ZSTK474

生物活性产品描述 ZSTK474能够抑制I型PI3K亚型，IC50为37 nM，对PI3Kδ作用最显著。Phase 1/2。 靶点 PI3Kδ [2] PI3Kα [2] PI3K [1] PI3Kβ [2] PI3Kγ [2] IC50 4.6 nM 16 nM 37 nM 44 nM 49 nM 体外研究 ZSTK474 at 1 μM potently reduces PI3K activity to 4.7% of the control level, whereas LY2194002 only reduces the activity to 44.6% of the control. ZSTK474 inhibits the activities of recombinant p110β, -γ, and -δ with IC50 of 17 nM, 53 nM, and 6 nM, respectively. ZSTK474 shows potent antiproliferative activity against a panel of 39 human cancer cell lines with mean GI50 of 0.32 μM, more effectively than that of LY294002 or wortmannin with mean GI50 of 7.4 μM or 10 μM, respectively. ZSTK474 treatment at 1 μM blocks membrane ruffling and generation of PIP3 induced by platelet-derived growth factor in murine embryonic fibroblasts (MEFs). ZSTK474 at 10 μM induces apoptosis in OVCAR3 cells, and induces complete G1-phase arrest but not apoptosis in A549 cells. ZSTK474 treatment at 0.5 μM significantly decreases the level of phosphorylated Akt and GSK-3β, as well as the cyclin D1 protein expression. ZSTK474 also inhibits the phosphorylation of other downstream signaling components that are involved in regulating cell proliferation including FKHRL1, FKHR, TSC-2, mTOR, and p70S6K in a dose-dependent manner. [1] ZSTK474 does not inhibit mTOR at 0.1 μM, and even at a concentration of 100 μM, ZSTK474 inhibits mTOR activity less than 40%. [2] ZSTK474 blocks VEGF-induced cell migration and the tube formation in human umbilical vein endothelial cells (HUVECs), and inhibits the expression of HIF-1α and secretion of VEGF in RXF-631L cells, exhibiting potent in vitro antiangiogenic activity. [3] ZSTK474 treatment inhibits the production of IFNγ and IL-17 in concanavalin A-activated T cells, and inhibits the proliferation and PGE(2) production by fibroblast-like synovial cells (FLS). [6] 体内研究 Oral administration of ZSTK474 inhibits the growth of subcutaneously implanted mouse B16F10 melanoma tumors in a dose-dependent manner, producing tumor regression of 28.5%, 7.1%, or 4.9% on day 14 at 100, 200, or 400 mg/kg, respectively, which is superior to that of the four major anticancer drugs irinotecan, cisplatin, doxorubicin, and 5-fluorouracil at their respective maximum tolerable doses with tumor regression of 96%, 35.7%, 24%, or 68.3%, respectively. ZSTK474 treatment at 400 mg/kg completely inhibits the growth of A549, PC-3, and WiDr xenografts in mice, and induces the regression of A549 xenograft tumors. [1] ZSTK474 significantly inhibits tumor growth in the RXF-631L xenograft model, correlated with a significantly reduced number of microvessels in the ZSTK474-treated mice. [3] Oral administration of ZSTK474 ameliorates the progression of adjuvant-induced arthritis (AIA) in rats. [6] 特征 First orally administered PI3K inhibitor used in vivo. 推荐的实验操作(此推荐来自于公开的文献所以Selleck并不保证其有效性) 激酶实验: [1] 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. 细胞试验: [1] 细胞系 MCF-7, HT-29, HCT-116, OVCAR3, A549, et al. 浓度 Dissolved in DMSO, final concentrations ~10 μM 处理时间 48 hours 方法 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. 