PROTAC molecule for targeted degradation of Lin28A / B based on oligonucleotide and application of PROTAC molecule

Abstract

The invention discloses an oligonucleotide PROTAC molecule based on targeted degradation of Lin28A / B protein and application of the oligonucleotide PROTAC molecule. The oligonucleotide PROTAC molecule disclosed by the invention is prepared by coupling an oligonucleotide sequence capable of being combined with Lin28A / B and a ligand capable of promoting the ubiquitination of Lin28A / B protein with connecting arms of different types or different lengths. In different tumor cells, the oligonucleotide PROTAC molecule can be used for degrading the Lin28A / B protein in a targeted manner, has remarkable degradation activity on the Lin28A / B protein and plays an anti-tumor function. The invention has application prospects in preparation of drugs or preparations for targeted degradation of Lin28A / B protein, or in preparation of antitumor drugs and preparations, and in preparation of antitumor drugs combined with micromolecules or macromolecules.

Description

Technical Field

[0001] The present invention relates to a protein degradation targeted chimera (PROTAC) and applications thereof, and in particular to an oligonucleotide PROTAC molecule for targeted degradation of Lin28A / B protein and applications thereof in the preparation of a drug, preparation or combination with an anti-tumor drug for targeted degradation of Lin28A / B protein, belonging to the field of protein degradation targeted chimeras and applications thereof. Background Art

[0002] Lin 28 is a highly conserved RNA binding protein with two RNA binding domains (cold shock domain and CCHC zinc finger domain). It was originally identified as a key regulator of developmental timing in nematodes. In mammals, Lin28 (Lin28A and Lin28B) can maintain cell stemness, is expressed in large quantities in embryonic cells, promotes rapid and massive cell proliferation, and plays an important role in embryogenesis, skeletal myogenesis, germ cell development, and gliogenesis. Increasing evidence suggests that Lin 28 may also be a major regulator controlling ES cell pluripotency. Together with OCT4, SOX2, and NANOG ("reprogramming factors"), LIN28 can reprogram somatic cells into induced pluripotent stem cells. LIN28 binds to mRNAs and regulates their stability and translation. In addition, Lin 28 can bind to the terminal loop of the miRNA let-7 precursor, thereby preventing let-7 from being processed into a mature form. In human tumors, Lin 28 has been shown to be overexpressed in a variety of malignant tumors, and can significantly promote the proliferation, migration and drug resistance of tumor cells. It plays a role as an oncogene for malignant transformation and tumor progression in tumors, and is closely related to the poor prognosis of tumor treatment. Lin 28 has two subtypes, Lin 28A and Lin 28B, which are located in the cytoplasm and nucleus of tumor cells, respectively. The relative amounts of Lin 28A and Lin 28B in cells of different tumor types are different, but both subtypes of Lin 28 have the effects of promoting the proliferation, migration and drug resistance of tumor cells. Therefore, inhibiting the expression of Lin28 is a new strategy for cancer treatment.

[0003] The protein degradation targeting chimera (PROTAC) technology originated from scientists' discovery of the protein degradation process regulated by ubiquitin (Ub). Eukaryotic cells are constantly striving to maintain appropriate protein levels, and they are producing and degrading thousands of proteins at every moment. The key factor in maintaining protein balance is a small protein molecule called ubiquitin. When it is linked to proteins, it causes these proteins to be transported to the proteasome for degradation. The protein hydrolysis targeting chimera (PROTAC) is a heterobifunctional molecule composed of the following three parts: (1) a ligand that binds to the target protein; (2) a ligand for recruiting E3 ubiquitin ligase to promote ubiquitination of the target protein; (3) a linker that connects these ligands. As a new and promising technology, PROTACs show great potential in the following aspects. First, PROTACs have special sensitivity to drug-resistant targets. Traditionally, chemotherapy is the main method for cancer treatment. Acquired resistance to chemotherapeutic drugs hinders clinical application and leads to disease recurrence. With the research progress of new targets and new drug discovery technologies, another powerful strategy is to directly and specifically inhibit the function of oncogenic proteins or receptors through small molecules. It is worth noting that the discovery of kinase inhibitors did not solve the problem of drug resistance in cancer treatment, while PROTACs affect protein function by eliminating the entire target, thereby deleting all functions of the target, including enzymatic activity and non-enzymatic functions, so PROTACs can solve the potential drug resistance faced by current treatments. In addition, PROTACs are less sensitive to increased target expression and target protein mutations, and because they are catalytic, only low doses of PROTACs are needed to achieve good results. Based on PROTACs, not only can the target protein be deeply degraded, with persistence and rapid and powerful inhibition of downstream signals, but also the occurrence and development of tumors can be inhibited for a long time in vitro, making the use of protein hydrolysis targeted chimeras to degrade proteins a hot method for current tumor treatment. Summary of the invention

[0004] One of the purposes of the present invention is to provide an oligonucleotide PROTAC molecule that targets and degrades Lin28A / B protein;

[0005] The second purpose of the present invention is to apply the oligonucleotide PROTAC molecule to the preparation of drugs or preparations for targeted degradation of Lin28A / B protein or anti-tumor and combination therapy.

[0006] To achieve the above object, the technical solution adopted by the present invention includes:

[0007] One aspect of the present invention is to provide an oligonucleotide PROTAC molecule for targeted degradation of Lin28A / B protein, wherein the oligonucleotide PROTAC molecule is obtained by coupling an oligonucleotide sequence capable of binding to Lin28A / B and a ligand capable of recruiting E3 ubiquitin ligase to promote ubiquitination of the target protein through a connecting arm; the coupling method can be to obtain a Lin28A / B binding oligonucleotide sequence by solid phase synthesis and the 3' end is amino-modified, and then cross-linked with the NHS active ester of the CRBN or VHL ligand connected with different connecting arms through the amino group to obtain the oligonucleotide PROTAC molecule.

[0008] In a preferred specific embodiment of the present invention, the oligonucleotide sequence capable of binding to Lin28A / B may be an oligonucleotide sequence capable of binding to both the CSD region and the ZKD region of the Lin28A / B protein, or an oligonucleotide sequence capable of binding only to the CSD region of the Lin28A / B protein, or an oligonucleotide sequence capable of binding only to the ZKD region of the Lin28A / B protein; preferably, the oligonucleotide sequence capable of binding to both the CSD region and the ZKD region of the Lin28A / B protein is selected from any one of the nucleotide sequences described in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13, SEQ ID No.15, SEQ ID No.17, SEQ ID No.19, SEQ ID No.21 or SEQ ID No.23; the oligonucleotide sequence capable of binding only to the CSD region is selected from any one of the nucleotide sequences described in SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID Any one of SEQ ID No.10, SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18, SEQ ID No.20, SEQ ID No.22 or SEQ ID No.24; or a nucleotide sequence having 80% or more homology with the above-mentioned nucleotide sequences, and still having the function or activity of binding to Lin28A / B protein.

[0009] The oligonucleotide sequence that binds only to the ZKD region of the Lin28A / B protein is selected from any one of the nucleotide sequences described in (1)-(12) below:

[0010] (1):5'-GGGAGAU-3'; (2):5'-GGGAGAU-3'; (3):5'-AUGGGAU-3'; (4):5'-GGAAGAU-3'; (5):5'-GGGAGUU-3'; (6):5'-AGGAGGU-3'; ( 7):5'-AGGAGAU-3'; (8):5'-AGGAGAU-3'; (9):5'-UGGAGAU-3'; (10):5'-AGGAGAU-3'; (11):5'-UGGAGAU-3'; (12):5'-AGAAGAU-3'.

[0011] Further preferably, any of the above-mentioned nucleotide sequences can be fully thio-modified; the nucleotide sequence is modified with different nucleotide analogs, including but not limited to modified nucleoside bases such as 5-methylcytosine, 6-methyladenine, ribavirin, pseudouracil and / or inosine; or the nucleotide or locked nucleotide obtained by modifying the 2' position of the sugar ring with methoxy, methylethoxy, fluorine, etc.

[0012] The ligand capable of recruiting E3 ubiquitin ligase to promote ubiquitination of target proteins described in the present invention is preferably a CRBN ligand (pomalidomide), a VHL (von Hippel-Lindau) ligand (VH032) or a derivative analog thereof; wherein the chemical name of the CRBN ligand small molecule is: 4-amino-2-(2,6-dioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione, and its structural formula is shown in Formula I:

[0013]

[0014] The chemical name of the VHL ligand small molecule described in the present invention is: (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutanyl)-4-hydroxy-n-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, and its structural formula is shown in the following formula II:

[0015]

[0016] The structural formula of the derivative analogue described in the present invention is shown in Formula III:

[0017]

[0018] The linker described in the present invention includes but is not limited to one or more of the following groups: alkylene, alkoxy, alkylamino, alkylthio, amide or amino carbonate bond, triazole derivatives;

[0019] Preferably, the linker is selected from any one of the following compounds of the general formula:

[0020]

[0021] Wherein, m or n is any integer from 0 to 10.

[0022] In a more preferred embodiment, the connecting arm includes but is not limited to any one of C3, C5, C6, C8, C12-1, C12-2, C16, P4 or P6:

[0023]

[0024] Another aspect of the present invention is to provide a method for preparing the oligonucleotide PROTAC molecule targeting the degradation of Lin28A / B protein, the method comprising:

[0025] (1) connecting a CRBN ligand or a VHL ligand to a linker of different types or lengths and then performing an activation reaction to obtain an NHS active ester of the CRBN or VHL ligand connected to the linker;

[0026] (2) coupling the oligonucleotide sequence with an amino group at the 3' end that can bind to Lin28A / B with the NHS active ester of the CRBN or VHL ligand connected with a linker obtained in step (1) to obtain.

[0027] As a preferred embodiment, in step (2), the oligonucleotide sequence with an amino group at the 3' end that can bind to Lin28A / B is dissolved in PBS buffer to obtain a mixed solution 1; the NHS active ester of the CRBN or VHL ligand connected with a linker is dissolved in dimethyl sulfoxide to obtain a mixed solution 2; the mixed solution 1 and the mixed solution 2 are mixed to perform a coupling reaction, and the reaction product is purified to obtain.

