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| Targets |
With antitumor activity, EMT Inhibitor-1 (C19) (0-10 μM; 24 hours) conjugates Hippo, TGF-β, and Wnt signaling dyes and couples migration, proliferation, and doxorubicin importance locally and topically[1].
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| ln Vitro |
With antitumor activity, EMT Inhibitor-1 (C19) (0-10 μM; 24 hours) conjugates Hippo, TGF-β, and Wnt signaling dyes and couples migration, proliferation, and doxorubicin importance locally and topically[1].
C19 inhibits the transcriptional activity of the Hippo, Wnt, and TGF-β pathways in a dose-dependent manner, as measured by luciferase reporter assays in 293 cells.[1] C19 induces phosphorylation (activation) of Mst1 and Lats1 kinases in a dose-dependent manner in WM266 melanoma cells.[1] C19 reduces protein levels of the Hippo transducer TAZ in the cytoplasm and nucleus of WM266 cells without affecting its mRNA levels, suggesting enhanced proteolysis. It does not affect protein levels of YAP and TEAD.[1] C19 reduces nuclear β-catenin and Smad levels and inhibits the expression of downstream pro-EMT genes (e.g., CTGF, fibronectin, smooth muscle actin, collagen A2) in WM266 cells.[1] C19 dose-dependently inhibits the expression of EMT-related genes (Zeb1, Snail, Hes, Myc, cyclin D) in WM266 cells.[1] C19 inhibits the migration of WM266 cells in a monolayer scratch assay.[1] C19 inhibits the proliferation of various cancer cell lines including melanoma (WM115, WM266, SK-Mel28), colon cancer (SW480), breast cancer (MCF7), and myeloma (8226) in MTT assays. Human dermal fibroblasts were less responsive.[1] C19 induces phosphorylation of AMPK at Thr172 in a dose- and time-dependent manner in multiple cell lines (WM266, WM115, SW480, MCF7, SKN-SH). It is about 10 times more potent than the known AMPK activator AICAR.[1] C19 induces phosphorylation of Ulk1, increases levels of the autophagy marker LC3-II, and causes cellular vacuolization in WM266 cells, suggesting induction of autophagy. It does not induce caspase-3 cleavage (apoptosis) under the tested conditions.[1] C19 attenuates doxorubicin resistance conferred by conditioned medium from belinostat-treated cells in WM266 cells.[1] The inhibitory effects of C19 on Hippo reporter activity and cell migration are dependent on TAZ degradation, while its induction of autophagy is mediated by AMPK activation and is independent of TAZ degradation.[1] |
| ln Vivo |
In mouse tumor models, EMT inhibitor-1 (C19) (ip; 5–20 mg/kg) demonstrates the strongest antitumor activity. Mechanistically, EMT inhibitor-1 activates the tumor suppressor AMPK and the Hippo buffer Mst/Lats upstream of the degradation complex, which causes GSK3-β-mediated degradation of the Hippo sensor TAZ [1]. The vehicle solution (100 μL DMEM with 5% DMEM) was used to dissolve C19.
In a nude mouse xenograft model inoculated with WM266 melanoma cells, intraperitoneal administration of C19 (5, 10, and 20 mg/kg, three injections separated by 3 days) dose-dependently inhibited tumor growth. The 20 mg/kg dose induced approximately 90% inhibition compared to the vehicle control.[1] Tumors from C19-treated mice showed reduced TAZ protein levels, increased phosphorylation of AMPK and Mst1, decreased phosphorylation of GSK3-β, and reduced expression of Zeb1 and cyclin D compared to tumors from control mice.[1] No significant changes in body weight or apparent signs of toxicity (eating habits, overall physical activity) were observed in mice treated with C19 throughout the experiment.[1] |
| Enzyme Assay |
An in vitro kinase assay was performed to assess the direct effect of C19 on GSK3-β activity. Immunoprecipitated GSK3-β from WM266 cells was incubated with its substrate in a kinase buffer containing [γ-³²P]ATP, in the absence or presence of C19 or the GSK3 inhibitor TWS119. After incubation, the reaction was stopped, spotted on filter paper, washed, and radioactivity was counted. C19 did not show a direct effect on GSK3-β activity in this assay.[1]
To assess direct effects on AMPK, Mst1, and Lats1, these kinases were immunoprecipitated from WM266 cells. The immunoprecipitates were resuspended in kinase buffer and incubated in the absence or presence of C19 or AICAR (for AMPK) without radioactive ATP. Reactions were stopped, and phosphorylation status was analyzed by Western blot using phospho-specific antibodies. C19 induced phosphorylation of AMPK under these conditions, suggesting a potential direct effect. No direct effect on phosphorylation of Mst1 or Lats1 was observed under the same assay conditions.[1] |
| Cell Assay |
For luciferase reporter assays, cells (e.g., 293) were transiently transfected with reporter constructs (8xGTIIC-luc for Hippo, 8xTCF-luc for Wnt, A3Lux for TGF-β) or a CMV-luc control using a lipofectamine-based transfection reagent. After 5 hours, the medium was replaced, and cells were treated with C19 or other inhibitors for 24 hours. Cells were then lysed, and luciferase activity in protein extracts was measured. Reporter activity was normalized to CMV-luc control activity.[1]
