Protein degradation targeting chimera based on G-quadruplex RNA as well as preparation method and application thereof

Abstract

The invention provides a protein degradation targeting chimera based on G-quadruplex RNA (Ribonucleic Acid) as well as a preparation method and application of the protein degradation targeting chimera. The structural general formula of the protein degradation targeting chimera is G-L-E, wherein G is G quadruplex RNA specifically combined with mammal fragile X hypophrenia protein (FMRP), E is a ligand of micromolecule E3 ubiquitination ligase, and L is a connecting arm between the G quadruplex RNA and the ligand of the micromolecule E3 ubiquitination ligase. The protein degradation targeting chimera provided by the invention has the advantages that the stability in an in-vivo environment and the capability of entering cells are greatly improved, and the protein degradation targeting chimera also has the capabilities of efficiently degrading FMRP and changing a secretion mode of cancer cells, so that a lot of convenience is provided for the development of nucleic acid PROTAC drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a protein-targeted degradation chimera based on G-quadruplex RNA, and a preparation method and application thereof. Background Art

[0002] Mammalian fragile X mental retardation protein (FMRP) is an RNA-binding protein that is highly expressed in neuronal synapses but relatively low in other organs. FMRP dysfunction has been shown to be associated with the neurodevelopmental disorders fragile X syndrome and autism. Recently, increasing evidence has shown that FMRP is abnormally highly expressed in many human tumors and is closely associated with tumor invasion, metastasis, and immune evasion. Transcriptome data show that FMRP significantly regulates hundreds of genes in cancer cells, among which interleukin-33 (IL-33), tumor secretory protein S (PROS1), chemokine CC motif ligand 7 (CCL7), and exosome-related genes have been shown to be most relevant to cancer. FMRP is now regarded as a tumor immunosuppressive regulatory protein and a potential tumor therapeutic target. However, there are currently no small molecule inhibitors for FMRP on the market. Inhibition of FMRP function can only be achieved through genetic means, such as siRNA and CRISPR-Cas9. Moreover, due to the disordered binding pockets and variable conformations of RNA-binding proteins, it is very difficult to screen small molecule ligands for RNA-binding proteins.

[0003] The proteolysis-targeting chimera (PROTAC) is a heterobifunctional molecule, one part of which is used to target E3 ubiquitin ligase and the other part is used to target the target protein. PROTAC, E3 ubiquitin ligase and target protein form a ternary complex and specifically degrade the target protein through the ubiquitin-proteasome system in the cell. PROTAC can be composed not only of small molecules, but also of nucleic acids, peptides or antibodies. This makes the design of PROTAC more diverse and provides new design ideas for many difficult-to-drug proteins. Taking advantage of the ability of RNA-binding proteins to recognize and bind to specific RNA sequences, PROTACs constructed based on RNA and peptides have shown strong potential in targeted degradation of RNA-binding proteins. However, RNA and peptides are very easy to degrade and difficult to enter cells, and the larger molecular weight also limits the possibility of PROTAC drugging based on RNA and peptides.

[0004] G-quadruplex is a higher-order structure formed by folding DNA or RNA rich in tandem repeats of guanine. The RGG domain of FMRP has been shown to bind to G-quadruplex RNA of a specific sequence. In PROTACs composed of G-quadruplex RNA and peptides (e.g., sc1-VHLL composed of G-quadruplex RNA (sc1) and a ligand peptide targeting VHL (von Hippel-Lindau) protein), G-quadruplex RNA is used to target FMRP, while the peptide is used to target E3 ubiquitin ligase. However, RNA and peptides are unstable and easily degraded in cells, which results in a very large amount required to degrade FMRP using sc1-VHLL. The peptide used to target E3 ligases also further increases the molecular weight of PROTAC, resulting in limited in vivo activity of sc1-VHLL, limiting its further clinical application and transformation.

