Oligonucleotide-based protac molecule for targeted degradation of lin28a / b and use thereof

Abstract

Disclosed in the present invention are an oligonucleotide PROTAC molecule based on the targeted degradation of Lin28A / B protein, and the use thereof. The oligonucleotide PROTAC molecule of the present invention is prepared by coupling an oligonucleotide sequence capable of binding to Lin28A / B with a ligand that promotes the ubiquitination of Lin28A / B protein via a linking arms of different types or different lengths. In different tumor cells, the oligonucleotide PROTAC molecule can be used for the targeted degradation of Lin28A / B protein, has a significant degradation activity on Lin28A / B protein, and exerts an anti-tumor function. The present invention has application prospects in the preparation of a drug or formulation for the targeted degradation of Lin28A / B protein, or the preparation of an anti-tumor drug or formulation, and the preparation of an anti-tumor drug combined with a small molecule or large molecule.

Description

Oligonucleotide-based PROTAC molecules targeting degradation of Lin28A / B and their applications Technical Field

[0001] The present invention relates to protein degradation targeting chimeras (PROTACs) and applications thereof, and in particular to oligonucleotide PROTAC molecules for targeted degradation of Lin28A / B proteins and applications thereof in the preparation of drugs, preparations for targeted degradation of Lin28A / B proteins or for anti-tumor use, or in combination with anti-tumor drugs, belonging to the field of protein degradation targeting chimeras and applications thereof. Background Art

[0002] Lin 28 is a highly conserved RNA-binding protein with two RNA-binding domains (a cold-shock domain and a CCHC zinc-finger domain). It was originally identified as a key regulator of developmental timing in nematodes. In mammals, Lin 28 (Lin28A and Lin28B) maintains stemness and is abundantly expressed in embryonic cells, promoting rapid and extensive cell proliferation. They play important roles in embryogenesis, myogenesis, germ cell development, and gliogenesis. Accumulating evidence suggests that Lin 28 may also be a master regulator of ES cell pluripotency. Together with OCT4, SOX2, and NANOG ("reprogramming factors"), LIN 28 can reprogram somatic cells into induced pluripotent stem cells. LIN 28 binds to mRNAs, regulating their stability and translation. Furthermore, Lin 28 can bind to the terminal loop of the miRNA let-7 precursor, thereby preventing let-7 from being processed into its mature form. In human tumors, Lin 28 has been shown to be overexpressed in a variety of malignant tumors, significantly promoting tumor cell proliferation, migration, and drug resistance. It acts as an oncogene in malignant transformation and tumor progression and is closely associated with poor prognosis in tumor treatment. Lin 28 has two isoforms, 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 vary across tumor types, but both isoforms promote tumor cell proliferation, migration, and drug resistance. Therefore, inhibiting Lin 28 expression is a novel 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 degradation targeting chimera (PROTAC) is a heterobifunctional molecule composed of 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 chemotherapy drugs hinders clinical application and leads to disease recurrence. With the progress of research on 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 has not solved 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 function. Therefore, 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. Because they have catalytic effects, only low doses of PROTACs are needed to achieve good results. Based on PROTACs, not only can the target protein be deeply degraded, with sustained and rapid and powerful inhibition of downstream signals, but also the occurrence and development of tumors can be suppressed for a long time in vitro, making the use of protein hydrolysis-targeted chimeras to degrade proteins a hot topic in current tumor treatment.

[0004] Summary of the Invention

[0005] One of the purposes of the present invention is to provide oligonucleotide PROTAC molecules that target and degrade Lin28A / B proteins;

[0006] A second object 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.

[0007] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0008] 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 linker; the coupling method can be to obtain a Lin28A / B binding oligonucleotide sequence by solid phase synthesis and modify the 3' end with an amino group, and then cross-link the oligonucleotide sequence with an NHS active ester of a CRBN or VHL ligand connected to a different linker through the amino group to obtain the oligonucleotide PROTAC molecule.

[0009] In a preferred 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 No. 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 nucleotide sequences, and still having the function or activity of binding to Lin28A / B protein.

[0010] The oligonucleotide sequence that only binds to the ZKD region of the Lin28A / B protein is selected from any one of the nucleotide sequences described in (1)-(12) below:

[0011] (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'.

[0012] Further preferably, any of the above-mentioned nucleotide sequences can be fully thiolated; 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 is obtained by modifying the 2' position of the sugar ring with methoxy, methylethoxy, fluorination, etc.

[0013] The ligand capable of recruiting E3 ubiquitin ligase to promote target protein ubiquitination 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:

[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 Formula II below:

[0015] The structural formula of the derivative analogue described in the present invention is shown in Formula III:

[0016] 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;

[0017] Preferably, the linker is selected from any one of the following compounds of the general formula:

[0018] Wherein, m or n is any integer from 0 to 10.

[0019] 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:

[0020] 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:

[0021] (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;

[0022] (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 the linker obtained in step (1) to obtain.

[0023] 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 mixture 1; the NHS active ester of the CRBN or VHL ligand connected to the linker is dissolved in dimethyl sulfoxide to obtain a mixture 2; the mixture 1 and the mixture 2 are mixed to perform a coupling reaction, and the reaction product is purified to obtain.

[0024] As a preferred embodiment, in step (2), the concentration 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 is 1:500.

[0025] The oligonucleotide PROTAC molecules targeted for degradation of Lin28A / B proteins provided by the present invention have 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.

[0026] According to the experimental results of the oligonucleotide PROTAC-mediated degradation of Lin28A / B or let-7 levels in miRNA, it can be seen that with the degradation of Lin28 protein, the expression levels of all mature let-7 are upregulated, and the expression level of miR-21 does not change, 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 levels is better than that of the Full-S ORN experimental group without pomalidomide and the Full-ORN mixed group with pomalidomide, and the experimental group with only pomalidomide added did not show a significant increase in let-7g levels.

[0027] 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. The targeting gene includes but is not limited to folic acid, polypeptides and nucleic acid aptamers.

[0028] 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.

[0029] Therefore, the oligonucleotide PROTAC provided by the present invention has the following uses:

[0030] (1) preparing drugs or preparations for targeted degradation of Lin28A / B proteins;

[0031] (2) A drug or preparation for preparing an anti-tumor drug; wherein Lin28A / B is overexpressed in the tumor cells;

[0032] (3) Combination use with anti-tumor small molecule drugs for anti-tumor use; wherein the anti-tumor small molecule drugs can be tamoxifen or paclitaxel, etc.

