PARP2 ( IC50 = 1 nM ); PARP1 ( IC50 = 2 nM ); TNKS1 ( IC50 = 50 nM ); TNKS2 ( IC50 = 50 nM )
Stenoparib (E7449) is a dual inhibitor of PARP1/2 and tankyrase1/2 (TNKS1/2, also known as PARP5a/5b) [1]

Stenoparib is a strong inhibitor of PARP1 and PARP2, as well as TNKS1 and TNKS2. It uses ³²P-NAD⁺ as substrate and has IC50 values of 2.0, 1.0, ∼50, and ∼50 nM for PARP1, PARP2, TNKS1, and TNKS2, respectively. There are no discernible inhibitory effects of stenoparib on PARP3 or PARPs 6–16. Stenoparib affects DNA repair pathways other than homologous recombination (HR) by binding to damaged DNA and trapping PARP1. Cells lacking in BRCA1 and 2, CtIP, and Rad54—components of the HR pathway—are most effectively suppressed by tenoparib. In SW480 cells, stenoparib (10 μM) inhibits Wnt signaling[1].

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Stenoparib (E7449) inhibited PARP enzymatic activity and trapped PARP1 onto damaged DNA, a mechanism that augments cytotoxicity. Cells deficient in DNA repair pathways beyond homologous recombination were sensitive to E7449 treatment, with the IC50 value of E7449 in wild-type DT40 cells being 3.2 μmol/L. In colon cancer cell line SW480, E7449 inhibited Wnt/β-catenin signaling by stabilizing axin and TNKS proteins, leading to β-catenin destabilization and significant alteration of Wnt target gene expression (10 μmol/L E7449 for 24 h altered the protein levels of axin2, total β-catenin, active β-catenin, and cyclin D1; 3/30 μmol/L E7449 for 72 h changed the expression of 17/30 Wnt-related genes). Additionally, E7449 potentiated the cytotoxicity of chemotherapeutic drugs (temozolomide, carboplatin) in vitro, and MDA-MB-436 (BRCA1 mutant), the most sensitive breast cancer cell line to E7449, showed impaired proliferation upon E7449 treatment [1]

Stenoparib moderately suppresses the growth of tumors at a dose of 100 mg/kg and dramatically increases the inhibition in the mouse melanoma B16-F10 isograft model when oral doses of 10, 30, and 100 mg/kg are combined with temozolomide (TMZ). In a BRCA mutant xenograft model, stenoparib (30 or 100 mg/kg, p.o.) inhibits PARP, exhibits anti-tumor activity, and is well-tolerated with no apparent body weight loss or fatalities. In C57BL/6 mice, tenoparib (30, 100, or 300 mg/kg, p.o.) inhibits Wnt signaling and dose-dependently suppresses hair regrowth. Breast tumors originally isolated from Wnt1 (int-1) transgenic mice demonstrate antitumor activity when stenoparib (100 mg/kg, p.o.) is combined with MEK inhibitor[1].

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Stenoparib (E7449) exhibited significant single-agent antitumor activity in BRCA-deficient MDA-MB-436 human breast cancer xenografts: oral administration once daily for 28 consecutive days significantly inhibited tumor growth on day 83 (P < 0.05 vs vehicle). A single dose of E7449 (30 mg/kg or 100 mg/kg) inhibited PARP activity in MDA-MB-436 tumor tissues at 1-36 hours post-administration, as measured by PAR levels. In NCI-H460 human lung cancer xenografts, E7449 dose-dependently (1-100 mg/kg) inhibited PARP activity in tumor tissues from 15 min to 36 hours post-treatment. In B16-F10 mouse melanoma isografts, E7449 combined with temozolomide enhanced antitumor efficacy (P < 0.05 vs TMZ alone on days 14 and 20). In the MX-1 human breast cancer orthotopic model, E7449 (100 mg/kg) combined with carboplatin (60 mg/kg, single IV dose) showed enhanced antitumor activity (P < 0.05 vs E7449 D4 + carboplatin D3 on day 19).
In the Wnt1 subcutaneous model, Stenoparib (E7449) lacked single-agent antitumor activity, but combination with the MEK inhibitor E6201 resulted in synergistic tumor growth inhibition. E7449 dose-dependently (30, 100, 300 mg/kg) inhibited Wnt signaling-mediated hair regrowth in C57BL/6 female mice after depilation. A pharmacodynamic effect of E7449 on Wnt target genes was observed in tumor tissues, with altered expression of CAR2, FZD9, LEF1, and VIL1 [1]

In summary, cells are lysed for 20 minutes 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) after being washed three times in ice-cold PBS within 24 to 48 hours after transfection. Lysates undergo a 30-minute ultracentrifugation process at 100,000 g. After lysates are cleared, they are incubated with pre-bound protein A magnetic beads and anti-GFP antibody (3E6) for one hour at 4°C. Following a 1-minute soak in CLB, beads are then washed for 3 minutes in CLB containing 1 M NaCl and 1 minute 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, and pepstatin). For 30 minutes at 25°C, NAD⁺ incorporation reactions are carried out in PRB containing 10 μM NAD⁺ supplemented with ³²P-NAD⁺ at a 1:20 ratio. NAD⁺ incorporation is carried out at a 1:5 ratio for one hour at 25°C for PARPs with low incorporation signals (PARP4, 5a, and 16). After that, the beads are once again suspended in Laemmli sample buffer, heated for ten minutes to 65°C, the beads are extracted with a magnet, and the supernatant is spotted onto Whatman paper. Phosphorimaging is used to analyze samples[1].

