TNKS2 ( IC50 = 0.83 μM ); TNKS1 ( IC50 = 1.9 μM )
Tankyrase 1 (TNKS1) (IC50 = 2.5 μM, recombinant tankyrase enzyme activity assay) [1]
Tankyrase 2 (TNKS2) (IC50 = 1.9 μM, recombinant tankyrase enzyme activity assay) [1]
β-Catenin (EC50 = 0.3 μM, TOPflash β-catenin reporter gene assay in SW480 cells) [1]

In vitro activity: JW55 suppresses the TNKS1/2 PARP domain, stabilizing AXIN2 and accelerating β-catenin degradation in the process. With an IC50 value of 470 nmol/L, JW55 inhibits Wnt3a-induced HEK293 cells that have a transiently transfected ST-Luc (SuperTop-luciferase) reporter. JW55 has an IC50 value of 1.9 μmol/L and 830 nmol/L, respectively, which decreases the auto-PARsylation of TNKS1/2 in vitro. In vitro, the CRC cell line SW480 exhibits decreased cell-cycle progression, proliferation, and colony formation when JW55-mediated canonical Wnt signaling inhibition is applied[1].

---

1. Inhibition of tankyrase enzymatic activity: JW 55 dose-dependently inhibited the ADP-ribosyltransferase activity of recombinant human Tankyrase 1 and Tankyrase 2, with IC50 values of 2.5 μM and 1.9 μM respectively. It showed no significant inhibition of other poly(ADP-ribose) polymerases (PARPs) including PARP1, PARP2, and PARP3 (inhibition rate < 10% at 10 μM), confirming selectivity for tankyrases [1]
2. Downregulation of canonical Wnt signaling: JW 55 (0.1-10 μM) dose-dependently suppressed β-catenin-mediated transcriptional activity in SW480 colon carcinoma cells transfected with the TOPflash luciferase reporter plasmid. At 1 μM, it reduced luciferase activity by 75% compared to the vehicle group. Western blot analysis showed decreased nuclear β-catenin protein levels (0.3 μM: 40% reduction; 1 μM: 65% reduction) and downregulated expression of Wnt target genes c-Myc (0.3 μM: 35% reduction; 1 μM: 58% reduction) and Cyclin D1 (0.3 μM: 30% reduction; 1 μM: 52% reduction) at the protein and mRNA levels (qRT-PCR) [1]
3. Antiproliferative activity in colon carcinoma cells: JW 55 inhibited the proliferation of APC-mutant colon carcinoma cell lines (SW480, HCT116, DLD-1) with IC50 values of 1.2 μM, 0.8 μM, and 1.5 μM respectively (72-hour CCK-8 assay). It had minimal effect on normal colonic epithelial cells (NCM460) with an IC50 > 10 μM, indicating cancer cell selectivity [1]
4. Induction of apoptosis: JW 55 (0.5-5 μM) dose-dependently induced apoptosis in HCT116 cells. Annexin V-FITC/PI staining showed apoptotic rates of 12% (0.5 μM), 28% (1 μM), and 45% (5 μM) after 48 hours, compared to 3% in the vehicle group. Western blot revealed increased cleavage of PARP and caspase-3, and upregulation of Bax/Bcl-2 ratio [1]
5. Inhibition of colony formation: JW 55 (0.1-1 μM) dose-dependently suppressed the clonogenic potential of SW480 and HCT116 cells. At 1 μM, colony formation rates were reduced by 70% (SW480) and 75% (HCT116) compared to the vehicle group [1]

JW55 inhibits the formation of polyposis in conditional APC mutant mice induced by tamoxifen and axis duplication in Xenopus embryos[1].

---

1. Tumor growth inhibition in conditional APC mutant mice: Conditional APC^min/+ mice (with intestinal adenoma formation due to Wnt pathway activation) were orally administered JW 55 (100 mg/kg/day) for 21 days. The drug significantly reduced the volume and weight of intestinal tumors: mean tumor volume decreased by 65% (from 120 mm³ to 42 mm³) and mean tumor weight decreased by 60% (from 180 mg to 72 mg) compared to the vehicle group. Immunohistochemical staining of tumor tissues showed decreased nuclear β-catenin expression (by 55%) and reduced Ki-67 proliferation index (from 65% to 25%). TUNEL staining revealed a 3.2-fold increase in apoptotic cells [1]
2. Regulation of Wnt target genes in vivo: qRT-PCR analysis of tumor tissues from JW 55-treated mice showed downregulated mRNA expression of c-Myc (58% reduction), Cyclin D1 (52% reduction), and AXIN2 (60% reduction), consistent with in vitro inhibition of canonical Wnt signaling [1]

JW 55, also known as JW55, is a strong and specific inhibitor of the Wnt pathway. JW55 exhibits inhibition of Wnt3a-induced HEK293 cells with an IC50 value of 470 nM, when the cells contain a transiently transfected ST-Luc (SuperTop-luciferase) reporter. JW55 exhibits efficacy within the concentration range of 1 to 5 μM in SW480 cells and 0.01 to 5 μM in HCT-15 cells. JW55 exhibits efficacy within the concentration range of 1 to 5 μM in SW480 cells and 0.01 to 5 μM in HCT-15 cells.