[0033] The present invention couples an oligonucleotide sequence capable of binding to Lin28A / B and a ligand capable of promoting ubiquitination of Lin28A / B proteins through connecting arms of different types or lengths to obtain an oligonucleotide PROTAC molecule; such oligonucleotide PROTAC molecules are used to selectively degrade Lin28A and / or Lin28B proteins in different tumor cells and exert anti-tumor effects; the present invention has application prospects in the preparation of drugs and preparations for targeted degradation of Lin28A / B proteins, or in 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

[0034] Figure 1 shows the degradation of Lin28A / B by PROTAC molecules made from different miRNA sequences targeting Lin28; a) Lin28A (T47D cells), b) Lin28B (H1299 cells), c) Lin28B (Huh7 cells).

[0035] Figure 2 shows the degradation of Lin28A protein in T47D cells based on the Let-7f-1 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.

[0036] Figure 3 shows the changes in Let-7 expression levels in T47D (ad) / H1299 (ef) cells after degradation of Lin28A / B protein by Let-7f-1 Full-SP4-C; a) Evaluation of the upregulation effect of all Let-7 in T47D cells, b) concentration dependence, c) time dependence, d) specificity; e) Evaluation of the upregulation effect of all Let-7 in H1299 cells, f) concentration dependence, g) time dependence, and h) specificity.

[0037] Figure 4 shows the anti-tumor activity of Let-7f-1 Full-SP4-C in T47D cells; a) cell proliferation, b) combined administration, c) cell cycle, d) colony formation, e) cell apoptosis, and f) cell migration.

[0038] Figure 5 shows the degradation of Lin28B protein in H1299 cells based on the Let-7f-1 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.

[0039] Figure 6 shows the anti-tumor activity of Let-7f-1 Full-SP4-C in H1299 cells; a) cell proliferation, b) colony formation, c) cell migration, d) cell invasion, and e) cell apoptosis.

[0040] Figure 7 shows the degradation of Lin28B protein in Huh7 cells based on the 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.

[0041] Figure 8 is the in vivo anti-tumor activity evaluation of the PROTAC molecule Let-7f-1 Full-S P4-C; a) mouse body weight changes, b) tumor volume growth curve, c) mouse normal tissue weight, d) mouse tumor anatomy, e) tumor weight, f) Lin28A expression in the tumor, g) tumor tissue section. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. 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, and such modifications and replacements fall within the scope of protection of the present invention.

[0043] Example 1 Design of oligonucleotide sequences capable of binding to Lin28A / B

[0044] The sequences of the oligonucleotides that can bind to Lin28A / B are as follows:

[0045] Let-7a-1 Full-S:5'-UUAGGUCACACCCACCACUGGGAGAU-3'(SEQ ID No.1)

[0046] Let-7a-1 CSD-S:5'-UUAGGUCACACCCACCACUG-3'(SEQ ID No.2)

[0047] Let-7a-2 Full-S:5'-UAGAAUUACAUCAAGGGAGAU-3'(SEQ ID No.3)

[0048] Let-7a-2 CSD-S:5'-UAGAAUUACAUCAAG-3'(SEQ ID No.4)

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

[0050] Let-7a-3 CSD-S:5'-UGGGGCUCUGCCCUGCUA-3'(SEQ ID No.6)

[0051] Let-7b Full-S:5'-UCAGGGCAGUGAUGUUGCCCCUCGGAAGAU-3'(SEQ ID No.7)

[0052] Let-7b CSD-S:5’-UCAGGGCAGUGAUGUUGCCCCUCG-3’(SEQ ID No.8)

[0053] Let-7c Full-S:5’-UAGAGUUACACCCUGGGAGUU-3’(SEQ ID No.9)

[0054] Let-7c CSD-S:5’-UAGAGUUACACCCUG-3’(SEQ ID No.10)

[0055] Let-7d Full-S:5’-UUAGGGCAGGGAUUUUGCCCACAAGGAGGU-3’(SEQ ID No.11)

[0056] Let-7d CSD-S:5’-UUAGGGCAGGGAUUUUGCCCACAA-3’(SEQ ID No.12)

[0057] Let-7e Full-S:5’-GAGGAGGACACCCAAGGAGAU-3’(SEQ ID No.13)

[0058] Let-7e CSD-S:5’-GAGGAGGACACCCAA-3’(SEQ ID No.14)

[0059] Let-7f-1 Full-S:5’-GUGGGGUAGUGAUUUUACCCUGUUCAGGAGAU-3’(SEQ ID No.15)

[0060] Let-7f-1 CSD-S:5’-GUGGGGUAGUGAUUUUACCCUGUUCA-3’(SEQ ID No.16)

[0061] Let-7f-2 Full-S:5’-UUAGGGUCAUACCCCAUCUUGGAGAU-3’(SEQ ID No.17)

[0062] Let-7f-2 CSD-S:5’-UUAGGGUCAUACCCCAUCUU-3’(SEQ ID No.18)

[0063] Let-7g Full-S:5’-UGAGGGUCUAUGAUACCACCCGGUACAGGAGAU-3’(SEQ ID No.19) <h2 style=";text-align:left;direction:ltr">

[0064] <h2 style=";text-align:left;direction:ltr"> Let-7g CSD-S:5'-UGAGGGUCUAUGAUACCACCCGGUACA-3'(SEQ ID No.20)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0065] <h2 style=";text-align:left;direction:ltr"> Let-7i Full-S:5'-GGUCGGGUUGUGACAUUGCCCGCUGUGGAGAU-3'(SEQ ID No.21)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0066] <h2 style=";text-align:left;direction:ltr"> Let-7i CSD-S:5'-GGUCGGGUUGUGACAUUGCCCGCUGU-3'(SEQ ID No.22)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0067] <h2 style=";text-align:left;direction:ltr"> miR98 Full-S:5'-GUGGGGGUAGGGAUAUUAGGCCCAAUUAGAAGAU-3'(SEQ ID No.23)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0068] <h2 style=";text-align:left;direction:ltr"> miR98 CSD-S:5'-GUGGGGGUAGGGAUAUUAGGCCCAAUU-3'(SEQ ID No.24)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0069] <h2 style=";text-align:left;direction:ltr"> Let-7a-1 ZKD-S:5'-GGGAGAU-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0070] <h2 style=";text-align:left;direction:ltr"> Let-7a-2 ZKD-S:5'-GGGAGAU-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0071] <h2 style=";text-align:left;direction:ltr"> Let-7a-3 ZKD-S:5'-AUGGGAU-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0072] <h2 style=";text-align:left;direction:ltr"> Let-7b ZKD-S:5'-GGAAGAU-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0073] <h2 style=";text-align:left;direction:ltr"> Let-7c ZKD-S:5'-GGGAGUU-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0074] <h2 style=";text-align:left;direction:ltr"> Let-7d ZKD-S:5'-AGGAGGU-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0075] <h2 style=";text-align:left;direction:ltr"> Let-7e ZKD-S:5'-AGGAGAU-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0076] <h2 style=";text-align:left;direction:ltr"> Let-7f-1 ZKD-S:5'-AGGAGAU-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0077] <h2 style=";text-align:left;direction:ltr"> Let-7f-2 ZKD-S:5'-UGGAGAU-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0078] <h2 style=";text-align:left;direction:ltr"> Let-7g ZKD-S:5'-AGGAGAU-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0079] Let-7i ZKD-S:5'-UGGAGAU-3'

