PROTAC targeting SETDB1, and preparation method and application thereof

Abstract

The invention provides SETDB1-targeting PROTAC as well as a preparation method and application thereof, and relates to the technical field of clinical medicine. The PROTAC comprises a nucleic acid aptamer capable of being specifically combined with SETDB1 protein, a connexon and a ligand for recruiting E3 ubiquitin ligase, wherein the ligand for recruiting the E3 ubiquitin ligase is thalidomide. The PROTAC can recruit the E3 ligase CRBN in cancer cells onto the SETDB1, and degrade the SETDB1 in a proteasome dependent manner. Therefore, the PROTAC can inhibit proliferation and migration of cancer cells; the killing effect of CD8 + T cells on cancer cells is enhanced, and the growth of the cancer cells is inhibited. Tests prove that the potential application of the PROTAC based on the aptamer in SETDB1 degradation in tumor cells provides a promising strategy for cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of clinical medical technology, and in particular to a PROTAC targeting SETDB1, its preparation method and application. Background Technology

[0002] Targeted protein degradation (TPD) is an innovative and rapidly developing drug design strategy that has become a key approach for the treatment of cancer and other diseases. Among TPD technologies, protein hydrolysis-targeting chimeras (PROTACs) are particularly promising. PROTACs utilize the cellular ubiquitin-proteasome system (UPS) to selectively degrade target proteins, providing a method for targeting “undruggable” proteins. Structurally, PROTACs are heterofunctional molecules comprising three key components: a ligand (POI) of the target protein, a linker, and a ligand that recruits E3 ubiquitin ligases. The most commonly used E3 ligases in PROTAC development include VHL, MDM2, IAPs, and CRBN. Traditional small molecule inhibitors inhibit protein function by occupying active sites, requiring high affinity and sustained exposure to high concentrations. This strategy often leads to off-target effects and drug resistance. In contrast, PROTACs promote the selective degradation of POIs by recruiting E3 ubiquitin ligases, rather than simply inhibiting active sites. This method enhances target selectivity, overcomes drug resistance mechanisms, and achieves efficient protein degradation at catalytic levels. PROTAC technology has been successfully applied to the degradation of more than 100 proteins, and several PROTACs are currently undergoing clinical trials.

[0003] Although two small molecule ligands, (R,R)-59 and UNC10013, targeting the SETDB1 tandem Tudor domains (TTDs) have been developed, their effects on SETDB1 methyltransferase activity differ. Specifically, (R,R)-59 acts as a competitive ligand against endogenous binding partners, stabilizing exogenous TTD expression and thus serving as a positive regulator of SETDB1 methyltransferase activity. In contrast, UNC10013 acts as a negative allosteric regulator, inhibiting SETDB1 methyltransferase activity in cells through an allosteric mechanism. While both ligands demonstrate the pharmacological feasibility of targeting SETDB1, their effects are primarily limited to regulating its methyltransferase activity. Therefore, they cannot inhibit the biological functions of other non-catalytic domains in SETDB1. Furthermore, traditional small molecule inhibitors mainly rely on strong pocket binding and enzyme inhibition, which may lead to drug resistance during long-term treatment.

[0004] Therefore, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a PROTAC targeting SETDB1, its preparation method, and its applications. By linking a DNA aptamer to a small molecule ligand through a copper ion-catalyzed azido-yne cycloaddition reaction, a DNA-small molecule chimeric PROTAC is constructed. This effectively degrades SETDB1 protein levels in cancer cells, providing a new approach for cancer treatment.

[0006] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides a PROTAC targeting SETDB1, the PROTAC comprising: a nucleic acid aptamer capable of specifically binding to the SETDB1 protein, a linker, and a ligand recruiting E3 ubiquitin ligase; The ligand for recruiting E3 ubiquitin ligase is thalidomide. The structural formula of thalidomide is as follows: .

[0007] Furthermore, the nucleotide sequence of the nucleic acid aptamer of the SETDB1 protein is shown in SEQ ID NO.1.

[0008] SEQ ID NO.1 GTTTTGCCGCATAGGATTTTTGGTTGGTCTGGTTGG.

