Peptides that specifically bind to the RNA-binding domain of the PRMT5 protein and their applications

Abstract

This invention discloses a polypeptide that specifically binds to the RNA-binding domain of the PRMT5 protein and its applications, belonging to the field of biomedical technology. The polypeptide specifically binds to the RNA-binding domain of the PRMT5 protein, and its amino acid sequence includes sequence A or sequence B. Sequence A is: LTNKKGFPVLSK; sequence B is: PGMFSWFPILFP. This polypeptide can precisely interfere with PRMT5-RNA interaction without affecting PRMT5 protein abundance, thereby specifically disrupting a key pathway for maintaining tumor protein translation. This polypeptide therapeutic agent has been shown to effectively inhibit tumor growth in preclinical models, validating the therapeutic feasibility of the "PRMT5-RBD targeting" strategy and providing a novel potential treatment option for patients (including those insensitive to or resistant to existing methylation inhibitors).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to polypeptides that specifically bind to the RNA-binding domain of the PRMT5 protein and their applications. Background Technology

[0002] PRMT5 has become a highly anticipated target in cancer therapy due to its overexpression and crucial role in various cancers. To date, technological development in this field has almost entirely focused on developing inhibitors of PRMT5 methyltransferase activity. Numerous pharmaceutical companies and research institutions worldwide have invested heavily in this area, resulting in several small molecule compounds that have entered clinical trials.

[0003] Existing technologies disclose small molecule inhibitors that target PRMT5 methylation function. For example: GSK3326595 (Pemrametostat): A potent, selective, SAM-competitively specific peptide developed by GlaxoSmithKline that binds to the PRMT5 protein. Clinical trials have been initiated for solid tumors and myelodysplastic syndromes. Its core patent base can be found in patents such as international publication number WO2016142680A1.

[0004] EPZ015666(GSK3235025) / EPZ015938(JNJ-64619178): Developed by Epizyme and later licensed to GlaxoSmithKline and Johnson & Johnson, these are also orally effective peptides that specifically bind to the PRMT5 protein. The relevant patent is WO2015038770A1.

[0005] PF-06939999: A clinical-stage peptide developed by Pfizer that specifically binds to the PRMT5 protein.

[0006] MRTX1719: A peptide developed by Mirati Therapeutics that specifically binds to the PRMT5 protein via MTA synergy (methionine synergy), representing a different inhibition strategy. The relevant patent involves WO2021154941A1.

[0007] These existing technologies constitute the main body of current PRMT5-targeted anti-tumor therapies, and their common feature is that they all aim to inhibit the enzymatic catalytic function of PRMT5.

[0008] Although the aforementioned inhibitors have shown some efficacy in preclinical and clinical studies, their inherent technical limitations restrict the maximization of their efficacy, specifically in the following ways: 1) Incomplete target coverage, resulting in therapeutic blind spots: Recent cutting-edge research (including the applicant's work) has clearly revealed that PRMT5 is not only a methyltransferase but also a non-classical RNA-binding protein (RBP). It directly stabilizes a series of tumor-promoting mRNAs (such as genes encoding translation machines) through an independent RNA-binding domain (RBD), a function completely independent of its methyltransferase activity. All existing technologies are unable to intervene in this newly discovered, crucial oncogenic pathway, leading to incomplete inhibition of PRMT5.

[0009] 2) Single-drug efficacy has a "ceiling": Since it can only block part of the function of PRMT5 (methylation) and cannot block the protein synthesis and tumor growth signals driven by its RNA binding, the anti-tumor effect of single methylation inhibitors is difficult to achieve the optimal level, and they show insufficient efficacy in many cancer models.

[0010] 3) Potential drug resistance risk: Tumor cells may compensate for the survival pressure after their methylation activity is inhibited by upregulating the RNA binding function of PRMT5 or becoming more dependent on this pathway, thereby leading to adaptive resistance to existing inhibitors.

[0011] In summary, the fundamental flaw of existing technologies lies in the limitation of their target, which targets only the "methylation function" of PRMT5 while completely ignoring its equally important "RNA binding function". Summary of the Invention

[0012] To address the shortcomings of existing technologies, the present invention aims to provide a polypeptide that specifically binds to the RNA-binding domain of the PRMT5 protein and its applications, thereby solving the problems in the prior art.