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1. PARP activity assay in tumor tissues: Tumor lysates from MDA-MB-436 or NCI-H460 xenografts were prepared, and PAR levels were measured to assess PARP activity. PAR levels were normalized by protein concentration, with the mean PAR level in vehicle-treated control animals set to 100% PARP activity. The inhibition rate of PARP activity by Stenoparib (E7449) at each time point was calculated using the average of all control replicates, with data from 2 experiments assayed in triplicate [1]
2. PARP1 trapping assay in DT40 cells: DT40 cells were treated with various concentrations of Stenoparib (E7449) for 30 min in the presence or absence of 0.05% MMS. Chromatin-bound proteins were extracted, and western blot was performed using antibodies against PARP1 and Histone H3 (a chromatin-bound marker). The signal intensity of PARP1 was quantified with Image Studio software on the LI-COR Odyssey imager, and the results were representative of 3 independent assays [1]

The 32 isogenic DT40 cell lines, each lacking a different DNA repair gene, are used as subjects for proliferation experiments. For two to three days (approximately eight cell cycles), cells are seeded and incubated with test compounds at different concentrations. Version 5.02 of the GraphPad Prism 5 software is used to compute IC50 values and evaluate cell growth using XTT. A minimum of three independent experiments are carried out, each carried out in duplicate. T47D, MDA-MB-157, MDA-MB-231, MDA-MB-468, MDA-MB-453, BT-20, and Hs578T are human breast cancer cell lines that are used. Cells are cultured and analyzed in RPMI 1640 or DMEM medium containing 10% FBS for cell line panel assays. Cells are plated in 96-well plates at a low density for proliferation assays. After adding tenoparib at different concentrations, the plates are incubated for eight days, during which time the compound and medium are replaced on day four. Utilizing the CellTiter-Glo cell viability assay, cell growth is evaluated. At least three independent experiments are carried out, and each experiment is carried out in duplicate[1].

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1. Sensitivity assay in DT40 cell lines: A panel of 32 isogenic DNA repair mutant DT40 cell lines were treated with Stenoparib (E7449), and the IC50 values were determined from at least 3 independent assays. The IC50 values were normalized to that of wild-type DT40 cells (3.2 μmol/L), and the sensitivity of each cell line was categorized by DNA repair function [1]
2. Wnt signaling-related protein detection in SW480 cells: SW480 cells were incubated with 10 μmol/L of Stenoparib (E7449) for 24 h. Cell lysates were subjected to SDS-PAGE and western blot, probed with antibodies against axin2, total β-catenin, active β-catenin, and cyclin D1 (tubulin as a loading control). The fluorescence intensity of bands was measured using Image Studio software, and the ratio of analyte to tubulin was calculated (representative of several independent experiments) [1]
3. Wnt target gene expression analysis in SW480 cells: SW480 cells were treated with 3 μmol/L or 30 μmol/L of Stenoparib (E7449) (or XAV939/olaparib) for 72 h. RNA was harvested, and gene expression profiling was performed using a custom TLDA. Wnt-related genes with a relative fold change of ≥ 1.5 (P > 0.05) versus DMSO control were subjected to hierarchical clustering (Manhattan distance) and complete linkage plotting [1]
4. Proliferation inhibition assay in breast cancer cell lines: Breast cancer cell lines were treated with Stenoparib (E7449), and the proliferation inhibition effect was assessed from at least 3 independent assays to determine the sensitivity of each cell line (MDA-MB-436 was the most sensitive) [1]

Combination of Temozolomide (TMZ) and B16-F10 isograft model: B16-F10 cells (2 × 10⁵) are subcutaneously injected into female C57BL/6 mice. One day after vaccination, medication treatment is started, with randomization based on body weight. Both TMZ and stenoparib are administered orally once daily and are both formulated in 0.5% methyl cellulose. Every day from day 1 to day 5, 50 mg/kg of TMZ is given either alone or in combination. Daily doses of 10, 30, and 100 mg/kg of tenoparib in combination with TMZ, as well as 100 mg/kg as a single agent, are given to patients on days 1 through 7. 0.5% methyl cellulose in water is the vehicle used to treat the control group. Animals receiving the combination are given TMZ after all animals have received their dose of stenoparib or the vehicle[1].

