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ZSTK474

ZSTK474 is a potent pan-class I PI3K inhibitor with IC50 of 37 nM.

Catalog No.S1072

2 Reviews 1 Product Citations

: Tel: +1-832-582-8158[email protected]

ZSTK474 Chemical Structure
Molecular Weight: 417.41

Validation & Quality Control

Product Citations(1)

J Biomol Screen, 2012, 17(8):1018-29

Customer Reviews(2) in vivo invitation

Quality Control & MSDS

Related Compound Libraries

ZSTK474 is available in the following compound libraries:

Cambridge Cancer Compound Library(96-well) Chemical Library (96-well) Inhibitor Library (96-well) Kinase Inhibitor Library (96-well)

Product Information

Compare PI3K Inhibitors

Cat.No.	Product Name	Solubility (25°C)
Cat.No.	Product Name	Water	DMSO	Ethanol
S1009	BEZ235 (NVP-BEZ235)	<1 mg/mL	1 mg/mL	<1 mg/mL
S1105	LY294002	<1 mg/mL	36 mg/mL	21 mg/mL
S1065	GDC-0941	<1 mg/mL	44 mg/mL	<1 mg/mL
S2226	CAL-101 (GS-1101)	<1 mg/mL	83 mg/mL	35 mg/mL
S2247	BKM120 (NVP-BKM120)	<1 mg/mL	82 mg/mL	2 mg/mL
S2638	NU7441 (KU-57788)	<1 mg/mL	3 mg/mL	<1 mg/mL
S2814	BYL719	<1 mg/mL	88 mg/mL	5 mg/mL
S1038	PI-103	<1 mg/mL	24 mg/mL	<1 mg/mL
S1169	TGX-221	<1 mg/mL	12 mg/mL	<1 mg/mL
S2758	Wortmannin	<1 mg/mL	86 mg/mL	<1 mg/mL
S2636	A66	<1 mg/mL	79 mg/mL	1 mg/mL
S1072	ZSTK474	<1 mg/mL	21 mg/mL	<1 mg/mL
S1118	XL147	<1 mg/mL	3 mg/mL	<1 mg/mL
S2767	3-Methyladenine	<1 mg/mL	4 mg/mL	4 mg/mL
S1268	IC-87114	<1 mg/mL	0.66 mg/mL	<1 mg/mL
S1410	AS-605240	<1 mg/mL	0.4 mg/mL	<1 mg/mL
S2671	AS-252424	<1 mg/mL	61 mg/mL	<1 mg/mL
S1523	XL765 (SAR245409)	<1 mg/mL	15 mg/mL	<1 mg/mL
S2658	GSK2126458	<1 mg/mL	101 mg/mL	<1 mg/mL
S1205	PIK-75	<1 mg/mL	3 mg/mL	<1 mg/mL
S2743	PF-04691502	<1 mg/mL	14 mg/mL	<1 mg/mL
S1462	AZD6482	<1 mg/mL	82 mg/mL	28 mg/mL
S2628	PF-05212384 (PKI-587)	<1 mg/mL	2 mg/mL	<1 mg/mL
S2696	GDC-0980 (RG7422)	<1 mg/mL	20 mg/mL	<1 mg/mL
S1489	PIK-93	<1 mg/mL	78 mg/mL	<1 mg/mL
S1187	PIK-90	<1 mg/mL	0.28 mg/mL	<1 mg/mL
S2802	BAY80-6946	<1 mg/mL	<1 mg/mL	<1 mg/mL
S1352	TG100-115	<1 mg/mL	9 mg/mL	<1 mg/mL
S1219	YM201636	<1 mg/mL	35 mg/mL	<1 mg/mL
S1360	GSK1059615	<1 mg/mL	2 mg/mL	<1 mg/mL
S2227	PIK-294	<1 mg/mL	98 mg/mL	<1 mg/mL
S2681	AS-604850	<1 mg/mL	57 mg/mL	5 mg/mL
S2699	CH5132799	<1 mg/mL	12 mg/mL	<1 mg/mL
S2682	CAY10505	<1 mg/mL	58 mg/mL	<1 mg/mL
S2749	NVP-BGT226	<1 mg/mL	30 mg/mL	<1 mg/mL
S2759	PI3K/HDAC InhibitorⅠ	<1 mg/mL	102 mg/mL	<1 mg/mL
S2207	PIK-293	<1 mg/mL	80 mg/mL	<1 mg/mL
S2739	PKI-402	<1 mg/mL	0.4 mg/mL	<1 mg/mL
S2870	TG 100713	<1 mg/mL	2 mg/mL	<1 mg/mL
S8002	GSK2636771	<1 mg/mL	87 mg/mL	19 mg/mL
S7028	IPI-145 (INK1197)	<1 mg/mL	84 mg/mL	<1 mg/mL

