| 名稱 | ZSTK474 |
| 描述 | PI3K Inhibitor ZSTK474 is an orally available, s-triazine derivative, ATP-competitive phosphatidylinositol 3-kinase (PI3K) inhibitor with potential antineoplastic activity. |
| 細胞實驗 | Cells are exposed to increasing concentrations of ZSTK474 for 48 hours. The inhibition of cell proliferation is assessed by measuring changes in total cellular protein by use of a sulforhodamine B assay. Apoptosis is assessed by chromatin condensation or by flow cytometry. For chromatin condensation assay, cells are stained with Hoechst 33342 and examined by fluorescence microscopy. Morphologic changes induced by ZSTK474, such as the condensation of chromatin, are indicative of apoptosis. For flow cytometry analysis, cells are harvested, washed with ice-cold PBS, and fixed in 70% ethanol. Cells are then washed twice with ice-cold PBS again, treated with RNase A (500 μg/mL) at 37 °C for 1 hour, and stained with propidium iodide (25 μg/mL). The DNA content of the cells is analyzed with a flow cytometer. (Only for Reference) |
| 激酶實驗 | Inhibition of PI3K activity: A549 cells are lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, and 1% Igepal CA-630, the lysates are centrifuged at 20,000 g and 4 °C for 10 minutes, and the supernatants are used as cell lysate (protein = 2-4 mg/mL). To immunoprecipitate PI3K, 200 μL of cell lysate are incubated with anti-p85 polyclonal antibody and protein G-agarose (5 μL). PI3Kα, PI3Kβ, and PI3Kδ can be immunoprecipitated by the anti-p85 polyclonal antibody. Agarose beads containing immunoprecipitates are washed twice with buffer A (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Igepal CA-630), once with buffer B (500 mM LiCl and 100 mM Tris-HCl at pH 7.5), once with distilled water, and once with buffer C (100 mM NaCl and 20 mM Tris-HCl at pH 7.5). Immunoprecipitates are suspended in 20 μL of buffer C containing phosphatidylinositol of 200 μg/mL. The mixture is preincubated with increasing concentrations of ZSTK474 at 25 °C for 5 minutes. [γ-32P]ATP (2 μCi per assay mixture; final concentration, 20 μM) and MgCl2 (final concentration, 20 mM) are added to start the reaction. The reaction mixture is incubated at 25 °C for 20 minutes. Phosphorylated products of phosphatidylinositol are separated by thin-layer chromatography and visualized by autoradiography. The phosphatidylinositol-3-phosphate region is scraped from the plate, and radioactivity is also measured with liquid scintillation spectroscopy. The level of inhibition for ZSTK474 is determined as the percentage of 32P counts per minute obtained without ZSTK474. |
| 體外活性 | ZSTK474以1μM的濃度顯著降低PI3K活性至對照水平的4.7%，而LY2194002只能將活性降低至44.6%。ZSTK474對重組的p110β、-γ和-δ的活性顯示出強大的抑制作用，IC50分別為17 nM、53 nM和6 nM。相比LY294002和wortmannin（其平均GI50分別為7.4 μM和10 μM），ZSTK474對39種人類癌細胞系展現(xiàn)出更強的抗增殖活性，平均GI50為0.32 μM。在1μM的濃度下，ZSTK474能夠阻斷由血小板衍生生長因子在小鼠胚胎成纖維細胞（MEFs）中引起的膜皺褶形成和PIP3的產(chǎn)生。10 μM的ZSTK474誘導(dǎo)OVCAR3細胞凋亡，并在A549細胞中引發(fā)完全的G1期阻滯但不誘導(dǎo)凋亡。0.5μM濃度的ZSTK474顯著降低磷酸化Akt和GSK-3β水平及cyclin D1蛋白表達，并以劑量依賴的方式抑制包括FKHRL1、FKHR、TSC-2、mTOR和p70S6K在內(nèi)的其它下游信號組分的磷酸化，這些組分參與調(diào)控細胞增殖。[1]在0.1μM濃度下，ZSTK474不抑制mTOR，即使?jié)舛冗_到100μM，ZSTK474對mTOR的活性抑制也不超過40%。[2]ZSTK474阻斷VEGF誘導(dǎo)的人臍靜脈內(nèi)皮細胞（HUVECs）的細胞遷移和管狀形成，并抑制RXF-631L細胞中HIF-1α的表達和VEGF的分泌，顯示出強大的體外抗血管生成活性。[3]ZSTK474治療抑制刀豆素A激活的T細胞中IFNγ和IL-17的產(chǎn)生，并抑制成纖維樣滑膜細胞（FLS）的增殖和PGE(2)產(chǎn)生。[6] |
| 體內(nèi)活性 | 通過口服給藥，ZSTK474以劑量依賴的方式抑制了小鼠皮下植入的B16F10黑色素瘤腫瘤的生長，在第14天時，分別以100, 200, 或400 mg/kg的劑量產(chǎn)生了28.5%, 7.1%, 或4.9%的腫瘤退縮率，優(yōu)于四種主要抗癌化合物irinotecan, cisplatin, doxorubicin, 以及5-fluorouracil在其各自的最大耐受劑量下的腫瘤退縮率，分別為96%, 35.7%, 24%, 或68.3%。400 mg/kg的ZSTK474治療完全抑制了小鼠體內(nèi)A549, PC-3, 和WiDr異種移植瘤的生長，并引起A549異種移植瘤腫瘤的退縮。ZSTK474顯著抑制了RXF-631L異種移植模型中的腫瘤生長，并與ZSTK474處理小鼠中顯著減少的微血管數(shù)量相關(guān)?？诜o藥的ZSTK474改善了大鼠佐劑誘導(dǎo)的關(guān)節(jié)炎(AIA)的進展。 |
| 存儲條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | DMSO : 8.75 mg/mL (20.96 mM), Sonication is recommended.
Ethanol : < 1 mg/mL (insoluble or slightly soluble)
H2O : < 1 mg/mL (insoluble or slightly soluble)
|
| 關(guān)鍵字 | PI3K | ZSTK-474 | Inhibitor | Phosphoinositide 3-kinase | ZSTK474 | inhibit | Autophagy | ZSTK 474 |
| 相關(guān)產(chǎn)品 | Guanidine hydrochloride | Naringin | Valproic Acid | Taurine | Gefitinib | Aceglutamide | Hydroxychloroquine | Curcumin | Stavudine | Salicylic acid | Paeonol | Sodium 4-phenylbutyrate |
| 相關(guān)庫 | 抑制劑庫 | 抗癌活性化合物庫 | 經(jīng)典已知活性庫 | 已知活性化合物庫 | 激酶抑制劑庫 | 抗衰老化合物庫 | 抗氧化化合物庫 | 藥物功能重定位化合物庫 | 抗癌臨床化合物庫 | 抗癌藥物庫 |