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1. B16-F10 melanoma isograft model: Mice were orally administered temozolomide once daily for 5 days, and Stenoparib (E7449) was orally co-administered once daily for 5 days (plus 2 additional days of single-agent E7449). Tumor growth and body weight were monitored, with statistical significance analyzed by one-way ANOVA followed by Dunnett's multiple comparison test [1]
2. MX-1 breast cancer orthotopic model: Mice received a single intravenous dose of carboplatin (60 mg/kg) on day 3 or 4, and Stenoparib (E7449) (100 mg/kg) was orally administered once daily starting on day 3 or 4. Tumor growth and body weight were recorded, and statistical significance was determined by one-way ANOVA [1]
3. MDA-MB-436 breast cancer xenograft model: Mice bearing MDA-MB-436 tumors were orally administered Stenoparib (E7449) once daily for 28 consecutive days to evaluate antitumor activity. For PARP activity detection, mice received a single dose of E7449 (30 mg/kg or 100 mg/kg), and tumors were harvested at 1-36 hours post-administration for PAR analysis [1]
4. NCI-H460 lung cancer xenograft model: Mice were given a single oral dose of Stenoparib (E7449) (1-100 mg/kg) or vehicle. Tumors were harvested at 15 min to 36 hours post-treatment, and PAR levels were measured by ELISA to assess PARP inhibition [1]
5. Hair regrowth inhibition model: C57BL/6 female mice were depilated, and Stenoparib (E7449) (30, 100, 300 mg/kg) was orally administered once daily for 12 days. Hair regrowth was monitored and recorded by photography [1]
6. Wnt1 subcutaneous model: Mice bearing Wnt1 tumors were orally administered Stenoparib (E7449) (100 mg/kg) once daily or Wnt-C59 (10 mg/kg) once daily to evaluate single-agent activity. For combination treatment, E7449 was orally administered once daily, and E6201 (MEK inhibitor) was intravenously administered Q4Dx3. Tumor growth and body weight were monitored, and tumor tissues were harvested after 7 days of treatment for Wnt target gene expression analysis [1]

Stenoparib (E7449) showed no intestinal toxicity reported as with other TNKS inhibitors, and overall toxicity was low. In the B16-F10 model, weight loss was observed on day 7 in the E7449 plus temozolomide group, but recovered after discontinuation of the drug. In the MX-1 model, no significant weight loss was observed in the E7449 plus carboplatin group. In the Wnt1 model, E7449 was well tolerated with very low toxicity (measured by weight loss). E6201 monotherapy resulted in weight loss (<10%), but recovered after treatment, and combination therapy with E7449 did not exacerbate this weight loss [1]

[1]. 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.

2X-121 is currently undergoing clinical trial NCT03562832 (investigating the antitumor activity and tolerability of the PARP inhibitor 2X-121 in metastatic breast cancer patients screened for 2X-121 drug response). The PARP/Tankyrase inhibitor 2X-121 is an orally administered small molecule inhibitor that inhibits ribozyme polymerase (ADP-ribose) 1 and 2, possessing potential antitumor activity. After administration, E7449 selectively binds to PARP 1 and 2, thereby blocking the base excision repair (BER) pathway for the repair of damaged DNA. This drug enhances the accumulation of single-stranded and double-stranded DNA breaks, promoting genomic instability and ultimately leading to apoptosis. The PARP 1/2 inhibitor E7449 may enhance the cytotoxicity of DNA damaging agents and radiotherapy. PARP catalyzes the post-translational ADP ribosylation of nucleoproteins, which signal and recruit other proteins to repair damaged DNA.

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Stenoparib (E7449) is a novel dual inhibitor of PARP1/2 and TNKS1/2. It not only impairs DNA damage repair by inhibiting PARP1/2 and capturing PARP1 on damaged DNA, but also antagonizes the classical Wnt/β-catenin signaling pathway by inhibiting TNKS1/2 [1]
Stenoparib (E7449) has monotherapy antitumor activity in BRCA-deficient xenograft tumors and can enhance the efficacy of chemotherapy; in Wnt-dependent tumor models, its antitumor activity is enhanced when used in combination with MEK inhibitors [1]
Stenoparib (E7449) is currently in the early stage of clinical development. Due to the lack of intestinal toxicity, it has a safety advantage compared with other TNKS inhibitors [1]

C18H15N5O

317.34

317.127

C, 68.13; H, 4.76; N, 22.07; O, 5.04

1140964-99-3

135565981

Light yellow to yellow solid powder

1.4±0.1 g/cm3

381.4±34.0 °C at 760 mmHg

184.5±25.7 °C

0.0±0.9 mmHg at 25°C

1.731

1.12

2

3

2

24

586

0

O=C1C2C([H])=C([H])C([H])=C3C=2C(=NN1[H])N=C(C([H])([H])N1C([H])([H])C2=C([H])C([H])=C([H])C([H])=C2C1([H])[H])N3[H]

JLFSBHQQXIAQEC-UHFFFAOYSA-N

InChI=1S/C18H15N5O/c24-18-13-6-3-7-14-16(13)17(21-22-18)20-15(19-14)10-23-8-11-4-1-2-5-12(11)9-23/h1-7H,8-10H2,(H,22,24)(H,19,20,21)

11-(1,3-dihydroisoindol-2-ylmethyl)-2,3,10,12-tetrazatricyclo[7.3.1.05,13]trideca-1,5(13),6,8,11-pentaen-4-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)