[0005] RNA chemical modification refers to replacing certain functional groups or chemical bonds on natural RNA with other types of functional groups or chemical bonds. Many types of RNA chemical modifications have been shown to effectively improve the stability of RNA and reduce the off-target effects of RNA-targeted drugs. Chemically modified sgRNAs required for chemically synthesized CRISPER-Cas9 gene editing can effectively improve the stability of sgRNAs and obtain higher CRISPER-Cas9 gene editing efficiency and lower off-target effects. Summary of the invention

[0006] In view of the problems existing in the above-mentioned prior art, the inventors have designed and prepared a novel PROTAC based on G-quadruplex RNA after long-term technical practice and exploration, and provided its application. The PROTAC provided by the present invention uses a small molecule E3 ubiquitin ligase ligand to target E3 ubiquitin ligase, and further selects chemical modification sites and uses G-quadruplex RNA chemically modified in an optimized manner to target FMRP, thereby achieving more efficient and more stable degradation of FMRP, thereby successfully exerting therapeutic efficacy against tumors.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In the first aspect, the present invention provides a protein degradation targeting chimera (PROTAC) having a general structural formula: GLE; wherein G is a G-quadruplex RNA that specifically binds to mammalian fragile X mental retardation protein (FMRP), E is a ligand of a small molecule E3 ubiquitin ligase, and L is a connecting arm between the G-quadruplex RNA and the ligand of the small molecule E3 ubiquitin ligase.

[0009] According to some embodiments of the present invention, the nucleotide sequence of the G quadruplex RNA comprises the nucleotide sequence shown in SEQ ID NO. 1.

[0010] SEQ ID NO. 1: 5'-GCU GCG GUG UGG AAG GAG UGG CUG GGU UGC GCA GC-3'.

[0011] According to some embodiments of the invention, the linker arm is -(O-CH 2 -CH 2 -O) n -, wherein n=1-4, more preferably, n=3, or according to some embodiments of the present invention, the linker arm is -(CH 2 ) m -, m=3-6, more preferably, m=5.

[0012] According to some embodiments of the present invention, the ligand of the small molecule E3 ubiquitin ligase is pomalidomide and / or (2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (hereinafter referred to as VH032).

[0013] Preferably, the ligand of the small molecule E3 ubiquitin ligase is pomalidomide, and the linker arm is -(O-CH 2 -CH 2 -O) 3 -; or the ligand of the small molecule E3 ubiquitin ligase is VH032, and the linker is -(CH 2 ) 5 -.

[0014] According to some embodiments of the invention, the linker is connected to the 3' end of the G-quadruplex RNA via a cross-linking group.

[0015] Preferably, the cross-linking group is formed by a click chemistry reaction between diphenylcyclooctyne (DBCO) and / or cyclopropanecyclooctyne (BCN) and an azide cross-linking agent; more preferably, the cross-linking group is formed by a click chemistry reaction between diphenylcyclooctyne and an azide cross-linking agent. For example, the structure of the cross-linking group is as follows:

[0016]

[0017] According to some embodiments of the present invention, the nucleotide sequence of the G quadruplex RNA comprises the nucleotide sequence shown in SEQ ID NO. 2, or the nucleotide sequence of the G quadruplex RNA is as shown in SEQ ID NO. 2 (hereinafter the G quadruplex RNA is represented by sc1).

[0018] SEQ ID NO. 2: 5'-GCG GCU GCG GUG UGG AAG GAG UGG CUG GGU UGC GCA GCUUG-3'.

[0019] According to some embodiments of the invention, at least the first 1, for example the first 1, 2 or 3 ribonucleotides at the 5' and 3' ends of the G-quadruplex RNA are chemically modified.

[0020] Preferably, the chemical modification is that the 2'-OH of the ribose of the ribonucleotide is replaced by a methoxy group, and / or the phosphate group is replaced by a phosphorothioate group.

[0021] More preferably, the 2'-OH of the ribose of the first three ribonucleotides at the 5' and 3' ends of the G-quadruplex RNA is replaced by a methoxy group, and the phosphate group is replaced by a phosphorothioate group.

[0022] According to some preferred embodiments of the present invention, the nucleotide sequence of the G quadruplex RNA comprises the nucleotide sequence shown in SEQ ID NO. 3, or the nucleotide sequence of the G quadruplex RNA is as shown in SEQ ID NO. 3 (hereinafter the G quadruplex RNA is represented by msc1).

[0023] SEQ ID NO. 3:5'-G(OCH 3 ) * C(OCH 3 ) * G(OCH 3 ) * GCU GCG GUG UGG AAG GAG UGGCUG GGU UGC GCA GCU(OCH 3 ) * U(OCH 3 ) * G(OCH 3 ) * -3'; among them, (OCH 3 ) indicates that the 2'-OH of the ribose of the ribonucleotide is replaced by a methoxy group, and * indicates phosphorothioate modification of the ribonucleotide.