[0028] As a preferred embodiment, in step (2), the ratio of the oligonucleotide sequence with an amino group at the 3' end capable of binding to Lin28A / B to the NHS active ester of the CRBN or VHL ligand connected to the linker arm is 1:500.

[0029] The oligonucleotide PROTAC molecule for targeted degradation of Lin28A / B protein provided by the present invention has significant degradation activity for Lin28A protein or Lin28B protein, and the degradation effect is significantly better than that of the Full-S ORN experimental group not coupled with pomalidomide and the Full-ORN and pomalidomide mixed group, and the experimental group with only pomalidomide added does not show obvious degradation of Lin28A protein, indicating that the degradation of Lin28A or Lin28B protein by the oligonucleotide PROTAC of the present invention is the specific degradation of Lin28A / B mediated by the oligonucleotide sequence that can bind to Lin28A / B and the derivative of pomalidomide or VH after coupling through the connecting arm, rather than the non-specific degradation mediated by a single part.

[0030] According to the experimental results of the oligonucleotide PROTAC-mediated degradation of Lin28A / B or let-7 level changes in miRNA, it can be seen that with the degradation of Lin28 protein, all mature let-7 expression levels are upregulated, and the expression level of miR-21 has not changed, indicating that targeted degradation of Lin28A / B specifically causes an increase in the expression level of tumor suppressor miRNA Let-7. At the same transfection concentration and action time, the effect of the oligonucleotide PROTAC of the present invention on the increase of let-7g level is better than that of the Full-S ORN experimental group without pomalidomide, the Full-ORN and pomalidomide mixed group, and the experimental group with only pomalidomide added did not show a significant increase in let-7g level.

[0031] In order to improve the targeting of oligonucleotide PROTAC molecules to tumor cells, the present invention can introduce a targeting gene at the end of the nucleic acid sequence of the oligonucleotide PROTAC molecule, and the targeting gene includes but is not limited to folic acid, polypeptides and nucleic acid aptamers.

[0032] The oligonucleotide PROTAC of the present invention can significantly inhibit the proliferation, clonal colony formation, migration and invasion ability of T47D or H1299 cells, and its inhibitory effect is more obvious when used in combination with small molecule drugs.

[0033] Therefore, the oligonucleotide PROTAC provided by the present invention has the following uses:

[0034] (1) preparing drugs or preparations for targeted degradation of Lin28A / B protein;

[0035] (2) A drug or preparation for preparing an anti-tumor drug; wherein Lin28A / B is overexpressed in the tumor cells;

[0036] (3) Combination with anti-tumor small molecule drugs for anti-tumor use; wherein the anti-tumor small molecule drugs can be tamoxifen or paclitaxel.

[0037] The present invention couples an oligonucleotide sequence that can bind to Lin28A / B and a ligand that can promote the ubiquitination of Lin28A / B protein through different types or different lengths of connecting arms to obtain an oligonucleotide PROTAC molecule; such oligonucleotide PROTAC molecules are used to selectively degrade Lin28A or / and Lin28B protein in different tumor cells and exert an anti-tumor effect; the present invention has application prospects in the preparation of drugs and preparations for targeted degradation of Lin28A / B protein or the preparation of anti-tumor drugs, or in combination with anti-tumor drugs such as small molecule or large molecule anti-tumor antibodies for anti-tumor use. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Degradation of Lin28A / B by PROTAC molecules made with different miRNA sequences targeting Lin28; a) Lin28A (T47D cells), b) Lin28B (H1299 cells), c) Lin28B (Huh7 cells).

[0039] Figure 2 Degradation of Lin28A protein in T47D cells based on the Let-7f-1PROTAC molecule; a) targeting the full-length sequence, b) targeting the CSD region sequence, c) targeting the ZKD region sequence, d) concentration dependence, e) time dependence, f) specificity.

[0040] Figure 3 Changes in the expression levels of Let-7 in T47D (ad) / H1299 (ef) cells after degradation of Lin28A / B protein by Let-7f-1Full-SP4-C; a) Evaluation of all Let-7 up-regulation effects in T47D cells, b) concentration dependence, c) time dependence, d) specificity, e) Evaluation of all Let-7 up-regulation effects in H1299 cells, f) concentration dependence, g) time dependence, h) specificity.

[0041] Figure 4 The antitumor activity of Let-7f-1Full-SP4-C in T47D cells; a) cell proliferation, b) combined administration, c) cell cycle, d) clonal colony formation, e) cell apoptosis, and f) cell migration.

[0042] Figure 5Degradation of Lin28B protein in H1299 cells based on the Let-7f-1PROTAC molecule; a) targeting the full-length sequence, b) targeting the CSD region sequence, c) targeting the ZKD region sequence, d) concentration dependence, e) time dependence, f) specificity.

[0043] Figure 6 The antitumor activity of Let-7f-1Full-SP4-C in H1299 cells; a) cell proliferation, b) clonal colony formation, c) cell migration, d) cell invasion, and e) cell apoptosis.

[0044] Figure 7 Degradation of Lin28B protein in Huh7 cells based on Let-7b PROTAC molecule; a) targeting the full-length sequence, b) targeting the CSD region sequence, c) targeting the ZKD region sequence, d) concentration dependence, e) time dependence, f) specificity.

[0045] Figure 8 Evaluation of the in vivo antitumor activity of the PROTAC molecule Let-7f-1Full-SP4-C; a) changes in mouse body weight, b) tumor volume growth curve, c) normal mouse tissue weight, d) actual image of mouse tumor anatomy, e) tumor weight, f) Lin28A expression in the tumor, g) tumor tissue section. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0047] Example 1 Design of oligonucleotide sequences capable of binding to Lin28A / B

[0048] The sequence of the oligonucleotide that can bind to Lin28A / B is as follows:

[0049] Let-7a-1Full-S:5'-UUAGGUCACACCCACCACUGGGAGAU-3'(SEQ ID No.1)

[0050] Let-7a-1CSD-S:5'-UUAGGUCACACCCACCACUG-3'(SEQ ID No.2)

[0051] Let-7a-2Full-S:5′-UAGAAUUACAUCAAGGGAGAU-3′(SEQ ID No.3)

[0052] Let-7a-2CSD-S:5′-UAGAAUUACAUCAAG-3′(SEQ ID No.4)

[0053] Let-7a-3Full-S:5′-UGGGGCUCUGCCCCUGCUAUGGGAU-3′(SEQ ID No.5)

[0054] Let-7a-3CSD-S:5′-UGGGGCUCUGCCCCUGCUA-3′(SEQ ID No.6)

[0055] Let-7b Full-S:5′-UCAGGGCAGUGAUGUUGCCCCUCGGAAGAU-3′(SEQ ID No.7)

[0056] Let-7b CSD-S:5′-UCAGGGCAGUGAUGUUGCCCCUCG-3′(SEQ ID No.8)

[0057] Let-7c Full-S:5'-3'(SEQ ID No.9)

[0058] Let-7c CSD-S:5′-UAGAGUUACACCCUG-3′(SEQ ID No.10)

[0059] Let-7d Full-S:5′-UUAGGGCAGGGAUUUUGCCCACAAGGAGGU-3′(SEQ ID No.11)

[0060] Let-7d CSD-S:5′-UUAGGGCAGGGAUUUUGCCCACAA-3′(SEQ ID No.12)

[0061] Let-7e Full-S:5′-GAGGAGACACCCAAGGAGAU-3′(SEQ ID No.13)

[0062] Let-7e CSD-S:5′-GAGGAGACACCCAA-3′(SEQ ID No.14)

[0063] Let-7f-1Full-S:5’-GUGGGGUAGUGAUUUUACCCUGUUCAGGAGAU-3’(SEQ ID No.15)

[0064] Let-7f-1CSD-S:5’-GUGGGGUAGUGAUUUUACCCUGUUCA-3’(SEQ ID No.16)

[0065] Let-7f-2Full-S:5’-UUAGGGUCAUACCCCAUCUUGGAGAU-3’(SEQ ID No.17)

[0066] Let-7f-2CSD-S:5’-UUAGGGUCAUACCCCAUCUU-3’(SEQ ID No.18)

[0067] Let-7g Full-S:5’-UGAGGGUCUAUGAUACCACCCGGUACAGGAGAU-3’(SEQ ID No.19)

[0068] Let-7g CSD-S:5’-UGAGGGUCUAUGAUACCACCCGGUACA-3’(SEQ ID No.20)

[0069] Let-7i Full-S:5’-GGUCGGGUUGUGACAUUGCCCGCUGUGGAGAU-3’(SEQ ID No.21)

[0070] Let-7i CSD-S:5’-GGUCGGGUUGUGACAUUGCCCGCUGU-3’(SEQ ID No.22)

[0071] miR98 Full-S:5’-GUGGGGUAGGGAUAUUAGGCCCCAAUUAGAAGAU-3’(SEQ ID No.23)

[0072] miR98 CSD-S:5’-GUGGGGUAGGGAUAUUAGGCCCCAAUU-3’(SEQ ID No.24)

[0073] Let-7a-1ZKD-S:5’-GGGAGAU-3’

[0074] Let-7a-2ZKD-S:5’-GGGAGAU-3’

[0075] Let-7a-3ZKD-S:5'-AUGGGAU-3'

[0076] Let-7b ZKD-S:5'-GGAAGAU-3'

[0077] Let-7c ZKD-S:5'-GGGAGUU-3'

[0078] Let-7d ZKD-S:5'-AGGAGGU-3'

[0079] Let-7e ZKD-S:5'-AGGAGAU-3'

[0080] Let-7f-1ZKD-S:5'-AGGAGAU-3'

[0081] Let-7f-2ZKD-S:5'-UGGAGAU-3'

[0082] Let-7g ZKD-S:5'-AGGAGAU-3'

[0083] Let-7i ZKD-S:5'-UGGAGAU-3'

[0084] miR98 ZKD-S:5′-AGAAGAU-3′;

[0085] Among them, the above oligonucleotide sequences can be fully thio-modified, that is, the non-bridging oxygen atoms in the original phosphate bonds are replaced with sulfur atoms.

