| Size | Price | Stock | Qty |
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| 25mg |
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| Targets |
The primary molecular target engaged by E3 Ligase Ligand-Linker Conjugates 2 is the cereblon (CRBN) E3 ubiquitin ligase complex. The compound contains the Pomalidomide-based cereblon ligand, which binds with high affinity to the CRBN protein, a substrate recognition component of the CRBN E3 ubiquitin ligase complex. This ligand directs the E3 ligase to ubiquitinate the target protein that is recruited by the complete PROTAC molecule. The PEG4 linker connects the cereblon ligand to a functional group (Ph-NH₂) that can be attached to a warhead (a target-binding moiety). When used in PROTACs, the compound enables the formation of a ternary complex between the target protein, the PROTAC, and the CRBN E3 ligase, leading to ubiquitination and proteasomal degradation of the target protein.
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| ln Vitro |
In vitro studies demonstrate that E3 Ligase Ligand-Linker Conjugates 2 can be incorporated into PROTAC molecules that induce targeted protein degradation. The compound enables the formation of a ternary complex between the target protein, the PROTAC, and the CRBN E3 ligase, leading to ubiquitination and subsequent proteasomal degradation of the target protein. When used in PROTACs targeting various proteins of interest, the conjugate enables potent degradation at low nanomolar concentrations. The PEG4 linker provides the appropriate length and flexibility for optimal ternary complex formation. The compound's activity is assessed by measuring target protein levels by Western blot and by evaluating downstream functional effects such as inhibition of cell proliferation and induction of apoptosis. The Pomalidomide-based ligand ensures high affinity binding to CRBN, enabling efficient target degradation.
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| ln Vivo |
In vivo studies of E3 Ligase Ligand-Linker Conjugates 2 are conducted in the context of PROTAC molecules incorporating this conjugate. PROTACs containing this linker have been evaluated in various animal models of cancer and other diseases. In xenograft models, PROTACs containing this conjugate demonstrate significant tumor growth inhibition through degradation of the target protein. The compounds are typically administered via intraperitoneal or intravenous injection at doses determined from pharmacokinetic and toxicology studies. Pharmacodynamic endpoints include measurement of target protein degradation in tumor tissues, assessment of downstream signaling pathway inhibition, and evaluation of anti-tumor efficacy. The PEG4 linker contributes to favorable pharmacokinetic properties of the resulting PROTACs, including improved solubility and reduced aggregation.
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| Enzyme Assay |
For CRBN binding assays, the affinity of the Pomalidomide-based ligand for CRBN is measured using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). Purified CRBN-DDB1 complex is immobilized on a sensor chip, and varying concentrations of the ligand-linker conjugate (0.001-100 µM) are injected over the chip surface. Binding kinetics (ka, kd) and affinity (KD) are calculated. For ternary complex formation assays, the ability of the complete PROTAC containing this conjugate to simultaneously bind to CRBN and the target protein is assessed by SPR or AlphaScreen. In SPR, the target protein is immobilized on the chip, and the PROTAC is injected in the presence or absence of CRBN complex. The formation of a ternary complex is indicated by enhanced binding or a change in binding kinetics. For ubiquitination assays, in vitro ubiquitination reactions are performed using purified E1, E2, CRBN E3 ligase, target protein, PROTAC, and ubiquitin, and ubiquitination is detected by Western blot.
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| Cell Assay |
For cellular degradation assays, cancer cells expressing the target protein are cultured in appropriate medium with 10% FBS and antibiotics. Cells are seeded in 6-well or 12-well plates and treated with PROTACs containing the conjugate at varying concentrations (0.001-10 µM) for 4-24 hours. Cells are lysed in RIPA buffer with protease inhibitors, and target protein levels are analyzed by Western blot using specific antibodies. Degradation efficiency is quantified by densitometry and expressed as DC50 (concentration for 50% degradation) and Dmax (maximum degradation). For cell viability assays, cells are treated with PROTACs for 48-72 hours and viability is assessed by MTT or CellTiter-Glo assays. Apoptosis is assessed by Annexin V/PI staining or caspase-3/7 activity assays. For selectivity studies, the effect of the PROTAC on other proteins is assessed by Western blot or proteomics. The Pomalidomide-based ligand may also have immunomodulatory effects, which should be considered when interpreting results.