---

1. Recombinant tankyrase activity assay: Recombinant human Tankyrase 1 or Tankyrase 2 protein was diluted in assay buffer containing Tris-HCl, MgCl₂, and DTT. Different concentrations of JW 55 (0.01-100 μM) were added to the reaction mixture, followed by the addition of biotinylated poly(ADP-ribose) substrate and NAD⁺ (including [³²P]-NAD⁺ for radioactive detection). The reaction was incubated at 37℃ for 60 minutes and terminated by adding SDS-PAGE sample buffer. ADP-ribosylated products were separated by SDS-PAGE, transferred to a membrane, and detected by autoradiography. The inhibition rate of tankyrase activity was calculated based on the intensity of radioactive bands, and IC50 values were derived from dose-response curves [1]
2. TOPflash β-catenin reporter gene assay: SW480 cells were seeded in 96-well plates and co-transfected with TOPflash luciferase reporter plasmid (containing β-catenin-responsive elements) and Renilla luciferase plasmid (internal control) using transfection reagent. After 24 hours of transfection, cells were treated with serial concentrations of JW 55 (0.01-10 μM) for 24 hours. Dual-luciferase activity was measured, and the ratio of firefly luciferase to Renilla luciferase activity was calculated to assess β-catenin transcriptional activity. EC50 values were determined from dose-response curves of activity inhibition [1]

In 96-well plates, 1,000 SW480 or RKO cells are seeded. The following day, solutions containing 0.1% DMSO or 10 μM JW55 for RKO cells and 0.1% DMSO or 10, 5, or 1 μM JW55 for SW480 cells are substituted for the original cell culture medium. Every sample has at least six duplicates. Within a cell culture incubator, the plate is incubated in an IncuCyte. Every two hours, pictures are taken to track proliferation.

---

1. Cell proliferation assay: Colon carcinoma cells (SW480, HCT116, DLD-1) and normal colonic epithelial cells (NCM460) were seeded in 96-well plates at a density of 2×10³ cells/well. After 24 hours of adherence, cells were treated with JW 55 (0.01-100 μM) for 72 hours. CCK-8 reagent was added, and after 2 hours of incubation, absorbance at 450 nm was measured to calculate cell viability and IC50 values [1]
2. Western blot assay for Wnt pathway proteins: HCT116 cells were seeded in 6-well plates (1×10⁶ cells/well) and treated with JW 55 (0.1-5 μM) for 24 hours. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Total protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against nuclear β-catenin, c-Myc, Cyclin D1, cleaved PARP, cleaved caspase-3, Bax, Bcl-2, and GAPDH (loading control). Chemiluminescent signals were detected and quantified [1]
3. qRT-PCR for Wnt target genes: SW480 cells were treated with JW 55 (0.3-1 μM) for 24 hours. Total RNA was extracted, reverse-transcribed into cDNA, and qRT-PCR was performed using specific primers for c-Myc, Cyclin D1, AXIN2, and GAPDH (internal control). Relative gene expression was calculated using the 2^(-ΔΔCt) method [1]
4. Apoptosis assay: HCT116 cells were seeded in 6-well plates (5×10⁵ cells/well) and treated with JW 55 (0.5-5 μM) for 48 hours. Cells were harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify apoptotic cells (early and late apoptosis) [1]
5. Colony formation assay: SW480 and HCT116 cells were seeded in 6-well plates (200 cells/well) and allowed to adhere for 24 hours. JW 55 (0.1-1 μM) was added, and cells were cultured for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted. The colony formation rate was calculated as (number of colonies in treatment group / number in control group) × 100% [1]

Mice: Seven 12-week-old female Apc^CKO/CKO/Lgr5-CreERT2 mice receive intraperitoneal injections of tamoxifen at a dose of 25 mg/kg, diluted in a 1:4 ratio of ethanol to corn oil. The mice are treated with JW55 (100 mg/kg) or a vehicle (DMSO) after being randomly assigned to two groups. The next day, daily oral applications were started, and they lasted for three weeks. The body weight of the mice is measured twice a week. Following the mice's sacrifice, the intestines are removed, cleaned in PBS, and fixed in formaldehyde (10% solution (v/v) in PBS). 10% paraformaldehyde (PFA)/PBS solution containing 1% methylene blue is used to stain the small intestines. Hematoxylin and eosin is used to stain small ileum Swiss-rolls that have been embedded in paraffin sections.

