ERα/estrogen receptor α

We designed another clozapine-based PROTAC targeting the nuclear receptor protein ERα as an endogenous therapeutic target. We used 4-OHT as the ERα ligand, PEG3 as the linker, and clozapine as the E3 ligand to synthesize the PROTAC Tamoxifen-PEG-Clozapine (10) (Figure 4A). The synthesized compound 10/Tamoxifen-PEG-Clozapine exhibited ERα binding activity comparable to that of the inhibitor 4-OHT (Figure S8). MCF-7 breast cancer cells were treated with 10 for 24 h, and the ERα protein levels were evaluated. The results demonstrated a significant decrease in ERα levels starting from 30 μM (Figure 4B). To verify that the reduction of ERα protein levels was due to the PROTAC, we conducted a cotreatment experiment with 4-OHT and clozapine. The results demonstrated that 10 significantly reduced ERα levels compared with treatments with 4-OHT alone, clozapine alone, or 4-OHT and clozapine together (Figure 4C). To analyze the mechanism of the PROTAC-induced reduction of ERα protein levels, we investigated the effects of UPS inhibitors. The compound 10-mediated reduction of ERα was abolished by cotreatment with the proteasome inhibitor MG132 or the ubiquitin-activating enzyme inhibitor MLN7243 (Figure 4D), suggesting that 10 induced UPS-mediated degradation of ERα protein. Similar to anti-estrogen drugs such as tamoxifen, which are known to increase ERα levels, (23,26,27) compound 10 incorporating 4-OHT as a ligand also seems to induce upregulation of ERα, particularly in low concentration ranges (0.03–10 μM) and when cotreated with MG132 (Figures 4B and 4D). [1]

Clozapine has been suggested to bind to the UBR-box of UBR family proteins involved in the N-degron pathway, but the specificity of its binding to the homologous UBR-box is unknown. Therefore, to identify the UBR proteins recruited by Tamoxifen-PEG-Clozapine/10, we investigated the effect of siRNA knockdown of each UBR protein on the compound 10-induced degradation of ERα. Knockdown of UBR1, UBR2, and UBR4 did not abolish the decrease in ERα induced by 10. In contrast, a slight but significant inhibition of ERα protein reduction was observed when UBR5 was knocked down (Figure 5). (ERα levels were increased from 16% to 45% at 30 μM and from 41% to 51% at 100 μM.) Considering that UBR5 siRNA does not completely suppress UBR5 expression and less than 50% of UBR5 remains, this result suggests that UBR5 partially but importantly contributed to the compound 10-mediated degradation of ERα. In the cell viability assay, compound 10 demonstrated concentration-dependent inhibitory effects on viability of ERα-positive breast cancer MCF-7 cells (Figure S9), which is consistent with the results of ERα degradation analyzed by Western blotting (Figure 4B).[1]

A previous report suggested that clozapine could be used as a new E3 ligand in PROTACs. Our research demonstrated that Tamoxifen-PEG-Clozapine (10) induces ERα degradation through the UPS pathway and UBR5 is involved in this degradation pathway. Clozapine expands the possible applications of PROTACs by increasing the repertoire of E3 ligands. Although further studies are needed to investigate the target specificity and binding stability of clozapine-based PROTAC, this study is the first to report a PROTAC design utilizing clozapine as an E3 ligand, potentially expanding the repertoire of E3 ligands available for PROTAC development and paving the way for new therapeutic strategies targeting disease-related proteins[1].

[1]. Clozapine as an E3 Ligand for PROTAC TechnologyJ. ACS Medicinal Chemistry Letters, 2025 Jan 8;16(2):258-262.