[0086] Example 2 Synthesis and characterization of CRBN ligand small molecules (pomalidomide) with different linker lengths

[0087]

[0088] The synthetic route is as follows:

[0089] 1) Synthesis of compound C: 2-(2,6-dioxopiperidin-3-yl)-4-fluoroindoline-1,3-dione (1 mmol, 1.0 equiv) was dissolved in N,N-dimethylacetamide (2 mL), and 3-amino-1-propanol (1.2 mmol, 1.2 equiv) or 6-amino-1-hexanol (1.2 mmol, 1.2 equiv) was added. The reaction mixture was then heated to 90° C. and stirred for 2 h. The reaction solution was extracted with ethyl acetate and water, and the crude product was purified by silica gel column chromatography to obtain compound C.

[0090] 2) Synthesis of compound D (NHS-active ester of CRBN ligand small molecule with connecting arms C3 and C6): Compound C (0.2 mmol, 1.0 equiv) and N,N'-disuccinimidyl carbonate (0.3 mmol, 1.5 equiv) were dissolved in anhydrous acetonitrile, and then triethylamine (0.6 mmol, 3.0 equiv) was added. The mixture was stirred at room temperature for 16 h, the solvent was evaporated, extracted with dichloromethane and water, washed with cold 5% sodium bicarbonate solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain compounds D1 and D2.

[0091] D1(C3-C): 1 H NMR (400MHz, CDCl3) δ8.07 (s, 1H), 7.57 (t, J=8.5, 7.2Hz, 1H), 7.16 (d, J=

[0092] 7.1Hz,1H),6.95(d,J=8.5Hz,1H),4.94(dd,J=12.1,5.3Hz,1H),4.49(t,J=5.9Hz,2H),3.50(t,J =6.8Hz,3H),2.96–2.89(m,1H),2.88(s,4H),2.85–2.78(m,2H),2.21–2.16(m,2H),2.13(m,3H). 13 C NMR (101MHz, CDCl3) δ171.27,169.55,168.54,167.66,146.98,136.13,132.50,116.70,1 11.43,109.88,62.72,48.88,42.59,32.52,31.42,29.15,26.72,25.48,22.81.ESI:[M+H] + cacld:473.1230,found:473.1303.[M+Na] + found:495.1122.

[0093] D2(C6-C): 1 H NMR (400MHz, CDCl3) δ8.07 (s, 1H), 7.52 (t, J=8.5, 7.2Hz, 1H), 7.11 (d, J=

[0094] 7.0Hz,1H),6.91(d,J=8.5Hz,1H),4.93(dd,J=12.1,5.3Hz,1H),4.36(t,J=6.5Hz,3H),3.31(d,J=4.6Hz,3H),2.95–2.8 8(m,1H),2.86(s,4H),2.82–2.72(m,2H),2.22–2.12(m,1H),1.86–1.77(m,3H),1.76–1.67(m,3H),1.49(m,5.4Hz,5H). 13 C NMR (101MHz, CDCl3) δ170.93,169.50,168.72,168.26,167.63,151.63,146.98,136.16,132.52,116. 67,111.48,109.97,71.34,48.89,42.45,31.42,29.00,28.28,26.42,25.49,25.22,22.82.ESI:[M+H] + cacld:515.1700,found:515.1769.[M+Na] + found:537.1597.

[0095] 3) Synthesis of Compound F: 2-(2,6-dioxopiperidin-3-yl)-4-fluoroindoline-1,3-dione (1 mmol, 1.0 equiv) was dissolved in N,N-dimethylacetamide (2 mL), and 1-amino-3,6,9-trioxa-11-undecanol or 17-amino-3,6,9,12,15-pentaoxaheptadecanol (1.2 mmol, 1.2 equiv) was added. The reaction mixture was then heated to 90° C. and stirred for 2 h. The reaction solution was extracted with ethyl acetate and water, and the crude product was purified by silica gel column chromatography to obtain Compound F.

[0096] 4) Synthesis of compound G (NHS-active ester of CRBN ligand small molecule with connecting arms P4 and P6): Compound F (0.2 mmol, 1.0 equiv) and N,N'-disuccinimidyl carbonate (0.3 mmol, 1.5 equiv) were dissolved in anhydrous acetonitrile, and then triethylamine (0.6 mmol, 3.0 equiv) was added. The mixture was stirred at room temperature for 16 h, the solvent was evaporated, extracted with dichloromethane and water, washed with cold 5% sodium bicarbonate solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain products G1 and G2.

[0097] G1(P4-C):G1:1H NMR(400MHz,CDCl3)δ8.30(s,1H),7.50(t,J=8.5,7.2Hz,1H),7.11(d,

[0098] J=7.1Hz,1H),6.94(d,J=8.5Hz,1H),4.93(dd,J=12.0,5.2Hz,1H),4.47(m,2H),3.82–3.78(m,2H),3.75(t,J=5.3Hz,2H),3.70(d,J=4.8Hz,6H),3.52–3.47(m,2H),2.91(s,1H),2.85(s,4H),2.81–2.71(m,2H),2.16–2.10(m,1H). 13 C NMR(101MHz,CDCl3)δ171.23,169.26,168.71,168.42,167.67,151.68,146.87,136.04,132.52,129.61,116.83,115.31,111.61,110.27,70.85,70.75,70.72,70.34,69.48,48.87,42.42,31.41,25.48,22.74.ESI:[M+H] + cacld:591.1860,found:591.1874.[M+Na] + found:613.1691.

[0099] G2(P6-C): 1 H NMR(400MHz,CDCl3)δ8.30(s,1H),7.51(t,J=8.5,7.2Hz,1H),7.12(d,J=

[0100] 7.1Hz,1H),6.95(d,J=8.5Hz,1H),4.94(dd,J=11.9,5.4Hz,1H),4.51–4.44(m,2H),3.82–3.76(m,2H),3.75(t,J=5.3Hz,2H),3.69(d,J=3.7Hz,12H),3.49(t,J=5.3Hz,2H),2.93–2.87(m,1H),2.86(s,4H),2.81–2.72(m,2H),2.24–2.07(m,1H). 13C NMR (101MHz, CDCl3) δ171.06,169.46–169.26(m),168.59,168.29,167.64,151.64,146.85,136.02,132.57,129.65,120.73,116.79 ,115.28,111.64,70.85,70.71,70.54,70.53–70.50(m),70.28,69.45,68.31,53.41,48.89,42.44,31.42,25.48,22.83.ESI:[M+H] + cacld:679.2385,found:679.2640.[M+NH4] + found:696.2909.[M+Na] + found:701.2466.

[0101] 5) Synthesis of Compound I: 2-(2,6-dioxopiperidin-3-yl)-4-fluoroindoline-1,3-dione (1 mmol, 1.0 equiv) was dissolved in N,N-dimethylacetamide (2 mL), and N-Boc-1,6-diaminohexane (1.2 mmol, 1.2 equiv) was added. The reaction mixture was then heated to 90° C. and stirred for 2 h. The reaction solution was extracted with ethyl acetate and water, and the crude product was purified by silica gel column chromatography to obtain Compound I.

[0102] 6) Synthesis of Compound J: Compound I (1 mmol, 1.0 equiv) was dissolved in dichloromethane (4 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature for 6 h. The mixture was concentrated in vacuo to dryness to obtain Compound J.

[0103] 7) Synthesis of compound L: 6-hydroxyhexanoic acid (0.5 mmol, 1.2 equiv) or 10-hydroxydecanoic acid (0.5 mmol, 1.2 equiv) and HATU (0.63 mmol, 1.5 equiv) were dissolved in N,N-dimethylformamide (2 mL), compound J (0.42 mmol, 1.0 equiv) and N,N-diisopropylethylamine (1.26 mmol, 3 equiv) were added, and stirred at room temperature for 6 h. The mixture was extracted with ethyl acetate and water, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain compound L.

[0104] 8) Synthesis of compound M (NHS active ester of CRBN ligand small molecule with connecting arms of C12-1 and C16): Compound L (0.2 mmol, 1.0 equiv) and N,N'-disuccinimidyl carbonate (0.3 mmol, 1.5 equiv) were dissolved in anhydrous acetonitrile, and triethylamine (0.6 mmol, 3.0 equiv) was added to obtain a mixture. The mixture was stirred at room temperature for 16 h, the solvent was evaporated, and the mixture was extracted with dichloromethane and water, washed with cold 5% sodium bicarbonate solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain products M1 and M2.

[0105] M1(C12-C): 1 H NMR (400MHz, CDCl3) δ8.46 (s, 1H), 7.50 (t, J = 8.5, 7.2Hz, 1H), 7.09 (d, J = 7.0

[0106] Hz,1H),6.89(d,J=8.5Hz,1H),5.81(s,1H),4.93(dd,J=11.9,5.4Hz,1H),4.33(t,J=6.4Hz,2H),3.32–3.18(m,4H),2.94–2.85(m,1H),2.84 (s,4H),2.78(m,2H),2.19(t,J=7.4Hz,2H),2.16–2.09(m,1H),1.82–1.73(m,2H),1.73–1.62(m,5H),1.55–1.50(m,2H),1.48–1.34(m,6H). 13 C NMR (101MHz, CDCl3) δ172.79,171.24,169.54,168.87,168.55,167.64,151.58,146.98,136.16,132.49,116.69,1 11.41,109.87,71.38,48.89,42.53,39.39,36.37,31.41,29.51,29.07,28.07,26.57,25.49,25.14,22.80.[M+H] + cacld:628.2540,found:628.2828.[M+Na] + found:650.2661.