[0024] In a second aspect, the present invention provides a method for preparing a PROTAC according to the first aspect of the present invention, comprising:

[0025] (1) Synthesizing G-quadruplex RNA with a linker;

[0026] (2) The G-quadruplex RNA with a linker synthesized in step (1) is coupled to a ligand of a small molecule E3 ubiquitination ligase through a click chemistry reaction.

[0027] According to some preferred embodiments of the present invention, the preparation method comprises:

[0028] (1) synthesizing a G-quadruplex RNA having a linker and at least the first one, for example, the first one, two or three ribonucleotides at the 5' and 3' ends are chemically modified;

[0029] (2) The chemically modified G-quadruplex RNA with a linker synthesized in step (1) is coupled to a ligand of a small molecule E3 ubiquitin ligase through a click chemistry reaction.

[0030] Preferably, the chemical modification is that the 2'-OH of the ribose of the ribonucleotide is replaced by a methoxy group, and / or the phosphate group is replaced by a phosphorothioate group.

[0031] More preferably, the 2'-OH of the ribose of the first three ribonucleotides at the 5' and 3' ends of the G-quadruplex RNA is replaced by a methoxy group, and the phosphate group is replaced by a phosphorothioate group.

[0032] According to some embodiments of the invention, the linker is connected to the 3' end of the G-quadruplex RNA via a cross-linking group.

[0033] Preferably, the cross-linking group is formed by a click chemistry reaction between diphenylcyclooctyne (DBCO) and / or cyclopropanecyclooctyne (BCN) and an azide cross-linking agent; more preferably, the cross-linking group is formed by a click chemistry reaction between diphenylcyclooctyne and an azide cross-linking agent. For example, the structure of the cross-linking group is as follows:

[0034]

[0035] In a third aspect, the present invention provides a liposome nanoparticle encapsulating the PROTAC according to the first aspect of the present invention.

[0036] In a fourth aspect, the present invention provides use of the PROTAC according to the first aspect of the present invention or the liposome nanoparticles according to the third aspect of the present invention in the preparation of a medicament for preventing and / or treating a disease associated with abnormal expression of FMRP.

[0037] According to some embodiments of the present invention, the disease associated with abnormal expression of FMRP is cancer.

[0038] Preferably, the cancer is selected from one or more of colon cancer, pancreatic ductal carcinoma, breast cancer, liver cancer, prostate cancer and lung cancer.

[0039] In a fifth aspect, the present invention provides a pharmaceutical composition for preventing and / or treating diseases associated with abnormal expression of FMRP, comprising a PROTAC according to the first aspect of the present invention or a liposome nanoparticle according to the third aspect of the present invention, and optionally a pharmaceutically acceptable excipient.

[0040] According to some embodiments of the present invention, the pharmaceutical composition further comprises other active pharmaceutical ingredients for preventing and / or treating diseases associated with abnormal expression of FMRP, such as immune checkpoint blockers.

[0041] Preferably, the immune checkpoint blocker is a PD-L1 / PD-1 and / or CTLA4 pathway inhibitor, such as one or more of pembrolizumab, carrelizumab, nivolumab, durvalumab and ipilimumab.

[0042] According to some embodiments of the present invention, the disease associated with abnormal expression of FMRP is cancer.

[0043] Preferably, the cancer is selected from one or more of colon cancer, pancreatic ductal carcinoma, breast cancer, liver cancer, prostate cancer and lung cancer.

[0044] It should be noted that the present invention provides the above technical solution, although only mentions exemplary G quadruplex RNA and RNA chemical modification methods to construct PROTAC for degrading FMRP, it is known that FMRP can also bind to other G quadruplex RNAs, RNA can also be modified in some other ways, and there are many kinds of ligands for constructing PROTAC's connecting arms and small molecule E3 ubiquitin ligases. Therefore, based on the concept of the present invention, PROTACs for degrading FMRP constructed by using other G quadruplex RNAs to target FMRP and using other RNA chemical modification methods, other connecting arms and small molecule E3 ubiquitin ligase ligase ligands also fall within the scope of protection of the present invention.

[0045] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0046] The PROTAC provided by the present invention utilizes G-quadruplex RNA and small molecule E3 ubiquitin ligase that specifically targets FMRP to develop a series of PROTACs targeting FMRP, and constructs a more stable and effective PROTAC by chemically modifying the G-quadruplex RNA at the preferred site. In addition, the present invention further improves the stability of PROTAC in the in vivo environment and its ability to enter cells by encapsulating PROTAC by liposome nanoparticles.