Choosing the right E3 ligase is crucial for PROTACs to achieve effective targeted protein degradation. Although the human genome encodes more than 600 E3 ligases, the development of PROTACs currently mainly uses a few E3 ligases, such as von Hippel-Lindau ligase, cereblon ligase, apoptosis inhibitor protein ligase and mouse two-microsome protein 2 ligase. Therefore, expanding the types of E3 ligases that can be used for PROTACs is crucial for the development of this technology. Several research teams have been searching for new E3 ligands, among which the aryl hydrocarbon receptor (ArHCR) was reported as a novel E3 ligase for PROTACs in 2019, and FEM1B and DCAF1 were reported in 2022 and 2024, respectively. [1] Degrons are unique amino acid sequences recognized by E3 ligases and have been found in a variety of organisms. Currently, research on degrons in budding yeast and mammals is still ongoing. The amino acid structure of degrons is used to develop E3 ligands in PROTACs. For example, N-degrader structures are primarily recognized by the N-recognition protein (UBR) subfamily (UBR1, UBR2, UBR4, and UBR5) of the ubiquitin protein ligase E3 component, leading to the degradation of their substrate proteins. PROTACs based on N-terminal degraders have been developed, utilizing UBR proteins as E3 ligases to degrade target proteins. Developing drug-active PROTACs requires small-molecule E3 ligands. Two examples of PROTACs targeting BCR-ABL and estrogen receptor α (ERα) employ a small-molecule E3 ligand consisting of a single arginine residue on the N-terminal degrader, which can be recognized by the UBR protein. Other potential small-molecule ligands have also been proposed, including amino acid mimicry heterovalent ligands targeting UBR1 and pyridinecarboxylic acid ligands that may bind to UBR4. Computer simulations show that the FDA-approved small-molecule antipsychotic clozapine should bind to the UBR protein and inhibit its physiological function, suggesting that clozapine could be used as a UBR E3 ligand. [1]
In this study, we explored the potential of clozapine as a novel E3 ligand for PROTAC. First, we designed and synthesized a clozapine-based PROTAC that targets the HaloTag protein as a proof-of-concept model and validated its effectiveness. We also designed and synthesized another clozapine-based PROTAC that targets ERα (a therapeutic target for breast cancer) and evaluated its ERα degradation activity and analyzed its degradation mechanism. [1]
Initially, we synthesized a clozapine-based PROTAC, namely chlorohexane-PEG-clozapine (5), which targets the HaloTag protein fused with Elerald luciferase (ELuc). ELuc is a luciferase from Brazil that catalyzes the oxidation of D-luciferin to generate electronically excited oxyluciferin, thereby producing bioluminescence. The PROTAC consists of chlorohexane, a PEG2 linker covalently bound to the HaloTag protein, and the E3 ligand clozapine (Figure 2A). Compound 5 was used to treat HEK293 cells stably expressing Halo-ELuc for 24 hours. Luciferase activity assays showed that the luminescence intensity of Halo-ELuc decreased in a concentration-dependent manner after treatment with compound 5 (Fig. 2A). Western blotting analysis showed that the Halo-ELuc protein level also decreased in a concentration-dependent manner, consistent with the change in luminescence intensity (Fig. 2B). [1]

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Novel ubiquitin ligands (E3) are crucial for developing proteolytic targeting chimeric (PROTACs) to induce target protein degradation. In this study, we developed a PROTAC using the antipsychotic drug clozapine as a novel E3 ligand. First, we synthesized a clozapine PROTAC targeting the model target protein HaloTag (Halo-PEG-clozapine), which induced the degradation of the HaloTag fusion protein in a cell culture system. Subsequently, we synthesized another clozapine PROTAC (tamoxifen-PEG-clozapine) targeting the cancer therapeutic target estrogen receptor α (ERα), which induced the degradation of ERα protein in MCF-7 breast cancer cells. Inhibitor and siRNA experiments showed that tamoxifen-PEG-clozapine degrades ERα via the ubiquitin-proteasome system, which utilizes the ubiquitin protein ligase E3 component N-recognition protein 5. These results suggest that clozapine is a promising E3 ligand that may expand the molecular design of PROTACs and contribute to drug development by promoting the degradation of disease-related proteins. [1]

C54H63CLN6O7

943.57

942.444676

172676875

Typically exists as solids at room temperature

7.8

3

10

24

68

1560

0

CC/C(=C(/C1=CC=C(C=C1)O)\C2=CC=C(C=C2)OCCN(C)C(=O)CCC(=O)NCCOCCOCCOCCN3CCN(CC3)C4=NC5=C(C=CC(=C5)Cl)NC6=CC=CC=C64)/C7=CC=CC=C7

CSMUZRSXZXLELT-YVUJEYQCSA-N

InChI=1S/C54H63ClN6O7/c1-3-46(40-9-5-4-6-10-40)53(41-13-18-44(62)19-14-41)42-15-20-45(21-16-42)68-34-30-59(2)52(64)24-23-51(63)56-25-32-65-35-37-67-38-36-66-33-31-60-26-28-61(29-27-60)54-47-11-7-8-12-48(47)57-49-22-17-43(55)39-50(49)58-54/h4-22,39,57,62H,3,23-38H2,1-2H3,(H,56,63)/b53-46+

N-[2-[2-[2-[2-[4-(3-chloro-11H-benzo[b][1,4]benzodiazepin-6-yl)piperazin-1-yl]ethoxy]ethoxy]ethoxy]ethyl]-N'-[2-[4-[(E)-1-(4-hydroxyphenyl)-2-phenylbut-1-enyl]phenoxy]ethyl]-N'-methylbutanediamide

Tamoxifen-PEG-Clozapine; compound 10

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.)