[0013] The objective of this invention can be achieved through the following technical solutions: A polypeptide that specifically binds to the RNA-binding domain of the PRMT5 protein, the amino acid sequence of the polypeptide comprising sequence A or sequence B; The sequence A is: LTNKKGFPVLSK; The sequence B is: PGMMFSWFPILFP.

[0014] Furthermore, the polypeptide also includes a cell-penetrating peptide sequence attached to the end of sequence A or sequence B.

[0015] Furthermore, the cell-penetrating peptide sequence is: YGRKKRRQRRR.

[0016] Furthermore, the polypeptide has an amidation modification at its end.

[0017] A polypeptide combination targeting the PRMT5 dual RNA binding domain, comprising: The first polypeptide is the polypeptide comprising sequence A as described in any one of claims 1-4; and, The second polypeptide is the polypeptide containing sequence B as described in any one of claims 1-4.

[0018] Furthermore, the molar concentration ratio of the first polypeptide to the second polypeptide is 1:1.

[0019] An antitumor drug composition comprising: The first active ingredient is: the aforementioned polypeptide, or a combination of the aforementioned polypeptides; and, The second active ingredient is a PRMT5 methyltransferase activity inhibitor.

[0020] Furthermore, the PRMT5 methyltransferase activity inhibitor is GSK3326595.

[0021] The application of the above-mentioned polypeptides, polypeptide combinations, or antitumor drug compositions in the preparation of antitumor drugs.

[0022] Furthermore, the mechanism of the antitumor drug includes blocking the binding of PRMT5 protein to oncogenic mRNA, wherein the oncogenic mRNA includes at least one of EIF3A, EIF3B, and EIF3D.

[0023] The beneficial effects of this invention are: 1. This invention, by identifying the RNA-binding domain (RBD) of PRMT5, provides for the first time a drug target that can intervene in the non-methylation-dependent oncogenic function of PRMT5. This makes it possible to fundamentally solve the deficiency of "incomplete target coverage" in existing technologies, laying an irreplaceable biological foundation for the development of peptides that specifically bind to the RNA-binding domain of the PRMT5 protein with novel mechanisms of action. This discovery directly explains why the efficacy of existing methylation inhibitors has a "ceiling," revealing the deep mechanism of their insufficient efficacy—namely, their inability to inhibit the parallel pro-tumor survival pathway driven by RNA-binding function. This provides crucial scientific evidence for optimizing clinical treatment strategies.

[0024] 2. This invention provides a novel therapeutic entity based on a novel RBD-targeting peptide designed with a new target, offering a mechanism of action completely independent of methylation inhibition. This peptide precisely interferes with PRMT5-RNA interactions without affecting PRMT5 protein abundance, thereby specifically disrupting a key pathway for maintaining tumor protein translation. Preclinical models have demonstrated the effectiveness of this peptide in inhibiting tumor growth, validating the therapeutic feasibility of the "PRMT5-RBD targeting" strategy and providing a novel potential treatment option for patients (including those insensitive to or resistant to existing methylation inhibitors).

[0025] 3. The core combination of this invention (RBD peptide + methylation inhibitor) has demonstrated significant synergistic effects in both in vitro and in vivo experiments. Its efficacy is significantly superior to any single-drug therapy and the theoretical summation effect, achieving a "full-pathway blockade" of PRMT5's oncogenic function, and is expected to overcome the efficacy bottleneck of existing single-drug therapies. By simultaneously inhibiting two independent key functions of PRMT5 that tumors rely on for survival, it greatly increases the difficulty for tumor cells to escape through functional compensation, thereby potentially delaying or even preventing the occurrence of acquired resistance to the greatest extent, and is expected to achieve more durable disease control. The synergistic effect allows for a potential reduction in the required dosage of each component while achieving equivalent or better efficacy. Due to the high specificity of the peptide and its different mechanism of action and metabolic pathway compared to existing small molecule inhibitors, this combination is expected to improve efficacy while maintaining or even improving safety profiles, resulting in a better therapeutic index. This combination strategy provides clinicians with a new weapon, especially for tumor types that may be highly dependent on PRMT5 RNA binding function or for refractory patients. Its modular design (the peptide can be used alone or in combination with different methylation inhibitors) also gives the treatment regimen a high degree of flexibility and scalability. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram illustrating the identification of PRMT5 RNA binding function and mutant verification of the present invention; Figure 2 This is a schematic diagram illustrating the functional and therapeutic efficacy verification of the inhibitory peptide targeting PRMT5-RBD of the present invention. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1 This embodiment describes the functional identification and verification of the RNA binding domain (RBD), a novel drug target of PRMT5. 1. Through systematic functional experiments (RNA binding function verification), two functional RNA binding domains on PRMT5 that can serve as drug targets were identified and verified.