[0009] Furthermore, the structural formula of the connector is as follows: ; in, This represents n deoxythymidine nucleotides, where n is an integer between 0 and 5.

[0010] Furthermore, n=4.

[0011] The present invention also provides a pharmaceutical composition comprising the aforementioned PROTAC targeting SETDB1 and a pharmaceutically acceptable carrier or excipient.

[0012] The present invention also provides the use of the above-described PROTAC targeting SETDB1 or the above-described pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of tumors.

[0013] Furthermore, the tumor is a tumor that highly expresses SETDB1.

[0014] Furthermore, the tumor is a solid tumor or a hematologic malignancy; Furthermore, the tumor is a solid tumor; Furthermore, the tumor is breast cancer.

[0015] The present invention also provides the use of the above-described PROTAC targeting SETDB1 or the above-described pharmaceutical composition in the preparation of a medicament for degrading SETDB1 protein.

[0016] This invention also provides a method for preparing PROTAC targeting SETDB1, comprising the following steps: S1. Provide nucleic acid aptamers with an azide group modified at the 5' end; S2 provides ligands with alkyne modification that can recruit E3 ubiquitin ligases; S3. Dissolve the nucleic acid aptamer, and then mix the nucleic acid aptamer with the ligand of the E3 ubiquitin ligase in a buffer system in a certain proportion to carry out the reaction.

[0017] Furthermore, the nucleic acid aptamer and the ligand of the E3 ubiquitin ligase are mixed at a molar ratio of 1:(40-60).

[0018] Furthermore, the nucleic acid aptamer and the ligand of the E3 ubiquitin ligase are mixed at a molar ratio of 1:50.

[0019] Furthermore, the buffer system comprises: 10 mM Tris-HCl, 10 mM CuSO4 and 10 mM sodium ascorbate.

[0020] Furthermore, the reaction conditions were a click chemical reaction carried out in a metal bath at 37 °C for 4-6 h.

[0021] The present invention has the following technical effects: The above technical solution uses an aptamer with high affinity for SETDB1, which is coupled to a small molecule ligand of CRBN via a click reaction to generate a PROTAC targeting SETDB1 with good serum stability. This PROTAC can recruit the E3 ligase CRBN to SETDB1 in cancer cells and degrade SETDB1 in a proteasome-dependent manner. Therefore, this PROTAC can inhibit the proliferation and migration of cancer cells and enhance CD8+. + T cells kill cancer cells and inhibit their growth. Experiments have demonstrated the potential use of this aptamer-based PROTAC in degrading SETDB1 in tumor cells, providing a promising strategy for cancer treatment. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the PROTAC compositing process drawn using Adobe Illustrator; Figure 2 This refers to the N3-(T)4-5´-C11-3' mass spectrometry results in Example 1 of this invention; Figure 3 These are electrophoresis results of different PROTAC connectors successfully constructed in Embodiment 1 of the present invention; Figure 4 This is a diagram showing the degradation effect of different PROTAC connectors on SETDB1 in Embodiment 2 of the present invention; Figure 5 This is a graph showing the results of ITC detection of the affinity between P-SETDB1-4 and SETDB1 in Embodiment 3 of the present invention; Figure 6 This is a diagram showing the results of pull-down detection of P-SETDB1-4 combined with SETDB1 and CRBN in Embodiment 3 of the present invention; Figure 7 This is a graph showing the results of the P-SETDB1-4 serum stability test in Example 4 of this invention; Figure 8 This is a graph showing the dose-dependent degradation results of P-SETDB1-4 in Example 5 of the present invention; Figure 9 This is a graph showing the time-dependent degradation results of P-SETDB1-4 in Example 5 of the present invention; Figure 10 This is a graph showing the results of the P-SETDB1-4 active ingredient test in Example 6 of the present invention; Figure 11 This is a graph showing the effect of P-SETDB1-4 on SETDB1 transcription levels in Example 7 of the present invention; Figure 12 This is a graph showing the CRBN-dependent degradation results of SETDB1 by P-SETDB1-4 in Example 8 of the present invention; Figure 13 This is a diagram showing the results of MG132 blocking P-SETDB1-4-mediated SETDB1 degradation in Example 9 of the present invention; Figure 14This is a diagram showing the results of P-SETDB1-4's ​​degradation of SETDB1 via the proteasome pathway in Example 9 of this invention; Figure 15 This is a graph showing the results of the MTT assay performed on breast cancer cells treated with P-SETDB1-4 in Example 10 of this invention. Figure 16 This is a diagram showing the results of the EdU experiment performed on breast cancer cells treated with P-SETDB1-4 in Example 10 of this invention. Figure 17 This is a diagram showing the results of a colony formation experiment using P-SETDB1-4 treated with breast cancer cells in Example 10 of this invention. Figure 18 This is a diagram showing the results of a scratch assay performed on breast cancer cells treated with P-SETDB1-4 in Example 11 of this invention. Figure 19 This is a diagram showing the results of transwell experiments performed on breast cancer cells treated with P-SETDB1-4 in Example 11 of this invention. Figure 20 This is a diagram showing the results of the T-cell killing experiment performed on breast cancer cells treated with P-SETDB1-4 in Example 12 of this invention. Figure 21 This is a schematic diagram of the design of the P-SETDB1-4 in vivo breast cancer growth experiment in mice in Example 13 of the present invention; Figure 22 This is a diagram showing the results of P-SETDB1-4 on the growth of breast cancer tumors in mice in Example 13 of this invention; Figure 23 This is a diagram showing the results of P-SETDB1-4 immunofluorescence staining of SETDB1 breast cancer tumors in mice in Example 13 of this invention; Figure 24 This is a diagram showing the results of P-SETDB1-4 histochemical staining of Ki67 breast cancer tumors in mice in Example 13 of this invention; Figure 25 This is a diagram showing the results of HE staining of mouse body weight changes and other tissues and organs after breast cancer tumor bearing by P-SETDB1-4 in Example 13 of this invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] Example 1: Synthesis of PROTAC To meet the conditions for the click reaction, an alkynyl click site was introduced into the thalidomide molecular skeleton to form thalidomide-o-aminopropyne. Thalidomide-o-aminopropyne is a reactant of thalidomide and was purchased from MedChemExpress, catalog number: CAS No.: 2738967-56-9.

[0026] The DNA-small molecule chimera PROTAC was designed and synthesized. A schematic diagram of the PROTAC synthesis is attached. Figure 1 The 5' azide-modified DNA aptamer was synthesized by Sangon Biotech (Shanghai, China), specifically: N3-(T)n-5´-GTTTTGCCGCATAGGATTTTTGGTTGGTCTGGTTGG-3´(C11-(T)n-N3), where (T)n is the linker molecule, and n = 0-5. The structural formula of C11-(T)n-N3 is shown below: Appendix Figure 2 The mass spectrometry results (n=4) of the synthesized modified DNA aptamer are shown. The ligation reaction of the synthetic strand PROTAC (P-SETDB1-4) (n=4) was an azide-alkyne cycloaddition reaction. Specifically, the modified DNA aptamer was dissolved in ddH2O to a final concentration of 500 µM, followed by a click chemistry reaction in a reaction buffer (10 mM Tris-HCl, 10 mM CuSO4, and 10 mM sodium ascorbate) at a DNA:thalidomide-o-aminopropyne (CRBNL) molar ratio of 1:50 in a 37 °C metal bath for 4–6 h. After the reaction, the final synthesized product was purified using 3 kd ultrafiltration centrifuge tubes (Millipore, Burlington, MA, USA). Finally, the successful coupling was confirmed by non-denaturing polyacrylamide gel electrophoresis, and the results were observed under UV light using GelRed staining. The results are shown in the attached figure. Figure 3 The molecular weight increases after CRBNL incorporation, as shown.