For Western blot analysis, proteins were extracted from monolayer cells using a lysis buffer. For nucleo-cytoplasmic fractionation, cells were resuspended in a hypotonic buffer, incubated on ice, lysed with Nonidet NP-40, and centrifuged to separate nuclei. Equal amounts of protein were separated by SDS-PAGE, transferred to membranes, and probed with specific primary and secondary antibodies. Detection was performed using chemiluminescence.[1] For quantitative PCR (qPCR), total RNA was extracted using a commercial kit, and first-strand cDNA was synthesized. Gene expression was measured by real-time PCR using Syber green Master Mix on a standard instrument. Gene expression was normalized to GAPDH.[1] For cell viability/proliferation assays, cells were seeded in 96-well plates and incubated with C19 for 96 hours. Viability was quantitatively determined by MTT assay. MTT solution was added to each well, incubated, the formed precipitate was solubilized, and optical density was measured at 570 nm with a reference at 650 nm. The percentage of viable cells was calculated relative to untreated controls.[1] For cell migration assays, a monolayer scratch assay was performed. Photographs were taken at different time points (e.g., day 1 and day 3) to assess wound closure.[1] For siRNA transfection, cells were transfected with siRNA duplexes using a commercial transfection reagent kit according to the manufacturer's protocol. After 48 hours, proteins were extracted for Western blot analysis.[1] |
| Animal Protocol |
Nude (nu/nu) mice (6 weeks old) were inoculated subcutaneously in the right flank with WM266 melanoma cells (10⁶ cells in 200 µL PBS). When tumors became palpable (approximately 5 mm in diameter), animals were randomized into control and treatment groups.[1]
C19 was dissolved in a vehicle solution consisting of DMEM containing 5% dimethyl sulfoxide (DMSO). The control group received the vehicle solution only.[1] Treatment groups received intraperitoneal injections of C19 at doses of 5, 10, or 20 mg/kg (7 animals per group). Injections were administered three times, with each injection separated by 3 days.[1] Mice were monitored daily for health. Tumor volumes were measured using the formula (Length × Width²)/2. The experiment was terminated 2 months after inoculation. Tumors were harvested for biochemical and gene expression analysis.[1] |
| Toxicity/Toxicokinetics |
In in vivo xenotransplantation studies, no significant changes in body weight or obvious signs of toxicity were observed in nude mice after treatment with C19 at doses up to 20 mg/kg (three intraperitoneal injections) (based on feeding habits and overall activity levels). Specific toxicological parameters, such as LD50, organ toxicity, drug interactions, or plasma protein binding rates, were not reported. [1]
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| References | |
| Additional Infomation |
C19 has been identified as a multi-target EMT pathway inhibitor that targets the Hippo, Wnt, and TGF-β signaling pathways. Its mechanism of action involves the activation of AMPK and Mst/Lats kinases, which leads to the degradation of the GSK3-β-mediated Hippo signaling molecule TAZ, thereby inhibiting the transcriptional activation function of β-catenin and Smads, ultimately inhibiting EMT. [1] Studies have shown that targeting multiple EMT pathways simultaneously with C19 may be a promising cancer treatment strategy. AMPK activation also opens the way for the potential application of C19 in metabolic-related diseases outside of cancer, such as diabetes and fibrotic diseases. [1] This compound showed strong antitumor activity in a mouse melanoma xenograft model without significant toxicity, and warrants further preclinical and clinical studies. [1]
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| Molecular Formula |
C12H12CL2N2O2S
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|---|---|
| Molecular Weight |
319.206879615784
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| Exact Mass |
317.999
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| CAS # |
1638526-21-2
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| PubChem CID |
90718214
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| Appearance |
White to off-white solid powder
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| LogP |
3.6
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
19
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| Complexity |
276
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1=C(C=CC(=C1)C1C(=NSN=1)OCCCCO)Cl
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| InChi Key |
FWMUIOJZHOPPDX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C12H12Cl2N2O2S/c13-9-4-3-8(7-10(9)14)11-12(16-19-15-11)18-6-2-1-5-17/h3-4,7,17H,1-2,5-6H2
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| Chemical Name |
4-[[4-(3,4-dichlorophenyl)-1,2,5-thiadiazol-3-yl]oxy]butan-1-ol
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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 |
| 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 Vitro) |
DMSO : ~100 mg/mL (~313.27 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.83 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 (7.83 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 (7.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1327 mL | 15.6637 mL | 31.3273 mL | |
| 5 mM | 0.6265 mL | 3.1327 mL | 6.2655 mL | |
| 10 mM | 0.3133 mL | 1.5664 mL | 3.1327 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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