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E7449 (Synonyms: E7449; 2X-121)

Catalog No.GC18172

E7449 is an inhibitor of poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 (IC50s = 1 and 1.2 nM, respectively) as well as tankyrase (TNKS) 1/2 (IC50s = 50-100 nM).

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E7449 Chemical Structure

Cas No.: 1140964-99-3

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Sample solution is provided at 25 µL, 10mM.

Product Documents

Quality Control & SDS

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Protocol

Kinase experiment:

Briefly, 24 to 48 h after transfection, cells are washed 3× in ice-cold PBS and lysed for 20 min on ice in cell lysis buffer (CLB: 50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT, 1% TritonX-100, 1 μg/mL leupeptin, aprotinin, pepstatin, PMSF). Lysates are subject to ultracentrifugation at 100,000 g for 30 min. Cleared lysates are incubated for 1 h at 4°C with anti-GFP antibody (3E6) and pre-bound protein A magnetic beads. Beads are then washed 1 × 5 min in CLB, followed by 3 × 10 min washes in CLB containing 1 M NaCl, and 1 × 5 min wash in PARP reaction buffer (PRB; 50 mM Tris, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.1% TritonX-100, 1 μg/mL leupeptin, aprotinin, pepstatin). NAD+ incorporation reactions are performed in PRB containing 10 μM NAD+ supplemented with 32P-NAD+ at 1:20 ratio for 30 min at 25°C. For PARPs with low incorporation signals (PARP4, 5a and 16), NAD+ incorporation is performed at 1:5 ratio for 1 h at 25°C. Beads are then re-suspended in Laemmli sample buffer, heated to 65°C for 10 min, the beads removed using a magnet, and the supernatant spotted onto Whatman paper. Samples are analyzed via phosphorimaging[1].

Cell experiment:

Proliferation assays are performed in a panel of 32 isogenic DT40 cell lines, in which each line is deficient in a distinct DNA repair gene. Cells are seeded and incubated with test compound at various concentrations for 2-3 days (∼ 8 cell cycles). Cell growth is assessed using XTT and IC50 values are calculated using the GraphPad Prism 5 software version 5.02. Each experiment is conducted in duplicate and a minimum of 3 separate experiments are performed. Human breast cancer cell lines, HCC1143, HCC70, HCC1806, MDA-MB-436, T47D, MDA-MB-157, MDA-MB-231, MDA-MB-468, MDA-MB-453, BT-20 and Hs578T are used. For cell line panel assays, cells are maintained and assayed in RPMI 1640 or DMEM medium containing 10% FBS. For proliferation assays cells are plated at low density in 96 well plates. E7449 is added at various concentrations and plates incubated for a total of 8 days; compound and medium are replenished on day 4. Cell growth is assessed using the CellTiter-Glo cell viability assay. Each experiment is conducted in duplicate and a minimum of 3 separate experiments are performed[1].

Animal experiment:

Temozolomide (TMZ) combination in B16-F10 isograft model: female C57BL/6 mice are inoculated subcutaneously with B16-F10 cells (2 × 105). Following randomization by body weight, drug treatment is initiated 1 day post-inoculation. Both E7449 and TMZ are formulated in 0.5% methyl cellulose and orally administrated once per day. TMZ is administered daily on days 1 to 5 at 50 mg/kg as a single agent or in combination. E7449 is administered daily on days 1 to 7 at doses of 10, 30 and 100 mg/kg in combination with TMZ and at a dose of 100 mg/kg as a single agent. The control group is treated with vehicle (0.5% methyl cellulose in water). E7449 or vehicle is administered first and when dosing of all animals is complete TMZ is administered to animals receiving the combination[1].

References:

[1]. McGonigle S, et al. E7449: A dual inhibitor of PARP1/2 and tankyrase1/2 inhibits growth of DNA repair deficient tumors and antagonizes Wnt signaling. Oncotarget. 2015 Dec 1;6(38):41307-23.

Background

E7449 is an inhibitor of poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 (IC50s = 1 and 1.2 nM, respectively) as well as tankyrase (TNKS) 1/2 (IC50s = 50-100 nM). It is selective for PARP1, PARP2, and TNKS1/2 over PARP 3 and PARP6-15 (IC50s = >3 μM). E7449 binds to chromatin in a concentration-dependent manner and inhibits growth of DT40 cells in a PARP-dependent manner (IC50s = 3.2 and >15 μM for wild-type and PARP-/- cells, respectively). It also decreases expression of the Wnt/β-catenin signaling pathway proteins axin2, total and active β-catenin, and cyclin D1 in SW480 cells. In vivo, E7449 enhances tumor growth inhibition induced by temozolomide in a B16/F10 isograft model as well as antitumor activity of the DNA-crosslinking agent carboplatin in an MX-1 mouse xenograft model. E7449 (30 and 100 mg/kg per day) inhibits tumor growth in a BRCA1 mutant MDA-MB-436 breast cancer mouse xenograft model and induces complete inhibition of tumor cell PARP activity in an NCI-H460 lung cancer mouse xenograft model when administered at a dose of 100 mg/kg. It also delays hair regrowth, a TNKS-dependent process, in mice following hair removal.

Chemical Properties

Cas No. 1140964-99-3 SDF
Synonyms E7449; 2X-121
Chemical Name 8-[(1,3-dihydro-2H-isoindol-2-yl)methyl]-1,2-dihydro-3H-pyridazino[3,4,5-de]quinazolin-3-one
Canonical SMILES O=C1C2=CC=CC3=C2C(N=C(CN4CC(C=CC=C5)=C5C4)N3)=NN1
Formula C18H15N5O M.Wt 317.3
Solubility DMSO : 6.4 mg/mL (20.17 mM) Storage Store at -20°C
General tips Please select the appropriate solvent to prepare the stock solution according to the solubility of the product in different solvents; once the solution is prepared, please store it in separate packages to avoid product failure caused by repeated freezing and thawing.Storage method and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored at -20°C, please use it within 1 month.
To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time.
Shipping Condition Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request.

Complete Stock Solution Preparation Table

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1 mg 5 mg 10 mg
1 mM 3.1516 mL 15.758 mL 31.5159 mL
5 mM 0.6303 mL 3.1516 mL 6.3032 mL
10 mM 0.3152 mL 1.5758 mL 3.1516 mL
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Average Rating: 5 ★★★★★ (Based on Reviews and 7 reference(s) in Google Scholar.)

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