[0047] After experimental verification, the PROTAC provided by the present invention has the ability to efficiently degrade FMRP and change the secretion pattern of cancer cells, among which 300nM PROTAC can effectively degrade FMRP and change the secretion pattern of cancer cells, which is twice as much as the amount required for the existing PROTAC for FMRP to achieve the same effect. At the same time, the optimized chemical modification also greatly improves the stability of G-quadruplex RNA PROTAC, and the use of small molecule E3 ubiquitin ligase ligands reduces the molecular weight of PROTAC to a certain extent. The use of liposome nanoparticles to encapsulate and deliver nucleic acid PROTACs also provides many conveniences for the development of nucleic acid PROTAC drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0049] Figure 1 The basic structure of PROTAC of the present invention is shown;

[0050] Figure 2 Shows chemical modifications in the chemical synthesis of the G quadruplex RNA of the present invention;

[0051] Figure 3 Showing the synthesis process of PROTAC of the present invention;

[0052] Figure 4 The chemical structure of the PROTAC of the present invention is shown (compared with sc1-VHLL);

[0053] Figure 5 Gel electrophoresis diagrams of different PROTACs obtained in the embodiments of the present invention;

[0054] Figure 6 Mass spectra of different PROTACs obtained in the embodiments of the present invention;

[0055] Figure 7 High performance liquid chromatography of different PROTACs obtained in the embodiments of the present invention;

[0056] Figure 8 This is an immunoblot image of different PROTACs obtained in the embodiment of the present invention degrading FMRP in cancer cells as the concentration changes;

[0057] Fig. 9 Showing the comparison of different PROTACs and sc1-VHLL obtained in the examples of the present invention in degrading FMRP in cancer cells;

[0058] Fig.10 Showing the comparison of different PROTACs and sc1-VHLL obtained in the examples of the present invention in changing the secretion pattern of tumor cells;

[0059] Fig.11 The encapsulated PROTAC (msc1-PEG 3 -CRBNL) liposome nanoparticles particle size characterization diagram;

[0060] Fig.12 The encapsulated PROTAC (msc1-PEG 3 -CRBNL) liposome nanoparticles and unencapsulated PROTAC (msc1-PEG 3 Fluorescence confocal image of PROTAC delivered into cancer cells by msc1-CRBNL; TD: bright field; Nucleus: blue fluorescence channel, blue fluorescence shows the cell nucleus; msc1-PEG 3 -CRBNL: green fluorescence channel, green shows FITC-msc1-PEG 3 -CRBNL; Merge: Overlay of the previous three channels;

[0061] Fig.13 The liposome nanoparticles (LNP@msc1-PEG) encapsulating PROTAC obtained in the embodiment of the present invention are shown. 3 -CRBNL) effectively degrades FMRP in cancer cells;

[0062] Fig.14 This is an immunoblot image of PROTAC constructed using sc1 and PROTAC constructed using other G-quadruplex RNAs in the present invention to degrade FMRP in cancer cells;

[0063] Fig.15 This is an immunoblot image of PROTAC constructed using msc1 and PROTAC constructed using sc1 modified with other chemicals to degrade FMRP in cancer cells in an embodiment of the present invention. DETAILED DESCRIPTION

[0064] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention but not for limiting the scope of the present invention.

[0065] The experimental methods and conditions used in the following examples are conventional methods and conditions unless otherwise specified. The reagents used in the following examples are commercially available products unless otherwise specified.

[0066] Example 1 Synthesis of PROTAC

[0067] A G quadruplex RNA with a DBCO 3' end and three bases modified with methoxy and phosphorothioate was designed and synthesized. Specifically, the nucleotide sequence of the G quadruplex RNA is: 5'-G(OCH3 ) * C(OCH 3 ) * G(OCH 3 ) * GCU GCG GUGUGG AAG GAG UGG CUG GGU UGC GCA GCU(OCH 3 ) * U(OCH 3 ) * G(OCH 3 ) * -DBCO-3' (msc1-DBCO), where (OCH 3 ) indicates that the 2'-OH of the ribose of the ribonucleotide is replaced by a methoxy group, and * indicates phosphorothioate modification of the ribonucleotide. Figure 1 The basic structure of the synthetic PROTAC is shown. Figure 2 Chemical modifications in the synthesis of G-quadruplex RNA are shown.