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| Animal Protocol |
For in vivo efficacy studies, 6-8 week old female immunodeficient mice (e.g., nude or SCID) are used. Mice are subcutaneously implanted with cancer cells expressing the target protein in the flank. When tumors reach approximately 100-200 mm³, animals are randomized into treatment groups (n = 6-10 per group). PROTACs containing the conjugate are formulated in vehicle (e.g., 10% DMSO, 40% PEG400, 50% PBS) and administered intravenously or intraperitoneally at doses of 1-50 mg/kg, typically on a q.d. or q.o.d. schedule for 2-3 weeks. Tumor volumes are measured twice weekly using calipers. Body weights are monitored for toxicity. At study termination, tumors are excised and processed for Western blot analysis of target protein degradation, histopathology, and biomarker analysis. Blood samples may be collected for pharmacokinetic analysis of the PROTAC.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of E3 Ligase Ligand-Linker Conjugates 2 are studied as part of the complete PROTAC molecule incorporating this conjugate. The PEG4 linker contributes to the overall pharmacokinetic profile by improving solubility and reducing aggregation, which is important for oral bioavailability and tissue distribution. PROTACs containing this conjugate typically show favorable pharmacokinetic properties, including moderate to good oral bioavailability, reasonable plasma half-life, and adequate tissue distribution. The conjugate's composition (Pomalidomide-PEG4-Ph-NH2) is designed to provide optimal properties for PROTAC drug development. However, specific pharmacokinetic data for the conjugate alone or for PROTACs containing this specific conjugate are not extensively reported in the public domain.
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| Toxicity/Toxicokinetics |
Toxicological data for E3 Ligase Ligand-Linker Conjugates 2 are derived from toxicology studies of PROTACs containing this conjugate, rather than studies of the conjugate alone. The conjugate is considered a non-toxic component of PROTAC molecules, as it is a synthetic chemical that is typically metabolized and cleared. The Pomalidomide-based ligand may have immunomodulatory effects, which could contribute to the pharmacology and toxicity of the resulting PROTACs. The safety and tolerability of PROTACs containing this conjugate are evaluated in animal toxicology studies. Parameters assessed include clinical observations, body weight, food consumption, clinical pathology, organ weights, and histopathology. The toxicity of PROTACs is primarily attributed to the warhead (the target-binding moiety) and the pharmacology of target degradation, rather than the conjugate itself. As with all research compounds, appropriate safety precautions should be taken when handling this conjugate.
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| References | |
| Additional Infomation |
E3 Ligase Ligand-Linker Conjugates 2 (CAS 1818885-63-0) is a synthetic compound designed for PROTAC development. It incorporates the Pomalidomide-based cereblon (CRBN) ligand and a 4-unit PEG linker. Also known as Pomalidomide-PEG4-Ph-NH2 and Cereblon Ligand-Linker Conjugates 9, this conjugate brings together the target protein and ubiquitinating machinery by linking the E3 ligase ligand to a PROTAC linker. The compound enables the recruitment of the CRBN E3 ubiquitin ligase to promote ubiquitination and degradation of target proteins. Its molecular formula is C₂₇H₃₂N₄O₈. The conjugate is used in PROTAC research to induce targeted protein degradation and is strictly for research use only.
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| Molecular Formula |
C27H32N4O8
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|---|---|
| Molecular Weight |
540.564987182617
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| Exact Mass |
540.221
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| CAS # |
1818885-63-0
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| PubChem CID |
118435234
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
792.0±60.0 °C at 760 mmHg
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| Flash Point |
432.8±32.9 °C
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| Vapour Pressure |
0.0±2.8 mmHg at 25°C
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| Index of Refraction |
1.625
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| LogP |
-0.71
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
15
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| Heavy Atom Count |
39
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| Complexity |
850
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CC(=O)NC(=O)C1N2C(=O)C3=C(C2=O)C(=CC=C3)NCCOCCOCCOCCOC4=CC=C(C=C4)N
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| InChi Key |
QTFDOBJYFQFDIO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H32N4O8/c28-18-4-6-19(7-5-18)39-17-16-38-15-14-37-13-12-36-11-10-29-21-3-1-2-20-24(21)27(35)31(26(20)34)22-8-9-23(32)30-25(22)33/h1-7,22,29H,8-17,28H2,(H,30,32,33)
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| Chemical Name |
4-[2-[2-[2-[2-(4-aminophenoxy)ethoxy]ethoxy]ethoxy]ethylamino]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione
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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 : ~50 mg/mL (~92.49 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 | 1.8499 mL | 9.2495 mL | 18.4990 mL | |
| 5 mM | 0.3700 mL | 1.8499 mL | 3.6998 mL | |
| 10 mM | 0.1850 mL | 0.9249 mL | 1.8499 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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