Myh2-fign interaction blocking amino acid compounds and uses thereof

Abstract

The application relates to a MYH2-FIGN interaction blocking amino acid compound and an application thereof, and relates to the fields of polypeptide drugs and tumor prevention and treatment. The MYH2-FIGN interaction blocking amino acid compound is an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:3 or a polypeptide derivative thereof. The application further discloses a pharmaceutical composition containing the MYH2-FIGN interaction blocking amino acid compound and a pharmaceutically acceptable carrier. The application first designs a polypeptide drug targeting the MYH2-FIGN interaction interface, and compared with traditional small molecules, the polypeptide drug has specificity for MYH2-FIGN interaction inhibition, and through fusion of an iRGD peptide, not only is the polypeptide cell penetration problem solved, but also the polypeptide drug is endowed with tumor targeting, the bioavailability of the drug is improved, and the systemic toxicity is reduced. The polypeptide drug can adopt various drug dosage forms, and the preparation process is simple.

Description

Technical Field

[0001] This invention relates to the fields of polypeptide drugs and tumor prevention and treatment, specifically to a MYH2-FIGN interaction-blocking amino acid compound and its application in the preparation of cancer therapeutic drugs. Background Technology

[0002] Extensive research and practice have been conducted on the treatment of malignant tumors, evolving from a single approach of "killing cancer cells" to multidisciplinary team (MDT) therapy. Current core treatment methods include surgical resection, radiation therapy, and drug therapy. With the development of precision medicine, molecular targeted therapy and immunotherapy have been introduced. Molecular targeted therapy works by attacking specific gene mutations or protein expressions in tumor cells. A typical example is EGFR-mutant non-small cell lung cancer patients who, with adjuvant therapy using third-generation targeted drugs (such as osimertinib), can achieve a 5-year survival rate of up to 88%. Immunotherapy (PD-1 / PD-L1) works by releasing the "brake" on immune T cells from the tumor, allowing the immune system to eliminate cancer cells on its own. Its applications include showing remarkable efficacy in melanoma, non-small cell lung cancer, and some sarcomas, even enabling long-term survival in some advanced-stage patients. However, these treatment methods still have significant room for development, and both efficacy and cost need further improvement.

[0003] Subcellular localization of proteins is a key factor determining their biological function. Fidgetin (FIGN) is a microtubule-cutting protein that is primarily located in the nucleus of normal cells. However, in various malignant tumor cells (such as breast cancer, liver cancer, and lung adenocarcinoma), FIGN is enriched in the cytoplasm. Clinical data indicate that FIGN's cytoplasmic localization is significantly associated with poor patient prognosis, but the specific correlation remains unclear. What causes this imbalance in FIGN's "nucleocytoplasmic shuttle" in cancer cells? Could controlling this shuttle inhibit the development and proliferation of cancer cells?

[0004] Furthermore, developing highly selective and highly active small molecule inhibitors that directly target specific protein-protein interaction (PPI) interfaces still faces significant challenges. Peptide drugs, due to their high specificity and relatively low toxicity, are an ideal choice for targeting PPIs. However, designing peptides that can precisely target specific PPI interfaces and addressing their cell membrane penetration issues are also key challenges for their clinical application.

[0005] In conclusion, there is an urgent need to develop a new shuttle blockade inhibitor and a corresponding drug to address the current deficiencies and shortcomings. Summary of the Invention

[0006] In view of this, the main objective of the present invention is to provide a MYH2-FIGN interaction blocking amino acid compound and its application, in order to at least partially solve the above-mentioned technical problems.

[0007] To achieve the above objectives, as a first aspect of the present invention, a MYH2-FIGN interaction blocking amino acid compound is provided, wherein the MYH2-FIGN interaction blocking amino acid compound is an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a polypeptide derivative thereof.

[0008] As a second aspect of the invention, a pharmaceutical composition comprising the MYH2-FIGN interaction-blocking amino acid compound as described above, and a pharmaceutically acceptable carrier is also provided.

[0009] As a third aspect of the present invention, a method for preparing the pharmaceutical composition as described above is also provided, comprising the following steps:

[0010] The MYH2-FIGN interaction-blocking amino acid compound described above is mixed with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0011] As a fourth aspect of the invention, the application of an amino acid compound that blocks MYH2-FIGN interaction as described above in the preparation of cancer therapeutic drugs is also proposed.

[0012] Based on the above technical solution, it can be seen that the MYH2-FIGN interaction blocking amino acid compound and its application of the present invention have at least one of the following beneficial effects compared with the prior art:

[0013] 1. Clear Mechanism: This invention is the first to design a peptide drug that targets the MYH2-FIGN interaction interface. Its mechanism of action is to competitively bind to MYH2 with FIGN, thereby relieving the cytoplasmic retention of FIGN by MYH2 and forcing FIGN nuclear translocation, thus significantly inhibiting FIGN-mediated oncogenic signals.