[0068] At the same time, a G quadruplex RNA with DBCO at the 3' end but no other chemical modifications is synthesized. Specifically, the nucleotide sequence of the G quadruplex RNA is: 5'-GCG GCU GCG GUG UGG AAG GAG UGG CUG GGU UGC GCA GCU UG-DBCO-3' (sc1-DBCO).

[0069] The 3'-end DBCO-modified msc1 and sc1 (i.e., msc1-DBCO and sc1-DBCO) were subjected to click chemistry reactions (strain-promoted azide-alkyne cycloaddition, SPAAC) with small molecule E3 ubiquitin ligase ligands. 3 CRBN ligands (N 3 -PEG 3 -CRBNL) (CRBNL is pomalidomide, 4-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione) or containing (CH 2 ) 5 VH032 ligand (N 3 -C 5 -VH032) binds to msc1-DBCO or sc1-DBCO, converting N 3 -PEG 3 -CRBNL (50 μM) or N 3 -(CH 2 ) 5-VH032 (50 μM) was incubated with msc1-DBCO (50 μM) or sc1-DBCO (50 μM) in water treated with diethyl pyrocarbonate (DEPC). The mixture was placed at room temperature for 6 hours, and the reaction products were purified by high-performance liquid chromatography (HPLC). The resulting PROTAC products were named msc1-PEG 3 -CRBNL, sc1-PEG 3 -CRBNL, msc1-C 5 -VH032 and sc1-C 5 -VH032. Specifically, the synthesized PROTAC was separated, purified and characterized using an Agilent InertslWP300Cis (150 x 3mm, 5μm) chromatographic column. First, 40mL of the prepared 2mol / L triethylamine acetate (TEAA) buffer was added with 760mL of ultrapure water to prepare a 0.1mol / L mobile phase A, and 800mL of analytically pure acetonitrile was taken as the mobile phase B. After that, PROTAC was characterized and purified by gradient elution (elution gradient is shown in Table 1). The mobile phase flow rate during the entire elution process was 1mL / min, and the detected ultraviolet absorption wavelength was 260nm (ultraviolet absorption wavelength of RNA). By comparing with the peak position of the unreacted RNA, the solution at the newly generated peak position was collected, and then the solvent was removed by freeze drying. The HPLC-purified PROTAC was characterized by time of flight mass spectrometry (TOF-MS) to obtain the corresponding molecular weight.

[0070] Table 1

[0071]

[0072] Figure 3 The synthetic process of the above PROTAC products is shown. Figure 4 The chemical structures of the above PROTAC products are shown and compared with the chemical structure of sc1-VHLL. Figure 5 Gel electrophoresis of the synthesized PROTAC product. Figure 6 Mass spectrum of the synthesized PROTAC product. Figure 7 This is the HPLC chromatogram of the synthesized PROTAC product after HPLC purification.

[0073] Example 2 Study on the Activity of PROTAC

[0074] In order to verify the msc1-PEG prepared in Example 1 3 -CRBNL and msc1-C 5-VH032 can degrade FMRP in cancer cells. HeLa cells were placed in 6-well plates until the cell confluence reached about 70%. Different concentrations (100, 200 and 300 nM) of msc1-PEG were injected into the wells of the cells using jetOPTIMUs (Polyplus). 3 -CRBNL and msc1-C 5 -VH032 was transfected into cells. Proteins were collected 24 hours after transfection and the intracellular FMRP content was detected by immunoblotting. Figure 8 As shown, msc1-PEG 3 -CRBNL and msc1-C 5 -VH032 can effectively degrade FMRP in cancer cells, and the degradation of FMRP is concentration-dependent, where the % of 0 nM represents that the gray value of the immunoblot band in each concentration group is the percentage of the gray value of the 0 nM concentration group, thereby analyzing the amount of FMRP in each concentration group relative to the 0 nM group.

[0075] In order to explore whether chemical modification can improve the ability of PROTAC to degrade FMRP in cancer cells, 300 nM sc1-VHLL, sc1-PEG 3 -CRBNL and msc1-PEG 3 -CRBNL were transfected into HeLa cells. Proteins were collected 24 hours after transfection and the intracellular FMRP content was detected by immunoblotting (the control group was cells without any treatment). Fig. 9 As shown in Figure 2, all three PROTACs can degrade FMRP in HeLa cells, and the degradation efficiency is msc1-PEG 3 -CRBNL>sc1-PEG 3 -CRBNL>sc1-VHLL, which indicates that the PROTAC with smaller molecular weight and chemical modification synthesized by the present invention can indeed improve the degradation efficiency of FMRP.