[0080] miR98 ZKD-S:5′-AGAAGAU-3′;

[0081] Among them, the above oligonucleotide sequences can be fully thiolated, that is, the non-bridging oxygen atoms in the original phosphate bonds are replaced with sulfur atoms.

[0082] Example 2 Synthesis and Characterization of CRBN Ligand Small Molecules (Pomalidomide) with Different Linker Lengths

[0083] The synthetic route is as follows:

[0084] 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 mixture was extracted with ethyl acetate and water, and the crude product was purified by silica gel column chromatography to obtain Compound C.

[0085] 2) Synthesis of Compound D (NHS-active ester of a small molecule CRBN ligand with linker 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, followed by the addition of triethylamine (0.6 mmol, 3.0 equiv). The mixture was stirred at room temperature for 16 h, the solvent was evaporated, and the mixture was extracted with dichloromethane and water. The mixture was washed with cold 5% sodium bicarbonate solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to provide compounds D1 and D2.

[0086] 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=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). 13C NMR(101MHz,CDCl3)δ171.27,169.55,168.54,167.66,146.98,136.13,132.50,116.70,111.43,109.88,62.72,48.88,42.59,32.52,31.42,29.15,26.72,25.48,22.81.ESI:[M+H] + cacld:473.1230,found:473.1303.[M+Na] + found:495.1122.

[0087] 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=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),2.95–2.88(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.

[0088] 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 mixture was extracted with ethyl acetate and water, and the crude product was purified by silica gel column chromatography to obtain Compound F.

[0089] 4) Synthesis of Compound G (NHS-active ester of a small molecule CRBN ligand with linker 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, followed by the addition of triethylamine (0.6 mmol, 3.0 equiv). The mixture was stirred at room temperature for 16 h, the solvent was evaporated, and the mixture was extracted with dichloromethane and water. The mixture was washed with cold 5% sodium bicarbonate solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give products G1 and G2.

[0090] G1(P4-C):G1:1H NMR (400MHz, CDCl3) δ8.30(s,1H),7.50(t,J=8.5,7.2Hz,1H),7.11(d,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.

[0091] 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 = 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=5.3 Hz,2H),2.93–2.87(m,1H),2.86(s,4H),2.81–2.72(m,2H),2.24–2.07(m,1H). 13 C 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 8) Synthesis of Compound M (NHS-active ester of a small molecule CRBN ligand with C12-1 and C16 linker arms): 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. The mixture was 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.

[0096] 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=7.0Hz,1H),6.8 9(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.

[0097] 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=7.1Hz,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.

[0098] Example 3 Synthesis and Identification of VHL Ligand Small Molecules with Linker Arms of Different Lengths

[0099] The synthetic route is as follows:

[0100] 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). 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.

[0101] 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.

[0102] 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). 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.

[0103] 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.

[0104] 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). 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.

[0105] 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.

[0106] 7) Synthesis of Compound 11 (NHS-active ester of VHL ligand with linker 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). 1-Ethyl-3-(3-dimethylpropylamino)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 afford Compounds 11-1, 11-2, and 11-3.

[0107] 11-1(C5-V): 1 H 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=15.5,5.7 Hz,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.

[0108] 11-2(C8-V): 1H 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). 13 C 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.

[0109] 11-3(C12-V): 1H 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), 4.6 8-4.48(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),2.1 7-2.08(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.

[0110] Example 4 Coupling of Nucleic Acids and CRBN / VHL Ligand Small Molecules with Linker Arms of Different Lengths, Characterization, and Effects on Lin28A / B Expression Levels

[0111] First, an oligonucleotide sequence ORN-C7-NH2 containing C7-NH2 at the 3' end was synthesized by solid phase synthesizer. Then, ORN-C7-NH2 (10.0 nmol, 1.0 equiv) was dissolved in 50 μL 1× PBS. The NHS active ester of the CRBN / VHL ligand small molecule with different linker lengths synthesized in Examples 2 and 3 (5.0 μmol, 500 equiv) was dissolved in 50 μL dimethyl sulfoxide. The two systems were then mixed and shaken at room temperature for 12 hours. 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 48 hours and 100 nM of each PROTAC are shown in Tables 2 and 3:

[0112] Table 2 Molecular structure and mass spectrometry characterization of each PROTAC

[0113] Table 3 Molecular structure and mass spectrometry characterization of each PROTAC

[0114] Experimental Example 1 Targeted degradation of Lin28A protein and subsequent biological activity evaluation experiment

[0115] 1 Experimental evaluation of the effect of targeted degradation of Lin28A

[0116] 1.1 Experimental methods

[0117] Cell culture and transfection: For the target Lin28A protein, T47D (human breast ductal carcinoma) cells were selected for cell-level activity evaluation. 2x10 5 Cells were plated to a density of 70-80% and cultured overnight in a 37°C, 5% CO2 incubator. Lipofectamine 2000 was used for transfection. Following the instructions, Lipofectamine 2000 was diluted with OptiMEM medium and incubated for 5 minutes. Lin28-PROTAC molecules and controls were removed at a dose of 100 nM per well and diluted with Opti-MEM. Lipofectamine 2000 was added to the Lin28-PROTAC and control solutions, pipetting evenly and incubating for 10 minutes to form a transfection complex. Fresh medium was replaced in the 12-well plate, and the transfection complex was added and incubated for 48 hours.

[0118] 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.