[0107] M2(C16-C): 1 H NMR (400MHz, CDCl3) δ8.42 (s, 1H), 7.50 (t, J = 8.4, 7.2Hz, 1H), 7.09 (d, J = 7.1

[0108] Hz,1H),6.89(d,J=8.7Hz,1H),5.80(s,1H),4.92(dd,J=11.8,5.4Hz,1H),4 .32(t,J=6.6Hz,2H),3.27(t,J=7.0Hz,4H),2.88(m,1H),2.85(s,4H),2.83– 2.70(m,2H),2.19(t,J=7.6Hz,2H),2.15–2.10(m,1H),1.79–1.70(m,2H),1 .70–1.59(m,4H),1.57–1.50(m,2H),1.44(m,2H),1.39(m,4H),1.30(s,8H). 13 C NMR (101MHz, CDCl3) δ173.47,171.21,169.54,168.85,168.51,167.64,151.62,146.97,136.16,132.49,129.51,116.68,115.39,111 .42,109.88,71.66,48.89,42.54,39.41,36.69,31.41,29.54,29.16,29.11,28.93,28.32,26.59,25.78,25.49,25.34,22.80.[M+H] + cacld:684.3166,found:684.3467.[M+Na] + found:706.3293.

[0109] Example 3 Synthesis and identification of VHL ligand small molecules with different linker lengths

[0110] The synthetic route is as follows:

[0111]

[0112] 1) Synthesis of compound 3: N-Boc-trans-4-hydroxy-L-proline methyl ester (1.2 mmol, 1.2 equiv) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 mmol, 1.5 equiv) were dissolved in N,N-dimethylformamide (3 mL), and 4-(4-methylthiazol-5-yl)benzylamine hydrochloride (1.0 mmol, 1.0 equiv) and N,N-diisopropylethylamine (3 mmol, 3 equiv) were added, and stirred at room temperature for 12 h. The mixture was extracted with ethyl acetate and water, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain compound 3.

[0113] 2) Synthesis of Compound 4: Compound 3 (0.84 mmol, 1.0 equiv) was dissolved in dichloromethane (4 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature for 6 h. The mixture was concentrated in vacuo to dryness to obtain Compound 4.

[0114] 3) Synthesis of compound 6: N-Boc-L-tert-leucine (1 mmol, 1.2 equiv) and HATU (1.5 mmol, 1.5 equiv) were dissolved in N,N-dimethylformamide (3 mL), and compound 4 (0.84 mmol, 1.0 equiv) and N,N-diisopropylethylamine (3 mmol, 3 equiv) were added, and stirred at room temperature for 12 h. The mixture was extracted with ethyl acetate and water, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain compound 6.

[0115] 4) Synthesis of Compound 7: Compound 6 (0.84 mmol, 1.0 equiv) was dissolved in dichloromethane (4 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature for 6 h. The mixture was concentrated in vacuo to dryness to obtain Compound 7.

[0116] 5) Synthesis of compound 9: Compound 8 (1 mmol, 1.2 equiv) and HATU (1.5 mmol, 1.5 equiv) were dissolved in N,N-dimethylformamide (3 mL), and compound 7 (0.84 mmol, 1.0 equiv) and N,N-diisopropylethylamine (3 mmol, 3 equiv) were added, and stirred at room temperature for 12 h. The mixture was extracted with ethyl acetate and water, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain compound 9.

[0117] 6) Synthesis of Compound 10: Compound 9 (0.84 mmol, 1.0 equiv) was dissolved in dichloromethane (4 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature for 6 h. The mixture was concentrated in vacuo to dryness to obtain Compound 10.

[0118] 7) Synthesis of compound 11 (VHL ligand NHS active ester with connecting arms C5, C8 and C12-2): Compound 10 (0.09 mmol, 1.0 equiv) and N-hydroxysuccinimide (0.14 mmol, 1.5 equiv) were dissolved in dichloromethane (2 mL), and 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (0.16 mmol, 1.8 equiv) was added, and stirred at room temperature for 12 h. The mixture was washed with water and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain compound 11-1, compound 11-2, and compound 11-3.

[0119] 11-1(C5-V): 1H NMR(400MHz,CD3CN)δ8.77(s,1H),7.48–7.39(m,4H),7.28(s,1H),6.75(d,J=8.8Hz,1H),4.58(d,J=8.9Hz,1H),4.54–4.42(m,3H),4.33(dd,J=15.5,5.7Hz,1H),3.86(d,J=11.0Hz,1H),3.70(d,J=11.0Hz,1H),3.44(s,1H),2.79(s,4H),2.49(s,3H),2.41–2.29(m,2H),2.25(s,4H),2.15(s,2H),0.98(s,9H). 13 C NMR(101MHz,CD3CN)δ171.82,171.70,170.83,170.26,168.88,150.74,148.37,139.30,130.47,129.10,127.70,69.80,59.09,57.22,56.56,54.23,42.18,37.36,35.02,33.76,29.74,28.74,25.76,25.41,20.66,15.43.[M+H] + cacld:642.2519,found:642.2651.[M+Na] + found:664.2474.

[0120] 11-2(C8-V): 1 H NMR(400MHz,CDCl3)δ8.73(s,1H),7.41–7.30(m,4H),6.34(d,J=8.9Hz,1H),4.69(t,J=8.1Hz,1H),4.62–4.51(m,3H),4.36(dd,J=15.0,5.3Hz,1H),4.11(d,J=11.4Hz,1H),3.64(dd,J=11.4,3.4Hz,1H),2.80(s,4H),2.59(t,J=7.2Hz,2H),2.54(s,3H),2.51–2.41(m,1H),2.30–2.22(m,2H),2.17(dd,J=13.5,8.0Hz,1H),1.79–1.69(m,2H),1.68–1.58(m,2H),1.46–1.38(m,2H),1.37–1.27(m,3H),0.97(s,9H). 13C NMR(101MHz,CDCl3)δ173.98,171.79,171.04,169.55,168.74,156.13,150.52,138.20,130.81,129.59,128.15,120.31,115.39,70.10,58.68,57.50,56.86,43.27,36.15,35.84,34.99,30.79,28.03,27.90,26.39,25.61,25.01,24.37,15.90.[M+H] + cacld:684.2989,found:684.2056.[M+Na] + found:706.2908.

[0121] 11-3(C12-V): 1 H NMR(400MHz,CDCl3)δ8.86(s,1H),7.39(s,4H),6.12(d,J=8.7Hz,1H),4.74(t,J=7.9Hz,1H),4.68-4.48(m,3H),4.37(dd,J=15.0,5.2Hz,1H),4.14(d,J=11.3Hz,1H),3.62(dd,J=11.4,3.6Hz,2H),2.85(s,4H),2.61(t,J=7.5Hz,2H),2.58(s,3H),2.57-2.53(m,1H),2.21(t,J=7.6Hz,2H),2.17-2.08(m,1H),1.81-1.71(m,2H),1.61(s,2H),1.40(t,J=7.8Hz,2H),1.29(s,10H),0.96(s,9H). 13 CNMR(101MHz,CDCl3)δ174.05,171.78,171.01,169.38,168.76,156.39,150.62,138.19,129.57,128.12,120.08,115.42,70.03,58.79,57.45,56.85,43.23,36.52,36.10,35.14,30.91,29.27,29.19,28.97,28.70,26.41,25.61,24.52,15.89.[M+H] + cacld:740.3615,found:740.3734.[M+Na] + found:762.3556.

[0122] Example 4 Coupling of nucleic acids and CRBN / VHL ligand small molecules with different linker lengths, characterization, and effects on the expression level of Lin28A / B

[0123]

[0124] First, the oligonucleotide sequence ORN-C7-NH2 containing C7-NH2 at the 3' end was synthesized by a solid phase synthesizer, and then ORN-C7-NH2 (10.0nmol, 1.0equiv) was dissolved in 50μL 1×PBS, and the NHS active ester (5.0μmol, 500equiv) of the CRBN / VHL ligand small molecule with different lengths of connecting arms synthesized in Example 2 and Example 3 was dissolved in 50μL dimethyl sulfoxide, and then the two systems were mixed and shaken at room temperature for 12h. After dialysis with a 1000D dialysis bag and purification by HPLC, the coupled oligonucleotide-based PROTAC molecule was obtained. The molecular structure composition, mass spectrometry characterization and expression levels of Lin28A / B at 48h and 100nM concentrations of each PROTAC are shown in Tables 2 and 3:

[0125] Table 2 Molecular structure composition and mass spectrometry characterization of each PROTAC

[0126]

[0127]

[0128] Table 3 Molecular structure composition and mass spectrometry characterization of each PROTAC

[0129]

[0130]

[0131] Experimental Example 1 Targeted degradation of Lin28A protein and subsequent biological activity evaluation experiment

[0132] 1 Experimental evaluation of the effect of targeted degradation of Lin28A

[0133] 1.1 Experimental methods

[0134] Cell culture and transfection: For the target Lin28A protein, T47D (human breast ductal carcinoma) cells were selected for cell-level activity evaluation. 2x10 5Cells were cultured to a cell density of 70-80% in an incubator at 37°C and 5% CO2 overnight. Lipofectamine 2000 was used for transfection. According to the instructions, Lipofectamine 2000 was diluted with OptiMEM medium and incubated for 5 minutes; Lin28-PROTAC molecules and controls were taken out at a dosage of 100nM per well and diluted with Opti-MEM; Lipofectamine 2000 was added to the Lin28-PROTAC molecule solution and the control molecule solution, blown evenly, incubated for 10 minutes to form a transfection complex; fresh culture medium was replaced in the 12-well plate, and the transfection complex was added and incubated for 48 hours.

[0135] For the time effect, the transfection concentration of Lin28-PROTAC molecules and controls was 100 nM, and the transfection time was 12 h, 24 h, 48 h, and 72 h; for the concentration effect, the transfection concentration of Lin28-PROTAC molecules and controls was 25 nM, 50 nM, 100 nM, and 200 nM, and the transfection time was 48 h;

[0136] Lin28A protein expression level detection: After culturing for an appropriate time, digest the cells and centrifuge them, add RIPA lysis buffer and lyse for 30 minutes. Centrifuge at 4°C and 12000 rpm for 15 minutes. Perform protein quantification according to the instructions of the BCA kit, prepare the required protein concentration, add SDS-PAGE loading buffer, and boil at 100°C for 5 minutes. Store at -20°C.