[0076] Similarly, 300 nM sc1-VHLL, sc1-C 5 -VH032 and msc1-C 5 -VH032's ability to degrade FMRP in HeLa cells (control group, i.e., cells without any treatment). The results showed that the efficiency of FMRP degradation was msc1-C 5 -VH032>sc1-C 5 -VH032>sc1-VHLL, which also shows that the PROTAC with smaller molecular weight and chemically modified synthesized by the present invention can indeed improve the degradation efficiency of FMRP.

[0077] In order to explore the effect of PROTAC degradation of FMRP on the secretion pattern of cancer cells, sc1-VHLL, sc1-PEG 3 -CRBNL and msc1-PEG 3 -HeLa protein after 36 hours of CRBNL treatment (control group is cells without any treatment). Fig.10 As shown in Figure 2, all three PROTACs can reduce the secretion of IL-33 and PROS1 by cancer cells, and this inhibitory effect is not significant in msc1-PEG 3 -CRBNL treatment was the most significant, followed by sc1-PEG 3 This suggests that PROTACs with smaller molecular weight and chemical modifications are more effective in changing the secretion pattern of cancer cells.

[0078] Similarly, collect sc1-VHLL, sc1-C 5 -VH032 and msc1-C 5 -HeLa protein after VH032 treatment for 36 hours (control group is cells without any treatment). msc1-C 5 -The decrease in IL-33 and PROS1 after VH032 treatment was the most significant, followed by sc1-C 5 -VH032. This also shows that the PROTAC with a smaller molecular weight and chemically modified by the present invention is more effective in changing the secretion pattern of cancer cells.

[0079] Both IL-33 and PROS1 are abnormally highly expressed in many tumors, and their high expression is often associated with the malignancy of the tumor and poor prognosis. IL-33 plays an important role in tumor occurrence, development and immunity. IL-33 promotes tumor proliferation and angiogenesis, and also creates an immunoregulatory tumor microenvironment, such as promoting the differentiation of regulatory T cells and myeloid-derived suppressor cells. In this environment, tumors are prone to immune escape. Therefore, reducing the amount of IL-33 secreted by cancer cells is very meaningful for the treatment of tumors. PROS1 depletes T cells in the tumor, promotes macrophage M2 polarization, and promotes the formation of an immunosuppressive microenvironment. In short, reducing the secretion of IL-33 and PROS1 will inhibit tumor growth and create a pro-inflammatory tumor microenvironment.

[0080] Example 3 In vivo delivery study of PROTAC

[0081] In order to better 3-CRBNL was delivered into cells to synthesize liposome nanoparticles encapsulating PROTAC. Specifically, lipid (4-(N,N-dimethylamino) butyric acid (dilinoleyl) methyl ester, DLin-MC3-DMA): distearoylphosphatidylcholine (DSPC): cholesterol: dimyristoylglycerol polyethylene glycol 2000 (DMG-PEG 2000 )) was dissolved in 100% ethanol at a molar ratio of 50: 10: 38.5: 1.5. 3 -CRBNL was diluted in 10 mM acetic acid (pH = 4) to achieve an N / P ratio of 6. The above solution was prepared into nanoparticles at a ratio of 1: 3 using a microfluidic device (Zhongxin Qiheng Cchippump-02 dual-channel split syringe pump and Zhongxin Qiheng micro-mixing liposome chip ZX-LS-31). Subsequently, the ethanol was removed by dialysis with PBS and the liposome nanoparticles were neutralized. The synthesized msc1-PEG coated 3 -CRBNL's liposomal nanoparticles are called LNP@msc1-PEG 3 -CRBNL. Fig.11 The results showed that the synthesized liposome nanoparticles had uniform particle size.

[0082] To investigate whether liposome nanoparticles can effectively deliver PROTACs into cancer cells, we synthesized a PROTAC modified with FITC at the 5' end of RNA (FITC-msc1-PEG 3 -CRBNL). HeLa cells were incubated with FITC-msc1-PEG 3 -CRBNL liposomal nanoparticles (LNP@FITC-msc1-PEG 3 -CRBNL) or unencapsulated FITC-msc1-PEG 3 -CRBNL were co-incubated for 6 h. Fig.12 The results showed that liposome nanoparticles can efficiently transfer msc1-PEG 3 -CRBNL delivery into cells.