[0119] Lin28A protein expression level assay: After incubation for the appropriate period, cells were digested and centrifuged, then lysed in RIPA buffer for 30 minutes. Centrifuged at 4°C, 12,000 rpm for 15 minutes. Protein was quantified according to the BCA assay kit instructions. The desired protein concentration was prepared, and SDS-PAGE loading buffer was added. The cells were boiled at 100°C for 5 minutes. Store at -20°C.

[0120] Prepare an SDS-PAGE gel of appropriate concentration, centrifuge the prepared protein sample, mix thoroughly, and load the sample into the SDS-PAGE gel loading well. Run the gel at 90V for 15 minutes, until the protein sample has run through the separating gel. Then adjust the voltage to 120V and continue electrophoresis. Terminate the electrophoresis after the appropriate time. After electrophoresis, remove the gel and transfer the membrane at a constant current of 300mA on ice for 60 minutes. Remove the NC membrane and block it with 5% skim milk powder. Wash three times with TBST buffer, then add the primary antibody at a moderate dilution and incubate at 4°C overnight. Wash three times with TBST buffer, shaking for 5 minutes each time. Discard the TBST buffer, add the diluted secondary antibody, and shake at room temperature for 1 hour. Discard the secondary antibody and wash three times with TBST buffer, shaking for 5 minutes each time. Finally, expose and image using the Odyssey dual-color infrared laser imaging system.

[0121] Ubiquitination effect detection: 5x10 cells were seeded per 6 cm dish. 5 After 12 hours, cells were transfected with the His-ub plasmid, and cultured for an additional 24 hours. Full-S ORN and Full-S P4-C samples were then transfected and cultured for an additional 24 hours. Four hours before harvesting, 20 μM MG132 was added. Cells were digested and centrifuged, washed with 1x PBS, and lysed on ice for 15 minutes using 500 μL of denaturing lysis buffer B (8 M urea, 100 mM NaH2PO4, 10 mM Tris, 25 mM Imidazole, pH 8.0). The cells were sonicated using an ultrasonic disruptor. After lysis, the cells were centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected and quantified using a BCA assay kit.

[0122] Wash 150 μL of nickel beads twice with PBS, centrifuging each time at 4°C, 550 g for 3 min. Remove the supernatant and wash twice with denaturing lysis buffer B, centrifuging each time at 4°C, 550 g for 3 min, and then add 150 μL of denaturing lysis buffer B. Add the aliquoted nickel beads to the quantified protein sample and incubate on a shaker at 4°C for 4-6 h. After incubation, centrifuge at 4°C, 550 g for 4 min, remove the supernatant, and wash three times with lysis buffer C (8 M urea, 100 mM NaH2PO4, 10 mM Tris, 25 mM Imidazole, pH = 6.3), centrifuging each time at 4°C, 550 g for 3 min. Finally, elute with buffer E (8 M urea, 100 mM NaH2PO4, 10 mM Tris, 250 mM Imidazole, pH = 4) twice, centrifuging each time at 4°C, 550 g for 10 min. Add 10 μL of 5x loading buffer and boil at 100°C for 5 min. Perform Western blotting according to the experimental procedures in 1.1.2.

[0123] 2 Experimental results

[0124] In T47D cells, the degradation effect of Lin28A was evaluated as follows:

[0125] All sequences capable of binding to the Lin28A protein, except Let-7a-3 P4-C (Let-7a-3 Full-S P4-C) and Let-7e P4-C (Let-7e Full-S P4-C), showed significant degradation activity after being conjugated to the pomalidomide small molecule via a PEG4 linker. The most pronounced effects were observed with Let-7b P4-C (Let-7b Full-S P4-C), Let-7c P4-C (Let-7c Full-S P4-C), and Let-7f-1 P4-C (Let-7f-1 Full-S P4-C). The Let-7f-1 sequence (SEQ ID No. 13) was selected for subsequent screening of linker lengths and E3 ubiquitin ligase ligands (Figure 1a).

[0126] At 48 h and 100 nM, except for Full-S C8-V (Let-7f-1 Full-S C8-V), all the Full-S sequences that can bind to both the CSD and ZKD regions showed significant degradation activity against Lin28A protein, among which Full-S P4-C (Let-7f-1 Full-S P4-C) and Full-S C12-V (Let-7f-1 Full-S C12-V) showed the best activities (Figure 2a). For CSD-S sequences that bind exclusively to the CSD region, CSD-S C3-C (Let-7f-1 CSD-S C3-C), CSD-S C6-C (Let-7f-1 CSD-S C6-C), CSD-S C16-C (Let-7f-1 CSD-S C16-C), and CSD-S C12-V (Let-7f-1 CSD-S C12-V) showed significant degradation activity against Lin28A (Figure 2b). However, for the ZKD-S sequence, which binds exclusively to the ZKD region, none of the samples showed significant degradation activity against Lin28A (Figure 2c). The Full-SP P4-C sample (Let-7f-1 Full-SP P4-C) was then used to evaluate the time- and concentration-dependent degradation of Lin28A. Full-SP P4-C showed a significant concentration-dependent degradation effect over 48 hours, reaching maximum degradation at 50 nM (Figure 2d). At a transfection concentration of 100 nM, Full-SP4-C achieved maximum degradation at 12 h (Figure 2e). Furthermore, the degradation effect of Full-SP4-C on Lin28A was superior to that of the Full-S ORN experimental group without pomalidomide and the Full-ORN and pomalidomide mixed group. Furthermore, the experimental group with pomalidomide alone showed no significant degradation of Lin28A protein, indicating that Full-SP4-C-mediated Lin28A degradation is a specific degradation mediated by the coupling of Full-S ORN and pomalidomide, rather than non-specific degradation mediated by a single component (Figure 2f). 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.

[0127] 2 Experimental study on changes in miRNA let-7 levels after Full-SP4-C mediated Lin28A degradation

[0128] 2.1 Experimental methods

[0129] Cell culture and transfection: 1x10 cells were seeded per well in a 24-well plate. 5 T47D cells were cultured overnight at a density of 70%-80% in a 37°C incubator containing 5% CO2. Transfection was performed as in 1.1.

