Home PI3K/Akt/mTOR PI3K inhibitor ZSTK474 For research use only.

ZSTK474

ZSTK474 inhibits class I PI3K isoforms with IC50 of 37 nM in a cell-free assay, mostly PI3Kδ. Phase1/2.

ZSTK474 Chemical Structure

ZSTK474 Chemical Structure

CAS No. 475110-96-4

Purity & Quality Control

ZSTK474 Related Products

Signaling Pathway

PI3K Signaling Pathways

PI3K Signaling Pathway

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
Sf21 insect cells Function assay 1 h Inhibition of bovine recombinant PI3K p110delta expressed in Sf21 insect cells using phosphatidylinositol as substrate after 1 hr by phosphoimaging, IC50=0.7 nM 21882832
human HCT116 cells Function assay 15 mins Inhibition of PIK3CA H1047R mutant-mediated cell signaling in human HCT116 cells expressing PTEN assessed as inhibition of insulin-induced pAkt/PKB phosphorylation at Thr308 treated for 15 mins before insulin challenge measured after 5 mins by immunoblotting, IC50=78 nM 21882832
human LNCAP cells Proliferation assay 3 days Antiproliferative activity against human LNCAP cells after 3 days by MTS assay, IC50=0.21 μM 20227881
human NZB5 cells Proliferation assay 5 days Antiproliferative activity against human NZB5 cells expressing wild type p110alpha assessed as incorporation of [3H]thymidine after 5 days, IC50=0.22 μM 21882832
human NZOV9 cells Proliferation assay 5 days Antiproliferative activity against human NZOV9 cells expressing p110alpha kinase Y1021C mutant assessed as incorporation of [3H]thymidine after 5 days, IC50=0.29 μM 21882832
human MDA-MB-468 cells Cytotoxic assay 48 h Cytotoxicity against PTEN-deficient human MDA-MB-468 cells assessed as inhibition of cell growth after 48 hrs by Cell Titer 96 assay 23795239
human A549 cells Function assay 10 μM 1 h Inhibition of PI3K in human A549 cells assessed as reduction in pAkt level at 10 uM after 1 hr by Western blotting analysis 25766633
human DMS114 cells Growth inhibition assay Growth inhibition of human DMS114 cells 22336246
human MKN74 cells Growth inhibition assay Growth inhibition of human MKN74 cells 22336246
human SNB78 cells Growth inhibition assay Growth inhibition of human SNB78 cells 22336246
human St-4 cells Growth inhibition assay Growth inhibition of human St-4 cells 22336246
human DU145 cells Growth inhibition assay Growth inhibition of human DU145 cells 22336246
human LOXIMVI cells Growth inhibition assay Growth inhibition of human LOXIMVI cells 22336246
human PC3 cells Growth inhibition assay Growth inhibition of human PC3 cells 22336246
human LOXIMVI cells Growth inhibition assay Growth inhibition of human LOXIMVI cells 22336246
human OVCAR3 cells Growth inhibition assay Growth inhibition of human OVCAR3 cells 22336246
human SKOV3 cells Growth inhibition assay Growth inhibition of human SKOV3 cells 22336246
human KM12 cells Growth inhibition assay Growth inhibition of human KM12 cells 22336246
human HT-29 cells Growth inhibition assay Growth inhibition of human HT-29 cells 22336246
human HCT15 cells Growth inhibition assay Growth inhibition of human HCT15 cells 22336246
human NCI-H226 cells Growth inhibition assay Growth inhibition of human NCI-H226 cells 22336246
human NCI-H522 cells  Growth inhibition assay Growth inhibition of human NCI-H522 cells  22336246
human A549 cells Growth inhibition assay Growth inhibition of human A549 cells 22336246
human HCC2998 cells Growth inhibition assay Growth inhibition of human HCC2998 cells 22336246
human SNB75 cells Growth inhibition assay Growth inhibition of human SNB75 cells 22336246
human OVCAR4 cells Growth inhibition assay Growth inhibition of human OVCAR4 cells 22336246
human OVCAR5 cells Growth inhibition assay Growth inhibition of human OVCAR5 cells 22336246
human OVCAR8 cells Growth inhibition assay Growth inhibition of human OVCAR8 cells 22336246
human SKOV3 cells Growth inhibition assay Growth inhibition of human SKOV3 cells 22336246
human ACHN cells Growth inhibition assay Growth inhibition of human ACHN cells 22336246
Click to View More Cell Line Experimental Data

Biological Activity

Description ZSTK474 inhibits class I PI3K isoforms with IC50 of 37 nM in a cell-free assay, mostly PI3Kδ. Phase1/2.
Features First orally administered PI3K inhibitor used in vivo.
Targets
PI3Kδ [2]
(Cell-free assay)		 PI3Kα [2]
(Cell-free assay)		 PI3K [1]
(Cell-free assay)		 PI3Kβ [2]
(Cell-free assay)		 PI3Kγ [2]
(Cell-free assay)
4.6 nM 16 nM 37 nM 44 nM 49 nM
In vitro
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]
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 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 Research 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.
Experimental Result Images Methods Biomarkers Images PMID
Western blot p-PDK1 / p-GSK3β / p-AKT / AKT P-gp / MRP1 p-Rb / p27 / Cyclin D1 HIF-1α / HIF-1β 28388564
Growth inhibition assay Cell viability 28388564
In Vivo
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 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]
Animal Research 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
Dosages ~400 mg/kg/day
Administration Orally

Chemical Information & Solubility

Molecular Weight 417.41 Formula

C19H21F2N7O2
CAS No. 475110-96-4 SDF Download ZSTK474 SDF
Smiles C1COCCN1C2=NC(=NC(=N2)N3C4=CC=CC=C4N=C3C(F)F)N5CCOCC5
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 15 mg/mL ( (35.93 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Water : Insoluble

Ethanol : Insoluble


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