| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| Targets |
MDM2
The targeting component of Nutlin-C1-amido-PEG4-C2-N3 is the Nutlin-3-based MDM2 ligand. This ligand binds with high affinity to MDM2, a RING finger E3 ubiquitin ligase that is the primary negative regulator of the tumor suppressor p53. By binding to MDM2, the ligand recruits the E3 ligase to a protein of interest (POI) when that POI is conjugated to the same molecule. The azide (N3) group at the other end of the linker can be conjugated to a target protein ligand via copper-catalyzed or strain-promoted azide-alkyne cycloaddition (click chemistry). This creates a bifunctional PROTAC molecule. The PROTAC brings the POI into proximity with MDM2, leading to ubiquitination and subsequent degradation of the POI by the proteasome. MDM2 is the E3 ligase used for targeted protein degradation. |
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| ln Vitro |
The in vitro activity of this compound alone (without a target-binding ligand attached) is not directly measured. Instead, its utility is demonstrated when conjugated to a target protein ligand. The parent Nutlin-3 is a potent inhibitor of the MDM2-p53 interaction (IC50 ∼ 90 nM). In PROTACs utilizing MDM2 as the E3 ligase, the chimeric molecule should induce degradation of the target protein in a concentration-dependent manner. For example, when conjugated to a BET bromodomain inhibitor (e.g., JQ1), the resulting PROTAC can degrade BRD4 protein in cells (e.g., IC50 for BRD4 degradation in the low nanomolar range after 6-24 hour treatment). The effectiveness of PROTACs varies depending on the target, linker length, and the specific conjugation chemistry. The PEG4 linker provides sufficient flexibility and solubility.
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| ln Vivo |
The in vivo activity of this compound is inferred from PROTACs developed using similar MDM2-recruiting conjugates. In mouse xenograft models, PROTACs that recruit MDM2 have been shown to induce degradation of target proteins (e.g., BRD4, ERalpha, AR) and inhibit tumor growth. For instance, a PROTAC targeting BRD4 using an MDM2 ligand has been reported to induce BRD4 degradation in tumors and reduce tumor volume after intravenous or intraperitoneal administration. The efficacy depends on the stability and bioavailability of the specific PROTAC molecule. The PEG linker is designed to improve water solubility and circulation time. The compound itself is a building block for PROTAC synthesis and is typically used in medicinal chemistry and chemical biology research.
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| Enzyme Assay |
Nutlin-C1-amido-PEG4-C2-N3 is not used in cell-free assays for target engagement. It is a chemical building block. However, after conjugation to a target ligand, a cell-free ubiquitination assay can be performed. The assay mixture (30-50 microL) contains 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM ATP, 1 mM DTT, E1 ubiquitin-activating enzyme (50 nM), E2 ubiquitin-conjugating enzyme (100 nM), MDM2 (as the E3 ligase, 50 nM), ubiquitin (10 microM), and the PROTAC molecule (0.01-10 microM). A recombinant target protein of interest (POI) is also included. The reaction is incubated at 37degC for 30-120 minutes. The reaction is stopped by adding SDS-PAGE loading buffer. Samples are analyzed by Western blotting using an anti-ubiquitin antibody or a tag-specific antibody (e.g., anti-His or anti-FLAG) to detect ubiquitinated POI. Formation of high molecular weight ubiquitin conjugates indicates successful PROTAC-mediated ubiquitination.
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| Cell Assay |
To validate the cellular activity of a PROTAC made from this conjugate, a cell-based degradation assay is performed. The target protein of interest is expressed in a cell line (e.g., HeLa, HEK293, or a cancer cell line with endogenous expression of the target). The PROTAC is synthesized by conjugating a target protein ligand (e.g., JQ1 for BRD4, fulvestrant for ERalpha, enzalutamide for AR) to the azide group using click chemistry. Cells are seeded in 6-well or 12-well plates (2-5×10⁵ cells/well). The next day, cells are treated with varying concentrations of the PROTAC (0.1 nM-10 microM) or DMSO vehicle for 4-24 hours. Cells are then harvested and lysed. The lysates are subjected to SDS-PAGE and Western blotting with an antibody against the target protein. Degradation is quantified by densitometry and normalized to a loading control (e.g., GAPDH, beta-actin). The half-maximal degradation concentration (DC50) is calculated. A negative control (PROTAC with an inactive ligand) should not induce degradation. The compound is an MDM2 ligand-linker conjugate.
