EZH2; CRBN

EZH2 is a member of PcG and can induce the occurrence of cancer when it is highly expressed. As an EZH2 inhibitor, Tazemetostat (EPZ6438) can inhibit the methylation catalytic activity of EZH2. However, many studies have shown that inhibition of EZH2 alone does not efficiently block tumor development. Therefore, in this study, proteolytic targeting chimera technology was employed to enhance the antiproliferative potency of EPZ6438 by degrading the oncogenic activity of EZH2. Several PROTACs have been synthesized by combining EPZ6438 with four E3 ligase ligands based on VHL, CRBN, MDM2, and cIAP E3 ligase systems. In our study, compound E-3P-MDM2 is the most active PROTAC molecule. It degraded EZH2 of the SU-DHL-6 cells in a concentration and dose-dependent manner and also degraded both EED and SUZ12 protein without affecting their mRNA levels, then significantly inhibited the expression of H3K27me3. The in vitro antiproliferative activity of E-3P-MDM2 was much stronger than that of EPZ6438. [1]

Determination of antiproliferative activity [1]
All cell viability assays were performed by CCK-8 method. Cells were harvested directly after proliferation to 80% in culture flasks. The collected cells were counted and inoculated in 96-well plates at 6000 cells per well. Cells were cultured overnight in 96-well plates, and then compounds to be tested were added. After 48 h of compound incubation, the CCK-8 reagent was added directly and then incubated at 37 °C. The absorbance was measured at 450 nm (0.8–2.0) and the inhibition rate was calculated.

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Western blot analysis [1]
Western Blot assays were used to detect changes in the expression of the proteins of interest. Cells were collected directly and counted when they proliferated to 80% in the culture flasks. Cells were inoculated into six-well plates at 500,000 cells per well and cultured overnight. Different compounds were added to act for 24–72 h. Collect the cells in the six-well plate at the end of the compound incubation time. Add RIPA lysate and centrifuge to collect protein supernatant. The concentration of the samples was determined using the BCA method. After the assay, the samples were added to loading buffer and heated to 95 °C for 5 min. Experiments were performed by electrophoresis using SDS-PAGE. After electrophoresis, the gel was converted to a PVDF membrane. The primary antibodies (EZH2, SUZ12, EED, GAPDH, Bcl-2, Bax, and caspase 3, caspase 9, cytochrome c (1:1000)) were incubated overnight after 5% milk closure for 1 h. The secondary antibody was incubated last and then exposed.

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Flow cytometry measure for cells apoptosis [1]
The apoptosis ratio was detected by the kit. Wait for cells to reach 80% fusion in the culture flask, collect them, and count them. Each well of a six-well plate was seeded with 500,000 cells and incubated overnight. Add the compound to be tested. Wait 48 h and collect the cells according to the kit steps. The sample to be assayed was added with 5 μL PI and V-FITC, incubated for 15 min at room temperature and protected from light. Finally, flow cytometry was used for detection.

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Detection of cell mitochondrial membrane potential [1]
The changes of mitochondrial membrane potential after compound treatment were detected using a kit. When the cells were in logarithmic growth phase, cells were collected and inoculated in 6-well plates (500,000 per well). After the cells were cultured overnight, the compound to be tested was added to the six-well plate and waited for 48 h. After compound action was complete, cells were collected by adding the JC-1 fluorescent probe and incubated at 37 °C for 30 min. Samples at the end of staining were washed twice using buffer. The final assay was performed using flow cytometry.

[1]. Design, synthesis and evaluation of EZH2-based PROTACs targeting PRC2 complex in lymphoma. Bioorg Chem. 2023 Nov;140:106762.

Our study reported the first MDM2 ligand-based PROTAC, which has potent antiproliferative activities. The length of linkers in this series of PROTACs affected the activities of these compounds. E-3P/4P-MDM2 markedly degraded the PRC2 complex (EZH2, EED, and SUZ12) without affecting their mRNA levels. Its degradation of the PRC2 complex is time-dose dependent. Compared with EPZ6438, which inhibits EZH2, E-3P/4P-MDM2 was more potent in blocking tumor development. Our data suggested that E-3P/4P-MDM2 promoted the degradation of the PRC2 complex through the proteasome pathway. It was implied that the compounds recruited the E3 ubiquitin ligase to the vicinity of the PRC2 complex, leading to ubiquitination and final degradation of all PRC2 subunits. MDM2 ligands have been increasingly used in PROTAC and MDM2 ligand-based PROTACs were also found to be able to stabilize p53. In this study, we found that the optimal PROTACs were based on MDM2 ligands. At the same time, we also found some degradation activities of cIAP ligand-based PROTACs, such as E-4W-3P-B5T. There are also no previous studies using Bestatin as an E3 ligand to degrade the PRC2 complex, and the cell growth inhibitory activities of this series of compounds were superior to that of EPZ6438. Therefore, cIAP ligand-based PROTACs still have great potential on the target of EZH2. [1]

C77H93CL2N13O12

1463.55

1461.6443

169450483

White to off-white solid powder

7.6

3

17

30

104

2910

2

CCN(C1CCOCC1)C2=CC(=CC(=C2C)C(=O)NCC3=C(C=C(NC3=O)C)C)C4=CC=C(C=C4)CN5CCN(CC5)C(=O)C6=CN(N=N6)CCOCCOCCOCCNC(=O)CN7CCN(CC7=O)C(=O)N8[C@H]([C@H](N=C8C9=C(C=C(C=C9)OC)OC(C)C)C1=CC=C(C=C1)Cl)C1=CC=C(C=C1)Cl

RRDJNHBEROQEFR-KPBBBUKSSA-N

InChI=1S/C77H93Cl2N13O12/c1-8-91(61-23-33-100-34-24-61)67-43-58(42-64(53(67)6)74(95)81-45-65-51(4)41-52(5)82-75(65)96)55-11-9-54(10-12-55)46-86-26-28-87(29-27-86)76(97)66-47-90(85-84-66)32-36-102-38-40-103-39-37-101-35-25-80-69(93)48-88-30-31-89(49-70(88)94)77(98)92-72(57-15-19-60(79)20-16-57)71(56-13-17-59(78)18-14-56)83-73(92)63-22-21-62(99-7)44-68(63)104-50(2)3/h9-22,41-44,47,50,61,71-72H,8,23-40,45-46,48-49H2,1-7H3,(H,80,93)(H,81,95)(H,82,96)/t71-,72+/m1/s1

5-[4-[[4-[1-[2-[2-[2-[2-[[2-[4-[(4R,5S)-4,5-bis(4-chlorophenyl)-2-(4-methoxy-2-propan-2-yloxyphenyl)-4,5-dihydroimidazole-1-carbonyl]-2-oxopiperazin-1-yl]acetyl]amino]ethoxy]ethoxy]ethoxy]ethyl]triazole-4-carbonyl]piperazin-1-yl]methyl]phenyl]-N-[(4,6-dimethyl-2-oxo-1H-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methylbenzamide

PROTAC EZH2 Degrader-2; PROTAC EZH2 Degrader 2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)