[0030] 1) In vivo orthogonal organic phase separation (OOPS) experiment The experimental procedure includes: The OOPS assay is used to capture UV-crosslinked protein-RNA complexes. A simplified procedure is as follows: HEK293T cells cultured in 10 cm culture dishes with 80-90% confluence were washed twice with pre-chilled PBS. The crosslinked group samples were placed on ice and irradiated with 254 nm UV light (400 mJ / cm²). The non-crosslinked control group underwent the same treatment except for the absence of UV irradiation. Immediately after treatment, 1 mL of TRIzol reagent (Invitrogen) was added to the culture dish to lyse the cells, and the mixture was incubated at room temperature for 5 minutes to dissociate non-covalent interactions. Then, 200 μL of chloroform was added, the mixture was vigorously shaken for 15 seconds, and centrifuged at 13,000 × g for 15 minutes at 4°C. The upper aqueous phase (containing free RNA) and the lower organic phase (containing free protein) were carefully removed, and the intermediate phase containing the covalently linked protein-RNA adduct was collected. A second round of TRIzol / chloroform extraction was then performed to remove residual impurities. The resulting intermediate phase was resuspended in 100 μL of elution buffer (containing 100 mM triethylammonium bicarbonate, 1 mM MgCl2, and 1% SDS), and 2 μg of RNase A / T1 mixture (2 mg / mL RNase A, 5000 U / mL RNase T1) was added. The mixture was digested at 37°C for 30 minutes to release the protein from the RNA. The protein was recovered by methanol-chloroform precipitation, dissolved in 1× loading buffer, and then subjected to Western blotting analysis.

[0031] Experimental results are as follows Figure 1 As shown in A, PRMT5 can bind to RNA.

[0032] 2) In vitro RNA binding experiment The experimental procedure includes: In vitro translation: In vitro protein synthesis was performed using the TNT® T7 Quick coupled transcription / translation system (Promega). A 50 μL reaction mixture containing 40 μL TNT Quick premix, 1 μL 1 mM methionine, and 1 μg plasmid DNA template with a T7 promoter and C-terminal FLAG tag was prepared on ice and incubated at 30°C for 90 minutes. To purify the protein, the reaction product was incubated with Anti-FLAG® M2 magnetic beads at 4°C with slow rotation for 1 hour. After washing three times with pre-chilled TBS-T buffer, TBS buffer containing 150 ng / μL 3× FLAG peptide was added, and competitive elution was performed at 4°C for 2 hours. The eluted product was used directly for subsequent RNA-protein binding experiments.

[0033] In vitro RNA-protein binding assay: The RNA-binding activity of the purified FLAG fusion protein was assessed using the Oligo(dT) magnetic bead pull-down method. Total RNA was extracted from HEK293T cells using TRIzol reagent. A fixed amount of purified protein (200 nM) and gradient concentrations of total RNA (0, 0.002, 0.2, 20 μg) were incubated in 1× protein-RNA binding buffer at 37°C for 30 min. All reaction mixtures were then transferred to pre-equilibrated Oligo(dT) buffer. 25 Poly(A)⁺ RNA and its binding proteins were captured by rotating and incubating in magnetic beads (NEB, #S1419S) at 4°C for 3 hours. After magnetic separation, the RNA was thoroughly washed five times with pre-cooled binding / washing buffer, and finally eluted by boiling with SDS-PAGE loading buffer. Proteins specifically bound to poly(A)⁺ RNA were detected by Western blotting.

[0034] Experimental results are as follows Figure 1 As shown in B, this further demonstrates that PRMT5 can directly bind to RNA.

[0035] 2. Identification of the RNA-binding region (RBD) of PRMT5: Based on previously published studies and bioinformatics analysis, it is speculated that the N-terminal region of PRMT5 and a region near an internally conserved "THW" motif may be involved in RNA binding. To verify this function, a corresponding loss-of-function mutant was designed in this embodiment.

