G-quadruplex RNA-based protein degradation targeting chimera, and preparation method and application thereof

Abstract

The application provides a G-quadruplex RNA-based protein degradation targeting chimera as well as a preparation method and application thereof. The structural general formula of the protein degradation targeting chimera is G-L-E; wherein G is G-quadruplex RNA that specifically binds to fragile X mental retardation protein (FMRP) of mammals, 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. The protein degradation targeting chimera provided by the application has greatly improved stability in an in-vivo environment and cell entry capability, and also has the capability of efficiently degrading FMRP and changing a secretion mode of cancer cells, thereby providing many conveniences for development of nucleic acid-based PROTAC drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically 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 linked to the neurodevelopmental disorders fragile X syndrome and autism. Recently, increasing evidence indicates that FMRP is abnormally overexpressed in many human tumors and is closely associated with tumor invasion, metastasis, and immune evasion. Transcriptome data indicate that FMRP significantly regulates hundreds of genes in cancer cells, with interleukin-33 (IL-33), tumor secreted protein S (PROS1), chemokine C-C motif ligand 7 (CCL7), and exosome-related genes being particularly relevant to cancer. FMRP is now considered a tumor immunosuppressive regulator and a potential therapeutic target. However, there are currently no marketed small molecule inhibitors targeting FMRP. Inhibition of FMRP function can only be achieved through genetic approaches, 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] A proteolysis-targeting chimera (PROTAC) is a heterobifunctional molecule, one part of which targets an E3 ubiquitin ligase and the other part targets a target protein. The PROTAC, E3 ubiquitin ligase, and target protein form a ternary complex that specifically degrades the target protein through the ubiquitin-proteasome system within the cell. PROTACs can be composed not only of small molecules but also of nucleic acids, peptides, or antibodies. This allows for greater diversity in PROTAC design and provides new design ideas for many difficult-to-drug proteins. Leveraging the ability of RNA-binding proteins to recognize and bind to specific RNA sequences, PROTACs based on RNA and peptides have shown strong potential for the targeted degradation of RNA-binding proteins. However, RNA and peptides are highly susceptible to degradation and difficult to enter cells, and their large molecular weight also limits the druggability of RNA- and peptide-based PROTACs.

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

[0005] RNA chemical modification involves replacing certain functional groups or chemical bonds on natural RNA with other types. Numerous RNA chemical modifications have been shown to effectively improve RNA stability and reduce the off-target effects of RNA-targeted therapeutics. Chemical modification of the synthesized sgRNA required for CRISPER-Cas9 gene editing can effectively improve sgRNA stability, resulting in higher CRISPER-Cas9 gene editing efficiency and reduced off-target effects. Summary of the Invention

[0006] In response to the problems existing in the above-mentioned prior art, the present inventors, after long-term technical practice and exploration, designed and prepared a novel PROTAC based on G-quadruplex RNA, 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 targets FMRP by selecting chemical modification sites and using G-quadruplex RNA chemically modified in an optimized manner, 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 a 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 for a small molecule E3 ubiquitin ligase, and L is a linker arm between the G-quadruplex RNA and the ligand for 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 present invention, the linker arm is -(O-CH2-CH2-O) n -, wherein n=1-4, more preferably, n=3, or according to some embodiments of the present invention, the linker arm is -(CH2) 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-CH2-CH2-O)3-; or the ligand of the small molecule E3 ubiquitin ligase is VH032, and the linker arm is -(CH2)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 crosslinking group is formed by a click chemistry reaction between diphenylcyclooctyne (DBCO) and / or cyclopropanecyclooctyne (BCN) and an azide crosslinker; more preferably, the crosslinking group is formed by a click chemistry reaction between diphenylcyclooctyne and an azide crosslinker. For example, the structure of the crosslinking group is shown below:

[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 one, for example the first one, two or three 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 group 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 groups of the ribose sugars of the first three ribonucleotides at the 5' and 3' ends of the G-quadruplex RNA are replaced by methoxy groups, and the phosphate groups are replaced by phosphorothioate groups.

[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(OCH3) * C(OCH3) * G(OCH3) * GCU GCG GUG UGG AAG GAG UGGCUG GGU UGC GCA GCU(OCH3) * U(OCH3) * G(OCH3) * -3'; wherein, (OCH3) 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) Synthesis of G-quadruplex RNA with linker arms;

[0026] (2) The 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.

[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 being 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 group 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 groups of the ribose sugars of the first three ribonucleotides at the 5' and 3' ends of the G-quadruplex RNA are replaced by methoxy groups, and the phosphate groups are replaced by phosphorothioate groups.

[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 crosslinking group is formed by a click chemistry reaction between diphenylcyclooctyne (DBCO) and / or cyclopropanecyclooctyne (BCN) and an azide crosslinker; more preferably, the crosslinking group is formed by a click chemistry reaction between diphenylcyclooctyne and an azide crosslinker. For example, the structure of the crosslinking group is shown below:

[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 nanoparticle according to the third aspect of the present invention in the preparation of a medicament for preventing and / or treating diseases associated with abnormal FMRP expression.

[0037] According to some embodiments of the present invention, the disease associated with abnormal FMRP expression 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 FMRP expression, 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 FMRP expression, 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 although the present invention provides the above technical solution, although only exemplary G-quadruplex RNA and RNA chemical modification methods are mentioned to construct PROTAC for degrading FMRP, it is known that FMRP can also bind to other G-quadruplex RNAs, RNA can also be modified by some other methods, and there are many kinds of tethers and small molecule E3 ubiquitin ligase ligands for constructing PROTAC. 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 tethers and small molecule E3 ubiquitin 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 PROTACs provided by this invention utilize G-quadruplex RNA that specifically targets FMRP and a small molecule E3 ubiquitin ligase to develop a series of PROTACs targeting FMRP. Furthermore, more stable and effective PROTACs are constructed by chemically modifying the G-quadruplex RNA at preferred sites. Furthermore, the present invention further enhances the PROTAC's stability and cellular entry in vivo by encapsulating the PROTAC in liposome nanoparticles.

[0047] Experimental verification has shown that the PROTACs provided by the present invention have the ability to efficiently degrade FMRP and alter the secretion pattern of cancer cells. A 300nM PROTAC can effectively degrade FMRP and alter the secretion pattern of cancer cells, which is half the amount required to achieve the same effect as existing PROTACs targeting FMRP. Furthermore, the optimized chemical modification greatly improves the stability of the G-quadruplex RNA PROTAC, and the use of a small molecule E3 ubiquitin ligase ligand reduces the molecular weight of the PROTAC to a certain extent. The use of liposome nanoparticles to encapsulate and deliver nucleic acid PROTACs also provides many advantages for the development of nucleic acid PROTAC drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

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

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

[0051] Figure 3 Showing the synthetic 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 examples of the present invention;

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

[0055] Figure 7 HPLC chromatograms of different PROTACs obtained in the examples of the present invention;

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

[0057] Figure 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] Figure 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] Figure 11 This is a particle size characterization diagram of liposome nanoparticles encapsulating PROTAC (msc1-PEG3-CRBNL) obtained in an embodiment of the present invention;

[0060] Figure 12Fluorescence confocal images of PROTAC (msc1-PEG3-CRBNL)-encapsulated liposome nanoparticles and unencapsulated PROTAC (msc1-PEG3-CRBNL) delivered into cancer cells obtained in the present invention; TD: bright field; Nucleus: blue fluorescence channel, blue fluorescence shows the cell nucleus; msc1-PEG3-CRBNL: green fluorescence channel, green shows FITC-msc1-PEG3-CRBNL; Merge: overlay of the previous three channels;

[0061] Figure 13 It shows that the PROTAC-encapsulated liposome nanoparticles (LNP@msc1-PEG3-CRBNL) obtained in the embodiment of the present invention effectively degrade FMRP in cancer cells;

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

[0063] Figure 15 This is an immunoblot image of the degradation of FMRP in cancer cells by PROTAC constructed using msc1 and PROTAC constructed using sc1 with other chemical modifications in the examples of the present invention. DETAILED DESCRIPTION

[0064] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given 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 methoxy and phosphorothioate modified first and last three bases was designed and synthesized. Specifically, the nucleotide sequence of the G-quadruplex RNA is: 5'-G(OCH3) * C(OCH3) * G(OCH3) * GCU GCG GUGUGG AAG GAG UGG CUG GGU UGC GCA GCU(OCH3) * U(OCH3) * G(OCH3) *-DBCO-3' (msc1-DBCO), where (OCH3) indicates that the 2'-OH group of the ribose sugar of the ribonucleotide is replaced by a methoxy group, and * indicates phosphorothioate modification of the ribonucleotide. Figure 1 The basic structure of the synthesized PROTAC is shown. Figure 2 Chemical modifications in the synthesis of G-quadruplex RNA are shown.

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

[0069] 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. To conjugate the azide-modified CRBN ligand containing a PEG3 linker (N3-PEG3-CRBNL) (CRBNL is pomalidomide, 4-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione) or the VH032 ligand containing a (CH2)5 linker (N3-C5-VH032) to msc1-DBCO or sc1-DBCO, N3-PEG3-CRBNL (50 μM) or N3-(CH2)5-VH032 (50 μM) was incubated with msc1-DBCO (50 μM) or sc1-DBCO (50 μM) in diethyl pyrocarbonate (DEPC)-treated water. The mixture was allowed to stand 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-PEG3-CRBNL, sc1-PEG3-CRBNL, msc1-C5-VH032, and sc1-C5-VH032, respectively. Specifically, an Agilent InertslWP300Cis (150 x 3 mm, 5 μm) column was used for separation, purification, and characterization of the synthesized PROTACs. First, 40 mL of 2 mol / L triethylamine acetate (TEAA) buffer was added to 760 mL of ultrapure water to prepare a 0.1 mol / L mobile phase A. 800 mL of analytical grade acetonitrile was used as mobile phase B. The PROTACs were then characterized and purified by gradient elution (elution gradient see Table 1). The mobile phase flow rate throughout the elution process was 1 mL / min, and the UV absorption wavelength for detection was 260 nm (the UV absorption wavelength of RNA). The newly generated peak was collected by comparing it with the peak position of unreacted RNA, and the solvent was removed by freeze-drying. The resulting 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 whether msc1-PEG3-CRBNL and msc1-C5-VH032 prepared in Example 1 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-PEG3-CRBNL and msc1-C5-VH032 were transfected into the cells using jetOPTIMUs (Polyplus). After 24 hours of transfection, proteins were collected and the content of intracellular FMRP was detected by immunoblotting. Figure 8 As shown in the figure, both msc1-PEG3-CRBNL and msc1-C5-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 under 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] To investigate whether chemical modification can enhance the ability of PROTAC to degrade FMRP in cancer cells, 300 nM of sc1-VHLL, sc1-PEG3-CRBNL, and msc1-PEG3-CRBNL were transfected into HeLa cells, respectively. 24 hours after transfection, proteins were collected and the intracellular FMRP content was detected by immunoblotting (the control group was cells without any treatment). Figure 9 As shown in the figure, all three PROTACs can degrade FMRP in HeLa cells, and the degradation efficiency is msc1-PEG3-CRBNL>sc1-PEG3-CRBNL>sc1-VHLL, which shows that the smaller molecular weight and chemically modified PROTAC synthesized by the present invention can indeed improve the degradation efficiency of FMRP.

[0076] Similarly, the ability of 300 nM sc1-VHLL, sc1-C5-VH032, and msc1-C5-VH032 to degrade FMRP in HeLa cells was compared (control group, i.e., cells without any treatment). The results showed that the efficiency of FMRP degradation was msc1-C5-VH032 > sc1-C5-VH032 > sc1-VHLL, which also shows that the smaller molecular weight and chemically modified PROTACs 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, the proteins of HeLa cells treated with sc1-VHLL, sc1-PEG3-CRBNL and msc1-PEG3-CRBNL for 36 hours were collected (the control group was cells without any treatment). Figure 10 As shown in the figure, all three PROTACs can reduce the secretion of IL-33 and PROS1 by cancer cells, and this inhibitory effect is most significant after treatment with msc1-PEG3-CRBNL, followed by sc1-PEG3-CRBNL. This indicates that PROTACs with smaller molecular weight and chemical modifications are more effective in changing the secretion pattern of cancer cells.

[0078] Similarly, HeLa cells were treated with sc1-VHLL, sc1-C5-VH032, and msc1-C5-VH032 for 36 hours (control group: untreated cells). msc1-C5-VH032 treatment produced the most significant decreases in IL-33 and PROS1, followed by sc1-C5-VH032. This suggests that the smaller molecular weight and chemically modified PROTACs synthesized by this invention are more effective in altering the secretion patterns of cancer cells.

[0079] Both IL-33 and PROS1 are abnormally overexpressed in many tumors, and their elevated expression is often associated with tumor malignancy and poor prognosis. IL-33 plays a crucial role in tumorigenesis, progression, and immunity. IL-33 promotes tumor proliferation and angiogenesis, and also creates an immunomodulatory tumor microenvironment, such as by promoting the differentiation of regulatory T cells and myeloid-derived suppressor cells. In this environment, tumors are susceptible to immune evasion. Therefore, reducing the amount of IL-33 secreted by cancer cells is of great significance for tumor treatment. PROS1 depletes T cells within the tumor, promotes M2 polarization of macrophages, and contributes to the formation of an immunosuppressive microenvironment. In summary, reducing the secretion of IL-33 and PROS1 inhibits tumor growth and creates a pro-inflammatory tumor microenvironment.

