ZSTK474

Catalog No.S1072
5 5 8 Product Citations

ZSTK474 inhibits class I PI3K isoforms with IC50 of 37 nM, mostly PI3Kδ. Phase1/2.

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ZSTK474 Chemical Structure

ZSTK474 Chemical Structure
Molecular Weight: 417.41

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Customer Reviews(5)

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  • Research Area
  • Inhibition Profile
  • ZSTK474 Mechanism

Product Description

Biological Activity

Description ZSTK474 inhibits class I PI3K isoforms with IC50 of 37 nM, mostly PI3Kδ. Phase1/2.
Targets PI3Kδ [2] PI3Kα [2] PI3K [1] PI3Kβ [2] PI3Kγ [2]
IC50 4.6 nM 16 nM 37 nM 44 nM 49 nM
In vitro 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]
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]
Features First orally administered PI3K inhibitor used in vivo.

Protocol(Only for Reference)

Kinase Assay: [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.

Cell Assay: [1]

Cell lines MCF-7, HT-29, HCT-116, OVCAR3, A549, et al.
Concentrations Dissolved in DMSO, final concentrations ~10 μM
Incubation Time 48 hours
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 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.

Animal Study: [1]

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
Formulation Suspended in 5% hydroxypropylcellulose in water as a solid dispersion form
Dosages ~400 mg/kg/day
Administration Orally
Solubility 0.5% hydroxyethyl cellulose, 30 mg/mL
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

SpeciesBaboonDogMonkeyRabbitGuinea pigRatHamsterMouse
Weight (kg)121031.80.40.150.080.02
Body Surface Area (m2)0.60.50.240.150.050.0250.020.007
Km factor202012128653
Animal A (mg/kg) = Animal B (mg/kg) multiplied by  Animal B Km
Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×  mouse Km(3)  = 11.2 mg/kg
rat Km(6)
1

References

[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.

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Clinical Trial Information( data from http://clinicaltrials.gov, updated on 2014-11-22)

NCT Number Recruitment Conditions Sponsor
/Collaborators
Start Date Phases
NCT01682473 Recruiting Neoplasms Zenyaku Kogyo Co., Ltd. September 2012 Phase 1|Phase 2
NCT01280487 Completed Neoplasms Zenyaku Kogyo Co., Ltd. January 2011 Phase 1

Chemical Information

Download ZSTK474 SDF
Molecular Weight (MW) 417.41
Formula

C19H21F2N7O2

CAS No. 475110-96-4
Storage 3 years -20℃Powder
6 months-80℃in DMSO
Synonyms
Solubility (25°C) * In vitro DMSO 21 mg/mL (50.31 mM)
Water <1 mg/mL (<1 mM)
Ethanol <1 mg/mL (<1 mM)
In vivo 0.5% hydroxyethyl cellulose 30 mg/mL
* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
Chemical Name 2-(difluoromethyl)-1-(4,6-dimorpholino-1,3,5-triazin-2-yl)-1H-benzo[d]imidazole

Research Area

Customer Reviews (5)


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Rating
Source PLoS One 2013 8(6), e66306. ZSTK474 purchased from Selleck
Method Immunofluorescence
Cell Lines ARPE-19 cells
Concentrations 250 nM
Incubation Time 1 h
Results 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.

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Rating
Source Invest New Drugs 2014 32(4), 626-35. ZSTK474 purchased from Selleck
Method Western blot
Cell Lines BCPAP, K1, 8505C cells
Concentrations 0-2.5 uM
Incubation Time 4 h
Results ZSTK474 alone produced a partially reduction in Akt phosphorylation in BCPAP cells while elicited a complete reduction in K1 and 8505C cells. ZSTK474 in combination with SB590885 or RAF265 demonstrated a complete or partial reduction of Akt phosphorylation in BCPAP, K1 and 8505C cells, respectively.

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Rating
Source Int J Oncol 2014 44(2), 557-62. ZSTK474 purchased from Selleck
Method Western blot
Cell Lines U87, U118 cells
Concentrations 2.5 uM
Incubation Time 24 h
Results When adding ZSTK474 (2.5 uM, 24 h), the phosphorylation of Akt, 4EBP1 and mTOR was markedly decreased in U87 and U118 cell lines. Cyclin D1, another protein central to cell proliferation in the PI3K/Akt pathway, was also upregulated in the presence of Velcade alone but downregulated when exposed in both ZSTK474 and Velcade.

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Rating
Source Dr. Zhang of Tianjin Medical University . ZSTK474 purchased from Selleck
Method Western blot
Cell Lines
Concentrations 0-20μM
Incubation Time
Results ZSTK474 treatment resulted in a reduction of Akt phosphorylation.

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Rating
Source Saraswati Sukumar of Johns Hopkins University School of Medicine. ZSTK474 purchased from Selleck
Method Western blot
Cell Lines T47D cells
Concentrations
Incubation Time 1 h
Results ZSTK474 treatment resulted in a reduction of Akt phosphorylation.

Product Citations (8)

Tech Support & FAQs

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