cells treated with high LET radiation. The purpose of this study is to investigate the effects of the ATR inhibitor VE-821 in human tumor and normal cells irradiated with high LET carbon ions. FINDINGS: HeLa, U2OS, and 1BR-hTERT (normal) cells were pre-treated with 1 muM VE-821 for 1 hour and irradiated with either high LET carbon ions or X-rays. Cell survival, cell cycle distribution, cell growth, and micronuclei formation were evaluated. VE-821 caused abrogation of G2/M checkpoint and forced irradiated cells to divide into daughter cells. We also found that carbon ions caused a higher number of multiple micronuclei than X-rays, leading to decreased cell survival in tumor cells when treated with VE-821, while the survival of irradiated normal cells were not significantly affected by this inhibitor. CONCLUSIONS: ATR inhibitor would be an effective tumor radiosensitizer with carbon ion irradiation.

ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase i inhibitors by disabling DNA replication initiation and fork elongation responses.[Pubmed:25269479]

Cancer Res. 2014 Dec 1;74(23):6968-79.

Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced gammaH2AX. In cells treated with the combination, the gammaH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials.