Setdb1 aptamer, protac constructed based on the aptamer and application

Abstract

The application provides a SETDB1 aptamer, a PROTAC constructed based on the aptamer and application, and relates to the technical field of biological medicines.The nucleotide sequence of the SETDB1 aptamer is any one of SEQ ID NO.1 or SEQ ID NO.2.Through SELEX screening, a DNA aptamer targeting SETDB1 is identified, and the DNA aptamer is specifically combined with a Tudor domain; based on the screened SETDB1 aptamer, a first PROTAC of SETDB1 is constructed.Single-chain PROTAC and double-chain PROTAC are synthesized, and the above-mentioned PROTACs are all targeted to degrade SETDB1 in tumors, and have no effect on non-tumor cells or tissues.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically to the SETDB1 aptamer, the PROTAC constructed based on the aptamer, and its applications. Background Technology

[0002] SETDB1 is a histone methyltransferase (HMT) primarily involved in H3K9 methylation for transcriptional silencing, and its expression is upregulated in various tumors, including brain cancer, melanoma, and breast cancer. As an oncogene, it promotes cancer development by transcriptionally inhibiting tumor suppressor factors such as P16, P21, and APOE. In addition, SETDB1 participates in chromatin remodeling, PML-NB-related functions, X chromosome inactivation, and immune regulation. SETDB1 interacts with PELP1, promoting resistance to tamoxifen endocrine therapy in breast cancer cells. Recent studies have shown that SETDB1 plays a crucial role in immune regulation, participating in B cell maturation, T cell activity regulation, endogenous retroviral (ERV) silencing leading to suppression of immune checkpoint blockade (ICB), and the expression of PD-L1 and MHC-I receptors. Overexpression of SETDB1 in tumor cells can disrupt the immune response by inhibiting tumor immunogenicity and promoting immune evasion, thus inhibiting the recruitment of immune cells to the tumor site. Given the crucial regulatory role of SETDB1 in tumorigenesis, progression, and tumor immune escape, targeting SETDB1 degradation is a promising strategy for targeted cancer therapy and immunotherapy. While numerous inhibitors exist for histone H3K9 methylation, research on small-molecule inhibitors specifically targeting SETDB1 is limited, and no such drugs are currently on the market. Therefore, developing drugs that target SETDB1 degradation is of great significance.

[0003] Aptamers are short, single-stranded RNA or DNA oligonucleotides capable of forming complex three-dimensional structures, including stem, loop, hairpin, and G-quadruplex structures. They are screened from random oligonucleotide libraries using the SELEX technique. Due to their high specificity and affinity for target molecules, aptamers are considered promising ligands and are widely used in biosensing, diagnostics, drug delivery systems, and therapeutic applications. Currently, no nucleic acid aptamers targeting SETDB1 have been reported, and PROTAC studies on SETDB1 have not yet been developed.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a SETDB1 aptamer, a PROTAC constructed based on the aptamer, and its applications. This invention identifies the first DNA aptamer targeting SETDB1 through screening, which specifically binds to its Tudor domain. Simultaneously, single-stranded or double-stranded PROTACs are synthesized based on this aptamer. All of the above PROTACs can target and degrade SETDB1 in tumor cells, while having no effect on non-tumor cells or tissues, thereby achieving targeted therapy for tumor cells.

[0006] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted:

[0007] The present invention provides a SETDB1 aptamer, wherein the nucleotide sequence of the SETDB1 aptamer is any one of SEQ ID NO.1 or SEQ ID NO.2.

[0008] SEQ ID NO.1

[0009] 5´-GTTTTGCCGCATAGGATTTTTGGTTGGTCTGGTTGG-3´

[0010] SEQ ID NO.2

[0011] 5´-TGCCGCATAGGATTTTTGGTTGGTCTGG-3´

[0012] The present invention also provides a single-stranded PROTAC constructed based on the above-mentioned SETDB1 aptamer, the single-stranded PROTAC comprising: a first DNA aptamer capable of specifically binding to the SETDB1 protein, and a linker capable of connecting the first DNA aptamer and the second DNA aptamer;

[0013] The second DNA aptamer is located at the 5' end and is connected to the first DNA aptamer located at the 3' end via the linker.

[0014] Preferably, the second DNA aptamer is AS1411.

[0015] Preferably, the linker is an oligonucleotide chain composed of 6, 10, or 15 deoxyadenosine nucleotides.

[0016] Preferably, the single-chain PROTAC is specifically 5´-GGTGGTGGTGGTTGTGGTGGTGGTGG-oligonucleotide chain-GTTTTGCCGCATAGGATTTTTGGTTGGTCTGGTTGG-3´.

[0017] The present invention also provides a double-chain PROTAC constructed based on the above-mentioned SETDB1 aptamer, the double-chain PROTAC comprising:

[0018] The first DNA aptamer and its first complementary sequence at the 3' end;

[0019] The second DNA aptamer capable of specifically binding to nucleolin and its second complementary sequence at the 3' end;

[0020] The first complementary sequence and the second complementary sequence form a connector;

[0021] Preferably, the first complementary sequence and the complementary sequence are rigid double-stranded structures;

[0022] Preferably, the sequence is 5'-AGCGCCGGCC-3' (SEQ ID NO.3), 5'-GGGATCCCGA-3' (SEQ ID NO.4), or 5'-CACGGGCAAC-3' (SEQ ID NO.5) and their reverse complementary sequences;

[0023] Preferably, the sequence is 5´-AGCGCCGGCC-3´ (SEQ ID NO.3);

[0024] Preferably, the second DNA aptamer is AS1411.

[0025] The present invention also provides a pharmaceutical composition comprising the above-described single-chain or double-chain PROTAC and a pharmaceutically acceptable carrier or excipient.

[0026] The present invention also provides the use of the above-mentioned single-chain PROTAC or double-chain PROTAC in the preparation of medicaments for the prevention and / or treatment of tumors;

[0027] Preferably, the tumor is a tumor that highly expresses SETDB1;

[0028] Preferably, the tumor is a solid tumor or a hematologic malignancy;

[0029] Preferably, the tumor is a solid tumor;

[0030] Preferably, the tumor is any one of breast cancer, lung cancer, liver cancer, or colorectal cancer;

[0031] Preferably, the tumor is breast cancer.

[0032] Preferably, the application of the above-mentioned single-chain PROTAC or double-chain PROTAC in inhibiting tumor cell proliferation.

[0033] Preferably, the application of the above-mentioned single-chain PROTAC or double-chain PROTAC in the degradation of SETDB1 protein.

[0034] Preferably, the application of the above-mentioned single-chain PROTAC or double-chain PROTAC in inhibiting the migration and invasion of tumor cells.

[0035] Preferably, the above-mentioned single-chain PROTAC or double-chain PROTAC has a positive effect on CD8. + Applications of T-cell killing.

[0036] Preferably, the drug is used to reverse or inhibit tumor resistance to tamoxifen;

[0037] Preferably, the drug is used in combination with tamoxifen.

[0038] The present invention also provides the use of the single-chain PROTAC or double-chain PROTAC or the pharmaceutical composition thereof in the preparation of a medicament for degrading SETDB1 protein.

[0039] The present invention also provides a method for preparing the single-chain PROTAC, comprising the following steps:

[0040] Based on the nucleotide sequence information in the single-chain PROTAC described above, the single-chain PROTAC was synthesized in one step using solid-phase synthesis technology.

[0041] Preferably, the solid-phase synthesis technique is the phosphoramidite method.

[0042] The present invention also provides a method for preparing the double-stranded PROTAC, comprising the following steps:

[0043] The first DNA aptamer and its first complementary sequence at the 3' end were synthesized separately.

[0044] The second DNA aptamer and its second complementary sequence at the 3' end were then synthesized;

[0045] The first and second strands are denatured and then renatured, allowing them to hybridize through complementary sequences to form the double-stranded PROTAC.

[0046] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0047] The above technical solution utilizes SELEX (Symplocative Elastic Ligand Systematic Evolution) to screen for nucleic acid aptamers with high affinity. These aptamers possess several desirable properties, such as low immunogenicity, high hydrophilicity, high thermal stability, small molecular weight (typically 10 to 100 nucleotides), ease of chemical synthesis, minimal batch-to-batch variation, and ease of modification. Based on these aptamers, a DNA-protein hydrolysis-targeting chimera is constructed. Leveraging the high expression of NCL (nucleolin) on the surface of various tumor cells, AS1411 is used as a ligand to recruit the E3 ubiquitin ligase MDM2, enabling specific targeting and internalization of tumor cells.

[0048] Experiments have demonstrated that the proteolytic targeting chimera based on nucleic acid aptamers involved in the above-mentioned technical solution exhibits good degradation effects in breast cancer, possesses anti-tumor activity, significantly inhibits tumor cell proliferation, migration, and invasion, and shows therapeutic potential against various tumors with high SETDB1 expression, demonstrating significant application potential and therapeutic prospects. It also enhances the sensitivity of tumor cells to tamoxifen-resistant endocrine therapy and promotes the growth of human CD8+. + The killing effect of T cells on breast cancer cells. Therefore, this proteolytic targeting chimera provides a new approach for targeted degradation of SETDB1, offering a new strategy for tumor treatment and tamoxifen endocrine therapy, and has good practical application value. Attached Figure Description

[0049] Figure 1 Example 1: A schematic diagram of the SELEX filtering process drawn using Adobe Illustrator;

[0050] Figure 2 : Binding affinity maps of candidate aptamers (C02, C08, C10, C11) containing linkers to SETDB1, determined using the SPR method in Example 1;

[0051] Figure 3 : Binding affinity map of SETDB1 for candidate aptamers (C02, C08, C10, C11) of the deleted linker, determined by ITC method in Example 1;

[0052] Figure 4 : Binding affinity map of SETDB1 for aptamers (C11-1, C11-2, C11-3) with further shortened C11 as determined by ITC method in Example 1;

[0053] Figure 5 Example 1: A schematic diagram of the synthesis of single-stranded PROTAC (AP-SETDB1-SnA) and double-stranded PROTAC (AP-SETDB1-D2) drawn using Adobe Illustrator;

[0054] Figure 6 Mass spectrometry results of single-chain PROTAC (AP-SETDB1-S6A) in Example 2;

[0055] Figure 7 Mass spectrometry results of the two DNA aptamers containing rigid complementary sequences used in the design and synthesis of double-stranded PROTAC (AP-SETDB1-D2) in Example 2;

[0056] Figure 8 Example 2: Non-denaturing gel image showing successful ligation of double-stranded PROTAC (AP-SETDB1-D2);

[0057] Figure 9 Example 3: Binding affinity maps of AP-SETDB1-S6A and AP-SETDB1-D2 to SETDB1 determined by ITC method;

[0058] Figure 10 Serum stability results of AP-SETDB1-S6A, AP-SETDB1-D1, AP-SETDB1-D2, and AP-SETDB1-D3 in Example 4;

[0059] Figure 11 Figure 5 shows the degradation results of SETDB1 in human breast cancer cells MCF-7 by AP-SETDB1-S6A, AP-SETDB1-S10A, and AP-SETDB1-S15A.

[0060] Figure 12 The degradation results of SETDB1 in human breast cancer cells MCF-7 by AP-SETDB1-D1, AP-SETDB1-D2 and AP-SETDB1-D3 in Example 5 are shown in Figure 5.

[0061] Figure 13 Example 5: Dose-dependent degradation results of SETDB1 by AP-SETDB1-S6A in different cells (one type of normal breast epithelial cell and three types of breast cancer cells);

[0062] Figure 14 Example 5: Dose-dependent degradation results of AP-SETDB1-D2 on SETDB1 in different cells (one type of normal breast epithelial cell and three types of breast cancer cells);

[0063] Figure 15 Example 5: Time-dependent degradation results of SETDB1 in different cells (one normal breast epithelial cell and three breast cancer cells) using AP-SETDB1-S6A;

[0064] Figure 16Example 5: Time-dependent degradation results of AP-SETDB1-D2 in different cell types (one normal breast epithelial cell and three breast cancer cell types);

[0065] Figure 17 Figure 6 shows the experimental results of AP-SETDB1-S6A and AP-SETDB1-D2 inhibiting breast cancer cell proliferation.

[0066] Figure 18 Figure 6 shows the experimental results of AP-SETDB1-S6A and AP-SETDB1-D2 inhibiting DNA replication in breast cancer cells.

[0067] Figure 19 Figure 6 shows the experimental results of AP-SETDB1-S6A and AP-SETDB1-D2 inhibiting the formation of breast cancer cell clones.

[0068] Figure 20 Figure 7 shows the experimental results of AP-SETDB1-S6A and AP-SETDB1-D2 inhibiting breast cancer cell migration.

[0069] Figure 21 Figure 7 shows the experimental results of AP-SETDB1-S6A and AP-SETDB1-D2 inhibiting breast cancer cell invasion.

[0070] Figure 22 Figure 8 shows the experimental results of AP-SETDB1-S6A and AP-SETDB1-D2 increasing the sensitivity of MCF-7-TamR and T47D-TamR cells to tamoxifen.

[0071] Figure 23 In Example 9, AP-SETDB1-D2 promoted human CD8 after acting on breast cancer cells. + Figure showing the experimental results of the killing effect of T cells on it;

[0072] Figure 24 The inhibitory effect of AP-SETDB1-S6A and AP-SETDB1-D2 on the proliferation of A549 cells in Example 10;

[0073] Figure 25 The inhibitory effect of AP-SETDB1-S6A and AP-SETDB1-D2 on the proliferation of HepG2 cells in Example 10;

[0074] Figure 26 Example 10: The inhibitory effect of AP-SETDB1-S6A and AP-SETDB1-D2 on the proliferation of HCT116 cells. Detailed Implementation

[0075] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0076] Example 1: Development of SETDB1 ligands

[0077] During the SELEX process, the SETDB1 fragment (aa 190-410) containing TTDs was used as the target; the screening process is detailed in the appendix. Figure 1 The single-stranded DNA library used contained a random sequence of 36 nucleotides, flanked by fixed primer regions (5'-TTCAGCACTCCACGCATAGC and CCTATGCGTGCTACCGTGAA-3'). After multiple rounds of screening, high-throughput sequencing was performed, and the specificity coefficients of the top 100 sequences were analyzed. Twelve highly specific enriched sequences were synthesized, and their affinity for SETDB1 TTDs was detected using surface plasmon resonance (SPR). Four of these sequences (C2-linker, C8-linker, C10-linker, and C11-linker) showed high affinity for SETDB1, as shown in the attached figure. Figure 2 As shown. The affinity of these sequence deletion flanking connective regions (named C2, C8, C10, and C11, respectively) for SETDB1 was further evaluated using isothermal titration calorimetry (ITC), and the results are attached. Figure 3 As shown, the DNA aptamer with the highest affinity was obtained (named C11), with the nucleotide sequence 5´-GTTTTGCCGCATAGGATTTTTGGTTGGTCTGGTTGG-3´; aptamer C11 was further shortened, and the affinity of the shortened sequence for SETDB1 was evaluated using ITC, with the results as shown. Figure 4 As shown, a DNA aptamer with good affinity was obtained (named C11-2), whose nucleotide sequence is 5´-TGCCGCATAGGATTTTTGGTTGGTCTGG-3´.

[0078] A PROTAC targeting SETDB1 was developed using DNA aptamers obtained through SELEX screening. A simulation diagram of the PROTAC is attached. Figure 5 For a single-stranded PROTAC, one end is a nucleic acid aptamer that specifically binds to SETDB1; the other end is the nucleic acid aptamer AS1411 of NCL, which can further recruit MDM2; and the middle is a linker molecule between the two nucleic acid aptamers. The linker molecule can be n deoxyadenine nucleotides (A), n=6, 10, or 15 (when n=6, the synthesized PROTAC is AP-SETDB1-S6A; when n=10, the synthesized PROTAC is AP-SETDB1-S10A; when n=15, the synthesized PROTAC is AP-SETDB1-S15A). The linker molecule can be linked to the 3' end of AS1411 and the 5' end of C11. For the synthesized single-stranded PROTAC (AP-SETDB1-SnA), it is directly synthesized into single-stranded DNA by Anstar according to the designed sequence. For constructing bistranded PROTACs (AP-SETDB1-D1, AP-SETDB1-D2, AP-SETDB1-D3), rigid complementary sequences were first designed at the ends of individual aptamers and then synthesized by Anstar. The inverse complementary rigid sequences were (1:5´-GGGATCCCGA-3´, 2:5´-AGCGCCGGCC-3´, 3:5´-CACGGGCAAC-3´). The two strands were renatured separately, mixed in a 1:1 molar ratio, and incubated at 4°C for 2 h to promote self-assembly. The two strands were then connected by the complementary sequences.

[0079] Example 2: Synthesis of PROTAC

[0080] (1) Design and synthesize a single-chain PROTAC (AP-SETDB1-SnA), specifically 5´-GGTGGTGGTGGTTGTGGTGGTGGTGG-(A)n-GTTTTGCCGCATAGGATTTTTGGTTGGTCTGGTTGG-3´, where (A)n is the linker molecule, n=6 / 10 / 15, and append... Figure 6 The mass spectrometry results for single-chain PROTAC (AP-SETDB1-S6A) are shown.

