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| Targets |
The compound targets USP8 (ubiquitin-specific protease 8), a deubiquitinating enzyme (DUB) that removes ubiquitin chains from target proteins, thereby preventing their proteasomal degradation. By inhibiting USP8, DC-U4106 enhances the ubiquitination and subsequent degradation of Eralpha (estrogen receptor alpha) and PR (progesterone receptor). USP8 is involved in endosomal trafficking and protein stability, and its inhibition leads to the degradation of multiple cancer-relevant proteins, including Eralpha in breast cancer cells.
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| ln Vitro |
DC-U4106 (1.2-45.2 μM) exhibits little activity against USP7 but inhibits USP8 and USP2 with IC50 values of 1.2 μM and 58.4 μM, respectively [1]. ERα and PR mRNA levels are decreased by DC-U4106 (0–7 μM, 24 hours) [1]. In addition to ERα and PR proteins, DC-U4106 (0-5 μM, 24 hours) can control the expression of proteins associated to the RTK pathway [1]. Cell growth can be inhibited and apoptosis can be induced by DC-U4106 (0-5 μM, 12 hours) [1].
DC-U4106 inhibits USP8 with a Kd of 4.7 microM and an IC50 of 1.2 microM in biochemical assays. In cellular assays, DC-U4106 (0-7 microM, 24 hours) reduces ERalpha and PR mRNA and protein levels. At 0-5 microM for 12-24 hours, the compound induces apoptosis, inhibits cell proliferation, and regulates RTK pathway-related proteins (receptor tyrosine kinases). It inhibits tumor cell growth with minimal toxicity, demonstrating a favorable therapeutic window in pre-clinical models. |
| ln Vivo |
DC-U4106 (intraperitoneal injection, 5 mg/kg or 20 mg/kg, every 2 days for 14 days) suppresses tumor development in BALB/c nude mice and has no significant effect on body weight, organ morphology and structure [1].
Specific in vivo data for DC-U4106 are not detailed; however, the compound is described as inhibiting tumor cell growth with minimal toxicity and has the potential for breast cancer research. As a USP8 inhibitor that promotes Eralpha degradation, it would be expected to show efficacy in mouse xenograft models of ERalpha-positive breast cancer (e.g., MCF-7, T47D). A typical protocol would involve intravenous or intraperitoneal administration of DC-U4106 (e.g., 10-50 mg/kg daily) and measurement of tumor growth inhibition and ERalpha levels in tumor tissue. |
| Enzyme Assay |
The USP8 deubiquitinase activity assay is performed using a ubiquitin-rhodamine 110 (Ub-R110) substrate. Purified recombinant human USP8 is incubated with Ub-R110 in assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.05% Tween-20) at room temperature. Varying concentrations of DC-U4106 (0.1-100 microM) are added, and the increase in fluorescence (Ex/Em = 485/535 nm) resulting from the cleavage of rhodamine 110 from ubiquitin is monitored continuously for 30-60 minutes. The IC50 is calculated from the dose-response curve. The Kd is determined using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC).
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: USP8 positive cell line MCF-7 Tested Concentrations: 0-5 μM Incubation Duration: 24 hrs (hours) Experimental Results: As the concentration increases, the expression of EGFR, ErbB2 and ErbB3 proteins decreases, and causes ERα and ErbB3 Degradation of proteins. PR proteins. RT-PCR[1] Cell Types: USP8 positive cell line MCF-7 Tested Concentrations: 0-7 μM Incubation Duration: 24 hrs (hours) Experimental Results: The mRNA levels of ERα and PR were diminished. Cell proliferation assay[1] Cell Types: USP8 positive cell line MCF-7 Tested Concentrations: 0-5 μM Incubation Duration: Experimental Results: Inhibition of cell growth in a dose-dependent manner. Apoptosis analysis [1] Cell Types: USP8 positive cell line MCF-7 Tested Concentrations: 0-5 μM Incubation Duration: 12 hrs (hours) Experimental Results: As the concentration increases, the proportion of apoptotic cells increases. Cellular assays are performed in ERalpha-positive breast cancer cell lines such as MCF-7 or T47D. Cells are seeded in 6-well plates and treated with varying concentrations of DC-U4106 (0-7 microM) for 24-48 hours. After treatment, cells are harvested, and ERalpha and PR protein levels are analyzed by Western blot using specific antibodies. For mRNA analysis, total RNA is extracted, and ERalpha and PR mRNA levels are measured by qRT-PCR. Apoptosis is assessed by measuring cleaved PARP and caspase-3 by Western blot, or by Annexin V/PI staining and flow cytometry. Cell proliferation is measured by MTT or CellTiter-Glo assays. RTK pathway protein expression is analyzed by phospho-RTK array or Western blot. |
| Animal Protocol |
Animal/Disease Models: BALB/c nude mice [1]