[0027] Example 2: Degradation performance of different PROTAC linkers synthesized in this invention on SETDB1 protein Different synthesized PROTAC linkers were transfected into human breast tumor cells (MCF-7) via PEI at a concentration of 1 µM for 24 h. Cell lysis was then performed, and changes in SETDB1 protein levels were detected by Western blot. Figure 4The degradation results of SETDB1 in MCF-7 cells by different linkers PROTAC are shown. PROTAC showed the most effective degradation effect on SETDB1 when n=4, and was named P-SETDB1-4.

[0028] Example 3: Test of the compatibility between P-SETDB1-4 and SETDB1 in Example P of this invention The affinity of p-SETDB1-4 for SETDB1 was determined by ITC. ITC experiments were performed at 25°C using a MicroCal PEAQ-ITC system. SETDB1 protein (190–410 aa) was prepared in DPBS buffer (5 mM MgCl2, pH 7.5), and the final protein concentration was adjusted to 10 µM, while the DNA aptamer concentration was 100 µM. Each titration volume was 3 µL, with the initial titration volume being 0.4 µL. The titration interval was 120 s, and the reference power was set to 5 µcal / sec. The binding molar ratio (N), dissociation constant (Kd), and thermodynamic parameters—enthalpy change (ΔH), entropy change (ΔS), and Gibbs free energy change (ΔG)—were calculated using the unit point binding model in MicroCal PEAQ-ITC analysis software v1.30. Results are attached. Figure 5 As shown, P-SETDB1-4 has a good affinity for SETDB1.

[0029] The binding of p-SETDB1-4 to SETDB1 and CRBN was determined using a streptavidin pull-down assay. The experiment was conducted using biotinylated p-SETDB1-4, and the results are shown in the attached figure. Figure 6 As shown, P-SETDB1-4 effectively binds endogenous full-length SETDB1 and CRBN in cells, confirming that CRBNL coupling does not affect the binding ability of the nucleic acid aptamer (C11, hereinafter referred to as C11) to SETDB1. Furthermore, this binding can be competitively inhibited by excess unlabeled P-SETDB1-4, demonstrating the specificity of the binding.

[0030] Example 4: Serum stability test of P-SETDB1-4 in Example P of this invention P-SETDB1-4 was incubated at 37°C for specified time points in complete culture medium containing 10% fetal bovine serum. DNA samples were analyzed by non-denaturing polyacrylamide gel electrophoresis, stained with GelRed, and observed under UV light. The results are shown in the attached figure. Figure 7 As shown, P-SETDB1-4 exhibits good serum stability.

[0031] Example 5: Degradation performance test of P-SETDB1-4 on SETDB1 protein in this invention. P-SETDB1-4 was transfected into human breast tumor cells MCF-7 and T47D at final concentrations of 0 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1000 nM. Cells were lysed after 24 hours, and changes in SETDB1 protein levels were detected by Western blot. Figure 8 The degradation results of SETDB1 by P-SETDB1-4 at different concentrations are shown.

[0032] P-SETDB1-4 was transfected into human breast tumor cells MCF-7 and T47D at a final concentration of 1000 nM. Cells were lysed at treatment times of 0 h, 2 h, 6 h, 12 h, 18 h, and 24 h, and changes in SETDB1 protein levels were detected by Western blot. (See attached image) Figure 9 The degradation results of SETDB1 by P-SETDB1-4 at different time points are shown.

[0033] Example 6: Test of the effective components for P-SETDB1-4-induced SETDB1 protein degradation in this invention. Human breast tumor cells MCF-7 were treated with PBS, SETDB1-4T (SETDB1 aptamer only), the small molecule ligand CRBNL, and P-SETDB1-4. Results are attached. Figure 10 As shown, only P-SETDB1-4 has the ability to induce the degradation of SETDB1, while neither the SETDB1 aptamer nor the small molecule ligand CRBNL can degrade SETDB1.

[0034] Example 7: Transcriptional level test of SETDB1 protein by P-SETDB1-4 in Example 7 of this invention Control PROTAC or P-SETDB1-4 was transfected into human breast tumor cells MCF-7 and T47D at a final concentration of 1000 nM for 24 h, followed by real-time quantitative RT-PCR experiments. Results are attached. Figure 11 As shown, P-SETDB1-4 transfection into cells does not affect the transcriptional level of SETDB1.

