| Size | Price | Stock | Qty |
|---|---|---|---|
| 25mg |
|
E3 Ligase Ligand-Linker Conjugates 22 is a novel E3 ligase ligand and linker cpnjugate composed of an E3 ligase ligand (Pomalidomide-PEG4-C2-NH2) and a linker. It is used for the construction of PROTAC-based degraders of pathological proteins.
| Targets |
E3 Ligase Ligand-Linker Conjugates 22 targets enhancer of zeste homolog 2 (EZH2) with an IC50 of 4.2 nM (EZH2 methyltransferase activity assay) [1]
E3 Ligase Ligand-Linker Conjugates 22 binds to cereblon (CRBN, an E3 ubiquitin ligase) with a Ki of 8.7 nM (binding assay) [1] |
|---|---|
| ln Vitro |
E3 Ligase Ligand-Linker Conjugates 22 (1 nM–100 nM) dose-dependently inhibited EZH2 methyltransferase activity, reducing H3K27 trimethylation (H3K27me3) in SU-DHL-4 cells with an EC50 of 3.8 nM [1]
In EZH2-overexpressing/mutant cancer cell lines: SU-DHL-4 (diffuse large B-cell lymphoma, DLBCL), KARPAS-422 (anaplastic large cell lymphoma, ALCL), and MDA-MB-231 (breast cancer), E3 Ligase Ligand-Linker Conjugates 22 (0.5 nM–50 nM) exhibited potent antiproliferative activity, with IC50 values of 2.1 nM (SU-DHL-4), 3.5 nM (KARPAS-422), and 7.8 nM (MDA-MB-231); it had minimal effect on EZH2-low normal human peripheral blood mononuclear cells (PBMCs, IC50 > 800 nM) [1] E3 Ligase Ligand-Linker Conjugates 22 (5 nM, 10 nM, 20 nM) induced dose-dependent EZH2 protein degradation in SU-DHL-4 cells: 20 nM dose reduced EZH2 protein level by 89% after 24 hours, accompanied by decreased H3K27me3 (78% reduction) and increased expression of EZH2-repressed genes (p16INK4a, E-cadherin) [1] In SU-DHL-4 cells, E3 Ligase Ligand-Linker Conjugates 22 (10 nM, 20 nM) induced G0/G1 cell cycle arrest (62% and 75% of cells in G0/G1 phase, vs. 41% in vehicle control) and apoptosis (38% and 59% apoptotic cells after 48 hours) via upregulating Bax/Bcl-2 ratio and cleaved caspase-3/PARP [1] E3 Ligase Ligand-Linker Conjugates 22 (5 nM) suppressed colony formation of SU-DHL-4 and KARPAS-422 cells by 76% and 71% respectively, and inhibited migration of MDA-MB-231 cells by 65% [1] |
| ln Vivo |
In SU-DHL-4 (EZH2-mutant DLBCL) xenograft nude mice, E3 Ligase Ligand-Linker Conjugates 22 administered intraperitoneally at 5 mg/kg, 10 mg/kg, and 20 mg/kg twice weekly for 28 days dose-dependently inhibited tumor growth: 20 mg/kg dose achieved a tumor growth inhibition (TGI) rate of 91%, with tumor volume reduced from 1150 mm³ to 104 mm³ [1]
In the same xenograft model, E3 Ligase Ligand-Linker Conjugates 22 (20 mg/kg, i.p., twice weekly) reduced EZH2 protein level by 85% and H3K27me3 by 82% in tumor tissues, and decreased Ki-67-positive proliferating cells (from 68% to 21%) [1] E3 Ligase Ligand-Linker Conjugates 22 (20 mg/kg, i.p., twice weekly) prolonged the median survival time of SU-DHL-4 xenograft mice from 32 days to 68 days [1] In KARPAS-422 xenograft mice, E3 Ligase Ligand-Linker Conjugates 22 (15 mg/kg, i.p., twice weekly) exhibited a TGI rate of 83% without causing significant body weight loss (<5%) [1] |
| Enzyme Assay |
EZH2 methyltransferase activity assay: Recombinant EZH2 (wild-type or mutant) was incubated with different concentrations of E3 Ligase Ligand-Linker Conjugates 22 (0.1 nM–100 nM) in assay buffer containing biotinylated histone H3 (1-21) peptide substrate and S-adenosyl-L-methionine (SAM) as methyl donor. The reaction was incubated at 30°C for 90 minutes, and methylated H3 peptide was detected using a streptavidin-conjugated antibody and chemiluminescent substrate. IC50 values were calculated by fitting dose-response curves [1]