Research Area
Research Area
ZSTK474 Mechanism
ZSTK474 Mechanism
PI3K-delta is a heterodimer consisting of a p110 catalytic subunit and a p85 regulatory subunit. The p85 subunit has a dual function including recruiting the p110 catalytic subunit to an activated upstream tyrosine kinase receptor, and inhibiting catalytic activity in the absence of this stimulatory interaction. The p110 catalytic subunit consists of an adaptor-binding domain (ABD), a Ras-binding domain (RBD), a putative membrane-binding domain (C2), a helical domain (accessory domain) and the kinase domain (catalytic domain). ^[1] Specifically, ABD, which is not shown in our figure, mediates the interaction of p110delta and its associated p85 regulatory subunit. The RBD mediates interaction with Ras in a GTP-dependent manner. The ATP-binding pocket is located in the cleft formed by N-lobe and C-lobe, and contains four regions: An ‘adenine’ pocket (hinge), a ‘specificity’ pocket, an ‘affinity’ pocket and the hydrophobic region II located at the mouth of the active-site. ^[2]
The crystal structure indicates that ZSTK474 binds to the ATP binding site by mimicking those in the ATP-PI3K complex. ZSTK474 is fairly flat, as an s-triazine derivative it does not open the ‘specificity’ pocket and shows little isotype selectivity. Specifically, the oxygen of one of the morpholino groups forms hydrogen bond as the acceptor to hinge residue, and the morpholino ring adopts a chair conformation to occupy a space created by Met804, Trp812, and Met953. Simultaneously, the benzimidazole group extends into the ‘affinity’ pocket and the nitrogen as a hydrogen bond acceptor interacts with the primary amine of Lys779. The difluoromethyl group extends towards Pro758 in the hydrophobic ‘affinity’ pocket. In addition, the second morpholino group also changes into a twisted chair conformation and extends out of the ATP binding pocket. The binding mode determines the high inhibitory potency against p110 delta. ^[2]
References
[1] Vanhaesebroeck B, et al. Exp Cell Res. 1999, 253(1), 239-254.
[2] Berndt A, et al. Nat Chem Biol. 2010, 6(2), 117-124.
Dr. Antonino Maria Sparta demonstrates a breakthrough in leukemia research by utilizing two inhibitors produced by Selleck Chemicals.

Product Description

Biological Activity Protocol Chemical Information Customer Reviews Product Citations Tech Support & FAQs

Biological Activity

Description	ZSTK474 is a potent pan-class I PI3K inhibitor with IC50 of 37 nM.
Targets	PI3K
IC50	37 nM ^[1]
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]
Clinical Trials	A Phase I study of ZSTK474 in subjects with advanced solid malignancies is currently ongoing.
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.

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

References

Chemical Information

Download ZSTK474 SDF

Molecular Weight (MW)	417.41
Formula	C₁₉H₂₁F₂N₇O₂
CAS No.	475110-96-4
Synonyms	N/A

Solubility (25°C) DMSO 21 mg/mL Water <1 mg/mL Ethanol <1 mg/mL
Storage	2 years -20°CPowder
	2 weeks4°Cin DMSO
	6 months-80°Cin DMSO

Chemical Name	2-(difluoromethyl)-1-(4,6-dimorpholino-1,3,5-triazin-2-yl)-1H-benzo[d]imidazole

Molarity Calculator Dilution Calculator Molecular Weight Calculator

Research Area

Customer Reviews (2)

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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.