[0081] (2) Design and synthesize double-stranded PROTACs (AP-SETDB1-D1, AP-SETDB1-D2, AP-SETDB1-D3), synthesize AS1411 aptamers with rigid sequences (1: 5´-TCGGGATCCC-3´, 2: 5´-GGCCGGCGCT-3´, 3: 5´-GTTGCCCGTG-3´) at the 3' end, and C11-2 aptamers with complementary sequences of the above rigid sequences at the 3' end (when rigid complementary sequence 1 is used, the synthesized double-stranded PROTAC is AP-SETDB1-D1; when rigid complementary sequence 2 is used, the synthesized double-stranded PROTAC is AP-SETDB1-D2; when rigid complementary sequence 3 is used, the synthesized double-stranded PROTAC is AP-SETDB1-D3), see appendix. Figure 7 The mass spectrometry results of the two DNA aptamers (AP-SETDB1-D2) are shown. The ligation reaction of the synthetic strands PROTAC (AP-SETDB1-D1, AP-SETDB1-D2, AP-SETDB1-D3) was performed as follows: First, individual aptamers were denatured in DPBS buffer (5 mmol / L MgCl2, pH 7.5) at 95°C for 10 min, then gradually cooled to 4°C. Then, complementary aptamers were mixed at a 1:1 molar ratio and incubated at 4°C for 2 h to promote self-assembly. The successful synthesis of AP-SETDB1-D1, AP-SETDB1-D2, and AP-SETDB1-D3 was confirmed by polyacrylamide gel electrophoresis. The results are attached. Figure 8 As shown.

[0082] Example 3: Affinity test of AP-SETDB1-S6A, AP-SETDB1-D2 and SETDB1

[0083] The affinity of AP-SETDB1-S6A, AP-SETDB1-D2 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 mg / mL MgCl2, pH 7.5), and the final protein concentration was adjusted to 10 µM. DNA aptamer concentrations were 50 or 100 µM, respectively. 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 9As shown, AP-SETDB1-S6A and AP-SETDB1-D2 have a good affinity for SETDB1.

[0084] Example 4: Serum stability test of AP-SETDB1-S6A, AP-SETDB1-D1, AP-SETDB1-D2, and AP-SETDB1-D3

[0085] AP-SETDB1-S6A, AP-SETDB1-D1, AP-SETDB1-D2, and AP-SETDB1-D3 were incubated at 37°C for specified time points in complete culture medium containing 10% fetal bovine serum. DNA samples were analyzed by 8% non-denaturing polyacrylamide gel electrophoresis (native PAGE), stained with GelRed, and observed under UV light. Results are shown in the attached figure. Figure 10 As shown, from the appendix Figure 10 As can be seen, the band of AP-SETDB1-D2 is clear and complete, and no obvious degradation was observed at different times. It can be seen that AP-SETDB1-D2 has better serum stability than AP-SETDB1-D1 and AP-SETDB1-D3.

[0086] Example 5: Degradation performance test of AP-SETDB1-S6A, AP-SETDB1-S10A, AP-SETDB1-S15A, AP-SETDB1-D1, AP-SETDB1-D2, and AP-SETDB1-D3 on SETDB1 protein

[0087] Solutions of AP-SETDB1-S6A, AP-SETDB1-S10A, and AP-SETDB1-S15A at concentrations of 1000 nM were prepared and added to MCF-7 human breast tumor cells. After incubation for 24 h, the cells were lysed, and changes in SETDB1 protein levels were detected by Western blot. Figure 11 The degradation results of SETDB1 in MCF-7 cells by AP-SETDB1-S6A, AP-SETDB1-S10A, and AP-SETDB1-S15A are shown in the attached figure. Figure 11 As shown, AP-SETDB1-S6A has a good degradation effect on SETDB1.

[0088] AP-SETDB1-D1, AP-SETDB1-D2, and AP-SETDB1-D3 solutions at concentrations of 1000 nM were prepared and added to MCF-7 human breast tumor cells transfected with PEI or incubated for 24 h before cell lysis. Changes in SETDB1 protein levels were detected by Western blot. Figure 12The degradation results of SETDB1 in MCF-7 cells by AP-SETDB1-D1, AP-SETDB1-D2, and AP-SETDB1-D3 are shown in the attached figure. Figure 12 As shown, both transfection and incubation lead to the degradation of SETDB1, and AP-SETDB1-D2 has a better degradation effect on SETDB1.

[0089] AP-SETDB1-S6A (AP-SETDB1-D2) solutions with concentration gradients of 0 nM, 100 nM (250 nM), 200 nM (500 nM), 500 nM (750 nM), 1000 nM, and 2000 nM were prepared and added to normal human breast epithelial cells MCF-10A and human breast tumor cells MCF-7, T47D, and MDA-MB-231, respectively. After incubation for 24 h, the cells were lysed, and changes in SETDB1 protein levels were detected by Western blot. Figure 13 and 14 The degradation results of SETDB1 in different cells by AP-SETDB1-S6A and AP-SETDB1-D2 are shown.

