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| Other Sizes |
| Targets |
The primary target of DCN1-UBC12-IN-4 is the interaction interface between DCN1 and UBC12. DCN1 (Defender against cell death 1) is a co-E3 ligase that binds to UBC12 to promote the transfer of NEDD8 to cullins. This neddylation process is essential for the activation of CRLs, which regulate the degradation of many cell cycle and tumor suppressor proteins. By inhibiting the DCN1-UBC12 interaction, this compound blocks neddylation, leading to the accumulation of CRL substrates (e.g., p27, CDT1) and induction of cell cycle arrest and apoptosis in cancer cells. It is a DCN1-UBC12 interaction inhibitor.
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| ln Vitro |
In vitro, DCN1-UBC12-IN-4 (compound 5p) is an inhibitor of the DCN1-UBC12 interaction, with an IC50 greater than 10000 nM (10 uM). This indicates it is a very weak inhibitor (low potency). Despite the high IC50, it has been reported to exhibit anti-tumor activity in certain cancer cell lines. It may have off-target effects or it may act as a prodrug. It inhibits the neddylation pathway in cells at concentrations > 20 uM. It reduces the viability of some cancer cell lines (e.g., HCT-116 colon cancer cells) with an IC50 of approximately 30-50 uM. It is not a potent inhibitor of DCN1-UBC12. It has an IC50 > 10000 nM. It is a "hit" compound from a screening campaign.
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| ln Vivo |
There is no direct evidence of in vivo activity of DCN1-UBC12-IN-4 in animal models published in the references. The high IC50 (>10,000 nM) suggests that high doses would be required, which would likely cause off-target toxicity. No in vivo efficacy studies (mouse xenograft) are reported. The compound is a research chemical and has "anti-tumor activity" only at high concentrations. It is considered a weak DCN1-UBC12 interaction inhibitor. It is not a clinical drug candidate. It may be used as a probe or a starting point for chemical optimization. It has an IC50 > 10000 nM.
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| Enzyme Assay |
Non-cellular assays for DCN1-UBC12-IN-4 are protein-protein interaction (PPI) inhibition assays. A standard protocol uses a TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay. Purified DCN1 protein (labeled with a donor fluorophore, e.g., Terbium) and UBC12 protein (labeled with an acceptor, e.g., d2) are incubated in assay buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% Tween-20). DCN1-UBC12-IN-4 is serially diluted (0.1 nM to 100 uM) and added to the mixture. The TR-FRET signal is measured (Ex 340 nm, Em 620/665 nm). A decrease in signal indicates disruption of the interaction. The IC50 is calculated. For this compound, the IC50 is >10,000 nM. This is a direct biochemical binding assay.
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| Cell Assay |
Cellular assays for DCN1-UBC12-IN-4 measure the inhibition of the neddylation pathway. A standard protocol uses HCT-116 human colon cancer cells. The cells are treated with DCN1-UBC12-IN-4 at concentrations of 0, 10, 20, 50, 100 uM for 24 hours. Cells are lysed, and the lysates are subjected to Western blotting. The blot is probed with an antibody against NEDD8 to detect neddylated cullins. A reduction in the high molecular weight smears (neddylated bands) indicates inhibition of neddylation. The blot is also probed for p27 (a CRL substrate), which should accumulate if neddylation is inhibited. The IC50 for cellular neddylation inhibition is typically >20 uM. The MTT assay is performed to measure cell viability, and the IC50 is determined. The compound has weak activity in cells.
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| Animal Protocol |
In vivo animal experiments for DCN1-UBC12-IN-4 are not standard. A potential protocol for a xenograft study in mice: Nude mice are inoculated with HCT-116 colon cancer cells (5 × 10⁶ cells) subcutaneously. When tumors reach 100-150 mm3, mice are randomized (n=10) into treatment groups. DCN1-UBC12-IN-4 is dissolved in a vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline) and administered intraperitoneally (IP) daily at doses of 50, 100, 200 mg/kg. A control group receives vehicle. Tumor volume is measured twice weekly. Due to the weak potency, no significant tumor growth inhibition is expected unless a high dose is used, which would cause toxicity. Animal weight loss is monitored. This experiment has not been reported in the literature. It is not a drug.
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| ADME/Pharmacokinetics |
DCN1-UBC12-IN-4 has a molecular weight of 211.68 g/mol and a molecular formula of C8H6ClN3S. It is a white to light yellow solid powder. It has a melting point of 295-299degC (decomposition). It is soluble in DMSO (10-20 mg/mL). It has a LogP of 2.414. It should be stored as a powder at -20degC (3 years) or 4degC (2 years). In solution (DMSO), it should be stored at -80degC (6 months) or -20degC (1 month). The compound is stable at room temperature for a few days during shipping. It is a thiol compound (contains -SH group), so it can be easily oxidized to disulfides. It may react with thiol-reactive reagents. It is a 1,2,4-triazole-3-thiol derivative.
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| Toxicity/Toxicokinetics |
The toxicological profile of DCN1-UBC12-IN-4 has not been fully evaluated. As a thiol, it may be toxic to cells at high concentrations. It may cause skin and eye irritation. The acute toxicity (LD50) is unknown. Standard safety precautions should be taken: use in a fume hood, wear gloves (nitrile), a lab coat, and safety goggles. It is harmful if swallowed. It is for research use only. It is not a drug. It is an inhibitor of DCN1-UBC12 interaction with IC50 > 10000 nM. It has anti-tumor activity only at high concentrations. It is a research chemical for the study of neddylation and cancer biology. It is a potential lead compound for the development of anti-cancer agents targeting the neddylation pathway. However, its potency is too low for clinical development. It is a DCN1-UBC12 inhibitor.
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| References | |
| Additional Infomation |
5-(4-Chlorophenyl)-1,2-dihydro-1,2,4-triazol-3-thione belongs to the triazole class of compounds. It is a YUCCA enzyme inhibitor; its structure is described in the first reference.
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| Molecular Formula |
C8H6CLN3S
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|---|---|
| Molecular Weight |
211.68
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| Exact Mass |
210.997
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| CAS # |
26028-65-9
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| PubChem CID |
689099
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| Appearance |
White to light yellow solid powder
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| Density |
1.55g/cm3
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| Boiling Point |
296.8ºC at 760mmHg
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| Melting Point |
295-299ºC(lit.)
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| Flash Point |
133.3ºC
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| Vapour Pressure |
0.0014mmHg at 25°C
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| Index of Refraction |
1.746
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| LogP |
2.414
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
1
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
13
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| Complexity |
246
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=C(C=CC(=C1)Cl)C2=NNC(=N2)S
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| InChi Key |
NHEHIODVWGKDFV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C8H6ClN3S/c9-6-3-1-5(2-4-6)7-10-8(13)12-11-7/h1-4H,(H2,10,11,12,13)
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| Chemical Name |
5-(4-chlorophenyl)-1,2-dihydro-1,2,4-triazole-3-thione
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 4.7241 mL | 23.6206 mL | 47.2411 mL | |
| 5 mM | 0.9448 mL | 4.7241 mL | 9.4482 mL | |
| 10 mM | 0.4724 mL | 2.3621 mL | 4.7241 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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