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.

Selecting the appropriate E3 ligase is important to achieve effective targeted protein degradation by PROTACs. Although the human genome encodes more than 600 E3 ligases, only a limited number of E3 ligases, such as von Hippel–Lindau, cereblon, inhibitor of apoptosis proteins, and mouse double minute protein 2 ligases, are mainly used in development of PROTACs. Expanding the repertoire of E3 ligases that can be used with PROTACs is critical to developing this technology. Various groups have been searching for new E3 ligands, with aryl hydrocarbon receptor reported as a new E3 ligase for PROTACs in 2019, FEM1B in 2022, DCAF1 in 2022, and KLHDC2 in 2024. [1]
Degrons, which are unique amino acid sequences recognized by E3 ligase, have been found in a variety of organisms, and searches for degrons in budding yeast and mammals are ongoing. The amino acid structure of the degron is used to develop the E3 ligands in PROTACs. For example, N-degron structures are recognized mainly by a subfamily of ubiquitin protein ligase E3 component N-recognin (UBR) proteins (UBR1, UBR2, UBR4, and UBR5), leading to the degradation of their substrate proteins. N-degron-based PROTACs have been developed, which use UBR proteins as E3 ligase to degrade target proteins. Small-molecule E3 ligands are required for developing drug-like PROTACs. Two examples of PROTACs targeting BCR-ABL and estrogen receptor α (ERα) employ a small-molecule E3 ligand consisting of a single arginine residue from the N-degron that is recognized by UBR proteins. Other potential small-molecule ligands have been suggested, including a heterovalent ligand of amino acid mimetics targeting UBR1 and a picolinic acid ligand potentially binding to UBR4. In silico modeling has shown that clozapine, an FDA-approved small-molecule antipsychotic, should bind to UBR proteins and inhibit their physiological functions, suggesting that clozapine could be used as a UBR E3 ligand. [1]
In this study, we investigated the potential of clozapine as a novel E3 ligand for PROTACs. First, we designed and synthesized a clozapine-based PROTAC targeting the HaloTag proteins as a proof-of-concept model and validated its efficacy. We also designed and synthesized another clozapine PROTAC targeting ERα, a therapeutic target for breast cancer, and evaluated its ERα degradation activity and analyzed the degradation mechanism. [1]
Initially, we synthesized a clozapine-based PROTAC, Chlorohexane-PEG-Clozapine (5), which targets the HaloTag protein fused with emerald luciferase (Halo-ELuc). ELuc is a luciferase from the Brazilian click beetle, an enzyme that catalyzes the oxidation of D-luciferin to produce the electronically excited state, oxyluciferin, resulting in bioluminescence. The PROTAC consisted of chlorohexane, which covalently bound to the HaloTag protein, a PEG2 linker, and the E3 ligand, clozapine (Figure 2A). Compound 5 was used to treat HEK293 cells stably expressing Halo-ELuc for 24 h. A luciferase assay showed a concentration-dependent decrease in Halo-ELuc luminescence with compound 5 treatment (Figure 2A). The protein levels of Halo-ELuc evaluated by Western blotting showed a concentration-dependent decrease, consistent with the changes in the luminescence (Figure 2B). [1]

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New ubiquitin ligase (E3) ligands are crucial for developing proteolysis-targeting chimeras (PROTACs) to induce the degradation of a target protein. In this study, we developed a PROTAC using the antipsychotic drug clozapine as a new E3 ligand. First, a clozapine PROTAC targeting a model target HaloTag protein (Halo-PEG-Clozapine) was synthesized, and the PROTAC induced degradation of the HaloTag-fused protein in a cell culture system. Another clozapine PROTAC targeting the cancer therapeutic target estrogen receptor α (ERα) (Tamoxifen-PEG-Clozapine) was synthesized and induced degradation of the ERα protein in MCF-7 breast cancer cells. Experiments with inhibitors and siRNAs showed that Tamoxifen-PEG-Clozapine degraded ERα via a ubiquitin-proteasome system that uses the ubiquitin protein ligase E3 component N-recognin 5. These results indicate that clozapine is a promising E3 ligand that may expand the molecular design of PROTACs, contributing to the advancement of drug discovery by facilitating 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.)