[0090] In three breast cancer cell lines, both PROTAC molecules exhibited a typical dose-response relationship: the protein band intensity of SETDB1 significantly decreased with increasing PROTAC concentration. Based on band grayscale values, the EC50 values ​​of AP-SETDB1-S6A and AP-SETDB1-D2 roughly fell within the range of 200 nM to 500 nM. This indicates that both PROTAC molecules possess efficient degradation capabilities at relatively low nanomolar concentrations, demonstrating significant killing effects on tumor cells while having no effect on normal cells.

[0091] A 1000 nM solution of AP-SETDB1-S6A (AP-SETDB1-D2) was prepared and added to normal human mammary epithelial MCF-10A cells and human breast tumor cells MCF-7, T47D, and MDA-MB-231, respectively, for incubation. Cells were lysed after incubation at time gradients of 0 h, 6 h, 12 h, 18 h, 24 h, and 48 h, and changes in SETDB1 protein levels were detected by Western blot. (See attached image) Figure 15 and 16 The degradation results of SETDB1 in different cells by AP-SETDB1-S6A and AP-SETDB1-D2 are shown.

[0092] In three types of breast cancer cell lines, the degradation effect of SETDB1 increased over time, typically reaching its maximum around 24 hours. The degradation effect remained stable over 48 hours, indicating that PROTAC has a sustained effect.

[0093] In normal MCF-10A cells, the SETDB1 protein level did not change significantly throughout the time period, further validating the selectivity of PROTAC for tumor cells.

[0094] Example 6: Testing of AP-SETDB1-S6A and AP-SETDB1-D2 on breast cancer cell proliferation

[0095] MCF-7 and T47D cells (2000 cells per well) were seeded into 96-well plates and treated with Veh (PBS), AS1411+C11 (Control), AP-SETDB1-S6A, or AP-SETDB1-D2 for 0, 2, 4, 6, and 8 days, respectively. The culture medium was changed every two days depending on the treatment group. At each time point, the medium was replaced with 10% MTT solution (serum-free DMEM containing 0.5 mg / mL MTT), and the cells were incubated at 37°C for 4 h. Subsequently, 110 µL of dimethyl sulfoxide (DMSO) was added to lyse the cells, and the cells were incubated at room temperature for 30 min. The absorbance was measured at 490 nM using a microplate reader, and the results are shown in the attached figure. Figure 17 As shown, AP-SETDB1-S6A and AP-SETDB1-D2 inhibit the proliferation of breast cancer cells.

[0096] MCF-7 and T47D cells were seeded at a density of 6000 cells per well in 96-well plates and incubated with Veh (PBS), AS1411+C11 (Control), AP-SETDB1-S6A, or AP-SETDB1-D2, respectively. After 48 h of incubation, the medium was replaced with EdU-containing medium, and incubation continued for 2 h. Cell proliferation was then assessed using the BeyoClick™ EdU Cell Proliferation Kit (C0078S or C0071S, Beyotime, Shanghai, China) containing Alexa Fluor 594 or Alexa Fluor 488. Cells were first fixed with 4% paraformaldehyde for 15 min, then washed three times with wash buffer (PBS containing 3% BSA), and then permeabilized with PBS containing 0.3% Triton X-100 for 10 min. 100 µL of Clickreaction solution was then added, and the cells were incubated at room temperature in the dark for 30 min. After three washes, the cells were incubated at room temperature in Hoechst 33342 in the dark for 10 min. 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 18 As shown, AP-SETDB1-S6A and AP-SETDB1-D2 inhibit DNA replication in breast cancer cells.

[0097] MCF-7 and T47D cells (500 cells per well) were seeded into each well of a 6-well plate and incubated every two days with Veh (PBS), AS1411+C11 (Control), AP-SETDB1-S6A, or AP-SETDB1-D2. After 14 days of incubation, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet solution (Solarbio, Beijing, China) for 15 min. Images were then taken, and the results are attached. Figure 19 As shown, AP-SETDB1-S6A and AP-SETDB1-D2 inhibit the clonogenic formation of breast cancer cells.

[0098] Example 7: Testing of AP-SETDB1-S6A and AP-SETDB1-D2 on breast cancer cell migration and invasion

[0099] MCF-7, T47D, and MDA-MB-231 cells were treated with Veh (PBS), AS1411+C11 (Control), AP-SETDB1-S6A, or AP-SETDB1-D2 for 48 h. Cells were then 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 20 As shown, AP-SETDB1-S6A and AP-SETDB1-D2 inhibit the migration of breast cancer cells.