[0083] To investigate whether PROTAC delivered by liposome nanoparticles can effectively degrade FMRP in cancer cells, HeLa cells were incubated with LNP@msc1-PEG 3 -CRBNL (0-300 nM, concentration according to the encapsulated msc1-PEG 3 -CRBNL) were incubated for 24 h, and then the protein was collected. Fig.13 Demonstrated effective delivery of msc1-PEG via liposomal nanoparticles 3 -CRBNL, thereby degrading FMRP in HeLa cells.

[0084] Example 4 Screening of G-quadruplex RNA and its chemical modification sites

[0085] In order to compare the differences in the ability of PROTACs constructed with different G-quadruplex RNAs that can target FMRP to degrade FMRP, DBCO-modified MAP1B, sc1, S3F, and S3F-shRNA were combined with N 3 -PEG 3 -CRBNL cross-linking to form different PROTACs. HeLa cells were placed in a 6-well plate until the cell confluence reached about 70%. 300nM PROTAC was transfected into the cells using jetOPTIMUs (Polyplus). Proteins were collected 24 hours after transfection and the intracellular FMRP content was detected by immunoblotting (the control group was cells without any treatment). Fig.14 As shown in the figure, PROTACs constructed with four G-quadruplex RNAs can degrade FMRP in cancer cells to a certain extent, but PROTAC constructed with sc1 showed the strongest ability to degrade FMRP.

[0086] SEQ ID NO. 4: GGC GCU GGG AGA GGG CGG AGG GGG AGG CGG CGC C (MAP1B)

[0087] SEQ ID NO. 5: GGG AGG GGG GUG AUU GGA AGG GAG GGA GGU GGC CUU CC (S3F)

[0088] SEQ ID NO. 6: GAU UGG AAG GGA GGG AGG UG (S3F-sh)

[0089] In order to compare the differences in the ability of PROTACs constructed with different chemical modifications of sc1 to degrade FMRP, the three (a total of 6) ribonucleotides near the 5' and 3' ends were modified with methoxy (M), phosphorothioate (S), or methoxy and phosphorothioate (MS) (the corresponding chemical modification structures are shown in Figure 2). Figure 2 As shown), the chemically modified sc1 containing DBCO is then combined with N 3 -PEG 3-CRBNL cross-linking to form different PROTACs. HeLa cells were placed in a 6-well plate until the cell confluence reached about 70%. 300nM PROTAC was transfected into the cells using jetOPTIMUs (Polyplus). Proteins were collected 24 hours after transfection and the intracellular FMRP content was detected by immunoblotting (the control group was cells without any treatment). Fig.15 As shown, PROTAC constructed with sc1 RNA dually modified with methoxyl group and phosphorothioate (MS) has the strongest ability to degrade FMRP in cancer cells.

[0090] The above are only several exemplary embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention is disclosed as above with preferred embodiments, they are not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, can make some changes or modifications to obtain equivalent or equivalent embodiments using the above disclosed technical content, which fall within the scope of the present invention.

Claims

1. A protein degradation targeting chimera, whose general structural formula is: GLE; wherein, G is a G-quadruplex RNA that specifically binds to mammalian fragile X mental retardation protein, E is a ligand of a small molecule E3 ubiquitin ligase, and L is a connecting arm between the G-quadruplex RNA and the ligand of the small molecule E3 ubiquitin ligase.

2. The protein degradation targeting chimera according to claim 1, wherein The nucleotide sequence of the G quadruplex RNA comprises the nucleotide sequence shown in SEQ ID NO.

1.

3. The protein degradation targeting chimera according to claim 1, wherein The connecting arm is -(O-CH2-CH2-O) n -, wherein n=1-4; or, the linker arm is -(CH2) m -, where m=3-6.

4. The protein degradation targeting chimera according to claim 1, wherein The ligand of the small molecule E3 ubiquitin ligase is pomalidomide and / or (2S, 4R)-1-((S)-2-acetamido-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.

5. The protein degradation targeting chimera according to claim 1, wherein The connecting arm is connected to the 3' end of the G quadruplex RNA via a cross-linking group; wherein the cross-linking group is formed by a click chemistry reaction between diphenylcyclooctyne and / or cyclopropanecyclooctyne and an azide cross-linking agent.