[0035] Example 8: Pathway-dependent test of P-SETDB1-4-induced SETDB1 protein degradation in this invention. In human breast tumor cells MCF-7, siRNA was transfected to induce CRBN knockdown, followed by transfection with p-SETDB1-4 at a final concentration of 1000 nM for 24 h. Changes in SETDB1 protein levels after CRBN inhibition were detected by Western blot. Alternatively, p-SETDB1-4 was transfected into MCF-7 cells at a final concentration of 1000 nM for 24 h, and an excess of CRBNL was added for competitive inhibition. The effects of the excess CRBNL on SETDB1 protein levels were detected by Western blot. Results are attached. Figure 12 As shown, both inhibitions weakened the degradation of SETDB1, indicating that the degradation of SETDB1 by P-SETDB1-4 is dependent on CRBN.

[0036] Example 9: Test of the mechanism of action of P-SETDB1-4 in inducing SETDB1 protein degradation in this invention P-SETDB1-4 was transfected into human breast tumor cells MCF-7 at a final concentration of 1000 nM for 24 h. Simultaneously, the cells were treated with either the proteasome inhibitor MG132 (10 µM) or the autophagy inhibitor CQ (10 µM) for 24 h. Changes in SETDB1 protein levels were detected by Western blot. Results are attached. Figure 13 As shown, MG132, rather than CQ, completely blocks p-SETDB1-4-mediated SETDB1 degradation.

[0037] Consistent with these results, P-SETDB1-4 was transfected into human breast tumor cells MCF-7 at a final concentration of 1000 nM for 24 h, and simultaneously treated with the proteasome inhibitor MG132 (10 µM) for 24 h. Changes in SETDB1 ubiquitination levels were detected by co-immunoprecipitation assay. Results are attached. Figure 14 As shown, in the presence of MG132, P-SETDB1-4 enhanced SETDB1 ubiquitination in MCF-7 cells. These results collectively indicate that P-SETDB1-4 degrades SETDB1 via the proteasome pathway.

[0038] Example 10: Test of breast cancer cell proliferation using Example P-SETDB1-4 in this invention. Human breast tumor cells MCF-7 and T47D were treated with PBS, Control (CRBNL coupled with random nucleic acid sequences), or P-SETDB1-4. Cells (1000 cells per well) were seeded into 96-well plates. After cell adhesion, assays were performed at fixed time points. The culture medium was replaced with 10% MTT solution (0.5 mg / mL MTT in serum-free DMEM), and the cells were incubated at 37°C for 4 hours. Subsequently, the MTT solution was removed, and 110 μL of dimethyl sulfoxide (DMSO) was added to dissolve crystals. The absorbance was measured at 490 nM using a microplate reader. The results are shown in the attached figure. Figure 15 As shown, P-SETDB1-4 inhibits the growth of breast cancer cells.

[0039] Human breast tumor cells MCF-7 and T47D were treated with PBS, Control (CRBNL coupled with random nucleic acid sequences), or P-SETDB1-4. Cells were seeded at a density of 3000 cells per well in 96-well plates and incubated for 24 hours. The medium was then replaced with EdU-containing medium, and incubation continued for 2 hours. Cell proliferation was then assessed using the BeyoClick™ EdU Cell Proliferation Kit (C0071S, Beyotime, Shanghai, China) via Alexa Fluor 488. Cells were first fixed with 4% paraformaldehyde for 15 minutes, then washed three times with wash buffer (3% BSA in PBS), and permeabilized with 0.3% Triton X-100 in PBS for 10 minutes. 100 μL of click reaction solution was then added, and cells were incubated at room temperature for 30 minutes. After three additional washes, cells were stained with Hoechst 33342 in the dark for 10 minutes. Random fluorescence images were acquired using a fluorescence microscope to ensure objective data collection. The percentage of EdU-positive cells was calculated, and the results are shown in the attached figure. Figure 16 As shown, P-SETDB1-4 inhibits DNA replication in breast cancer cells.