[0130] Detection of miRNA let-7 expression level:

[0131] MiRNA Extraction: Lyse cells with Lysis Buffer MZ for 5 minutes, transfer to a 1.5 mL centrifuge tube, and incubate at room temperature for 5 minutes. Add 40 μL of chloroform and shake vigorously for 15 seconds. Allow to stand at room temperature for 5 minutes. Centrifuge at 12,000 rpm at 4°C 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 to the tube and mix thoroughly. Transfer the resulting solution and precipitate to a miRspin column and centrifuge at 12,000 rpm at 4°C for 30 seconds. Retain the flow-through. Slowly add 0.75 times the volume of the flow-through to the tube and mix thoroughly. Transfer the resulting solution and precipitate to a miRelute column and centrifuge at 12,000 rpm at 4°C for 30 seconds. Discard the flow-through and retain the miRspin column. Add 500 μL of Deproteinized Buffer MRD to the miRelute column, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm at 4°C for 30 seconds. Discard the flow-through and retain the miRspin column. Add 500 μL of Rinse Buffer (RW), let stand at room temperature for 2 minutes, centrifuge at 4°C, 12,000 rpm for 30 seconds, and discard the waste liquid. Repeat this procedure twice. Place the miRelute adsorption column in a 2 mL collection tube, centrifuge at 4°C, 12,000 rpm for 1 minute, remove any remaining liquid, and let stand at room temperature for a while. Transfer the miRelute adsorption column to a new 1.5 mL centrifuge tube, add 15-30 μL of RNase-Free ddH2O, let stand at room temperature for 2 minutes, and centrifuge at 4°C, 12,000 rpm for 2 minutes.

[0132] Reverse transcription: After quantifying the extracted miRNA, prepare a 40 ng / μL solution. Add the miRNA and reverse transcription reagents (Total RNA: 8 μL; 2x miRNA RT Reaction Buffer: 10 μL; miRNA RT Enzyme Mix: 2 μL) to an RNase-free centrifuge tube. Perform reverse transcription according to the protocol (42°C for 60 minutes; 95°C for 3 minutes).

[0133] RT-PCR assay: The primers used are listed in Table 4. Add cDNA and RT-PCR reagents (10 μL of 2x miRcute Plus miRNA PreMix, 2 μL of 2 μM Forward Primer, 2 μL of 2 μM Reverse Primer, and 6 μL of miRNA first-strand cDNA) to an ice-cold RNase-free microcentrifuge tube. The RT-PCR protocol was as follows: 1 cycle at 95°C for 15 min, followed by 40 cycles at 94°C for 20 s and 60°C for 34 s.

[0134] Table 4 RT-PCR primers

[0135] 2.2 Experimental Results

[0136] The expression levels of miRNA Let-7 are shown in Figure 3. Following the degradation of Lin28A, the expression levels of all mature let-7 proteins were upregulated by more than 1.5-fold, while the expression level of miR-21 remained unchanged, indicating that Full-SP4-C specifically increases Let-7 expression levels after targeted degradation of Lin28A (Figure 3a). Over 48 hours, as the concentration of Full-SP4-C increases, the expression level of Let-7g gradually increases, reaching its peak at 100 nM (Figure 3b). At a transfection concentration of 100 nM, let-7g levels increased significantly with prolonged incubation time, reaching a significant level by 48 hours, and continued to increase slightly over time (Figure 3c). At the same transfection concentration and exposure time, the effect of the experimental group Full-S P4-C on the increase of let-7g levels 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 levels (Figure 3d).

[0137] 3 Inhibitory effect of Full-S P4-C combined with tamoxifen on T47D cell proliferation

[0138] 3.1 Experimental methods

[0139] MTT assay procedure: 1.5 x 10 4 Cells were plated at 100 μL per well and cultured overnight in a 37°C, 5% CO2 incubator. Following transfection, cells were cultured for 24, 48, and 72 hours. 10 μL of MTT solution (5 mg / mL) was added to each well. Cultures were continued for another 4 hours, and the supernatant was carefully discarded. 150 μL of DMSO was then added to each well. The cells were shaken for 10 minutes, and the absorbance at 490 nm was monitored.

[0140] For MTT assays in combination with tamoxifen: 5 x 10 5 Cells were grown in a 2 mL volume per well at 37°C in a 5% CO2 incubator overnight. Transfection was the same as before. After 12 h of culture, the cells in the six-well plate were digested and centrifuged, and each well was re-counted. 1.5 x 10 cells were seeded into each well of a 96-well plate. 4 Cells were cultured overnight in a 37°C, 5% CO2 incubator at 100 μL per well. Tamoxifen was added at varying concentrations and incubated for 48 hours. After the appropriate incubation period, MTT solution (5 mg / mL) was added to each well. After 4 hours of incubation, the supernatant was carefully discarded. DMSO (150 μL) was added to each well and shaken for 10 minutes. The absorbance of each well was monitored at 490 nm.

[0141] 3.2 Experimental Results

[0142] 100 nM Full-S P4-C (Let-7f-1 Full-S P4-C) significantly inhibited the proliferation of T47D cells, and the inhibitory effect became more obvious with time, and its effect was stronger than that of Full-S ORN without pomalidomide ( Figure 4 a ).

[0143] At a concentration of 100 nM, the survival rate of T47D cells in the Full-SP4-C (Let-7f-1 Full-SP4-C) group administered alone was close to 40%. At 25 μM, the survival rate of T47D cells in the Tamoxifen group was close to 40%. The combination of Full-SP4-C (Let-7f-1 Full-SP4-C) and Tamoxifen significantly enhanced the inhibitory effect on T47D cell proliferation, with a cell survival rate of approximately 10% (Figure 4b).

[0144] 4 Inhibitory effect of Full-SP4-C combined with tamoxifen on T47D cell colony formation

[0145] 4.1 Experimental methods

[0146] 5x10 cells were seeded per well in a 6-well plate. 5Cells were plated in 2 mL per well and cultured overnight at 37°C in a 5% CO2 incubator. Full-S P4-C (Let-7f-1 Full-S P4-C) and Full-S ORNs were transfected using Lipofectamine 2000. For wells treated with tamoxifen alone and in combination with tamoxifen and Full-S P4-C (Let-7f-1 Full-S P4-C), 25 μM tamoxifen was added after 24 hours of culture and culture continued for another 24 hours. Cells in the six-well plates were digested and centrifuged, and each well was recounted. Then, 1,000 cells were seeded per well of a six-well plate and cultured for three weeks, with the medium changed every three days and the cells observed.

[0147] 4.2 Experimental Results

[0148] Consistent with the MTT assay, 100 nM Full-S P4-C (Let-7f-1 Full-S P4-C) significantly inhibited the colony formation ability of T47D cells, while the inhibitory efficiency of Full-S ORN without pomalidomide was relatively low ( Figure 4 d ).