6. The protein degradation targeting chimera according to any one of claims 1 to 5, wherein The nucleotide sequence of the G quadruplex RNA comprises the nucleotide sequence shown in SEQ ID NO. 2, or the nucleotide sequence of the G quadruplex RNA is as shown in SEQ ID NO.

2.

7. The protein degradation targeting chimera according to claim 6, wherein: The first 1, 2 or 3 ribonucleotides at the 5' and 3' ends of the G quadruplex RNA are chemically modified; wherein the chemical modification is that the 2'-OH of the ribose of the ribonucleotide is replaced by a methoxy group, and / or the phosphate group is replaced by a thiophosphate group.

8. The protein degradation targeting chimera according to claim 7, wherein: The 2'-OH of the ribose of the first three ribonucleotides at the 5' and 3' ends of the G-quadruplex RNA is replaced by a methoxy group, and the phosphate group is replaced by a phosphorothioate group.

9. The protein degradation targeting chimera according to claim 8, wherein: The nucleotide sequence of the G quadruplex RNA comprises the nucleotide sequence shown in SEQ ID NO. 3, or the nucleotide sequence of the G quadruplex RNA is as shown in SEQ ID NO.

3.

10. The method for preparing a protein degradation targeting chimera according to any one of claims 1 to 9, comprising: (1) Synthesizing G-quadruplex RNA with a linker; (2) The G-quadruplex RNA with a linker synthesized in step (1) is coupled to a ligand of a small molecule E3 ubiquitination ligase through a click chemistry reaction.

11. The preparation method according to claim 10, comprising: (1) synthesizing a G-quadruplex RNA having a linker and the first 1, 2 or 3 ribonucleotides at the 5' and 3' ends being chemically modified; (2) coupling the chemically modified G-quadruplex RNA with a linker synthesized in step (1) with a ligand of a small molecule E3 ubiquitin ligase through a click chemistry reaction; The chemical modification is that the 2'-OH of the ribose of the ribonucleotide is replaced by a methoxy group, and / or the phosphate group is replaced by a thiophosphate group.

12. The preparation method according to claim 11, wherein The 2'-OH of the ribose of the first three ribonucleotides at the 5' and 3' ends of the G-quadruplex RNA is replaced by a methoxy group, and the phosphate group is replaced by a phosphorothioate group.

13. The preparation method according to any one of claims 10 to 12, wherein The connecting arm is connected to the 3' end of the G quadruplex RNA via a cross-linking group; wherein the cross-linking group is formed by a click chemistry reaction between diphenylcyclooctyne and / or cyclopropanecyclooctyne and an azide cross-linking agent.

14. A liposome nanoparticle encapsulating the protein degradation targeting chimera according to any one of claims 1 to 9.

15. Use of the protein degradation targeted chimera according to any one of claims 1 to 9 or the liposome nanoparticle according to claim 14 in the preparation of a drug for preventing and / or treating a disease associated with abnormal expression of fragile X mental retardation protein in mammals.

16. The use according to claim 15, wherein: The disease associated with abnormal expression of fragile X mental retardation protein in mammals is cancer; wherein the cancer is selected from one or more of colon cancer, pancreatic ductal carcinoma, breast cancer, liver cancer, prostate cancer and lung cancer.

17. A pharmaceutical composition for preventing and / or treating diseases associated with abnormal expression of fragile X mental retardation protein in mammals, comprising the protein degradation targeting chimera according to any one of claims 1 to 9 or the liposome nanoparticle according to claim 14, and optional pharmaceutically acceptable excipients.

18. The pharmaceutical composition according to claim 17, further comprising other active pharmaceutical ingredients for preventing and / or treating diseases associated with abnormal expression of fragile X mental retardation protein in mammals.

19. The pharmaceutical composition according to claim 18, wherein The active ingredient of the drug is an immune checkpoint blocker, and the immune checkpoint blocker is a PD-L1 / PD-1 and / or CTLA4 pathway inhibitor.

20. The pharmaceutical composition according to any one of claims 17 to 19, wherein The disease associated with abnormal expression of fragile X mental retardation protein in mammals is cancer; wherein the cancer is selected from one or more of colon cancer, pancreatic ductal carcinoma, breast cancer, liver cancer, prostate cancer and lung cancer.

Images