Click to enlarge

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.

Product Citations (1)

A Yeast-Based In Vivo Bioassay to Screen for Class I Phosphatidylinositol 3-Kinase Specific Inhibitors. [Fernández-Acero T, et al. J Biomol Screen 2012;17(8):1018-29]
PubMed: 22706348

Tech Support & FAQs

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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Related PI3K Inhibitors

BEZ235 (NVP-BEZ235)

BEZ235 (NVP-BEZ235) is a dual ATP-competitive PI3K and mTOR inhibitor of p110α, p110γ, p110δ and p110β with IC50 of 4 nM, 5 nM, 7 nM and 75 nM, respectively, and also inhibits ATR with IC50 of 21 nM.
PI-103

PI-103 is a potent, ATP-competitive PI3K inhibitor of DNA-PK, p110α, mTORC1, PI3KC2β, p110δ, mTORC2, p110β, and p110γ with IC50 of 2 nM, 8 nM, 20 nM, 26 nM, 48 nM, 83 nM, 88 nM and 150 nM, respectively.
GDC-0941

GDC-0941 is a potent inhibitor of PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ with IC50 of 3 nM, 33 nM, 3 nM and 75 nM, respectively.
LY294002

LY294002 is a PI3K inhibitor for p110α, p110δ and p110β with IC50 of 0.5 μM, 0.57 μM and 0.97 μM, respectively.
XL147

XL147 (SAR245408) is a selective and reversible class I PI3K inhibitor for wild type and mutant p110α with IC50 of 40 nM and 40 nM, respectively.
TGX-221

TGX-221 is a potent, selective PI3K inhibitor for p110β with IC50 of 8.5 nM.
PIK-90

PIK-90 is a potent and cell permeable inhibitor of p110α, p110β, p110γ and p110δ with IC50 of 11 nM, 350 nM, 18 nM and 58 nM, respectively.
PIK-75

PIK-75 is a selective and competitive inhibitor of p110α with IC50 of 5.8 nM and also potently inhibits DNA-PK with IC50 of 2 nM.
YM201636
YM201636 is a selective PIKfyve inhibitor with IC50 of 33 nM.
IC-87114

IC-87114 is a selective inhibitor of PI3Kδ with IC50 of 0.5 μM.

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ZSTK474

Validation & Quality Control

Product Citations(1)

Customer Reviews(2) in vivo invitation

Quality Control & MSDS

Related Compound Libraries

ZSTK474 is available in the following compound libraries:

Product Information

Compare PI3K Inhibitors

Research Area

ZSTK474 Mechanism

Product Description

Biological Activity

Protocol(Only for Reference)

Kinase Assay: ^[1]

Cell Assay: ^[1]

Animal Study: ^[1]

References

Chemical Information

Research Area

Customer Reviews (2)

Product Citations (1)

Tech Support & FAQs

If you have any other enquiries, please leave a message.

Related PI3K Inhibitors

PI3K/Akt/mTOR Pathway Products

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Choose Your Country or Region

Product Categories

ZSTK474

Validation & Quality Control

Product Citations(1)

Customer Reviews(2) in vivo invitation

Quality Control & MSDS

Related Compound Libraries

ZSTK474 is available in the following compound libraries:

Product Information

Compare PI3K Inhibitors

Research Area

ZSTK474 Mechanism

Product Description

Biological Activity

Protocol(Only for Reference)

Kinase Assay: [1]

Cell Assay: [1]

Animal Study: [1]

References

Chemical Information

Research Area

Customer Reviews (2)

Product Citations (1)

Tech Support & FAQs

If you have any other enquiries, please leave a message.

Related PI3K Inhibitors

PI3K/Akt/mTOR Pathway Products

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Kinase Assay: ^[1]

Cell Assay: ^[1]

Animal Study: ^[1]