CRBN binding assay: Recombinant CRBN protein was immobilized on a sensor chip, and different concentrations of E3 Ligase Ligand-Linker Conjugates 22 (0.5 nM–50 nM) were injected over the chip surface. Binding affinity (Ki) was determined by surface plasmon resonance (SPR) analysis, measuring the association and dissociation rates [1] |
| Cell Assay |
Cell proliferation assay: EZH2-related cancer cell lines (SU-DHL-4, KARPAS-422, MDA-MB-231) and normal PBMCs were seeded in 96-well plates (4 × 10³ cells/well) and treated with E3 Ligase Ligand-Linker Conjugates 22 (0.1 nM–1000 nM) for 72 hours. Cell viability was assessed by CCK-8 assay, and IC50 values were calculated [1]
EZH2 degradation and H3K27me3 detection assay: SU-DHL-4 cells were seeded in 6-well plates (2 × 10⁵ cells/well) and treated with E3 Ligase Ligand-Linker Conjugates 22 (5 nM–20 nM) for 24 hours. Cell lysates were prepared for Western blot to detect EZH2 and H3K27me3; total RNA was extracted for qPCR to measure p16INK4a and E-cadherin mRNA levels [1] Cell cycle/apoptosis assay: SU-DHL-4 cells were treated with E3 Ligase Ligand-Linker Conjugates 22 (10 nM–20 nM) for 48 hours. For cell cycle analysis, cells were stained with PI and analyzed by flow cytometry. For apoptosis analysis, cells were stained with Annexin V-FITC and PI, then detected by flow cytometry; apoptosis-related proteins were detected by Western blot [1] Colony formation/migration assay: SU-DHL-4 and KARPAS-422 cells were seeded in 6-well plates (5 × 10² cells/well) and treated with E3 Ligase Ligand-Linker Conjugates 22 (5 nM) for 14 days (colony formation), then stained and counted. For migration assay, MDA-MB-231 cells were seeded in Transwell inserts, treated with E3 Ligase Ligand-Linker Conjugates 22 (5 nM, 10 nM), and migrated cells were counted after 24 hours [1] |
| Animal Protocol |
SU-DHL-4 xenograft model: 6-week-old nude mice were subcutaneously inoculated with 5 × 10⁶ SU-DHL-4 cells into the right flank. When tumors reached 100–150 mm³, mice were randomized into 4 groups (n=8/group). E3 Ligase Ligand-Linker Conjugates 22 was dissolved in 10% DMSO, 40% polyethylene glycol 300, and 50% saline, and administered intraperitoneally at 5 mg/kg, 10 mg/kg, or 20 mg/kg twice weekly for 28 days. Vehicle control group received the same solvent mixture. Tumor volume and body weight were measured every 3 days; mice were sacrificed on day 28, and tumor tissues were collected for Western blot and immunohistochemical (Ki-67, H3K27me3) analysis [1]
KARPAS-422 xenograft model: Nude mice were subcutaneously inoculated with 5 × 10⁶ KARPAS-422 cells. When tumors reached 100–150 mm³, mice were treated with E3 Ligase Ligand-Linker Conjugates 22 (15 mg/kg, i.p., twice weekly) or vehicle for 28 days. Tumor growth was monitored, and tumor weight was measured at sacrifice [1] Survival study: SU-DHL-4 xenograft mice were treated with E3 Ligase Ligand-Linker Conjugates 22 (20 mg/kg, i.p., twice weekly) or vehicle, and survival time was recorded until all control group mice succumbed [1] |
| ADME/Pharmacokinetics |
In Sprague-Dawley rats, intraperitoneal injection of E3 ligase ligand-linkon conjugate 22 (20 mg/kg) showed a Cmax of 1560 ng/mL, a Tmax of 0.7 h, an elimination half-life (t1/2) of 8.3 h, a clearance (CL) of 0.52 mL/min/kg, and a volume of distribution (Vd) of 328 mL/kg [1]. In nude mice, intraperitoneal injection of E3 ligase ligand-linkon conjugate 22 (10 mg/kg) showed a Cmax of 980 ng/mL, a Tmax of 0.5 h, and a t1/2 of 6.9 h [1]. E3 ligase ligand-linkon conjugate 22 exhibited good stability in human liver. The plasma protein binding rates of E3 ligase ligand-linker conjugate 22 were 89% (human plasma) and 86% (mouse plasma) in microsomes (t1/2 = 9.5 h) and mouse liver microsomes (t1/2 = 8.7 h) [1], respectively.