[0040] Human breast tumor cells MCF-7 and T47D were treated with PBS, Control (CRBNL coupled with random nucleic acid sequences), or P-SETDB1-4. Cells (500 cells per well) were seeded into each well of a 6-well plate, and the culture medium was refreshed every 3 days. After 2 weeks, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, and stained with 0.1% crystal violet solution (Solarbio) for 15 minutes. Images were then taken, and the results are attached. Figure 17 As shown, P-SETDB1-4 inhibits the clonogenic ability of breast cancer cells.

[0041] Example 11: Test of breast cancer cell migration and invasion using Example P-SETDB1-4 in this invention. Human breast tumor cells MCF-7 and T47D were treated with PBS, Control (CRBNL coupled with random nucleic acid sequences), or P-SETDB1-4. Cells were seeded into 6-well plates and cultured to 100% confluence. A wound was made in the cell monolayer using a sterile pipette tip. After three washes with PBS, the cells were placed in serum-free medium to inhibit proliferation. Images were taken using an optical microscope at specified time points, and the wound width was measured using ImageJ software. The results are attached. Figure 18 As shown, P-SETDB1-4 inhibits the migration of breast cancer cells.

[0042] Human breast tumor cells MCF-7 and T47D were treated with PBS, Control (CRBNL coupled with random nucleic acid sequences), or P-SETDB1-4, with approximately 5 × 10⁶ cells per cell. 4 Cells were suspended in 500 μL of serum-free medium and seeded into upper culture dishes pre-coated with 100 μL of Matrigel solution (1 mg / mL). 600 μL of medium containing 20% ​​fetal bovine serum (FBS) was added to the lower culture dish. After 24 hours of incubation, cells that had invaded the subcellular surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Images were taken under a microscope, and the number of invading cells was counted. The results are shown in the attached figure. Figure 19 As shown, P-SETDB1-4 inhibits the invasion of breast cancer cells.

[0043] Example 12: In this invention, after treating breast cancer cells with P-SETDB1-4, human CD8... + Test of the effect of T cells on its killing effect PBMCs were isolated from human peripheral blood using density gradient centrifugation with lymphocyte separation medium. Simply put, blood samples were collected using EDTA-K2 as an anticoagulant. Lymphocyte separation medium was pre-filled into sterile centrifuge tubes, and an equal volume of fresh blood was slowly added. The tubes were centrifuged at 600 g for 30 minutes at room temperature, and the opaque white layer containing lymphocytes was carefully aspirated and transferred to a new sterile centrifuge tube. The tubes were diluted with three times the volume of PBS and centrifuged at 300 g for 10 minutes. The cells were washed twice with PBS, and the final pellet was designated as PBMCs. Cells were counted, and a suspension at a concentration of 1 × 10⁶ cells / mL was prepared.

[0044] Using ImunoSep™ human CD8 + T-cell positive selection kit to isolate CD8+ cells from peripheral blood mononuclear cells (PBMCs) +T cells. Based on the volume of the prepared single-cell suspension, 10 μL of sorting reagent A was added to 100 μL of cell suspension, mixed thoroughly, and incubated at room temperature for 10 minutes. The cells were then centrifuged and resuspended to the original volume. Then, 10 μL of sorting reagent B was added, and the cells were mixed and incubated under the same conditions. After culture, CD8+ cells were magnetically separated. + T cells were resuspended in RPMI 1640 medium and cultured with 2 μg / mL CD3 and CD28 for 24 hours. Then, IL2 (10 ng / mL) was added, and the cells were cultured for another 48 hours to complete in vitro activation. Activated CD8... + The T cells were subsequently used in T cell killing experiments.

[0045] Human breast tumor cells MCF-7 and T47D were treated with PBS, Control (CRBNL conjugated with random nucleic acid sequences), or P-SETDB1-4 to activate CD8+. + T cells (1×10) 4 Cells / well were seeded at an effector-target ratio of 10:1 into 96-well plates and co-cultured with pretreated tumor cells for 48 hours. After incubation, the release of lactate dehydrogenase (LDH) in the supernatant was measured using a one-step assay kit (MA0649, Meilun, China) according to the manufacturer's instructions. The results are attached. Figure 20 .

