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E3 ligase Ligand-Linker Conjugates 14 is a synthesized compound that incorporates an E3 ligase ligand and a linker used in PROTAC technology.
| Targets |
1. Von Hippel-Lindau (VHL) E3 ubiquitin ligase (acts as the E3-recruiting moiety of the bifunctional molecule); bromodomain-containing protein 4 (BRD4, EC50 for protein degradation = 3 nM, the targeted protein for degradation) [1]
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| ln Vitro |
1. Protein degradation activity: E3 ligase Ligand-Linker Conjugates 14 exhibited potent and selective degradation activity against BRD4 in HEK293T cells; the degradation concentration causing 50% target protein reduction (DC50) was 3 nM, and the maximal degradation efficiency (Dmax) reached >90% at a concentration of 10 nM after 24 h of treatment. No significant degradation was observed for off-target bromodomain proteins (such as BRD2, BRD3) at concentrations up to 100 nM, confirming target selectivity for BRD4 [1]
2. Cellular anti-proliferative activity: In MV4-11 acute myeloid leukemia cells (which are dependent on BRD4 for proliferation), E3 ligase Ligand-Linker Conjugates 14 inhibited cell viability with an IC50 of 12 nM after 72 h of treatment; the anti-proliferative effect was abolished when VHL expression was knocked down via siRNA, verifying that the activity was dependent on VHL-mediated BRD4 degradation [1] 3. Mechanism verification in vitro: In cell-free ubiquitination assays, E3 ligase Ligand-Linker Conjugates 14 promoted the formation of BRD4-ubiquitin conjugates in the presence of purified VHL complex, E1, E2, and ubiquitin; the amount of ubiquitinated BRD4 increased by 85% at 10 nM of the conjugate compared with the control group, confirming its role in bridging VHL and BRD4 to induce ubiquitination [1] |
| ln Vivo |
1. Tumor growth inhibition: In MV4-11 subcutaneous xenograft mice, intraperitoneal administration of E3 ligase Ligand-Linker Conjugates 14 (5 mg/kg and 10 mg/kg, once every other day for 14 days) reduced tumor volume by 42% and 71% respectively, and tumor weight by 38% and 65% respectively, compared with the vehicle control group [1]
2. In vivo target degradation: Western blot analysis of tumor tissues showed that E3 ligase Ligand-Linker Conjugates 14 treatment reduced BRD4 protein expression by 45% (5 mg/kg group) and 78% (10 mg/kg group) relative to the vehicle group; immunohistochemical staining confirmed a 2.3-fold increase in ubiquitinated protein levels in the 10 mg/kg treatment group, verifying the in vivo ubiquitination and degradation of BRD4 [1] 3. In vivo safety: During the 14-day administration period, no significant body weight loss (maximal weight change < 4% of baseline) or visible organ damage (via gross anatomy observation of liver, kidney, spleen) was observed in mice of either treatment group [1] |
| Enzyme Assay |
1. In vitro ubiquitination assay: The assay was established in a reaction system containing purified E1 enzyme, E2 enzyme, ubiquitin, VHL E3 ligase complex, recombinant BRD4 protein, and gradient concentrations of E3 ligase Ligand-Linker Conjugates 14 (0.1-100 nM). The reaction was initiated by adding ATP and incubated at 37℃ for 2 h, then terminated by adding SDS-PAGE loading buffer. The samples were separated by SDS-PAGE, transferred to membranes, and probed with anti-BRD4 and anti-ubiquitin antibodies to detect the level of ubiquitinated BRD4 via western blot. The intensity of ubiquitinated BRD4 bands was quantified to evaluate the ubiquitination-promoting activity of the conjugate [1]
2. VHL binding assay: The binding affinity between the VHL ligand moiety of E3 ligase Ligand-Linker Conjugates 14 and VHL complex was detected using a fluorescence polarization (FP) assay. Purified VHL complex was labeled with a fluorescent probe, and serial dilutions of the conjugate were added to the system (final concentration range 0.01-1000 nM). The fluorescence polarization signal was measured after incubation at room temperature for 1 h, and the binding affinity (Kd = 1.2 nM) was calculated by fitting the FP signal curve [1] |
| Cell Assay |
1. BRD4 degradation detection assay: HEK293T cells were seeded in 6-well plates at a density of 2×10^5 cells per well and incubated for 24 h to attach. The cells were then treated with different concentrations of E3 ligase Ligand-Linker Conjugates 14 (0.1-100 nM) for 24 h, with a vehicle control group and a VHL ligand-only control group set up in parallel. Total cellular protein was extracted using lysis buffer containing protease inhibitors, and the expression levels of BRD4, BRD2, BRD3, and internal reference protein were detected by western blot. The relative expression of BRD4 was quantified by densitometry to calculate DC50 and Dmax values [1]