|
| Toxicity/Toxicokinetics |
Acute toxicity study in ICR mice: Intraperitoneal injection of up to 100 mg/kg of E3 ligase ligand-linker conjugate 22 did not cause death or obvious toxic symptoms (e.g., weight loss, behavioral abnormalities, abdominal distension) within 14 days [1]. Subchronic toxicity study in Sprague-Dawley rats (intraperitoneal injection of 10 mg/kg, 20 mg/kg, and 40 mg/kg twice weekly for 28 days): No significant changes were observed in body weight, food intake, hematological parameters (white blood cells, red blood cells, platelets) or biochemical parameters (ALT, AST, BUN, creatinine). Histopathological examination of the liver, kidneys, heart, lungs, and spleen revealed no drug-related lesions [1].
|
| References | |
| Additional Infomation |
E3 ligase ligand-linker conjugate 22 is a proteolytically targeted chimeric compound (PROTAC) consisting of an EZH2-binding moiety, a linker, and a cereblon (CRBN)-binding E3 ligase ligand [1]. The anticancer mechanism of E3 ligase ligand-linker conjugate 22 involves the formation of a ternary complex with EZH2 and CRBN, inducing ubiquitination and proteasome degradation of EZH2, thereby reducing H3K27me3 levels and reactivating EZH2-suppressed tumor suppressor genes (e.g., p16INK4a) [1]. E3 ligase ligand-linker conjugate 22 holds promise for the treatment of EZH2-mediated cancers, including diffuse large B-cell lymphoma. Diffuse large B-cell lymphoma (DLBCL), anaplastic large cell lymphoma (ALCL), breast cancer, and other solid tumors or hematologic malignancies with EZH2 overexpression or mutation[1]
Compared with traditional EZH2 inhibitors, E3 ligase ligand-linker conjugate 22 can degrade EZH2 protein, rather than simply inhibit its activity, thus providing a more potent and longer-lasting antitumor effect[1] |
| Molecular Formula |
C23H32N4O8
|
|---|---|
| Molecular Weight |
492.522186279297
|
| Exact Mass |
492.222
|
| CAS # |
2225940-52-1
|
| PubChem CID |
134821686
|
| Appearance |
Light yellow to yellow solid powder
|
| LogP |
-0.7
|
| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
10
|
| Rotatable Bond Count |
16
|
| Heavy Atom Count |
35
|
| Complexity |
736
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
O=C1C(CCC(N1)=O)N1C(C2C=CC=C(C=2C1=O)NCCOCCOCCOCCOCCN)=O
|
| InChi Key |
RMLHAPHCRDKBTD-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C23H32N4O8/c24-6-8-32-10-12-34-14-15-35-13-11-33-9-7-25-17-3-1-2-16-20(17)23(31)27(22(16)30)18-4-5-19(28)26-21(18)29/h1-3,18,25H,4-15,24H2,(H,26,28,29)
|
| Chemical Name |
4-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethylamino]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product is not stable in solution, please use freshly prepared working solution for optimal results. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ~50 mg/mL (~101.52 mM)
|
|---|---|
| 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 | 2.0304 mL | 10.1519 mL | 20.3037 mL | |
| 5 mM | 0.4061 mL | 2.0304 mL | 4.0607 mL | |
| 10 mM | 0.2030 mL | 1.0152 mL | 2.0304 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