[0036] 1) Mutant construction The construction process included the following: the construction of plasmids RBD#1_mut (containing N239A / K240D / F243A mutations) and RBD#2_mut (W579A / F580A mutations) was completed by Suzhou Genewiz Biotechnology Co., Ltd.

[0037] like Figure 1As shown in the upper half of C in the figure, point mutants targeting the two speculative regions mentioned above were constructed: RBD#1_mut (containing N239A / K240D / F243A mutations) targeting the N-terminal region and RBD#2_mut (W579A / F580A mutations) targeting the “THW” motif.

[0038] 2) Verification of intracellular interactions The OOPS assay is used to capture UV-crosslinked protein-RNA complexes. A simplified procedure is as follows: HEK293T cells cultured in 10 cm culture dishes with 80-90% confluence were washed twice with pre-chilled PBS. The crosslinked group samples were placed on ice and irradiated with 254 nm UV light (400 mJ / cm²). The non-crosslinked control group underwent the same treatment except for the absence of UV irradiation. Immediately after treatment, 1 mL of TRIzol reagent (Invitrogen) was added to the culture dish to lyse the cells, and the mixture was incubated at room temperature for 5 minutes to dissociate non-covalent interactions. Then, 200 μL of chloroform was added, the mixture was vigorously shaken for 15 seconds, and centrifuged at 13,000 × g for 15 minutes at 4°C. The upper aqueous phase (containing free RNA) and the lower organic phase (containing free protein) were carefully removed, and the intermediate phase containing the covalently linked protein-RNA adduct was collected. A second round of TRIzol / chloroform extraction was then performed to remove residual impurities. The resulting intermediate phase was resuspended in 100 μL of elution buffer (containing 100 mM triethylammonium bicarbonate, 1 mM MgCl2, and 1% SDS), and 2 μg of RNase A / T1 mixture (2 mg / mL RNase A, 5000 U / mL RNase T1) was added. The mixture was digested at 37°C for 30 minutes to release the protein from the RNA. The protein was recovered by methanol-chloroform precipitation, dissolved in 1× loading buffer, and then subjected to Western blotting analysis.

[0039] Western blotting analysis, such as Figure 1 As shown in the lower half of C in the figure, the results indicate that the enrichment of RBD#1_mut and RBD#2_mut mutants in the RNA-protein complex is significantly reduced, confirming their defective RNA binding function in the cellular environment.

[0040] 3) In vitro direct binding verification In vitro RNA pull-down experiments were performed using purified wild-type (WT) and the aforementioned mutant PRMT5 protein. The procedure included: purifying polyA mRNA using oligodT magnetic beads, followed by incubation with prokaryotically expressed GST-PRMT5 protein at 37°C for 30 minutes; enriching GST-PRMT5 protein with GST magnetic beads; removing unbound mRNA by washing with PBS; and then extracting and recovering the mRNA using TRIzol / chloroform and identifying it by agarose gel electrophoresis. Results are as follows: Figure 1 As shown in D in the figure, it can be seen that compared with the WT protein, the RBD#1_mut and RBD#2_mut mutants have significantly lost their ability to bind to the target RNA probe.

[0041] 4) In vivo functional verification Wild-type and RBD mutant PRMT5 were overexpressed in HepG2 cells, and a nude mouse subcutaneous tumor model was constructed. Tumor growth was observed at different time points. Specifically, the experimental procedure included: overexpressing wild-type and RBD mutant PRMT5 in HepG2 cells, followed by cell line 1 10 7 Cells were inoculated into the lateral ventral region of the armpit of nude mice, and tumor growth was monitored at different time points.

[0042] Figure 1 E, F, and G in the figure reflect the changes in tumor volume, growth curve, and endpoint weight of the RBD mutant PRMT5 overexpressing group compared to the wild-type overexpressing group in the HepG2 xenograft model. It can be seen that the tumor size, growth, and weight of the RBD mutant overexpressing group were significantly lower than those of the wild-type overexpressing group.

[0043] Example 2 This embodiment describes the design, preparation, and functional characterization of inhibitory peptides targeting RBD; To achieve pharmacological intervention on the aforementioned new functional modules, this embodiment designed a specific inhibitory peptide.

[0044] 1. Peptide design and preparation process like Figure 2 As shown in A, based on the key amino acid region identified in the functional experiments of Example 1, this example designs two core repressive peptides: The sequence of Peptide#1 originates from the key functional region of RBD#1 and is designed to competitively occupy this site; The sequence of Peptide#2 contains the core “THW” motif of RBD#2 or its key residues, designed to mimic or interfere with the interface.

