| 名稱(chēng) | AZ20 |
| 描述 | AZ20 is an effective and specific inhibitor of ATR kinase (IC50: 5 nM, in a cell-free assay), 8-fold selectivity over mTOR. |
| 細(xì)胞實(shí)驗(yàn) | AZ20 is dissolved in 100% DMSO. Compound dose ranges are created by diluting in 100% DMSO and then further into assay medium (EMEM, 10% FCS, 1% glutamine) using a Labcyte Echo acoustic dispensing instrument. Cells are plated in 384-well Costar plates at 9×104?cells per mL in 40 μL of EMEM, 10% FCS, 1% glutamine and grown for 24 h. Following addition of compound the cells are incubated for 60 min. A final concentration of 3 μM?4NQO?(prepared in 100% DMSO) is then added using the Labcyte Echo, and the cells are incubated for a further 60 min. The cells are fixed by adding 40 μL of 3.7% v/v formaldehyde solution for 20 min. After removal of fix, cells are washed with PBS and permeabilized in 40 μL of PBS containing 0.1% Triton X-100. The cells are then washed, and 15 μL primary antibody solution (pChk1 Ser345) is added. The plates are incubated at 4°C overnight. The primary antibody is then washed off, and 20 μL of secondary antibody solution and 1 μM Hoechst 33258 added for 90 min at room temperature. The plates are washed and left in 40 μL of PBS. Plates are then read on an ArrayScan VTI instrument to determine staining intensities, and dose responses are obtained and used to determine the IC50?values for the compounds. |
| 激酶實(shí)驗(yàn) | ATR for use in the in vitro enzyme assay is obtained from HeLa nuclear extract by immunoprecipitation with rabbit polyclonal antiserum raised to amino acids 400-480 of ATR contained in the following buffer: 25 mM HEPES (pH 7.4), 2 mM MgCl2, 250 mM NaCl, 0.5 mM EDTA, 0.1 mM Na3VO4, 10% v/v glycerol, and 0.01% v/v Tween 20. ATR-antibody complexes are isolated from nuclear extract by incubating with protein A-Sepharose beads for 1 h and then through centrifugation to recover the beads. In the well of a 96-well plate, 10 μL ATR-containing Sepharose beads are incubated with 1 μg of substrate glutathione?S-transferase-p53N66 (NH2-terminal 66 amino acids of p53 fused to glutathione?S-transferase are expressed in?E. coli) in ATR assay buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 6 mM MgCl2, 4 mM MnCl2, 0.1 mM Na3VO4, 0.1 mM DTT, and 10% (v/v) glycerol) at 37°C in the presence or absence of inhibitor. After 10 min with gentle shaking, ATP is added to a final concentration of 3 μM and the reaction continued at 37°C for an additional 1 h. The reaction is stopped by addition of 100 μL of PBS, and the reaction is transferred to a white opaque glutathione coated 96-well plate and incubated overnight at 4°C. This plate is then washed with PBS/0.05% (v/v) Tween 20, blotted dry, and analyzed by a standard ELISA technique with a phosphoserine 15 p53 antibody. The detection of phosphorylated glutathione?S-transferase-p53N66 substrate is performed in combination with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody. Enhanced chemiluminescence solution is used to produce a signal, and chemiluminescent detection is carried out via a TopCount plate reader. The resulting calculated % enzyme activity is then used to determine the IC50?values for the compounds (IC50?taken as the concentration at which 50% of the enzyme activity is inhibited). |
| 體外活性 | AZ20（10 μM）可使CYP3A4介導(dǎo)的咪達(dá)唑侖代謝的50％被抑制.LoVo腫瘤的雌性裸鼠中,服用13天AZ20（25 mg/kg,2次/天,p.o.;50 mg/kg/day,p.o. ）可使腫瘤生長(zhǎng)受到顯著抑制,這與異種移植組織中γH2AX核染色的持續(xù)廣泛升高有關(guān). |
| 體內(nèi)活性 | 在體外,AZ20濃度依賴(lài)性地降低pChk1 Ser345/317/296水平。延長(zhǎng)AZ20的處理時(shí)間可增加γH2AX核染色,這是因?yàn)榇嬖趶?fù)制壓力,且與S期阻滯和組蛋白H3的磷酸化水平增加有關(guān)。AZ20對(duì)體外生長(zhǎng)抑制和細(xì)胞死亡的誘導(dǎo)作用是完全不同于其它細(xì)胞毒性藥物的。AZ2與ATM 抑制劑KU-60019聯(lián)用會(huì)增加細(xì)胞毒性。 |
| 存儲(chǔ)條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | Ethanol : 3 mg/mL (7.27 mM)
H2O : < 1 mg/mL (insoluble or slightly soluble)
DMSO : 50 mg/mL (121.21 mM), Sonication is recommended.
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| 關(guān)鍵字 | AZ20 | Ataxia telangiectasia mutated | inhibit | AZ-20 | ATM and RAD3 related | Inhibitor | ATM/ATR | AZ 20 |
| 相關(guān)產(chǎn)品 | Schisandrin B | α-Hydroxyglutaric Acid Lithium | P-2281 | Capivasertib | Rapamycin | Elimusertib | Bimiralisib | L-Leucine | GDC0084 | Tacrolimus | PI3K/Akt/mTOR-IN-2 | Sapanisertib |
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