| 名稱(chēng) | Ipatasertib |
| 描述 | Ipatasertib (GDC-0068) is a selective, ATP-competitive pan-Akt inhibitor that inhibits Akt1 (IC50:5 nM), Akt2 (IC50:18 nM), and Akt3 (IC50:8 nM). Ipatasertib (GDC-0068) can lead to p53-independent PUMA activation by inhibiting Akt, thereby activating FoxO3a and NF-κB simultaneously, directly binding to the PUMA promoter, upregulating PUMA transcription and Bax-mediated intrinsic mitochondrial apoptosis. |
| 細(xì)胞實(shí)驗(yàn) | GDC-0068 is prepared in DMSO and stored, and then diluted with appropriate medium before use[2]. The 384-well plates are seeded with 2,000 cells per well in a volume of 54 μL per well followed by incubation at 37°C under 5% CO2 overnight (~16 hours). Compounds (e.g., GDC-0068) are diluted in DMSO to generate the desired stock concentrations then added in a volume of 6 μL per well. All treatments are tested in quadruplicates. After 4 days incubation, relative numbers of viable cells are estimated using CellTiter-Glo and total luminescence is measured on a Wallac Multilabel Reader. The concentration of drug resulting in IC50 is calculated from a 4-parameter curve analysis (XLfit) and is determined from a minimum of 3 experiments. For cell lines that failed to achieve an IC50, the highest concentration tested (10 μM) is listed[2]. |
| 激酶實(shí)驗(yàn) | Kinase Assay: The fluorescence polarization assay for ATP competitive inhibition is done as follows: mPI3Kα dilution solution (90 nM) is prepared in fresh assay buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 5 mM DTT, 0.05% CHAPS) and kept on ice. The enzyme reaction contains 0.5 nM mouse PI3Kα (p110α/p85α complex purified from insect cells), 30 μM PIP2, PF-04691502 (0, 1, 4, and 8 nM), 5 mM MgCl2, and 2-fold serial dilutions of ATP (0–800 μM). Final dimethyl sulfoxide is 2.5%. The reaction is initiated by the addition of ATP and terminated after 30 minutes with 10 mM EDTA. In a detection plate, 15 uL of detector/probe mixture containing 480 nM GST-Grp1PH domain and 12 nM TAMRA tagged fluorescent PIP3 in assay buffer is mixed with 15 uL of kinase reaction mixture. The plate is shaken for 3 minutes, and incubated for 35 to 40 minutes before reading on an LJL Analyst HT. |
| 體外活性 | 方法：在 Ipatasertib (GDC-0068) （1-20 μM） 處理 HCT116 細(xì)胞后的 0、3、6、12 和 24 小時(shí)，通過(guò) CCK-8 在 HCT116 中檢測(cè)細(xì)胞活力，研究 ipatasertib 如何影響腫瘤進(jìn)展。
結(jié)果：HCT116 細(xì)胞活力隨著劑量或時(shí)間的增加而顯著下降， Ipatasertib (GDC-0068) 可以以劑量和時(shí)間依賴(lài)性方式抑制細(xì)胞增殖。[1]
方法： Ipatasertib (GDC-0068) （10μM）處理HCT116細(xì)胞，通過(guò)蛋白質(zhì)印跡分析 HCT116 WT 和 p53?/? 中 p53 或 PUMA 的表達(dá)；通過(guò)實(shí)時(shí) qPCR 分析 WT、p53?/?HCT116 和 DLD1 中 Ipatasertib (GDC-0068) 誘導(dǎo)的 PUMA mRNA，并將其標(biāo)準(zhǔn)化為管家基因 β-actin。
結(jié)果：Ipatasertib (GDC-0068) 處理隨著劑量的增加，PUMA的表達(dá)水平逐漸上調(diào)；在 WT （HCT116， RKO）、p53 突變體 （DLD1， HT29） 和 p53 中均觀察到這種上調(diào)；Ipatasertib (GDC-0068) 可導(dǎo)致 PUMA 的 p53 非依賴(lài)性轉(zhuǎn)錄激活并抑制細(xì)胞增殖。[1] |
| 體內(nèi)活性 | 方法：裸鼠皮下注射HCT116 WT 或 PUMA?/?，模型小鼠服用Ipatasertib (GDC-0068) （30mg / kg，口服，15天）， 計(jì)算實(shí)驗(yàn)結(jié)束時(shí)的代表性腫瘤、腫瘤重量和治療后指定時(shí)間點(diǎn)的 c 腫瘤體積，研究PUMA 介導(dǎo)的細(xì)胞凋亡對(duì)于 ipatasertib 的抗腫瘤活性是否是必需的。
結(jié)果：Ipatasertib (GDC-0068) 顯著抑制了 WT 腫瘤的生長(zhǎng)；免疫組化染色顯示，WT和PUMA中P-Akt的表達(dá)均降低；Ki67在WT腫瘤中明顯降低，但在PUMA中無(wú)較大變化；C-Caspase3在WT腫瘤中明顯增加，在PUMA中略有增加；Ipatasertib (GDC-0068) 在結(jié)腸癌中具有 PUMA 依賴(lài)性的抗腫瘤作用。[1] |
| 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 | DMSO : 85 mg/mL (185.6 mM)
Ethanol : 85 mg/mL (185.6 mM)
H2O : < 1 mg/mL (insoluble or slightly soluble)
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| 關(guān)鍵字 | Protein kinase B | RG-7440 | Ipatasertib | Inhibitor | Akt | PKB | GDC 0068 | inhibit | GDC0068 | RG 7440 |
| 相關(guān)產(chǎn)品 | Honokiol | Capivasertib | Methyl-Hesperidin | AKT Kinase Inhibitor | Artemisinin | 2,3-Butanediol | PI3K/Akt/mTOR-IN-2 | Oridonin | Ethyl gallate | SKLB-163 |
| 相關(guān)庫(kù) | 抑制劑庫(kù) | 經(jīng)典已知活性庫(kù) | 抗癌活性化合物庫(kù) | 已知活性化合物庫(kù) | 激酶抑制劑庫(kù) | 高選擇性抑制劑庫(kù) | 抗衰老化合物庫(kù) | 藥物功能重定位化合物庫(kù) | 抗癌臨床化合物庫(kù) | 抗癌藥物庫(kù) |