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| Animal Protocol |
This compound is a building block for PROTAC synthesis and is not typically administered in animals. For in vivo efficacy studies of a PROTAC molecule, the fully synthesized PROTAC is used. For example, a PROTAC targeting BRD4 (using this Nutlin-based MDM2 ligand) might be evaluated in a mouse xenograft model. Female BALB/c nude mice bearing subcutaneous tumors (e.g., HeLa or 22Rv1) of 150-200 mm3 are randomized into treatment groups (n=8-10 per group). The PROTAC is formulated in an appropriate vehicle (e.g., 10% DMSO, 10% Cremophor EL, 80% saline or 0.5% methylcellulose). The PROTAC is administered by intraperitoneal (IP) or intravenous (IV) injection at doses of 1-30 mg/kg, once daily or every other day, for 14-21 days. Tumor volume is measured twice weekly with calipers. At study termination, tumors are excised and processed for Western blotting to confirm target degradation. Pharmacokinetic parameters are also measured. This compound is not administered alone; it requires conjugation to a target-binding ligand to be functional.
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| ADME/Pharmacokinetics |
Nutlin-C1-amido-PEG4-C2-N3 has a molecular formula of C42H52Cl2N8O9 and a molecular weight of 883.83 g/mol. The compound is supplied as a solid (purity >97%) and should be stored at -20degC in a sealed container, protected from light and moisture, where it is stable for up to 3 years. For solution storage, stock solutions in DMSO (10-20 mM) should be stored at -80degC for up to 6 months. The compound is soluble in DMSO (10-20 mg/mL). The PEG4 linker improves solubility in aqueous buffers. The azide group is reactive and can be used for copper-catalyzed click chemistry (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC) with alkyne-containing target protein ligands. The design incorporates an amide bond and a short PEG linker. This product is for research use only.
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| Toxicity/Toxicokinetics |
This product is for research use only and is not for human therapeutic use. The compound is an MDM2 ligand-linker conjugate. Nutlin-3, the parent ligand, is not acutely toxic at nanomolar concentrations in cell culture but can activate the p53 pathway, leading to cell cycle arrest and apoptosis in p53 wild-type cells. The compound should be handled as a potential biohazard. Standard laboratory safety practices (gloves, lab coat, safety glasses) should be followed. Avoid inhalation of powder and contact with skin and eyes. The compound is not an FDA-approved drug. It is a chemical tool for research purposes only.
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| References | |
| Additional Infomation |
PROTACs (proteolysis-targeting chimeras) are a new class of therapeutic modalities that induce the degradation of disease-relevant proteins rather than inhibiting them. MDM2 is one of several E3 ubiquitin ligases (others include CRBN, VHL, IAP) that have been successfully recruited for PROTAC-based protein degradation. The Nutlin-based MDM2 ligand binds to the p53-binding pocket of MDM2 with high affinity (Ki ∼ 30-50 nM). The PEG4 linker is hydrophilic and flexible, improving the water solubility of the PROTAC molecule and allowing the two binding moieties to adopt optimal conformations for ternary complex formation. The azide group is a bioorthogonal handle for selective conjugation. This conjugate is MDM2 Ligand-Linker Conjugate 1. It is intended for research use only. Supplier information must not be included.
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| Molecular Formula |
C42H52CL2N8O9
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| Appearance |
Typically exists as solid at room temperature
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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. |
| 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 :~200 mg/mL (~226.29 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.) |
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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