[0149] Compared with either drug alone, the combined use of Full-SP4-C (Let-7f-1 Full-SP4-C) and tamoxifen further enhanced the inhibition of T47D cell colony formation. The combined treatment increased the inhibitory effect of Full-SP4-C (Let-7f-1 Full-SP4-C) and tamoxifen on T47D cell colony formation by approximately 5-6 times compared with either drug alone (Figure 4d).

[0150] 5 Experiment on apoptosis induction of T47D cells by combination of Full-S P4-C and tamoxifen

[0151] 5.1 Experimental methods

[0152] Apoptosis experiment operation process: 2x10 cells were seeded per well in a 12-well plate. 5 Cells were cultured overnight at 37°C in a 5% CO2 incubator to a cell density of 70-80%. Transfection was performed according to step 1.1.1. After 24 hours of culture, various concentrations of tamoxifen were added and cultured for an additional 48 hours. All cells were harvested with 0.25% trypsin, washed with PBS, and assayed using the Annexin V-FITC Cell Apoptosis Detection Kit: resuspend cells in 195 μL Binding Buffer; add 5 μL Annexin V-FITC and mix thoroughly; then add 10 μL PI and mix gently. Protect from light and incubate at room temperature for 15 minutes. Cell apoptosis was assayed by flow cytometry.

[0153] 5.2 Experimental Results

[0154] At a concentration of 100 nM, the Full-SP4-C (Let-7f-1 Full-SP4-C) group significantly outperformed the Full-S ORN group in inducing apoptosis in T47D cells. Tamoxifen alone, administered at a concentration of 25 nM, induced apoptosis in T47D cells at a comparable efficiency to the Full-SP4-C (Let-7f-1 Full-SP4-C) group, with an apoptosis rate of approximately 30%. The combination of Full-SP4-C (Let-7f-1 Full-SP4-C) and tamoxifen significantly increased apoptosis in T47D cells, with an apoptosis rate of approximately 70% (Figure 4e).

[0155] 6 Full-S P4-C combined with tamoxifen induces cell cycle arrest in T47D cells

[0156] 6.1 Experimental methods

[0157] Cell cycle experiment operation process: 2x10 cells were seeded per well in a 12-well plate. 5 Cells were cultured to a density of 70-80% and incubated overnight at 37°C in a 5% CO2 incubator. Transfection was performed according to step 1.1.1. After 24 hours of incubation, various concentrations of tamoxifen were added and incubation continued for 48 hours. All cells were harvested 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 analysis was performed using a cell cycle detection kit: 500 μL staining buffer, 25 μL propidium iodide staining solution, and 10 μL RNase A were added, and the cell pellet was slowly and thoroughly resuspended. Incubated at 37°C in the dark for 30 minutes. Cell cycle analysis was performed using flow cytometry.

[0158] 6.2 Experimental Results

[0159] At a concentration of 100 nM, the Full-SP4-C (Let-7f-1 Full-SP4-C) group, when administered alone, slightly increased the number of T47D cells in the G0 / G1 phase, from 71% to 76%, while the number of cells in the G2 / M phase slightly decreased, from 10% to 5%. In the tamoxifen group, the number of T47D cells in the G0 / G1 and G2 / M phases remained similar to that in the Full-SP4-C (Let-7f-1 Full-SP4-C) group. Following combined administration, the number of T47D cells in the G0 / G1 phase increased to 81%, while the number in the G2 / M phase decreased to less than 1%. Therefore, the combination of Full-SP4-C (Let-7f-1 Full-SP4-C) and tamoxifen effectively arrested T47D cells in the G0 / G1 phase (Figure 4c).

[0160] 7.Full-SP4-C combined with tamoxifen inhibits T47D cell migration

[0161] 7.1 Experimental Methods

[0162] 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 cultured for 12 hours. Culture-Insert was placed in a six-well plate, cells were digested and centrifuged to prepare 5x10 5 100 μL of a cell suspension (100 μL / mL) was added to each of the two wells of a Culture-Insert. The cells were incubated at 37°C and 5% CO2 for 24 hours. Gently remove the two wells of the Culture-Insert using sterile forceps, wash the cell layer with cell-free culture medium or PBS to remove cell debris and non-adherent cells, add 2 mL of fresh culture medium (in the tamoxifen group, culture medium containing tamoxifen was added), and continue incubation for the appropriate time. Cell images were captured using an automated inverted fluorescence microscope at 0, 12, 24, and 48 hours, and the results were analyzed using Image J software.

[0163] 7.2 Experimental Results

[0164] At a concentration of 100 nM, the Full-SP4-C (Let-7f-1 Full-SP4-C) group significantly inhibited T47D cell migration compared to the Full-SORN group. Tamoxifen alone, at a concentration of 25 μM, inhibited T47D cell migration comparable to that of the Full-SP4-C (Let-7f-1 Full-SP4-C) group, with a T47D cell migration rate of approximately 20%. The combination of Full-SP4-C (Let-7f-1 Full-SP4-C) and tamoxifen significantly increased the inhibition of T47D cell migration, reducing the migration rate to 0 (Figure 4f).

[0165] Experimental Example 2 Targeted degradation of Lin28B protein in H1299 cells and subsequent biological activity evaluation

[0166] 1 Experiment to evaluate the degradation effect of Lin28B protein

[0167] 1.1 Experimental methods

[0168] Cell culture and transfection: The cell culture and transfection were the same as those in Experimental Example 1, except that the transfection reagent was replaced with Lipofectamine 3000.

[0169] Lin28B protein expression level detection and ubiquitination effect detection: The experimental operations of Lin28B protein expression level detection and ubiquitination effect detection are the same as those in Experimental Example 1.

[0170] 1.2 Experimental Results

[0171] In H1299 cells, the degradation effect of Lin28B was evaluated as follows:

[0172] For all sequences that bind to the Lin28B protein, 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), and Let-7i P4-C (Let-7i Full-S P4-C) all showed comparable degradation activity after being conjugated to the pomalidomide small molecule via a PEG4 linker. The Let-7f-1 sequence (SEQ ID No. 13) was selected for subsequent screening of linker lengths and E3 ubiquitin ligase ligands (Figure 1b).