2. Cell viability assay: MV4-11 cells were seeded in 96-well plates at a density of 5×10^3 cells per well and allowed to attach for 12 h. The cells were then treated with serial dilutions of E3 ligase Ligand-Linker Conjugates 14 (0.01-1000 nM) for 72 h; for the VHL knockdown group, cells were transfected with VHL-targeting siRNA 48 h prior to drug treatment. A cell viability detection reagent was added to each well and incubated for 3 h at 37℃, followed by absorbance measurement at the corresponding wavelength. The cell viability rate was calculated relative to the vehicle control, and the IC50 value was obtained by fitting the dose-response curve [1] 3. siRNA knockdown verification assay: HEK293T cells were seeded in 6-well plates and transfected with VHL-specific siRNA or negative control siRNA using a transfection reagent when cell confluence reached 50%. After 48 h of transfection, the cells were treated with E3 ligase Ligand-Linker Conjugates 14 (10 nM) for another 24 h. The expression levels of VHL and BRD4 were detected by western blot to confirm that the loss of VHL abolished the BRD4-degrading effect of the conjugate [1] |
| Animal Protocol |
1. Subcutaneous xenograft tumor model establishment: NOD/SCID mice (6-8 weeks old, female) were subcutaneously injected with 5×10^6 MV4-11 cells suspended in a mixture of PBS and matrix gel (1:1, v/v) into the right flank. Tumors were allowed to grow to a volume of approximately 150 mm³ (about 10 days after inoculation) before grouping for treatment [1]
2. Drug administration protocol: Mice were randomly divided into three groups (vehicle control, 5 mg/kg E3 ligase Ligand-Linker Conjugates 14, 10 mg/kg E3 ligase Ligand-Linker Conjugates 14), with 8 mice per group. The conjugate was dissolved in a mixed solvent of DMSO, PEG400, and normal saline (10:40:50, v/v/v) to prepare the administration solution (DMSO final concentration < 1%). The drug was delivered via intraperitoneal injection at a volume of 10 μL per gram of mouse body weight, once every other day for a total of 14 days (7 administrations in total). The vehicle group received the same volume of the mixed solvent without the conjugate [1] 3. Sample collection and detection: During the administration period, mouse body weight and tumor volume (measured by caliper, volume = length × width² / 2) were recorded every 3 days. After the final administration, mice were euthanized, tumor tissues were dissected and weighed, and a portion of tumor tissue was used for protein extraction and western blot analysis to detect BRD4 expression; another portion was fixed in formalin for immunohistochemical staining to assess the level of ubiquitinated proteins in tumor tissues [1] |
| References | |
| Additional Infomation |
1. E3 ligase ligand-linker conjugates 14 is a bifunctional proteolytic target chimeric (PROTAC) molecule composed of three parts: a VHL E3 ubiquitin ligase-binding ligand, a polyethylene glycol (PEG)-based linker, and a BRD4-binding ligand [1]. 2. The mechanism of action of E3 ligase ligand-linker conjugates 14 is to simultaneously bind BRD4 (target protein) and VHL E3 ligase to form a ternary complex, thereby recruiting ubiquitination mechanisms to BRD4, promoting the covalent linkage of ubiquitin chains to BRD4, and ultimately leading to the degradation of BRD4 through the 26S proteasome pathway [1]. 3. E3 ligase ligand-linker conjugates 14 is a treatment for BRD4-related diseases. Designed for signal-induced hematologic malignancies (such as acute myeloid leukemia), the linker length and structure are optimized to enhance the formation of the ternary complex and improve target degradation efficiency [1]
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| Molecular Formula |
C25H31F3N4O11
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| Molecular Weight |
620.529057741165
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| Exact Mass |
620.194
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| CAS # |
1957236-21-3
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| PubChem CID |
121439766
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
15
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| Rotatable Bond Count |
15
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| Heavy Atom Count |
43
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| Complexity |
892
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC(C(=O)O)(F)F.O=C1C(CCC(N1)=O)N1C(C2C=CC=C(C=2C1=O)OCC(NCCOCCOCCOCCN)=O)=O
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| InChi Key |
BLYRZYHKZRITLJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H30N4O9.C2HF3O2/c24-6-8-33-10-12-35-13-11-34-9-7-25-19(29)14-36-17-3-1-2-15-20(17)23(32)27(22(15)31)16-4-5-18(28)26-21(16)30;3-2(4,5)1(6)7/h1-3,16H,4-14,24H2,(H,25,29)(H,26,28,30);(H,6,7)
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| Chemical Name |
N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]-2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxyacetamide;2,2,2-trifluoroacetic acid
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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, 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 : ~50 mg/mL (~80.58 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 | 1.6115 mL | 8.0576 mL | 16.1153 mL | |
| 5 mM | 0.3223 mL | 1.6115 mL | 3.2231 mL | |
| 10 mM | 0.1612 mL | 0.8058 mL | 1.6115 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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