Doses: 5mg/kg, 20mg/kg Route of Administration: intraperitoneal (ip) injection, once every 2 days for 14 days. Experimental Results: Tumor growth was Dramatically inhibited at the concentration of 20mg/kg. In vivo efficacy is evaluated in a mouse xenograft model of ERalpha-positive breast cancer. Female athymic nude mice are injected subcutaneously with MCF-7 cells (in Matrigel) and estrogen pellets are implanted to support tumor growth. When tumors reach approximately 100-150 mm3, mice are randomized into treatment groups. DC-U4106 is administered via intraperitoneal injection at doses of 10, 25, or 50 mg/kg daily for 2-4 weeks. Tumor volume is measured by calipers twice weekly. On study termination, tumors are harvested for Western blot analysis of ERalpha and PR, immunohistochemistry for Ki-67 and ERalpha, and histopathological assessment. Mouse body weight is monitored for toxicity assessment. |
| ADME/Pharmacokinetics |
Specific PK parameters for DC-U4106 are not detailed. As a small-molecule USP8 inhibitor (MW 525.56), the compound is designed for in vivo administration. Key PK properties such as bioavailability, half-life, and tissue distribution would require empirical determination. The compound's ability to reduce ERalpha levels in cellular assays suggests it has adequate cell permeability. In vivo, it is likely administered via intraperitoneal injection.
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| Toxicity/Toxicokinetics |
DC-U4106 is described as having minimal toxicity in pre-clinical models. Specific toxicological data are not detailed, but the compound's mechanism of action-promoting Eralpha degradation-is expected to have a favorable safety profile in ERalpha-positive breast cancer because normal cells are less dependent on ERalpha for survival. Additionally, USP8 is involved in various cellular processes, and its inhibition might have broader effects. However, the compound's minimal toxicity in studies suggests a good therapeutic window. Standard toxicological endpoints (body weight, organ histopathology) would be assessed in animal studies.
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| References | |
| Additional Infomation |
DC-U4106 is a research-grade chemical tool for studying USP8 biology and ERalpha-positive breast cancer. USP8 is a deubiquitinating enzyme that plays a role in endosomal recycling and protein stability. By inhibiting USP8, DC-U4106 promotes the degradation of ERalpha via the ubiquitin-proteasome pathway, representing an alternative strategy to direct ERalpha antagonists (e.g., tamoxifen) or aromatase inhibitors. This mechanism may be effective in tamoxifen-resistant breast cancers. As of the latest updates, the compound has not been approved for clinical use and is exclusively available for pre-clinical research.
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| Molecular Formula |
C29H27N5O5
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| Molecular Weight |
525.56
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| Exact Mass |
525.201
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| CAS # |
2410534-62-0
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| PubChem CID |
155301443
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| Appearance |
Yellow to brown solid powder
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| LogP |
4.6
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
39
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| Complexity |
889
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1(C2=CC=C(N3CCN(C4=NC=CC=C4)CC3)C=C2)OC2=C(OC3=C(C)NN=C3C)C(O)=CC(O)=C2C(=O)C=1
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| InChi Key |
VNWOJNLRTJFWRQ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C29H27N5O5/c1-17-27(18(2)32-31-17)39-28-23(37)15-21(35)26-22(36)16-24(38-29(26)28)19-6-8-20(9-7-19)33-11-13-34(14-12-33)25-5-3-4-10-30-25/h3-10,15-16,35,37H,11-14H2,1-2H3,(H,31,32)
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| Chemical Name |
8-[(3,5-dimethyl-1H-pyrazol-4-yl)oxy]-5,7-dihydroxy-2-[4-(4-pyridin-2-ylpiperazin-1-yl)phenyl]chromen-4-one
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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 (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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) |
THF : 10 mg/mL (~19.03 mM)
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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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9027 mL | 9.5137 mL | 19.0273 mL | |
| 5 mM | 0.3805 mL | 1.9027 mL | 3.8055 mL | |
| 10 mM | 0.1903 mL | 0.9514 mL | 1.9027 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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