[0045] Both peptides are fused with a proven cell-penetrating peptide (CPP) to ensure their transmembrane delivery efficiency.

[0046] The amino acid sequence of Peptide #1 is shown in SEQ ID NO. 1: LTNKKGFPVLSK; The amino acid sequence of Peptide #2 is shown in SEQ ID NO.2: PGMFSWFPILFP; The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.3: YGRKKRRQRRR.

[0047] The preparation process of the peptide is as follows: The targeted RBD peptide was synthesized by Jier Biochemical (Shanghai) and the HPLC purity was >95%.

[0048] In addition, such as Figure 2 As shown in A, this embodiment modifies the terminal of the peptide. Specifically, the C-terminus of the peptide is amidated (-CONH2) to eliminate the terminal negative charge and enhance its resistance to protease degradation. This modification helps to enhance the efficiency of the peptide in penetrating the cell membrane and its targeted binding affinity to the RNA binding domain of PRMT5.

[0049] 2. Immunofluorescence experiment The experimental procedure included: after treating cells with 15 µM Peptide #1 or Peptide #2 for 4 hours, adding hochest dye (Beyotime) to stain the cell nuclei for 10 minutes, and then acquiring images using a laser confocal microscope.

[0050] from Figure 2 As can be seen in B and C, immunofluorescence images confirm that FITC-labeled Peptide #1 and Peptide #2 can be effectively delivered into cells.

[0051] In addition, after treating cancer cells with 15 µM Peptide #1 or Peptide #2 for 4 hours, 1,500–2,000 cells were seeded into 96-well cell culture plates, and cell counts were performed at different time points using CTG (Promega); the results are as follows. Figure 2 As shown in D, it can be seen that treatment with either Peptide#1 or Peptide#2 alone can significantly inhibit cancer cell proliferation; thus, it can be concluded that disrupting any one of the RNA binding interfaces is sufficient to weaken the oncogenic function of PRMT5.

[0052] 3. To maximally disrupt the RNA binding function of PRMT5, Peptide #1 and Peptide #2 were mixed in a 1:1 ratio (7.5 µM: 7.5 µM) (named RBD-Peptides) for all subsequent studies.

[0053] The experimental procedure includes: Figure 2 In step E, after treating cells with Peptide #1 and Peptide #2 for 4 hours, the protein-RNA complexes used to capture UV crosslinks were determined using an OOPS assay. A simplified procedure is as follows: HEK293T cells cultured in 10 cm culture dishes with 80-90% confluence were washed twice with pre-chilled PBS. The crosslinked samples were placed on ice and irradiated with 254 nm UV light (400 mJ / cm²). Immediately after treatment, 1 mL of TRIzol reagent (Invitrogen) was added to the culture dish to lyse the cells, and incubated at room temperature for 5 minutes to dissociate non-covalent interactions. Then, 200 μL of chloroform was added, the mixture was vigorously shaken for 15 seconds, and centrifuged at 13,000 × g for 15 minutes at 4°C. The upper aqueous phase (containing free RNA) and the lower organic phase (containing free protein) were carefully removed, and the intermediate phase containing the covalent protein-RNA adduct was collected. A second round of TRIzol / chloroform extraction was performed to remove residual impurities. The resulting intermediate phase was resuspended in 100 μL of elution buffer (containing 100 mM triethylammonium bicarbonate, 1 mM MgCl2, and 1% SDS), and 2 μg of RNase A / T1 mixture (2 mg / mL RNase A, 5000 U / mL RNase T1) was added. The mixture was digested at 37°C for 30 minutes to release the protein from the RNA. The protein was recovered by methanol-chloroform precipitation, dissolved in 1× loading buffer, and then subjected to Western blot analysis.

[0054] Figure 2 In the F-cell assay, cells were treated with Peptide #1 and Peptide #2 every 12 hours. After 24 hours, the cells were collected for immunoblotting analysis.

[0055] Figure 2 In G, cells were treated with Peptide #1 and Peptide #2 every 12 hours. After 24 hours, cells were collected for RIP-qCPR analysis.