[0137] Prepare SDS-PAGE gel of appropriate concentration, centrifuge the prepared protein sample, mix well, and load it into the SDS-PAGE gel loading hole. The voltage is 90V, and the electrophoresis is performed for 15min until the protein sample runs into the separation gel, and then the voltage is adjusted to 120V to continue the electrophoresis. After the appropriate time, the electrophoresis is terminated. After the electrophoresis is completed, remove the gel and transfer the membrane at 300mA constant flow on ice for 60min. Take out the NC membrane and block it with 5% skim milk powder. Wash it 3 times with TBST buffer, then add the primary antibody at a moderate dilution ratio and incubate it at 4℃ overnight. Wash it 3 times with TBST buffer, oscillating for 5min each time. Discard the TBST buffer, add the diluted secondary antibody, and oscillate on a shaker at room temperature for 1h. Discard the secondary antibody, wash it 3 times with TBST buffer, oscillating for 5min each time. Finally, expose and image it with the Odyssey dual-color infrared laser imaging system.

[0138] Ubiquitination effect detection: 5x10 cells were seeded per 6 cm dish. 5Cells were transfected with His-ub plasmid after 12 hours of cell attachment. After 24 hours of continuous culture, Full-S ORN and Full-S P4-C samples were transfected and cultured for another 24 hours. 20μM MG132 was added 4 hours before harvesting cells. Digested and centrifuged cells, washed with 1xPBS, added with 500μL denaturing lysis buffer B (8M urea, 100mM NaH2PO4, 10mM Tris, 25mM Imidazole, pH=8.0), lysed on ice for 15 minutes, and lysed with ultrasonic disruptor. After lysis, centrifuged at 4°C and 12000rpm for 15 minutes. The supernatant was taken and quantified with BCA kit.

[0139] Take 150μL of nickel beads and wash them twice with PBS, centrifuge them at 4℃ and 550g for 3min each time, remove the supernatant, wash them twice with denaturing lysis buffer B, centrifuge them at 4℃ and 550g for 3min each time and add 150μL of denaturing lysis buffer B. Add the equally divided nickel beads to the quantified protein sample and incubate them on a shaker at 4℃ for 4-6h. After incubation, centrifuge them at 4℃ and 550g for 4min, remove the supernatant, wash them three times with lysis buffer C (8M urea, 100mM NaH2PO4, 10mM Tris, 25mM Imidazole, pH=6.3), centrifuge them at 4℃ and 550g for 3min each time. Finally, use solution E (8M urea, 100mM NaH2PO 4 , 10mM Tris, 250mM Imidazole, pH=4) for two times, 25ul each time, centrifuge at 4℃, 550g for 10min. Then add 10μL 5xloading buffer and boil at 100℃ for 5min. Then perform WB operation according to the experimental operation in 1.1.2.

[0140] 2 Experimental results

[0141] In T47D cells, the degradation effect of Lin28A was evaluated as follows:

[0142] For all sequences that can bind to Lin28A protein, after coupling with pomalidomide small molecules through PEG4 linker, except Let-7a-3P4-C (Let-7a-3Full-S P4-C) and Let-7e P4-C (Let-7e Full-S P4-C), all showed obvious degradation activity; among them, Let-7b P4-C (Let-7b Full-S P4-C), Let-7c P4-C (Let-7cFull-S P4-C), and Let-7f-1P4-C (Let-7f-1Full-S P4-C) had the most obvious effects. The Let-7f-1 sequence (SEQ ID No. 13) was selected for subsequent screening of linker arm length and E3 ubiquitin ligase ligands ( Figure 1 a).

[0143] At 48h and 100nM, except for Full-S C8-V (Let-7f-1Full-S C8-V), the remaining samples showed obvious degradation activity on Lin28A protein, among which Full-S P4-C (Let-7f-1Full-S P4-C) and Full-S C12-V (Let-7f-1Full-S C12-V) showed the best activity ( Figure 2 a). For CSD-S sequences that can only bind to the CSD region, CSD-S C3-C (Let-7f-1CSD-SC3-C), CSD-S C6-C (Let-7f-1CSD-S C6-C), CSD-S C16-C (Let-7f-1CSD-S C16-C), and CSD-SC12-V (Let-7f-1CSD-S C12-V) showed obvious degradation activity against Lin28A protein ( Figure 2 b). For the ZKD-S sequence that can only bind to the ZKD region, all samples showed no obvious degradation activity against Lin28A protein ( Figure 2 c). Then, the Full-SP4-C sample (Let-7f-1Full-SP4-C) was selected to evaluate the time effect and concentration effect of Lin28A protein degradation. Under 48h conditions, Full-SP4-C had a significant concentration effect, and the maximum degradation effect was achieved at 50nM ( Figure 2 d). At a transfection concentration of 100 nM, Full-SP4-C achieved the maximum degradation effect at 12 h ( Figure 2e). At the same time, the degradation effect of Lin28A in the experimental group Full-S P4-C was better than that in the Full-S ORN experimental group without Pomalidomide and the Full-ORN and Pomalidomide mixed group, and the experimental group with only Pomalidomide added did not show obvious degradation of Lin28A protein, indicating that the degradation of Lin28A mediated by Full-S P4-C was a specific degradation mediated by the coupling of Full-S ORN and Pomalidomide, rather than a non-specific degradation mediated by a single part ( Figure 2 f). Ubiquitination experiments showed that Full-SP4-C treatment increased the polyubiquitination of Lin28A protein in T47D cells. At the same time, the addition of protease inhibitor MG132 greatly weakened the degradation effect of Full-SP4-C on Lin28A, indicating that Full-SP4-C mediates the degradation of Lin28A through the proteasome pathway.

[0144] 2 Experiment on changes in miRNA let-7 levels after Full-SP4-C mediated Lin28A degradation

[0145] 2.1 Experimental methods

[0146] Cell culture and transfection: 1x10 cells were seeded per well in a 24-well plate. 5 T47D cells were cultured overnight at 37°C in an incubator containing 5% CO2 to increase the cell density to 70%-80%. Transfection was the same as in 1.1.

[0147] Detection of miRNA let-7 expression level:

[0148] Extraction of miRNA: Lyse the cells with lysis buffer MZ. After 5 minutes, transfer to a 1.5mL centrifuge tube, let stand at room temperature for 5 minutes, add 40μL chloroform and shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 4℃ and 12000rpm for 15 minutes, and transfer the top aqueous phase to a new centrifuge tube. Slowly add 0.43 times the volume of the transfer buffer and mix well. Transfer the obtained solution and precipitate to the adsorption column miRspin, centrifuge at 4℃ and 12000rpm for 30 seconds, and retain the effluent. Slowly add 0.75 times the volume of the effluent and mix well. Transfer the obtained solution and precipitate to the adsorption column miRelute, centrifuge at 4℃ and 12000rpm for 30 seconds, discard the effluent after centrifugation, and retain the miRspin adsorption column. Add 500μL of deproteinized solution MRD to the adsorption column miRelute, let stand at room temperature for 2 minutes, centrifuge at 4℃ and 12000rpm for 30 seconds, and discard the waste liquid. Add 500μL of rinse solution RW, let stand at room temperature for 2min, centrifuge at 4℃, 12000rpm for 30s, and discard the waste liquid. Repeat this operation twice. Place the adsorption column miRelute in a 2mL collection tube, centrifuge at 4℃, 12000rpm for 1min, remove the residual liquid, and let it stand at room temperature for a while. Transfer the adsorption column miRelute to a new 1.5mL centrifuge tube, add 15-30μL RNase-Free ddH2O, let stand at room temperature for 2min, and centrifuge at 4℃, 12000rpm for 2min.

[0149] Reverse transcription experiment: After the extracted miRNA is quantified, it is prepared into a 40ng / μL solution. Add miRNA and reverse transcription reagent (Total RNA: 8μL; 2x miRNA RT Reaction Buffer: 10μL; miRNA RT Enzyme Mix: 2μL) into an RNase Free centrifuge tube. Then perform reverse transcription according to the reverse transcription program (42℃60min; 95℃, 3min).

[0150] RT-PCR detection: The primers used are shown in Table 4. Add cDNA and RT-PCR reagents (2x miRcute Plus miRNA PreMix: 10μL; 2μM Forward Primer: 2μL; 2μM Reverse Primer: 2μL; miRNA first-strand cDNA: 6μL) to a pre-cooled RNase Free centrifuge tube on ice. The RT-PCR program is: 95℃15min, 1 cycle; 94℃20s, 60℃34s, 40 cycles.

[0151] Table 4 RT-PCR detection primers

[0152]

[0153] 2.2 Experimental Results

[0154] miRNA Let-7 expression levels Figure 3 As shown in the figure, with the degradation of Lin28A protein, the expression levels of all mature let-7 proteins were upregulated by more than 1.5 times, and the expression level of miR-21 did not change, indicating that Full-SP4-C targeted degradation of Lin28A specifically caused an increase in the expression level of Let-7 ( Figure 3 a). Under 48h conditions, as the concentration of Full-SP4-C increased, the expression level of Let-7g gradually increased, and reached the highest level at 100nM ( Figure 3 b) At a transfection concentration of 100 nM, as the incubation time increased, by 48 h, the level of let-7g increased significantly, and as time went on, let-7g also increased slightly ( Figure 3 c). Under the same transfection concentration and action time, the effect of the experimental group Full-S P4-C on the increase of let-7g level was better than that of the Full-S ORN experimental group without pomalidomide and the Full-ORN and pomalidomide mixed group, and the experimental group with only pomalidomide added did not show a significant increase in let-7g level ( Figure 3 d).

[0155] 3Inhibitory effect of Full-SP4-C combined with tamoxifen on T47D cell proliferation

[0156] 3.1 Experimental methods

[0157] MTT assay procedure: Inoculate 1.5x10 4 Cells / well (volume 100uL), and cultured overnight in an incubator at 37°C with 5% CO2. Transfection was the same as before, and cells were cultured for 24h, 48h, and 72h, respectively, and 10μL MTT solution (5mg / mL) was added to each well. Continue to culture for 4h, and carefully discard the culture supernatant in the well. Add 150μL DMSO to each well, shake for 10min, and monitor the absorbance of each well at a wavelength of 490nm.