[0100] T47D and MDA-MB-231 cells were incubated for 48 hours with Veh (PBS), AS1411+C11 (Control), AP-SETDB1-S6A, or AP-SETDB1-D2, respectively. Approximately 1×10⁻⁶ cells were produced. 4 Cells were suspended in 200 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 h 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 21 As shown, AP-SETDB1-S6A and AP-SETDB1-D2 inhibit the invasion of breast cancer cells.

[0101] Example 8: Efficacy test of AP-SETDB1-S6A, AP-SETDB1-D2 in combination with tamoxifen against tamoxifen-resistant breast cancer cells.

[0102] MCF-7-TamR and T47D-TamR cells were cultured for 6 months in medium supplemented with 1 μmol / L 4OH-tamoxifen to induce drug resistance. 2000 MCF-7-TamR or T47D-TamR cells were added to each well of a 96-well plate and cultured at 37°C in a 5% CO2 incubator for 24 hours. Cells were then treated with Veh (PBS), 1 μM AP-SETDB1-S6A or AP-SETDB1-D2, and a gradient of increasing concentrations of 4OH-tamoxifen. After 48 hours of culture at 37°C in a 5% CO2 incubator, the medium was replaced with 10% MTT solution (serum-free DMEM containing 0.5 mg / mL MTT), and the cells were incubated at 37°C for 4 hours. Subsequently, 110 µL of dimethyl sulfoxide (DMSO) was added to lyse the cells, and the cells were incubated at room temperature for 30 minutes. The absorbance was measured at 490 nM using an ELISA reader, and the results are shown in the attached figure. Figure 22 As shown, AP-SETDB1-S6A and AP-SETDB1-D2 can increase the sensitivity of MCF-7-TamR and T47D-TamR cells to tamoxifen.

[0103] Example 9: Treatment of breast cancer cells with AP-SETDB1-D2, human CD8 + Test of the killing effect of T cells

[0104] The process of isolating peripheral blood mononuclear cells (PBMCs) from human peripheral blood involves density gradient centrifugation followed by separation using a lymphocyte separation medium. Specifically, blood samples are collected using EDTA-K2 as an anticoagulant. Lymphocyte separation medium is pre-loaded into sterile centrifuge tubes, and an equal volume of fresh blood is gently added. The sample is centrifuged at 600g for 30 minutes at room temperature. The opaque white layer in the middle is carefully aspirated and transferred to a new sterile centrifuge tube. Three volumes of phosphate-buffered saline (PBS) are added for dilution, followed by centrifugation at 300g for 10 minutes. The cells are washed twice with PBS, and the resulting precipitate is the PBMC. The cells are counted, and a concentration of 1×10⁻⁶ cells is prepared. 8 Cell suspension of cells / mL.

[0105] Using ImunoSep™ Human CD8 + The cell positive selection kit isolates CD8 cells from peripheral blood mononuclear cells (PBMCs). +T cell preparation was performed according to the manufacturer's instructions. Based on the volume of the prepared single-cell suspension, 10 µL of sorting reagent A was added to 100 µL of the cell suspension, mixed thoroughly, and then incubated at room temperature for 10 min. The cells were then centrifuged and restored to their initial volume. Subsequently, 10 µL of sorting reagent B was added, mixed, and incubated under the same conditions. After incubation, magnetic separation was performed to isolate CD8+. + T cells. The isolated cells were resuspended in RPMI 1640 medium and cultured for 24 hours with 2 µg / mL of CD3 and CD28 cytokines added. Then, IL-2 (10 ng / mL) was added, and the cells were cultured for another 48 hours to complete the in vitro activation process. Activated CD8 cells... + The T cells were subsequently used in T cell killing experiments.

[0106] MCF-7 and T47D cells were incubated with Veh (PBS), Control, or AP-SETDB1-D2 for 48 h, respectively. 1×10 4 Activation of CD8 per cell / well + T cells were seeded into 96-well plates and co-cultured with pretreated tumor cells at an effector-to-target cell ratio of 10:1 for 48 h. After culture, the release of lactate dehydrogenase (LDH) in the supernatant was determined using a one-step LDH in vitro toxicity assay kit, following the manufacturer's instructions. During experiment setup, 10 µL of lysis buffer was added to the high control wells, and 10 µL of culture medium was added to the experimental group, blank sample, low control, and background blank wells. The culture plates were incubated in a cell culture incubator for 30 min. The LDH detection working solution was prepared by mixing Catalyst Solution and Dye Solution at a volume ratio of 1:9. Then, 100 µL of the working solution was added to each well and incubated at room temperature in the dark. After color formation, 50 µL of stop solution was added immediately, and the absorbance was measured at 490 nm using a microplate reader. The percentage of cytotoxicity was calculated according to the provided formula, and the results are shown in the attached figure. Figure 23 As shown, AP-SETDB1-D2 promotes human CD8 after acting on breast cancer cells. + The killing effect of T cells.

