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ZSTK474

ZSTK474

Item no.	S1072-1000
Manufacturer	Selleckchem
CASRN	475110-96-4
Amount	1 g
Quantity options	10 mg 100 mg 1 g 10 g 10 mM/1 mL 200 mg 50 mg 5 g
Category	Oncology Inhibitors & Compound Libraries Small Molecules By Organ Skin Cancer Bladder Cancer Colorectal Cancer Testicular & Prostate Cancer
Type	Inhibitors
Specific against	other
Smiles	C1COCCN1C2=NC(=NC(=N2)N3C4=CC=CC=C4N=C3C(F)F)N5CCOCC5
ECLASS 10.1	32160490
ECLASS 11.0	32160490
UNSPSC	12000000
Alias	475110-96-4'
Similar products	ZSTK474
Available
Manufacturer - Targets	PI3K
Storage Conditions	2 years -80 in solvent
Molecular Weight	417, 41
Administration	Orally
Animal Models	Male BDF1 mice injected subcutaneously with B16F10 cells, and female BALB/c nude mice inoculated subcutaneously with A549, PC-3, or WiDr cells
Cell lines	MCF-7, HT-29, HCT-116, OVCAR3, A549, et al.
Clinical Trials	A Phase I study of ZSTK474 in subjects with advanced solid malignancies is currently ongoing.
Concentrations	Dissolved in DMSO, final concentrations ca.10 uM
Dosages	ca.400 mg/kg/day
Formulation	Suspended in 5% hydroxypropylcellulose in water as a solid dispersion form
IC50	37 nM [1], 37 nM [1], 37 nM [1], 37 nM [1], 37 nM [1], 37 nM [1]
In vitro	ZSTK474 at 1 uM 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 p110beta, -gamma, and -delta 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 uM, more effectively than that of LY294002 or wortmannin with mean GI50 of 7.4 uM or 10 uM, respectively. ZSTK474 treatment at 1 uM blocks membrane ruffling and generation of PIP3 induced by platelet-derived growth factor in murine embryonic fibroblasts (MEFs). ZSTK474 at 10 uM induces apoptosis in OVCAR3 cells, and induces complete G1-phase arrest but not apoptosis in A549 cells. ZSTK474 treatment at 0.5 uM significantly decreases the level of phosphorylated Akt and GSK-3beta, 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 uM, and even at a concentration of 100 uM, 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-1alpha and secretion of VEGF in RXF-631L cells, exhibiting potent in vitro antiangiogenic activity. [3] ZSTK474 treatment inhibits the production of IFNgamma and IL-17 in concanavalin A-activated T cells, and inhibits the proliferation and PGE(2) production by fibroblast-like synovial cells (FLS). [6]
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. [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]
Incubation Time	48 hours
Kinase Assay	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 uL of cell lysate are incubated with anti-p85 polyclonal antibody and protein G-agarose (5 uL). PI3Kalpha, PI3Kbeta, and PI3Kdelta 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 uL of buffer C containing phosphatidylinositol of 200 ug/mL. The mixture is preincubated with increasing concentrations of ZSTK474 at 25 C for 5 minutes. [gamma-32P]ATP (2 uCi per assay mixture, final concentration, 20 uM) 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.
Method	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 ug/mL) at 37 C for 1 hour, and stained with propidium iodide (25 ug/mL). The DNA content of the cells is analyzed with a flow cytometer.
Solubility (25C)	DMSO 21 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL
Information	ZSTK474 inhibits class I PI3K isoforms with IC50 of 37 nM in a cell-free assay, mostly PI3Kδ. Phase1/2.
Chemical Name	2-(difluoromethyl)-1-(4, 6-dimorpholino-1, 3, 5-triazin-2-yl)-1H-benzo[d]imidazole
Features	First orally administered PI3K inhibitor used in vivo.

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