[0158] For MTT assays with tamoxifen: seed 5x10 cells / well in a 6-well plate 5 The volume of each well was 2 mL, and the cells were cultured overnight in an incubator at 37°C with 5% CO2. The transfection was the same as before. After 12 hours of culture, the cells in the six-well plate were digested and centrifuged, and each well was re-counted. 1.5x10 4Cells, 100 μL per well, were cultured overnight in an incubator at 37°C with 5% CO2. Tamoxifen at different concentrations was added and culture was continued for 48 hours. After the cells were cultured for the appropriate time, MTT solution (5 mg / mL) was added to each well. After 4 hours of culture, the culture supernatant in the well was carefully discarded. 150 μL DMSO was added to each well and shaken for 10 minutes. The absorbance of each well was monitored at a wavelength of 490 nm.

[0159] 3.2 Experimental Results

[0160] 100nM Full-S P4-C (Let-7f-1Full-S P4-C) significantly inhibited the proliferation of T47D cells, and the inhibitory effect became more obvious as time went by. Its effect was stronger than that of Full-S ORN ( Figure 4 a).

[0161] At a concentration of 100 nM, the survival rate of T47D cells in the group of Full-SP4-C (Let-7f-1Full-SP4-C) administered alone was close to 40%. At 25 μM, the survival rate of T47D cells in the group of tamoxifen administered alone was close to 40%. The combination of Full-SP4-C (Let-7f-1Full-SP4-C) and tamoxifen significantly enhanced the inhibitory effect on T47D cell proliferation, and the cell survival rate was about 10% ( Figure 4 b).

[0162] 4Inhibitory effect of Full-S P4-C combined with tamoxifen on T47D cell colony formation

[0163] 4.1 Experimental methods

[0164] 5x10 cells were seeded per well in a 6-well plate. 5 Cells were added in a volume of 2 mL per well and cultured overnight in an incubator at 37°C with 5% CO2. Full-SP4-C (Let-7f-1Full-SP4-C) and Full-S ORN were transfected with Lipofectamine 2000. For wells where tamoxifen was administered alone and wells where tamoxifen and Full-SP4-C (Let-7f-1Full-SP4-C) were administered in combination, 25 μM tamoxifen was added after 24 hours of culture and culture was continued for 24 hours. The cells in the six-well plate were digested and centrifuged, and each well was recounted. Then, 1000 cells were inoculated per well in the 6-well plate and cultured for three weeks. The medium was changed every three days and the cell status was observed.

[0165] 4.2 Experimental Results

[0166] Consistent with the MTT experiment, 100 nM Full-S P4-C (Let-7f-1Full-S P4-C) significantly inhibited the colony formation ability of T47D cells, while the inhibition efficiency of Full-S ORN without pomalidomide was relatively low ( Figure 4 d).

[0167] Compared with the single-dose group, the combined use of Full-SP4-C (Let-7f-1Full-SP4-C) and tamoxifen further enhanced the inhibition of T47D cell clonal colony formation. The combined administration of tamoxifen increased the inhibition of T47D cell clonal colony formation by about 5-6 times compared with single-dose group ( Figure 4 d).

[0168] 5Full-S P4-C combined with tamoxifen to induce apoptosis of T47D cells

[0169] 5.1 Experimental Methods

[0170] Cell apoptosis experiment operation process: 2x10 cells were inoculated in each well of a 12-well plate. 5 Cells were cultured overnight at 37°C in an incubator containing 5% CO2. Transfection was performed according to 1.1.1. After 24 hours of culture, different concentrations of tamoxifen were added and culture was continued for 48 hours. All cells were collected with 0.25% trypsin, washed with PBS, and tested with Annexin V-FITC apoptosis detection kit: 195 μL Binding Buffer was added to resuspend the cells; 5 μL Annexin V-FITC was added and mixed; then 10 μL PI was added and mixed gently; protected from light and placed at room temperature for 15 minutes. Cell apoptosis was detected by flow cytometry.

[0171] 5.2 Experimental Results

[0172] At a concentration of 100 nM, the efficiency of the Full-SP4-C (Let-7f-1Full-SP4-C) group in inducing apoptosis of T47D cells was significantly better than that of the Full-S ORN group. The efficiency of the tamoxifen group, which was administered alone, at a concentration of 25 nM, inducing apoptosis of T47D cells was comparable to that of the Full-SP4-C (Let-7f-1Full-SP4-C) group, and the apoptosis rate of T47D cells was about 30%. The combination of Full-SP4-C (Let-7f-1Full-SP4-C) and tamoxifen significantly increased the apoptosis of T47D cells, with an apoptosis rate of about 70% ( Figure 4 e).

[0173] 6Full-S P4-C combined with tamoxifen induces cell cycle arrest in T47D cells

[0174] 6.1 Experimental methods

[0175] Cell cycle experiment operation process: 2x10 cells were seeded per well in a 12-well plate. 5 Cells were cultured overnight at 37°C in an incubator containing 5% CO2. Transfection was performed according to 1.1.1. After 24 hours of culture, different concentrations of tamoxifen were added and culture was continued for 48 hours. All cells were collected with 0.25% trypsin, washed with PBS, fixed with pre-cooled 70% ethanol at 4°C overnight, and then washed once with pre-cooled PBS at 4°C. Cell cycle detection kit was used for detection: 500 μL staining buffer, 25 μL propidium iodide staining solution, and 10 μL RNaseA were added, and the cell pellet was slowly and fully resuspended, and incubated at 37°C in the dark for 30 minutes. Cell cycle was detected by flow cytometry.

[0176] 6.2 Experimental Results

[0177] At a concentration of 100 nM, in the group where Full-SP4-C (Let-7f-1Full-SP4-C) was administered alone, the number of T47D cells in the G0 / G1 phase increased slightly, from 71% to 76%, while the number of cells in the G2 / M phase decreased slightly, from 10% to 5%. In the group where tamoxifen was administered alone, the number of T47D cells in the G0 / G1 phase and the number of cells in the G2 / M phase were the same as those in the group where Full-SP4-C (Let-7f-1Full-SP4-C) was administered alone. After combined administration, the number of T47D cells in the G0 / G1 phase increased to 81%, while the number of cells in the G2 / M phase decreased to less than 1%. Therefore, the combination of Full-SP4-C (Let-7f-1Full-SP4-C) and tamoxifen can effectively block T47D cells in the G0 / G1 phase ( Figure 4 c).

[0178] 7.Full-SP4-C combined with tamoxifen inhibits T47D cell migration

[0179] 7.1 Experimental Methods

[0180] 5x10 cells were seeded per well in a 6-well plate. 5 T47D cells were cultured overnight in a 37°C, 5% CO2 incubator. Transfection was performed according to 1.1.1. Culture was performed for 12 hours. Culture-Insert was placed in a six-well plate, and the cells were digested and centrifuged to prepare 5x10 5Cells / mL of cell suspension were added to each of the Culture-Insert 2 wells, and the cells were incubated at 37°C and 5% CO2 for 24h. The Culture-Insert 2 wells were gently removed with sterile tweezers, the cell layer was washed with cell-free culture medium or PBS to remove cell debris and non-attached cells, 2mL of fresh culture medium was added (the culture medium containing tamoxifen was added to the tamoxifen experimental group), and the culture was continued for an appropriate time. Cell images were taken with an automatic inverted fluorescence microscope at 0h, 12h, 24h, and 48h, and the results were analyzed using Image J software.

[0181] 7.2 Experimental Results

[0182] At a concentration of 100 nM, the Full-SP4-C (Let-7f-1Full-SP4-C) group had a significantly better ability to inhibit T47D cell migration than the Full-SORN group. The tamoxifen group, which was administered alone, had an ability to inhibit T47D cell migration comparable to that of the Full-SP4-C (Let-7f-1Full-SP4-C) group at a concentration of 25 μM, with a T47D cell migration rate of about 20%. The combination of Full-SP4-C (Let-7f-1Full-SP4-C) and tamoxifen significantly increased the inhibition of T47D cell migration, with the migration rate reduced to 0 ( Figure 4 f).

[0183] Experimental Example 2 Targeted degradation of Lin28B protein in H1299 cells and subsequent biological activity evaluation experiment

[0184] 1 Experiment to evaluate the degradation effect of Lin28B protein

[0185] 1.1 Experimental methods

[0186] Cell culture and transfection: Cell culture and transfection were the same as those in Experimental Example 1, except that the transfection reagent was replaced with Lipofectamine3000.

[0187] Lin28B protein expression level detection and ubiquitination effect detection: The experimental operation of Lin28B protein expression level detection and ubiquitination effect detection is the same as the relevant content in Experimental Example 1.

[0188] 1.2 Experimental Results

[0189] In H1299 cells, the degradation effect of Lin28B was evaluated as follows:

[0190] For all sequences that can bind to Lin28B protein, after coupling with pomalidomide small molecules through PEG4 linker, Let-7b P4-C (Let-7b Full-S P4-C), Let-7d P4-C (Let-7d Full-S P4-C), Let-7f-1 P4-C (Let-7f-1 Full-S P4-C), Let-7g P4-C (Let-7g Full-S P4-C), Let-7i P4-C (Let-7i Full-S P4-C) all showed comparable degradation activity. The Let-7f-1 sequence (SEQ ID No. 13) was selected for subsequent screening of linker arm length and E3 ubiquitin ligase ligands ( Figure 1 b).