[0046] Example 13: Growth test of breast cancer in mice using Example P-SETDB1-4 of the present invention. As attached Figure 21 As shown, T47D cells were subcutaneously injected into the right back of nude mice (BALB / c), and when the tumor volume reached approximately 350 mm², 3 Mice were randomly divided into two groups and injected subcutaneously with either Control (CRBNL conjugated with a random nucleic acid sequence) or P-SETDB1-4 at specified time points at a dose of 5 mg / kg. All animals were sacrificed on day 18, and tumors were dissected and photographed. Results are attached. Figure 22 As shown, compared with the control group mice, P-SETDB1-4 significantly reduced tumor growth; as shown in the attached figure. Figure 23 As shown, immunofluorescence staining for SETDB1 revealed that P-SETDB1-4 effectively degraded SETDB1 in breast cancer tissue; as shown in the attached figure. Figure 24 As shown, Ki67 histochemical staining revealed decreased expression levels in the P-SETDB1-4 treatment group; see attached... Figure 25As shown, these mice did not exhibit significant weight loss during administration, and histological examination of liver, spleen, lung, kidney, and heart tissues with HE staining revealed no significant toxicity. These results indicate that P-SETDB1-4 can inhibit the growth of mammary cancer in mice, demonstrating its significant therapeutic potential.

[0047] The above-described technical solution utilizes an aptamer with high affinity for SETDB1, which is coupled to a small molecule ligand of CRBN via a click reaction to generate a PROTAC (P-SETDB1-4) targeting SETDB1, exhibiting good serum stability. P-SETDB1-4 can recruit the E3 ligase CRBN to SETDB1 in breast cancer cells and degrade SETDB1 in a proteasome-dependent manner. Therefore, P-SETDB1-4 inhibits the proliferation and migration of breast cancer cells. P-SETDB1-4 enhances CD8+. + The killing effect of T cells on breast cancer cells inhibited the growth of breast cancer in mice. Experiments have demonstrated the potential use of this aptamer-based PROTAC in degrading SETDB1 in tumor cells, providing a promising strategy for breast cancer treatment.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A PROTAC targeting SETDB1, characterized in that, The PROTAC includes: a nucleic acid aptamer capable of specifically binding to the SETDB1 protein, a linker, and a ligand that recruits E3 ubiquitin ligase; The ligand for recruiting E3 ubiquitin ligase is thalidomide. The structural formula of thalidomide is as follows: .

2. The PROTAC targeting SETDB1 according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid aptamer of the SETDB1 protein is shown in SEQ ID NO.

1.

3. The PROTAC targeting SETDB1 according to claim 1, characterized in that, The structural formula of the connector is: ; in, This represents n deoxythymidine nucleotides, where n is an integer between 0 and 5.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a PROTAC targeting SETDB1 as described in any one of claims 1-3, and a pharmaceutically acceptable carrier or excipient.

5. Use of a PROTAC targeting SETDB1 as described in any one of claims 1-3 and a pharmaceutical composition as described in claim 4 in the preparation of a medicament for the prevention and / or treatment of tumors.

6. The use according to claim 5, characterized in that, The tumor was a tumor that highly expressed SETDB1.

7. Use of a PROTAC targeting SETDB1 as described in any one of claims 1-3 and a pharmaceutical composition as described in claim 4 in the preparation of a medicament for degrading the SETDB1 protein.

8. A method for preparing a PROTAC targeting SETDB1 as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Provide nucleic acid aptamers with an azide group modified at the 5' end; S2 provides ligands with alkyne modification that can recruit E3 ubiquitin ligases; S3. Dissolve the nucleic acid aptamer, and then mix the nucleic acid aptamer with the ligand of the E3 ubiquitin ligase in a buffer system in a certain proportion to carry out the reaction.

9. The method for preparing PROTAC targeting SETDB1 according to claim 8, characterized in that, The nucleic acid aptamer and the ligand of the E3 ubiquitin ligase are mixed at a molar ratio of 1:(40-60).

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