[0173] For the Full-S sequence, which binds to both the CSD and ZKD regions, Full-SP4-C showed significant degradation activity at 48 h and 100 nM. For the CSD-S sequence, which binds exclusively to the CSD region, and the ZKD-S sequence, which binds exclusively to the ZKD region, none of the samples showed significant degradation activity against Lin28B (Figures 5a, 5b, 5c). At 100 nM, Full-SP4-C achieved maximum degradation at 48 h (Figure 5d). At a transfection concentration of 100 nM, Full-SP4-C achieved maximum degradation at 24 h (Figure 5e). Furthermore, the degradation of Lin28B by Full-SP4-C was more effective than that achieved by the Full-SORN group without pomalidomide or the Full-ORN / pomalidomide combination. The pomalidomide-only group showed no significant degradation of Lin28B, indicating that Full-SP4-C-mediated Lin28B degradation is a specific degradation mediated by both Full-SORN and pomalidomide, rather than nonspecific degradation mediated by either component alone (Figure 5f). Ubiquitination assays demonstrated that Full-SP4-C (Let-7f-1 Full-SP4-C) treatment effectively increased the polyubiquitination of Lin28B in H1299 cells. Simultaneous addition of the protease inhibitor MG132 significantly attenuated the degradation of Lin28B, indicating that Full-SP4-C-mediated Lin28B degradation occurs through the proteasome.

[0174] 2 Experimental study on changes in miRNA let-7 levels after Full-SP4-C mediated Lin28B degradation

[0175] 2.1 Experimental methods

[0176] Cell seeding: 6 x 10 cells per well in a 24-well plate 4 H1299 cells were cultured overnight at a density of 70%-80% in an incubator at 37°C containing 5% CO2. Transfection was performed in the same manner as in 1.1.1 of Experimental Example 1 above.

[0177] miRNA let-7 expression detection: The experimental operation of let-7 expression level detection is the same as that in Experimental Example 1.

[0178] 2.2 Experimental Results

[0179] The expression levels of miRNA Let-7 are shown in Figure 3. Following degradation of Lin28B, the expression levels of all mature let-7 proteins were upregulated by approximately 1.7-fold, while the expression level of mirR-21 remained unchanged, indicating that Full-SP4-C (Let-7f-1 Full-SP4-C) specifically increases Let-7 expression levels after targeted degradation of Lin28B (Figure 3e). Over 48 hours, as the concentration of Full-SP4-C increases, the expression level of Let-7g gradually increases, reaching its highest level at 100 nM (Figure 3f). At a transfection concentration of 100 nM, let-7g levels increase slightly with prolonged incubation time, reaching their maximum level at 24 hours and significantly increasing to a maximum level at 48 hours (Figure 3g). At the same transfection concentration and exposure time, the effect of the experimental group Full-S P4-C on the increase of let-7g levels 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 levels ( Figure 3h ).

[0180] 3 Inhibitory experiments on H1299 cell proliferation and cell clone colony formation

[0181] 3.1 Experimental Methods The MTT and colony formation assays were performed in the same manner as in Experimental Example 1.

[0182] 3.2 Experimental Results

[0183] 100 nM Full-S P4-C (Let-7f-1 Full-S P4-C) inhibited the proliferation of H1299 cells, and its effect was stronger than that of Full-S ORN without pomalidomide ( Figure 6 a ).

[0184] Consistent with the MTT assay, 100 nM Full-S P4-C (Let-7f-1 Full-S P4-C) significantly inhibited the colony formation ability of H1299 cells, while the inhibitory efficiency of Full-S ORN without pomalidomide was relatively low ( Figure 6 b ).

[0185] 4 Inhibitory experiments on H1299 cell migration and invasion

[0186] 4.1 Experimental methods

[0187] Experimental steps: 4x10 cells were seeded per well in a 6-well plate. 5Cells were cultured overnight in a 37°C, 5% CO2 incubator with 2 mL per well. Transfection was performed according to step 1.2, and culture was continued for 48 hours. Serum-free culture medium and Matrigel were diluted 1:5, mixed, and added to the upper chamber of a 24-well Transwell chamber (100 μL per well). The cells were placed in a 37°C incubator and incubated for 1-2 hours. After transfection and 48 hours of culture, the cells were digested and counted to prepare a cell suspension (100,000 / mL). 200 μL of cell suspension was added to each well, and 500 μL of culture medium containing 10% FBS was added to the lower chamber. The cells were incubated in a 37°C incubator for 24 hours. The cells were washed twice with PBS, fixed with paraformaldehyde, and stained with 0.1% crystal violet solution. The cells were washed twice with PBS, and the upper surface of the cells was wiped off with a cotton ball. The cells were observed and photographed under a microscope. Matrigel was not added for the cell migration assay; the remaining procedures were the same as for the cell invasion assay.

[0188] 4.2 Experimental Results

[0189] 100 nM Full-S P4-C (Let-7f-1 Full-S P4-C) significantly inhibited the migration and invasion ability of H1299 cells, and was superior to the inhibitory effect of Full-S ORN (Figures 6c, 6d).

[0190] 5 Experiment on apoptosis of H1299 cells induced by combination of Full-SP4-C and paclitaxel

[0191] 5.1 Experimental Methods The apoptosis experiment was performed in the same manner as in Experimental Example 1.

[0192] 5.2 Experimental Results

[0193] At a concentration of 100 nM, the Full-SP P4-C (Let-7f-1 Full-SP P4-C) group significantly outperformed the Full-S ORN group in inducing apoptosis in H1299 cells. Paclitaxel alone, at a concentration of 50 μM, induced approximately 10% apoptosis in H1299 cells. The combination of Full-SP P4-C (Let-7f-1 Full-SP P4-C) and paclitaxel significantly increased apoptosis in H1299 cells, reaching an apoptotic rate of approximately 30% (Figure 6e).

[0194] Experimental Example 3 Targeted degradation of Lin28B protein in Huh7 cells and subsequent biological activity evaluation

[0195] 1 Experiment to evaluate the degradation effect of Lin28B protein

[0196] 1.1 Experimental methods

[0197] 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 Lipofectamine 3000.

[0198] Lin28B protein expression level detection and ubiquitination effect detection: The experimental operations of Lin28B protein expression level detection and ubiquitination effect detection are the same as those in Experimental Example 1.

[0199] 1.2 Experimental Results

[0200] In Huh7 cells, the degradation effect of Lin28B was evaluated as follows:

[0201] For all sequences that can bind to the Lin28B protein, after coupling the pomalidomide small molecule through the PEG4 linker, all PROTAC molecules showed varying degrees of degradation activity. Among them, Let-7b P4-C (Let-7b Full-S P4-C) and Let-7f-1 P4-C (Let-7f-1 Full-S P4-C) had the best degradation activity, which could degrade the Lin28B expression level 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 1c).