[0191] For the Full-S sequence that can bind to both the CSD region and the ZKD region, Full-SP4-C showed a significant degradation effect at 48 h and 100 nM. For the CSD-S sequence that can only bind to the CSD region and the ZKD-S sequence that can only bind to the ZKD region, all samples did not show significant degradation activity against the Lin28B protein ( Figure 5 a, 5b, 5c). Under 48h conditions, Full-SP4-C reached the maximum degradation effect at 100nM ( Figure 5 d). At a transfection concentration of 100 nM, Full-SP4-C achieved the maximum degradation effect at 24 h ( Figure 5 e). At the same time, the degradation effect of Lin28B in the experimental group Full-SP4-C was better than that in the Full-SORN experimental group without pomalidomide and the Full-ORN and pomalidomide mixed group, and the experimental group with only pomalidomide added did not show obvious degradation of Lin28B protein, indicating that the degradation of Lin28B mediated by Full-SP4-C was a specific degradation mediated by the coupling of Full-S ORN and pomalidomide, rather than a non-specific degradation mediated by a single part ( Figure 5 f). Ubiquitination experiments showed that Full-SP4-C (Let-7f-1Full-SP4-C) treatment effectively increased the polyubiquitination of Lin28B protein in H1299 cells. The addition of protease inhibitor MG132 greatly weakened the degradation effect of Lin28B, indicating that the degradation of Lin28B protein mediated by Full-SP4-C is through the proteasome pathway.

[0192] 2 Experiment on the changes of miRNAlet-7 levels after Full-SP4-C mediated Lin28B degradation

[0193] 2.1 Experimental methods

[0194] Cell seeding: 6 x 10 cells per well in a 24-well plate 4 H1299 cells were cultured overnight at 37°C in an incubator containing 5% CO2 to a cell density of 70%-80%. Transfection was performed in the same manner as in 1.1.1 of Experimental Example 1 above.

[0195] miRNA let-7 expression detection: The experimental operation of let-7 expression level detection is the same as the relevant content in Experimental Example 1.

[0196] 2.2 Experimental Results

[0197] miRNA Let-7 expression levels Figure 3 As shown in the figure, with the degradation of Lin28B protein, the expression levels of all mature let-7 were upregulated by about 1.7 times, and the expression level of mirR-21 did not change, indicating that Full-SP4-C (Let-7f-1Full-SP4-C) targeted degradation of Lin28B specifically caused the increase of Let-7 expression level ( Figure 3 e). Under 48h conditions, as the concentration of Full-SP4-C increased, the expression level of Let-7g gradually increased, and reached the highest level at 100nM ( Figure 3 f). At a transfection concentration of 100 nM, as the incubation time increased, the level of let-7g slightly increased at 24 h, and at 48 h, the level of let-7g increased significantly to reach the maximum level ( Figure 3 g). Under the same transfection concentration and action time, the effect of the experimental group Full-S P4-C on the increase of let-7g level was better than that of the Full-S ORN experimental group without pomalidomide and the Full-ORN and pomalidomide mixed group, and the experimental group with only pomalidomide added did not show a significant increase in let-7g level ( Figure 3 h).

[0198] 3 Inhibition experiment on H1299 cell proliferation and cell clone colony formation

[0199] 3.1 Experimental Methods The MTT and colony formation assays were performed in the same manner as in Experimental Example 1.

[0200] 3.2 Experimental Results

[0201] 100nM Full-SP4-C (Let-7f-1Full-SP4-C) inhibited the proliferation of H1299 cells, and its effect was stronger than that of Full-S ORN ( Figure 6 a).

[0202] Consistent with the MTT experiment, 100 nM Full-S P4-C (Let-7f-1Full-S P4-C) significantly inhibited the colony formation ability of H1299 cells, while the inhibition efficiency of Full-S ORN without pomalidomide was relatively low ( Figure 6 b).

[0203] 4 Inhibition experiment on H1299 cell migration and invasion ability

[0204] 4.1 Experimental methods

[0205] Experimental steps: 4x10 cells were inoculated per well in a 6-well plate. 5 Cells were added to each well with a volume of 2 mL, and cultured overnight in an incubator at 37°C with 5% CO2. Transfection was performed according to 1.2 and cultured for 48 hours. Serum-free culture medium and matrix gel were diluted 1:5, mixed, added to the upper chamber of the 24-Transwell chamber (100uL per well), placed in a 37°C incubator, and incubated for 1-2 hours. After the above transfection and cultured for 48 hours, the above cells were digested and counted to prepare a cell suspension (100,000 / mL). 200μL of cell suspension was added to each well, and 500μL of culture medium containing 10% FBS was added to the lower chamber. Incubated in a 37°C incubator for 24 hours. Washed twice with PBS, fixed with paraformaldehyde, and stained with 0.1% crystal violet staining solution. Washed twice with PBS, wiped the upper surface cells with a cotton ball, observed under a microscope and photographed. No matrix gel was added to the cell migration experiment, and the rest of the operation steps were the same as the cell invasion experiment.

[0206] 4.2 Experimental Results

[0207] 100nM Full-SP4-C (Let-7f-1Full-SP4-C) significantly inhibited the migration and invasion of H1299 cells, and was superior to the inhibitory effect of Full-S ORN ( Figure 6 c,6d).

[0208] 5Full-SP4-C combined with paclitaxel to induce apoptosis in H1299 cells

[0209] 5.1 Experimental Methods The apoptosis experiment was performed in the same manner as in Experimental Example 1.

[0210] 5.2 Experimental Results

[0211] At a concentration of 100 nM, the efficiency of Full-SP4-C (Let-7f-1Full-SP4-C) in inducing apoptosis of H1299 cells was significantly better than that of Full-S ORN. The efficiency of paclitaxel alone in inducing apoptosis of H1299 cells at a concentration of 50 μM was about 10%. The combination of Full-SP4-C (Let-7f-1Full-SP4-C) and paclitaxel significantly increased the apoptosis of H1299 cells, with the apoptosis rate reaching about 30% ( Figure 6 e).

[0212] Experimental Example 3 Targeted degradation of Lin28B protein in Huh7 cells and subsequent biological activity evaluation experiment

[0213] 1 Experiment to evaluate the degradation effect of Lin28B protein

[0214] 1.1 Experimental methods

[0215] Cell culture and transfection: Cell culture and transfection were the same as those in Experimental Example 1, except that the transfection reagent was replaced with Lipofectamine3000.

[0216] Lin28B protein expression level detection and ubiquitination effect detection: The experimental operation of Lin28B protein expression level detection and ubiquitination effect detection is the same as the relevant content in Experimental Example 1.

[0217] 1.2 Experimental Results

[0218] In Huh7 cells, the degradation effect of Lin28B was evaluated as follows:

[0219] For all sequences that can bind to Lin28B protein, after coupling the pomalidomide small molecule through the PEG4 linker, all PROTAC molecules showed different degrees of degradation activity. Among them, Let-7b P4-C (Let-7b Full-S P4-C) and Let-7f-1P4-C (Let-7f-1Full-S P4-C) had the best degradation activity, which could degrade the expression level of Lin28B to less than 10%. The Let-7b sequence (SEQ ID No.7) was selected for subsequent screening of linker arm length and E3 ubiquitin ligase ligands ( Figure 1 c).

[0220] At 48h and 100nM, PROTAC molecules prepared with sequences that can simultaneously bind to the CSD region and ZKD region of Lin28B protein all showed significant degradation activity against Lin28B protein, among which let-7b Full-S C12-C, let-7b Full-SP4-C and let-7b Full-S P6-C had the best activity, which could degrade the expression level of Lin28B to about 10% ( Figure 7 a). For sequences that can only bind to the CSD region of Lin28B protein, let-7b CSD-S C3-C, let-7bCSD-S C6-C, let-7b CSD-S C12-C, let-7b CSD-S C16-C, let-7b CSD-S P4-C and let-7bCSD-S P6-C can effectively degrade Lin28B and its expression level can be degraded to about 10%-20% ( Figure 7 b). For sequences that can only bind to the ZKD region of Lin28B protein, all samples showed no obvious degradation activity on Lin28B protein ( Figure 7 c).

[0221] Then let-7b Full-S P6-C was selected to evaluate the time and concentration effects. Under 48h conditions, let-7b Full-S P6-C molecules had a significant degradation effect on Lin28B at a concentration of 25nM, and the maximum degradation effect was achieved at 100nM ( Figure 7 d). At a transfection concentration of 100 nM, let-7b Full-SP6-C molecules significantly degraded Lin28B at 8 h, and maintained its efficient degradation activity until 48 h ( Figure 7 e). At the same time, the degradation effect of let-7b Full-S P6-C on Lin28B was better than that of the Full-S ORN experimental group without pomalidomide and the Full-S ORN and pomalidomide mixed group, and the experimental group with only pomalidomide did not show obvious degradation of Lin28B protein, indicating that the degradation of Lin28B mediated by let-7b Full-S P6-C was a specific degradation mediated by Full-S ORN and pomalidomide after coupling, rather than a non-specific degradation mediated by a single part ( Figure 7f). Ubiquitination experiments showed that let-7b Full-SP6-C treatment increased the polyubiquitination of Lin28B protein in Huh-7 cells. At the same time, the addition of protease inhibitor MG132 greatly weakened the degradation effect of let-7b Full-SP6-C on Lin28B, indicating that let-7b Full-SP6-C mediates the degradation of Lin28B through the proteasome pathway.

[0222] Experimental Example 4 Evaluation of PROTAC molecules’ anti-tumor activity in vivo

[0223] 1. Experimental Methods

[0224] This experiment selected 6-week-old BALB / c female nude mice with a body weight of 16-20g. They were kept in a barrier environment and ordered one week in advance to adapt them to the experimental environment and maintain adequate food and water, as well as a normal life rhythm. Normally proliferating T47D cells were digested with 0.25% trypsin at 37°C for 3 minutes, serum-containing medium was added to terminate the digestion, and the cells were washed with 1×PBS. The cells were resuspended in serum-free 1640 medium, and the same amount of matrix gel as the medium was added. The cells were evenly mixed and injected subcutaneously with 1×10 7 To stimulate tumor growth, 0.1 ml of β-estradiol 17-valerate (5 mg / ml, dissolved in sesame oil) was injected subcutaneously in the scapular region of the mice, and the growth of the tumor was observed regularly. When the tumor volume reached 50 mm 3 At about 1:10 p.m., T47D mice were randomly divided into 5 groups. The first group was injected intratumorally with 0.9% saline. The second group was injected intratumorally with Full-S ORN (6nmol / mouse). The third group was injected intratumorally with Full-S P4-C (Let-7f-1Full-S P4-C) (6nmol / mouse). The fourth group was injected intragastricly with tamoxifen (5mg / kg, dissolved in corn oil). The fifth group was injected intratumorally with Full-S P4-C (Let-7f-1Full-S P4-C) (6nmol / mouse) and intragastricly with tamoxifen (5mg / kg). The nucleic acid was administered 8 times on days 1, 4, 7, 10, 13, 16, 19, and 22, respectively, and the tamoxifen was administered 8 times on days 2, 5, 8, 11, 14, 17, 20, and 23, respectively. During the experiment, the body weight of nude mice was monitored every three days, and the tumor size was monitored by measuring the large and small diameters of the tumors, and using the formula 1 / 2 × a × b 2Tumor volume was calculated. Two days after the last administration, the tumor was removed and photographed and weighed. Western Blot technology was then used to characterize the level of Lin28A protein in the tumor. Paraffin sections of the tumor were made and the tumor tissue sections were immunohistochemically stained to characterize the distribution of Lin28A protein at the tumor tissue level. This experiment also used hematoxylin-eosin staining to stain tissue sections and analyze the pathological conditions in the tumor tissue.