[0080] Example 3 In vivo delivery study of PROTAC

[0081] In order to better deliver msc1-PEG3-CRBNL into cells, liposome nanoparticles encapsulating PROTAC were synthesized. 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. msc1-PEG3-CRBNL was diluted in 10 mM acetic acid (pH 4) to achieve an N / P ratio of 6. Nanoparticles were prepared from this solution at a 1:3 ratio using a microfluidic device (a Zhongxin Qiheng Cchippump-02 dual-channel split syringe pump and a Zhongxin Qiheng micro-mixing liposome chip ZX-LS-31). Subsequently, the ethanol was removed and the liposome nanoparticles were neutralized by dialysis against PBS. The synthesized liposome nanoparticles encapsulated with msc1-PEG3-CRBNL are referred to as LNP@msc1-PEG3-CRBNL. Figure 11 The results showed that the synthesized liposome nanoparticles had uniform particle size.

[0082] To investigate whether liposomal nanoparticles can effectively deliver PROTACs into cancer cells, a PROTAC modified with FITC at the 5' end of RNA (FITC-msc1-PEG3-CRBNL) was synthesized. HeLa cells were incubated with liposomal nanoparticles encapsulating FITC-msc1-PEG3-CRBNL (LNP@FITC-msc1-PEG3-CRBNL) or unencapsulated FITC-msc1-PEG3-CRBNL for 6 hours. Figure 12 It was shown that liposome nanoparticles can efficiently deliver msc1-PEG3-CRBNL 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-PEG3-CRBNL (0-300 nM, concentration calculated according to the amount of encapsulated msc1-PEG3-CRBNL) for 24 h, and then their proteins were collected. Figure 13 It was shown that msc1-PEG3-CRBNL could be effectively delivered via liposome nanoparticles, 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-sh RNA were cross-linked with N3-PEG3-CRBNL to form different PROTACs. HeLa cells were placed in 6-well plates until the cell confluence reached approximately 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). Figure 14 As shown in the figure, PROTACs constructed with four types of G-quadruplex RNA can degrade FMRP in cancer cells to a certain extent, but PROTAC constructed with sc1 shows 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 (total 6) ribonucleotides near the 5' and 3' ends were modified with methoxy (M), phosphorothioate (S), or methoxy and phosphorothioate dual modifications (MS) (the corresponding chemical modification structures are shown in Figure 2). Figure 2 As shown), the chemically modified sc1 containing DBCO was cross-linked with N3-PEG3-CRBNL to form different PROTACs. HeLa cells were placed in 6-well plates 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). Figure 15 As shown, PROTAC constructed with sc1 RNA dually modified with methoxyl and thiophosphate (MS) has the strongest ability to degrade FMRP in cancer cells.

[0090] The above descriptions are merely exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention is disclosed above with reference to preferred embodiments, they are not intended to limit the present invention. Any person skilled in the art who, without departing from the scope of the present invention, may make slight changes or modifications to the above-disclosed technical contents to obtain equivalent or equivalent embodiments falls within the scope of the present invention.

Claims

1. A protein degradation targeting chimera, having the general structural formula: GLE; wherein, G is a G-quadruplex RNA that specifically binds to the mammalian fragile X mental retardation protein, E is a ligand for a small molecule E3 ubiquitin ligase, and L is a linker arm between the G-quadruplex RNA and the ligand for the small molecule E3 ubiquitin ligase; Wherein, the nucleotide sequence of the G quadruplex RNA is shown in SEQ ID NO. 3; Among them, SEQ ID NO. 3 is 5'-G(OCH3 C(OCH3 G(OCH3 GCU GCG GUG UGG AAG GAGUGG CUG GGU UGC GCA GCU(OCH3 U(OCH3 G(OCH3 -3'; wherein (OCH3) indicates that the 2'-OH of the ribose of the ribonucleotide is replaced by a methoxy group, Indicates phosphorothioate modification of a ribonucleotide.

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

3. 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.

4. 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.

5. The method for preparing a protein degradation targeted chimera according to any one of claims 1 to 4, comprising: (1) Synthesizing a G-quadruplex RNA with a linker and chemically modified first three ribonucleotides at the 5' and 3' ends; (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.

6. The preparation method according to claim 5, 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. 7 . A liposome nanoparticle encapsulating the protein degradation targeting chimera according to any one of claims 1 to 4 .

8. Use of the protein degradation targeting chimera according to any one of claims 1 to 4 or the liposome nanoparticle according to claim 7 in the preparation of a drug for preventing and / or treating cancer; in, The cancer is selected from one or more of colon cancer, pancreatic ductal carcinoma, breast cancer, liver cancer, prostate cancer and lung cancer.

9. 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 4 or the liposome nanoparticle according to claim 7, and optionally a pharmaceutically acceptable excipient.

10. The pharmaceutical composition according to claim 9, further comprising other pharmaceutically active ingredients; in, 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.

11. The pharmaceutical composition according to claim 9 or 10, 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.

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