---

1. Conditional APC mutant mouse tumor model: Male conditional APC^min/+ mice (6-8 weeks old, 20-25 g) were randomly divided into 2 groups (n=10/group): vehicle control (0.5% carboxymethylcellulose sodium, CMC-Na) and JW 55 100 mg/kg/day group. JW 55 was suspended in 0.5% CMC-Na and administered orally by gavage once daily for 21 days. The vehicle group received the same volume of 0.5% CMC-Na. Tumor volume was measured every 3 days using a digital caliper (calculated as length × width² / 2). At the end of the experiment, mice were sacrificed by cervical dislocation, and intestinal tumors were excised, weighed, and fixed in 4% paraformaldehyde for immunohistochemical staining (β-catenin, Ki-67) and TUNEL staining. A portion of tumor tissue was frozen in liquid nitrogen for qRT-PCR analysis of Wnt target genes [1]

1. Acute toxicity: No significant death or toxic symptoms (e.g., lethargy, weight loss, gastrointestinal discomfort) were observed in mice after a single oral dose of up to 500 mg/kg of JW 55 during a 14-day observation period.[1]
2. Chronic toxicity: Mice were given JW 55 (100 mg/kg/day) orally for 21 consecutive days. Compared with the control group, there was no significant change in body weight. Histopathological analysis of major organs (liver, kidney, heart, lung, spleen) revealed no abnormal lesions. Liver function (ALT, AST) and kidney function (BUN, creatinine) indicators were all within the normal range.[1]

[1]. A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice. Cancer Res. 2012 Jun 1;72(11):2822-32.

N-[4-[[[4-(4-methoxyphenyl)-4-oxacyclohexyl]methylamino]-oxomethyl]phenyl]-2-furan carboxamide belongs to the furan class of compounds and is an aromatic amide. 1. JW 55 is a novel small molecule inhibitor that inhibits Tankyrase 1 and Tankyrase 2, which are key enzymes in the classical Wnt signaling pathway. Tankyrase regulates the stability of AXIN, a negative regulator of β-catenin; inhibition of Tankyrase leads to AXIN stabilization, promotes β-catenin degradation, and inhibits Wnt pathway activation [1]. 2. The classical Wnt pathway is aberrantly activated in most colorectal cancers (e.g., due to APC mutations), making Tankyrase an attractive therapeutic target. JW 55 exhibits potent antiproliferative and proapoptotic effects in APC mutant colon cancer cells and significantly inhibits tumor growth in preclinical mouse models, supporting its potential as a targeted therapy for colon cancer [1]. 3. JW 55 is highly selective for tankyrase, superior to other PARP family members, thereby minimizing off-target effects associated with nonspecific PARP inhibition (e.g., DNA damage-related toxicity). Its good safety profile in preclinical studies (no significant organ toxicity and minimal impact on normal cells) further supports its clinical development [1]. 4. The mechanism of action of JW 55 involves direct binding to the catalytic domain of tankyrase, thereby blocking its ADP-ribosyltransferase activity. This leads to downstream inhibition of β-catenin-mediated pro-proliferative and anti-apoptotic gene transcription, ultimately inhibiting cancer cell growth and inducing apoptosis [1].

C₂₅H₂₆N₂O₅

434.48

434.184

C, 69.11; H, 6.03; N, 6.45; O, 18.41

664993-53-7

2946601

White solid powder

1.2±0.1 g/cm3

572.6±50.0 °C at 760 mmHg

300.1±30.1 °C

0.0±1.6 mmHg at 25°C

1.601

3.54

2

5

7

32

620

0

O1C([H])([H])C([H])([H])C(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])(C([H])([H])N([H])C(C2C([H])=C([H])C(=C([H])C=2[H])N([H])C(C2=C([H])C([H])=C([H])O2)=O)=O)C([H])([H])C1([H])[H]

ZJZWZIXSGNFWQQ-UHFFFAOYSA-N

InChI=1S/C25H26N2O5/c1-30-21-10-6-19(7-11-21)25(12-15-31-16-13-25)17-26-23(28)18-4-8-20(9-5-18)27-24(29)22-3-2-14-32-22/h2-11,14H,12-13,15-17H2,1H3,(H,26,28)(H,27,29)

N-[4-[[4-(4-methoxyphenyl)oxan-4-yl]methylcarbamoyl]phenyl]furan-2-carboxamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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