[0225] 2. Experimental results

[0226] During the administration period, there was no significant change in body weight between the several administration groups and the NC group, and the nude mice in each group were in normal growth and living conditions, indicating that PROTAC molecules Full-SP4-C (Let-7f-1Full-SP4-C) and tamoxifen had no obvious toxicity ( Figure 8 a). The growth of breast cancer tumors in the Full-S ORN group was not significantly different from that in the NC group. Both Full-S P4-C (Let-7f-1Full-SP4-C) and tamoxifen administered alone could effectively inhibit the growth of breast cancer tumors. Compared with single administration, combined administration could further inhibit the growth of breast cancer tumors, and the tumors tended to regress ( Figure 8 b). By dissecting and weighing breast cancer tumors, it was found that the tumor weight of the Full-S ORN group was similar to that of the NC group, the tumor weight of the Full-S P4-C (Let-7f-1Full-S P4-C) and tamoxifen groups administered alone was significantly smaller than that of the NC group, and the tumor weight of the combined administration group was smaller than that of the single administration group ( Figure 8 d, 8e). Compared with the NC group, the weights of heart, liver, spleen, lung and kidney did not change significantly in the several drug-treated groups ( Figure 8 c), further demonstrating that Full-SP4-C (Let-7f-1Full-SP4-C) and tamoxifen have no obvious toxicity. WB results showed that Full-SP4-C (Let-7f-1Full-SP4-C) can effectively degrade Lin28A protein in breast cancer tumor tissues ( Figure 8 f). H&E staining results showed that the NC group and the Full-S ORN group showed typical tumor tissue morphology. The Full-SP4-C (Let-7f-1Full-SP4-C) and tamoxifen groups administered alone showed obvious cell death and nuclear condensation phenotypes, and the cancer cell density was much lower than that of the NC group and the Full-S ORN group. The cancer cell tissue density of the combined Full-SP4-C (Let-7f-1Full-SP4-C) and tamoxifen group was much lower than that of the single-drug group.

[0227] TUNEL staining results showed that no obvious apoptosis fluorescence signals were observed in the NC group and the Full-S ORN group. Obvious apoptosis fluorescence signals were observed in the Full-S P4-C (Let-7f-1Full-S P4-C) and tamoxifen groups administered alone, while a large area of ​​apoptosis fluorescence signals appeared in the combined administration group. IHC results showed that both the NC group and the Full-S ORN group showed a strong positive phenotype of Lin28A. The Lin28A positivity of the Full-S P4-C (Let-7f-1Full-S P4-C) group administered alone was significantly weakened. Although the tamoxifen group administered alone showed changes in tissue morphology, it still maintained a strong Lin28A positive phenotype. The positive phenotype of the combined administration group was further weakened, which was consistent with the Western Blot results of tumor tissue proteins ( Figure 8 g). The above results collectively indicate that the PROTAC molecule Full-SP4-C (Let-7f-1Full-SP4-C) can effectively inhibit the growth of breast cancer tumors, and can further exert a powerful synergistic anti-tumor effect when combined with tamoxifen.

Claims

1. An oligonucleotide PROTAC molecule targeting degradation of Lin28A / B protein, characterized in that: The oligonucleotide PROTAC molecule is obtained by coupling an oligonucleotide sequence that can bind to the Lin28A / B protein and a ligand that can promote the ubiquitination of the Lin28A / B protein through a connecting arm.

2. The oligonucleotide PROTAC molecule targeting degradation of Lin28A / B protein according to claim 1, characterized in that The ligand capable of promoting the ubiquitination of Lin28A / B protein is pomalidomide, VHL ligand VH032 or their derivative analogs; Wherein, the chemical structural formula of pomalidomide is shown in the following formula I: The structural formula of the VHL ligand VH032 is shown in the following formula II: The structural formula of the derivative analogue is shown in Formula III:

3. The oligonucleotide PROTAC molecule targeting degradation of Lin28A / B protein according to claim 1, characterized in that The oligonucleotide sequence capable of binding to the Lin28A / B protein is an oligonucleotide sequence capable of binding to both the CSD region and the ZKD region of the Lin28A / B protein, or an oligonucleotide sequence that only binds to the CSD region of the Lin28A / B protein, or an oligonucleotide sequence that only binds to the ZKD region of the Lin28A / B protein; preferably, the backbone of the oligonucleotide sequence is thio-modified or modified or substituted with different nucleotide analogs, including but not limited to the following modified nucleoside bases: 5-methylcytosine, 6-methyladenine, pseudouracil or inosine; or a nucleotide or locked nucleotide with methoxy, methylethoxy or fluoro modification on the 2' position of the sugar ring.

4. The oligonucleotide PROTAC molecule targeting degradation of Lin28A / B protein according to claim 3, characterized in that The oligonucleotide sequence capable of simultaneously binding to the CSD region and the ZKD region of the Lin28A / B protein is selected from any one of the nucleotide sequences described in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13, SEQ ID No.15, SEQ ID No.17, SEQ ID No.19, SEQ ID No.21 or SEQ ID No.23; the oligonucleotide sequence that can only bind to the CSD region of the Lin28A / B protein is selected from SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18, SEQ ID No.20, SEQ ID No.22 or SEQ ID No.24; or a nucleotide sequence having more than 80% homology with the above-mentioned nucleotide sequence, and the nucleotide sequence still has the function or activity of binding to Lin28A / B protein; The oligonucleotide sequence that binds only to the ZKD region of the Lin28A / B protein is selected from any one of the nucleotide sequences described in (1)-(12) below: (1):5'-GGGAGAU-3'; (2):5'-GGGAGAU-3'; (3):5'-AUGGGAU-3'; (4):5'-GGAAGAU-3'; (5):5'-GGGAGUU-3'; (6):5'-AGGAGGU-3'; ( 7):5'-AGGAGAU-3'; (8):5'-AGGAGAU-3'; (9):5'-UGGAGAU-3'; (10):5'-AGGAGAU-3'; (11):5'-UGGAGAU-3'; (12):5'-AGAAGAU-3'; Preferably, any of the above oligonucleotide sequences is subjected to backbone thiolation modification.

5. The oligonucleotide PROTAC molecule targeting degradation of Lin28A / B protein according to any one of claims 1 to 4, characterized in that: A ligand molecule targeting tumor cells is connected to the 5' end of the oligonucleotide nucleic acid sequence; the ligand molecule includes but is not limited to folic acid, polypeptide or nucleic acid aptamer.

6. The oligonucleotide PROTAC molecule targeting degradation of Lin28A / B protein according to claim 1, characterized in that The connecting arm is selected from one or more of the following groups: alkylene, alkoxy, alkylamino, alkylthio, amide, aminocarbonate bond or triazole derivatives; Preferably, the linker arm is selected from any one of the following compounds of the general formula: Wherein, m or n is any integer from 0 to 10; More preferably, the connecting arm includes but is not limited to any one of C3, C5, C6, C8, C12-1, C12-2, C16, P4 or P6; wherein the structural formula of C3 is shown in Formula IV, the structural formula of C5 is shown in Formula V, the structural formula of C6 is shown in Formula VI, the structural formula of C8 is shown in Formula VII, the structural formula of C12-1 is shown in Formula VIII, the structural formula of C12-2 is shown in Formula IX, the structural formula of C16 is shown in Formula X, the structural formula of P4 is shown in Formula XI, and the structural formula of P6 is shown in Formula XII:

7. A method for preparing an oligonucleotide PROTAC molecule for targeting degradation of Lin28A / B protein according to any one of claims 1 to 4, characterized in that: include: (1) connecting a CRBN ligand or a VHL ligand to a linker of different types or lengths and then performing an activation reaction to obtain an NHS active ester of the CRBN or VHL ligand connected to the linker; (2) coupling the oligonucleotide sequence with an amino group at the 3' end that can bind to Lin28A / B with the NHS active ester of the CRBN or VHL ligand connected with a linker obtained in step (1) to obtain.

8. The method according to claim 7, characterized in that In step (2), the oligonucleotide sequence with an amino group at the 3' end that can bind to Lin28A / B is dissolved in PBS buffer to obtain a mixture 1; the NHS active ester of the CRBN or VHL ligand connected with a linker is dissolved in dimethyl sulfoxide to obtain a mixture 2; the mixture 1 and the mixture 2 are mixed for coupling reaction, and the reaction product is purified to obtain.

9. Use of the oligonucleotide PROTAC molecule targeting the degradation of Lin28A / B protein according to any one of claims 1 to 4, or its combination with a small molecule anti-tumor drug in the preparation of a drug or preparation targeting the degradation of Lin28A / B protein; preferably, the small molecule anti-tumor drug is tamoxifen or paclitaxel.

10. The oligonucleotide PROTAC molecule targeting degradation of Lin28A / B protein according to any one of claims 1-4, or its use in combination with a small molecule or large molecule protein antibody anti-tumor drug in the preparation of an anti-tumor drug or preparation; preferably, the tumor cells of the tumor have overexpression of Lin28A / B protein; the small molecule anti-tumor drug is tamoxifen or paclitaxel.

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