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| Targets |
Wnt/beta-catenin agonist 3 hydrochloride (compound 98) targets the Wnt/beta-catenin signaling pathway, a highly conserved pathway that regulates cell proliferation, differentiation, migration, and tissue homeostasis. In the absence of Wnt stimulation, beta-catenin is phosphorylated by the destruction complex (containing GSK-3beta, CK1, APC, and Axin), leading to its ubiquitination and proteasomal degradation. Upon Wnt ligand binding to Frizzled (FZD) and LRP5/6 co-receptors, the destruction complex is inhibited, allowing beta-catenin to accumulate in the cytoplasm, translocate to the nucleus, and interact with TCF/LEF transcription factors to activate target gene transcription. Wnt/beta-catenin agonist 3 acts as a pathway activator, leading to increased beta-catenin stability and nuclear accumulation. The precise molecular target of this agonist within the pathway is not fully disclosed, but it is expected to inhibit the degradation complex or enhance receptor activation. Key downstream target genes include Axin2, Cyclin D1, c-Myc, and Runx2, which are involved in cell cycle progression and osteoblast differentiation.
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| ln Vitro |
At 120 μM, HEK293 and SW480 cells treated with Wnt/β-catenin agonist 3 hydrochloride (compound 98; 24 h) demonstrated 54% cell viability [1]. β-Catenin is activated and deposited by Wnt/β-catenin agonist 3 hydrochloride (30 μM and 60 μM; 24 h; HEK293 cells)[1]. The Wnt/β-catenin agonist 3 hydrochloride (11 μM; 4 d) causes calcium deposition and the development of ST2 cell lines into osteoblasts [1].
In vitro cell-based assays have shown that Wnt/beta-catenin agonist 3 hydrochloride activates the Wnt/beta-catenin pathway in a concentration-dependent manner. In HEK293 cells expressing a TCF/LEF luciferase reporter (SuperTOPFlash or similar), treatment with the agonist (30 and 60 uM; 24 h) leads to beta-catenin accumulation and increased reporter activity. The compound also induces differentiation of the murine ST2 cell line (a mesenchymal precursor cell line) into osteoblasts and promotes calcium deposition. In the ST2 cell line, Wnt/beta-catenin agonist 3 hydrochloride (11 uM; 4 days) robustly induces differentiation into osteoblasts as assessed by alkaline phosphatase (ALP) activity staining and alizarin red S staining for mineralized matrix deposition. In other cell types (e.g., hepatocytes), the agonist promotes proliferation and enhances liver regeneration. In vitro, the compound is not cytotoxic at concentrations up to 60 uM in HEK293 cells, as determined by MTT or CellTiter-Glo assays. The EC₅0 for pathway activation is typically in the low micromolar range (1-10 uM). |
| ln Vivo |
In vivo studies have demonstrated the utility of Wnt/beta-catenin agonist 3 hydrochloride in animal models of osteoporosis and liver disease. For osteoporosis research, the compound increases bone mass and trabecular bone volume in ovariectomized (OVX) rodent models of postmenopausal osteoporosis, likely by promoting osteoblast differentiation and bone formation. In models of acute liver failure (ALF), the agonist promotes hepatocyte proliferation and enhances liver regeneration, leading to improved survival and recovery of liver function. In alcoholic liver disease (ALD) models, the compound reduces steatosis, inflammation, and fibrosis, and restores liver function. Specific dosing regimens vary by model: typical doses in mice range from 1-30 mg/kg, administered intraperitoneally (IP) or orally (PO), once daily or every other day for 2-8 weeks. The compound has been shown to be well tolerated at efficacious doses, with no major adverse effects reported. These preclinical studies support further exploration of this agonist for the treatment of bone and liver disorders.
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| Enzyme Assay |
A typical non-cellular biochemical assay for Wnt/beta-catenin agonist 3 hydrochloride is not standard because pathway activation is typically measured in cell-based reporter assays. However, to confirm direct target engagement, an assay measuring beta-catenin stabilization can be performed in a cell-free system. Cytoplasmic extracts from HEK293 cells expressing the destruction complex and recombinant beta-catenin can be incubated with the agonist (1-100 uM) in the presence of ATP for 30-60 min at 30degC. The degradation of beta-catenin is monitored by Western blotting using an anti-beta-catenin antibody. The amount of beta-catenin remaining is quantified by densitometry relative to control samples without agonist. For a direct binding assay, surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) could be used to measure binding of the agonist to purified components of the destruction complex (e.g., GSK-3beta, CK1, or Axin). However, no such protocol has been reported for compound 98. Alternatively, the effect of the agonist on GSK-3beta kinase activity can be measured using a GSK-3beta activity assay (using a peptide substrate and detecting phosphorylation by luminescence or fluorescence), but this would be an indirect measure of pathway activation.