[0202] At 48h, 100nM, PROTAC molecules prepared with sequences that can simultaneously bind to the CSD region and ZKD region of the Lin28B protein showed significant degradation activity against the 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 7a). For sequences that can only bind to the CSD region of the Lin28B protein, let-7b CSD-S C3-C, let-7b CSD-S C6-C, let-7b CSD-S C12-C, let-7b CSD-S C16-C, let-7b CSD-S P4-C and let-7b CSD-S P6-C can all effectively degrade Lin28B, and its expression level can be degraded to about 10%-20% (Figure 7b). However, for sequences that can only bind to the ZKD region of the Lin28B protein, all samples showed no obvious degradation activity against the Lin28B protein ( FIG7 c ).

[0203] Next, let-7b Full-S P6-C was selected for time- and concentration-dependent evaluation. Over 48 hours, let-7b Full-S P6-C significantly degraded Lin28B at a concentration of 25 nM, reaching maximum degradation at 100 nM (Figure 7d). At a transfection concentration of 100 nM, let-7b Full-S P6-C significantly degraded Lin28B at 8 hours and maintained its efficient degradation activity up to 48 hours (Figure 7e). Furthermore, the degradation of Lin28B by let-7b Full-SP6-C was more effective than that by the Full-S ORN group without pomalidomide or the Full-S ORN mixed group. The group treated with pomalidomide alone showed no significant degradation of Lin28B, indicating that let-7b Full-SP6-C-mediated Lin28B degradation is a specific degradation mediated by both Full-S ORN and pomalidomide, rather than nonspecific degradation mediated by either component alone (Figure 7f). Ubiquitination experiments demonstrated that let-7b Full-SP6-C treatment increased the polyubiquitination of Lin28B in Huh-7 cells. Furthermore, the addition of the protease inhibitor MG132 significantly attenuated the degradation of Lin28B by let-7b Full-SP6-C, indicating that let-7b Full-SP6-C mediates Lin28B degradation through the proteasome pathway.

[0204] Experimental Example 4 Evaluation of the Anti-tumor Activity of PROTAC Molecules in Vivo

[0205] 1. Experimental Methods

[0206] This experiment selected BALB / c female nude mice about 6 weeks old, with a body weight of 16-20g, and housed in a barrier environment. They were ordered one week in advance to adapt to the experimental environment, maintain adequate food and water, and a normal life rhythm. Normally proliferating T47D cells were digested with 0.25% trypsin at 37°C for 3 minutes, and serum-containing medium was added to terminate the digestion. The cells were washed with 1×PBS. The cells were then resuspended in serum-free 1640 medium, and an equal amount of matrix gel was added to the medium. 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 mice, and the growth of the tumor was observed regularly. When the tumor volume reached 50 mm 3At approximately 14 days, T47D mice were randomly divided into five groups. The first group received 0.9% saline intratumorally. The second group received Full-S ORN (6 nmol / mouse) intratumorally. The third group received Full-S P4-C (Let-7f-1 Full-S P4-C) (6 nmol / mouse) intratumorally. The fourth group received tamoxifen (5 mg / kg, dissolved in corn oil) by oral gavage. The fifth group received Full-S P4-C (Let-7f-1 Full-S P4-C) (6 nmol / mouse) intratumorally and tamoxifen (5 mg / kg) by oral gavage. The nucleic acid was administered eight times on days 1, 4, 7, 10, 13, 16, 19, and 22, respectively, and the tamoxifen was administered eight 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 tumor major diameter a and minor diameter b, and using the formula 1 / 2×a×b 2 Tumor volume was calculated. Two days after the last dose, tumors were excised, photographed, and weighed. Western blotting was then used to characterize Lin28A protein levels in the tumors. Paraffin sections were also prepared and immunohistochemical staining was performed on these sections to characterize the distribution of Lin28A protein within the tumor tissue. Hematoxylin-eosin staining was also used to analyze the pathological changes in the tumor tissue.

[0207] 2. Experimental Results

[0208] During the administration period, there was no significant change in body weight compared with the NC group in several administration groups, and the nude mice in each group were in normal growth and living conditions, indicating that the PROTAC molecule Full-SP4-C (Let-7f-1 Full-SP4-C) and tamoxifen had no obvious toxicity (Figure 8a). The breast cancer tumor growth in the Full-S ORN group was not significantly different from that in the NC group. Both Full-SP4-C (Let-7f-1 Full-SP4-C) and tamoxifen administered alone effectively inhibited the growth of breast cancer tumors. Compared with single administration, combined administration further inhibited the growth of breast cancer tumors, and the tumor showed a trend of regression (Figure 8b). By dissecting and weighing the breast cancer tumors, it was also 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-SP4-C (Let-7f-1 Full-SP4-C) and tamoxifen 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 (Figures 8d, 8e). Compared with the NC group, the weights of the heart, liver, spleen, lung, and kidney did not change significantly in the several treatment groups (Figure 8c), further demonstrating that Full-SP4-C (Let-7f-1 Full-SP4-C) and tamoxifen are not significantly toxic. Western blot results showed that Full-SP4-C (Let-7f-1 Full-SP4-C) effectively degraded Lin28A protein in breast cancer tumor tissue (Figure 8f). H&E staining results showed that the NC and Full-S ORN groups exhibited typical tumor tissue morphology. The Full-SP4-C (Let-7f-1 Full-SP4-C) and tamoxifen groups, when administered alone, showed significant cell death and nuclear condensation, with cancer cell density significantly lower than that in the NC and Full-S ORN groups. The combined Full-SP4-C and tamoxifen group exhibited even lower cancer cell tissue density than either the NC or Full-S ORN groups.

[0209] TUNEL staining results showed that no obvious apoptosis fluorescence signals were observed in the NC and Full-S ORN groups. Significant apoptosis fluorescence signals were observed in both the Full-S P4-C (Let-7f-1 Full-S P4-C) and tamoxifen groups administered alone, while a large area of ​​apoptosis fluorescence signals was observed in the combined administration group. IHC results showed that both the NC and Full-S ORN groups showed a strong positive phenotype for Lin28A. Lin28A positivity was significantly weakened in the Full-S P4-C (Let-7f-1 Full-S P4-C) group administered alone. Although the tamoxifen group administered alone showed changes in tissue morphology, it still maintained a strong Lin28A-positive phenotype. The positive phenotype in the combined administration group was further weakened, which was consistent with the Western Blot results of tumor tissue proteins (Figure 8g). The above results collectively indicate that the PROTAC molecule Full-SP4-C (Let-7f-1 Full-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 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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