[0107] Example 10: Testing of AP-SETDB1-S6A and AP-SETDB1-D2 on the proliferation of other tumor cells

[0108] Lung adenocarcinoma cell line A549, liver cancer cell line HepG2, and colorectal cancer cell line HCT116 (2000 cells per well) were seeded into 96-well plates and treated with Veh (PBS), AS1411+C11 (Control), AP-SETDB1-S6A, or AP-SETDB1-D2 for 0, 2, 4, and 6 days, respectively. The culture medium was changed every two days according to the treatment group. At each time point, the culture medium was replaced with 10% MTT solution (serum-free DMEM containing 0.5 mg / mL MTT) and the cells were incubated at 37°C for 4 h. Subsequently, 110 µL of dimethyl sulfoxide (DMSO) was added to lyse the cells, and the cells were incubated at room temperature for 30 min. The absorbance was measured at 490 nM using a microplate reader, and the results are shown in the attached figure. Figure 24-26 As shown, AP-SETDB1-S6A and AP-SETDB1-D2 inhibited the proliferation of three types of tumor cells.

[0109] The experimental data above show that both PROTAC molecules (AP-SETDB1-S6A and AP-SETDB1-D2) significantly inhibited the proliferation of solid tumor cells (A549, HepG2, and HCT116) from three different sources. This indicates that the anti-tumor effects of PROTACs constructed based on the SETDB1 aptamer are not limited to breast cancer, but may have therapeutic potential for multiple cancer types with high SETDB1 expression.

[0110] Meanwhile, over time (0, 2, 4, 6 days), compared with the Veh group and the AS1411+C11 control group, the cell proliferation activity of the two PROTAC treatment groups was significantly reduced, and the inhibitory effect became more pronounced with the extension of treatment time, indicating that the anti-cancer effect of PROTAC is continuous and cumulative.

[0111] It is evident that this invention not only provides a new solution for breast cancer treatment, but also demonstrates its enormous application potential and market prospects in the treatment of various solid tumors.

[0112] 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 scope of the technical solutions of the embodiments of the present invention.

Claims

1. A SETDB1 aptamer, characterized in that, The nucleotide sequence of the SETDB1 aptamer is any one of SEQ ID NO. 1 or SEQ ID NO.

2.

2. A single-chain PROTAC constructed based on the SETDB1 aptamer of claim 1, characterized in that, The single-chain PROTAC comprises: a first DNA aptamer capable of specifically binding to SETDB1 protein, a second DNA aptamer capable of specifically binding to nucleolin, and a linker connecting the first DNA aptamer and the second DNA aptamer. The second DNA aptamer is located at the 5' end and is connected to the first DNA aptamer at the 3' end through the linker. The linker is an oligonucleotide chain, and the oligonucleotide chain is composed of 6 deoxyadenine nucleotides.

3. The SETDB1 aptamer constructed single-chain PROTAC of claim 2, characterized in that, The single-chain PROTAC is specifically 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-oligonucleotide chain-GTTTTGCCGCATAGGATTTTTGGTTGGTCTGGTTGG-3'.

4. A double-stranded PROTAC constructed based on the SETDB1 aptamer of claim 1, characterized in that, The double-chain PROTAC comprises: The first DNA aptamer and the first complementary sequence at the 3' end thereof; The second DNA aptamer capable of specifically binding to nucleolin and the second complementary sequence at the 3' end thereof; The first complementary sequence and the second complementary sequence form a linker; The first complementary sequence and the second complementary sequence are SEQ ID NO. 3 and its reverse complementary sequence.

5. A pharmaceutical composition, characterized by, The single-chain PROTAC of claim 2 or 3 or the double-chain PROTAC of claim 4 and a pharmaceutically acceptable carrier or excipient.

6. Use of the single-chain PROTAC of claim 2 or 3 or the double-chain PROTAC of claim 4 in the preparation of a drug for preventing and / or treating tumors.

7. Use according to claim 6, characterized in that, The drug is used to reverse or inhibit the drug resistance of tumors to tamoxifen.

8. A method of preparing a single-chain PROTAC according to claim 3, characterized in that, The method comprises the following steps: According to the nucleotide sequence information of claim 3, the single-chain PROTAC is synthesized by solid-phase synthesis technology at one time.

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