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ZSTK474
本产品不向个人销售,仅用作科学研究,不用于任何人体实验及非科研性质的动物实验。
ZSTK474图片
CAS NO:475110-96-4
规格:≥98%
包装与价格:
包装价格(元)
5mg电议
50mg电议
100mg电议
250mg电议
500mg电议
1g电议
2g电议

产品介绍
理化性质和储存条件

Molecular Weight (MW)

417.41

Formula

C19H21F2N7O2

CAS No.

19545-26-7

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 21 mg/mL (50.31 mM)

Water:<1 mg/mL (slightly soluble or insoluble)

Ethanol: <1 mg/mL

Solubility (In vivo)

0.5% hydroxyethyl cellulose: 30mg/mL

Chemical Name/Synonyms

ZSTK474; ZSTK-474; ZSTK 474; 4-[4-[2-(difluoromethyl)benzimidazol-1-yl]-6-morpholin-4-yl-1,3,5-triazin-2-yl]morpholine

实验参考方法

In Vitro

Kinase Assay: 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.

Cell Assay: Cells (MCF-7, HT-29, HCT-116, OVCAR3, A549, et al.) 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.

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. ZSTK474 does not inhibit mTOR at 0.1 μM, and even at a concentration of 100 μM, ZSTK474 inhibits mTOR activity less than 40%.

In Vivo

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. ZSTK474 significantly inhibits tumor growth in the RXF-631L xenograft model, correlated with a significantly reduced number of microvessels in the ZSTK474-treated mice. Oral administration of ZSTK474 ameliorates the progression of adjuvant-induced arthritis (AIA) in rats.

Animal model

Male BDF1 mice injected subcutaneously with B16F10 cells, and female BALB/c nude mice inoculated subcutaneously with A549, PC-3, or WiDr cells

Formulation & Dosage

Suspended in 5% hydroxypropylcellulose in water as a solid dispersion form; mg400/kg; oral

References

[1] Yaguchi S, et al. J Natl Cancer Inst, 2006, 98(8), 545-556.

生物活性


ZSTK474 alleviates neurological deficits and reduces infarct volume in a model of ischemic reperfusion. J Neuroinflammation. 2016; 13(1): 192.


ZSTK474 inhibits the expression of Iba-1 and GFAP at 72 h after cerebral reperfusion injury. J Neuroinflammation. 2016; 13(1): 192.


Double immunofluorescent staining for the microglial/macrophage marker Iba-1 and detrimental or restorative state markers. J Neuroinflammation. 2016; 13(1): 192.


ZSTK474 inhibits pro-inflammatory cytokines and promotes anti-inflammatory cytokines. J Neuroinflammation. 2016; 13(1): 192.


ZSTK474 reduces P-AKT and P-p70S6k protein levels. ZSTK474 suppresses phosphorylation of AKT and other downstream compounds. J Neuroinflammation. 2016; 13(1): 192.


Summary diagram illustrates a hypothesis for how ZSTK474 exerts its effects on ischemic reperfusion injury. J Neuroinflammation. 2016; 13(1): 192.