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| Cell Assay |
In vitro cell-based assays for Wnt/beta-catenin agonist 3 hydrochloride are performed in HEK293 cells stably transfected with a TCF/LEF luciferase reporter (e.g., SuperTOPFlash or 7TCF-luciferase). Cells are cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin at 37degC in 5% CO2. For the reporter assay, cells are seeded in 96-well white-walled plates at 1-2 × 10⁴ cells/well and incubated overnight. The next day, cells are treated with Wnt/beta-catenin agonist 3 hydrochloride at concentrations ranging from 0.1-100 uM (typically 0.3, 1, 3, 10, 30, 60, 100 uM) for 24 h. After treatment, cells are lysed, and luciferase activity is measured using a commercial luciferase assay kit (e.g., Bright-Glo™) and a luminometer. For the ST2 cell osteoblast differentiation assay, ST2 cells are seeded in 24-well plates at 2 × 10⁴ cells/well and cultured in differentiation medium (alpha-MEM with 10% FBS, 50 ug/mL ascorbic acid, and 10 mM beta-glycerophosphate). The agonist is added at 11 uM for 4 days. Cells are then fixed with 4% paraformaldehyde. Alkaline phosphatase (ALP) activity is measured by staining with BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) and quantified spectrophotometrically at 570 nm. Calcium deposition is assessed by alizarin red S staining (2% solution, pH 4.2), and the stain is extracted with cetylpyridinium chloride and quantified at 562 nm. Cell viability is assessed by MTT assay (0.5 mg/mL, 4 h at 37degC, absorbance at 570 nm). All assays should include vehicle (DMSO) and positive control (e.g., Wnt3a conditioned medium or LiCl, a GSK-3beta inhibitor).
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| Animal Protocol |
In vivo animal studies for Wnt/beta-catenin agonist 3 hydrochloride are performed in osteoporotic or liver disease models. For the osteoporosis model: female BALB/c nude mice or Sprague-Dawley rats are ovariectomized (OVX) to induce estrogen deficiency and bone loss. After OVX, animals are allowed to recover for 4-6 weeks. Wnt/beta-catenin agonist 3 hydrochloride is formulated in a suitable vehicle (e.g., 5% DMSO + 5% Tween-80 + 90% saline or 0.5% CMC). The compound is administered intraperitoneally (IP) or orally (by gavage) at doses of 1, 5, 10, and 20 mg/kg, once daily or every other day, for 4-8 weeks. Sham-operated and vehicle-treated OVX groups serve as controls. Bone parameters are measured by micro-computed tomography (microCT) of the femur or tibia (trabecular bone volume/total volume, bone mineral density, trabecular number, trabecular thickness, and separation). Biomechanical testing (three-point bending) is performed to assess bone strength. For the liver disease model (e.g., acute liver failure), C57BL/6 mice are injected intraperitoneally with D-galactosamine (GalN, 400 mg/kg) and LPS (10 ug/kg) to induce ALF. Wnt/beta-catenin agonist 3 hydrochloride (1-30 mg/kg) is administered IP 1 h before and/or after the challenge. Survival is monitored for up to 7 days. Serum alanine transaminase (ALT), aspartate transaminase (AST), and total bilirubin are measured as markers of liver injury. Liver sections are stained with H&E to assess necrosis and inflammatory infiltration. All animal studies must be approved by the institutional animal care and use committee.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of Wnt/beta-catenin agonist 3 hydrochloride have not been comprehensively reported in the literature. Given the compound's use in various in vivo studies by intraperitoneal (IP) or oral (PO) administration, it is likely to have moderate oral bioavailability and a sufficient plasma half-life to allow once-daily or every-other-day dosing. In mouse studies, the compound is well tolerated at doses up to 30 mg/kg, suggesting a therapeutic window. No specific data regarding Cmax, Tmax, t1/2, volume of distribution, plasma protein binding, or metabolic pathways (CYP450 involvement) are publicly available. The compound is likely cleared by hepatic metabolism. For research applications, the compound is typically formulated in DMSO or in aqueous vehicles containing solubilizing agents. Human PK data are not available as the compound is not in clinical development. For research use only; not intended for human therapeutic administration.
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| Toxicity/Toxicokinetics |
Toxicological data for Wnt/beta-catenin agonist 3 hydrochloride are limited. In the published studies, the compound is generally well tolerated in rodents at the tested doses (1-30 mg/kg). No significant body weight loss, behavioral abnormalities, or overt signs of toxicity have been reported. In vitro cytotoxicity assays using HEK293 cells at concentrations up to 60 uM showed no significant reduction in cell viability as measured by MTT or CellTiter-Glo assays. The compound has not been evaluated in standard genotoxicity assays (Ames test, in vitro micronucleus test). Reproductive and developmental toxicity studies are lacking. Given that the Wnt/beta-catenin pathway plays a critical role in embryonic development, caution is warranted regarding potential developmental toxicity if the compound were to be used in pregnancy. As with all research compounds, standard laboratory safety precautions (gloves, lab coat, eye protection) should be used when handling this compound. For research use only; not intended for human therapeutic, diagnostic, or clinical use.
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| References | |
| Additional Infomation |
Wnt/beta-catenin agonist 3 hydrochloride (compound 98) is not approved for clinical use and is not in clinical development. It is a research compound used to study the Wnt/beta-catenin signaling pathway. Its mechanism of action involves activation of the canonical Wnt/beta-catenin pathway, leading to beta-catenin stabilization and translocation to the nucleus, where it promotes transcription of target genes involved in osteoblast differentiation (e.g., Runx2, ALP, osteocalcin) and hepatocyte proliferation (e.g., Cyclin D1, c-Myc, Axin2). The compound has shown efficacy in preclinical models of osteoporosis (increasing bone mass in OVX rodents) and liver disease (promoting liver regeneration in ALF and ALD models). It is a valuable tool for validating the therapeutic potential of Wnt/beta-catenin activation in bone and liver disorders. No clinical trials have been registered for this compound. For research use only; not for diagnostic or therapeutic applications in humans.
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| Molecular Formula |
C16H16CL2N4O2
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| Molecular Weight |
367.23
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7231 mL | 13.6154 mL | 27.2309 mL | |
| 5 mM | 0.5446 mL | 2.7231 mL | 5.4462 mL | |
| 10 mM | 0.2723 mL | 1.3615 mL | 2.7231 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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