动物实验: [1] 动物模型 Male BDF1 mice injected subcutaneously with B16F10 cells, and female BALB/c nude mice inoculated subcutaneously with A549, PC-3, or WiDr cells 配制 Suspended in 5% hydroxypropylcellulose in water as a solid dispersion form 剂量 ~400 mg/kg/day 给药处理 Orally 溶解度 0.5% hydroxyethyl cellulose 30 mg/mL * 溶解度检测是由Selleck技术部门检测的，可能会和文献中提供的溶解度有所差异，这是由于生产工艺和批次不同产生的正常现象。 不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南） 小鼠 大鼠 兔 豚鼠 仓鼠 狗 猴 狒狒 重量 (kg) 0.02 0.15 1.8 0.4 0.08 10 3 12 体表面积 (m2) 0.007 0.025 0.15 0.05 0.02 0.5 0.24 0.6 Km 系数 3 6 12 8 5 20 12 20 动物A (mg/kg) = 动物B (mg/kg) × 动物B的Km系数 动物A的Km系数 例如，依据体表面积折算法，将白藜芦醇用于小鼠的剂量22.4 mg/kg 换算成大鼠的剂量，需要将22.4 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到白藜芦醇用于大鼠的等效剂量为11.2 mg/kg。 大鼠剂量 (mg/kg) = 小鼠剂量 (22.4 mg/kg) × 小鼠的Km系数(3) = 11.2 mg/kg 大鼠的Km系数(6) 1 参考文献 [1] Yaguchi S, et al. J Natl Cancer Inst, 2006, 98(8), 545-556. [2] Kong D, et al. Cancer Sci, 2007, 98(10), 1638-1642. view more [3] Kong D, et al. Eur J Cancer, 2009, 45(5), 857-865. [4] Marone R, et al. Mol Cancer Res, 2009, 7(4), 601-613. [5] Yang S, et al. PLoS One, 2011, 6(10), e26343. [6] Haruta K, et al. Inflamm Res, 2012, 61(6), 551-562. Clinical Trial Information( data from http://clinicaltrials.gov) NCT Number Recruitment Conditions Sponsor /Collaborators Start Date Phases NCT01682473 Recruiting Neoplasms Zenyaku Kogyo Co., Ltd. September 2012 Phase 1 NCT01280487 Completed Neoplasms Zenyaku Kogyo Co., Ltd. January 2011 Phase 1 化学数据点击下载ZSTK474 (SDF格式文件) 分子量 417.41 化学式 C19H21F2N7O2 CAS号 475110-96-4 稳定性 3年 -20℃粉状 6个月-80℃溶于溶剂 别名 N/A 溶解性 (25°C) * 体外 DMSO 21 mg/mL (50.31 mM) 水 <1 mg/mL (<1 mM) 乙醇 <1 mg/mL (<1 mM) 体内 0.5% hydroxyethyl cellulose 30 mg/mL * <1 mg/ml 指产品微溶或不溶 * 溶解度检测是由Selleck技术部门检测的，可能会和文献中提供的溶解度有所差异，这是由于生产工艺和批次不同产生的正常现象。 . Chemical Name 2-(difluoromethyl)-1-(4,6-dimorpholino-1,3,5-triazin-2-yl)-1H-benzo[d]imidazole 制备储备液 1 mg 5 mg 10 mg 1 mM 2.3957 mL 11.9786 mL 23.9573 mL 5 mM 0.4791 mL 2.3957 mL 4.7915 mL 10 mM 0.2396 mL 1.1979 mL 2.3957 mL 50 mM 0.0479 mL 0.2396 mL 0.4791 mL 摩尔浓度计算器 稀释计算器 分子量计算器 Research Area 客户使用Selleck产品的实验数据(5) 点击放大 The proliferation of tachyzoites in ARPE-19 cells was examined by fluorescence microscopy. Cells were pre-incubated with PI3K inhibitors, 250 nM GDC-0941 and 10 nM ZSTK474 for 1 h. After washing, the cells were then infected with T. gondii at moi of 5 for 24 h. Cells were fixed and stained with Texas Red?X phalloidin for labeling F-actin (red), and nuclei were stained with DAPI (blue). Data are representative of three independent experiments. Scale bar = 100 uM. 评级 数据来源于 PLoS One, 2013, 8(6), e66306. ZSTK474购于Selleck 方法 Immunofluorescence 细胞系 ARPE-19 cells 浓度 250 nM 处理时间 1 h 结果 PI3K-specific inhibitors, GDC-0941 and ZSTK474, showed effect as on T. gondii proliferation that inhibition of the PI3K/Akt pathway reduced the rate of T. gondii division, and most PVs only contained one or two T. gondii. Proliferation rate observation at different time point (12 h) also showed that PI3K inhibitors delay the proliferation of T. gondii compared to untreated.