[0056] Figure 2 In the H phase, cells were treated with Peptide #1 and Peptide #2 every 12 hours. After 24 hours, actinomycin D (5 μg / mL) was added. Cells were collected at 0 and 4 hours for qCPR analysis.

[0057] Figure 2In step I, cells were treated with Peptide #1 and Peptide #2 every 12 hours. After 24 hours, cells were treated with 1 μg / mL puromycin (Sigma) for 1 hour, and then the cells were collected for immunoblotting analysis.

[0058] from Figure 2 As can be seen from E and F, RBD-Peptides treatment reduced the binding of PRMT5 to total RNA without affecting PRMT5 protein levels. From... Figure 2 As can be seen from G, RBD-Peptides treatment specifically weakens the binding of PRMT5 to EIF3A, EIF3B, and EIF3D mRNA. This disruptive effect leads to a decrease in the mRNA and protein levels of the target genes (e.g., Figure 2 As shown in F), and accelerated the degradation of mRNA (e.g. Figure 2 (As shown in H in the figure). Ultimately, RBD-Peptides treatment significantly inhibited overall cellular protein synthesis (e.g., ...). Figure 2 As shown in I).

[0059] Example 3 In this embodiment, the synergistic anti-tumor combination therapy (RBD-Peptides+GSK3326595) was validated. The verification process includes: taking HepG2 (1×10) 7 (Number of tumors per mouse) were subcutaneously injected into the abdomen of five-week-old nude mice to establish a xenograft tumor model. After tumor formation, mice were randomly divided into a control group, an RBD peptide monotherapy group, a GSK3326595 monotherapy group, and a combination therapy group, with each group receiving the medication every other day. RBD peptide was administered intraperitoneally (15 mg / kg), and GSK3326595 was prepared in 0.5% DMSO and administered by gavage at a dose of 50 mg / kg. Tumor size was measured simultaneously using the formula (width...). 2 Calculate the volume by (×length) / 2, and monitor it until the end of the experiment.

[0060] The verification results are as follows Figure 2 As shown in JL, these correspond to the endpoint tumor photographs, growth curves, and weight statistics, respectively. It can be seen that in the xenograft tumor model, RBD-Peptides monotherapy can inhibit tumor growth; its combination with the catalytic inhibitor GSK3326595 exhibits a stronger anti-tumor effect. P <0.01); This demonstrates that the dual-targeting strategy, which simultaneously targets the methyltransferase activity and RNA binding activity of PRMT5, has superior therapeutic effects.

[0061] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0062] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A polypeptide that specifically binds to the RNA-binding domain of the PRMT5 protein, characterized in that, The amino acid sequence of the polypeptide includes sequence A or sequence B; The sequence A is: LTNKKGFPVLSK; The sequence B is: PGMMFSWFPILFP.

2. The polypeptide according to claim 1, characterized in that, The polypeptide also includes a cell-penetrating peptide sequence attached to the end of sequence A or sequence B.

3. The polypeptide according to claim 2, characterized in that, The cell-penetrating peptide sequence is: YGRKKRRQRRR.

4. The polypeptide according to any one of claims 1-3, characterized in that, The polypeptide has an amidation modification at its end.

5. A polypeptide combination targeting the PRMT5 dual RNA binding domain, characterized in that, include: The first polypeptide is the polypeptide containing sequence A as described in any one of claims 1-4; as well as, The second polypeptide is the polypeptide containing sequence B as described in any one of claims 1-4.

6. The polypeptide combination according to claim 5, characterized in that, The molar ratio of the first polypeptide to the second polypeptide is 1:

1.

7. An antitumor drug composition, characterized in that, include: The first active ingredient is: the polypeptide according to any one of claims 1-4, or a combination of polypeptides according to any one of claims 5-6; as well as, The second active ingredient is a PRMT5 methyltransferase activity inhibitor.

8. The antitumor pharmaceutical composition according to claim 7, characterized in that, The PRMT5 methyltransferase activity inhibitor is GSK3326595.

9. The use of the polypeptide of any one of claims 1-4, the polypeptide combination of any one of claims 5-6, or the antitumor drug composition of any one of claims 7-8 in the preparation of an antitumor drug.

10. The application according to claim 9, characterized in that, The mechanism of action of the antitumor drug includes blocking the binding of PRMT5 protein to oncogenic mRNA, wherein the oncogenic mRNA includes at least one of EIF3A, EIF3B and EIF3D.

Images