[0014] 2. High efficacy and specificity: Peptides designed based on protein-protein interaction interfaces have higher specificity and efficacy in inhibiting MYH2-FIGN interactions compared to traditional small molecules.

[0015] 3. High delivery efficiency: By fusing iRGD peptide, not only is the cell penetration problem of peptides solved, but also tumor targeting is endowed, improving drug bioavailability and reducing systemic toxicity.

[0016] 4. Broad-spectrum anti-cancer potential: Given the universal role of cytoplasmic FIGN in various cancers, the fusion peptide provided by this invention has broad prospects for treating a variety of cancer types.

[0017] 5. The polypeptide small molecule drug of the present invention can be used in a variety of drug dosage forms and the preparation process is simple. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 This is a schematic diagram of the three-dimensional structure of the interaction interface between the MYH2 protein and FIGN protein, as predicted by molecular docking simulation, highlighting the key functional residue F321 of the FIGN protein.

[0020] Figure 2 The experimental results map shows the effect of the FIGN protein F321 mutation (F321S) on its binding ability to MYH2 protein by co-immunoprecipitation (Co-IP) and Western blotting techniques.

[0021] Figure 3 This is a Western blot analysis of the protein binding assay for peptide competitive binding; among which, Figure 3 A and 3B are Western blot patterns obtained from competitive binding assays of peptides with IP values ​​of MYH2 or FIGN, respectively.

[0022] Figure 4 Images from immunofluorescence experiments show the subcellular localization of FIGN protein in Huh7 cells in different treatment groups (control, F321S-iRGD, WT-iRGD).

[0023] Figure 5 This is a set of experimental results showing the effects of the WT-iRGD fusion peptide on the β-catenin signaling pathway; among them, Figure 5 A is a map showing the detection of β-catenin protein levels using Western blot; Figure 5 B is a bar chart showing the qPCR detection of β-catenin mRNA levels; Figure 5 C is a line graph showing the stability of β-catenin mRNA under Actinomycin D treatment; Figure 5 D, 5E, and 5F are bar charts showing the mRNA levels of downstream target genes c-MYC, Cyclin D1, and MMP7 detected by qPCR.

[0024] Figure 6 This is a set of figures showing the experimental results regarding the effect of the WT-iRGD fusion peptide on the proliferation ability of Huh7 cells; among them, Figure 6 A is a line graph showing cell viability as detected by the CCK-8 assay; Figure 6 B is a line graph of the direct cell counting experiment; Figure 6 C is a representative photograph from a cell clonogenic experiment; Figure 6 D is the correct answer. Figure 6 A bar chart showing the number of clones formed in C;

[0025] Figure 7 This is a set of figures showing the experimental results regarding the effects of the WT-iRGD fusion peptide on the migration and invasion abilities of Huh7 cells; among them, Figure 7 A shows representative photographs of the cell scratch healing experiment at different time points; Figure 7 B is the correct answer. Figure 7 A statistical bar chart of the area where scratches have healed; Figure 7 C is a representative photograph of the Transwell cell invasion assay (stained with crystal violet); Figure 7 D and 7E are opposites Figure 7 A bar chart showing the number of C-cells that have permeated the membrane;

[0026] Figure 8 This is a collection of in vivo experimental results showing the inhibitory effect of the WT-iRGD fusion peptide on tumor growth in a BALB / c nude mouse orthotopic liver cancer model; among them, Figure 8 A represents the gross liver photographs of representative mice in each group at the experimental endpoint, high-power field images of HE-stained liver tissue sections, and immunohistochemical (IHC) staining images of Ki-67, β-catenin, and FIGN in tumor tissues. Figure 8 B is a violin plot showing the ratio of mouse liver weight to body weight (liver-to-body ratio); Figure 8 C is a statistical bar chart showing the number of tumor nodules on the liver surface; Figure 8 D is a statistical bar chart of the Ki-67 positive cell index in tumor tissue; Figure 8 E is a scatter plot of the β-catenin IHC staining score in tumor tissue; Figure 8 F is a scatter plot of the ratio of FIGN protein nucleoplasmic staining intensity in tumor cells;

[0027] Figure 9 This is a collection of in vivo experimental results showing the inhibitory effect of the WT-iRGD fusion peptide on tumor metastasis in a BALB / c nude mouse tail vein lung metastasis model; among them, Figure 9 A represents the lungs of representative mice in each group at the experimental endpoint, a panoramic HE-stained lung tissue section, and a high-power HE-stained image of metastatic lesions. Figure 9 B is a bar chart showing the number of lung metastases. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0029] The terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of the invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0030] The meanings of some terms in this invention are as follows:

[0031] Peptide derivatives are peptides that have been modified in ways other than sequence alterations. These modifications include chemical modifications (such as introducing PEG (polyethylene glycol) to increase solubility and half-life), conjugates (linking peptides to toxins, fluorescent dyes, or drug molecules), and isomers (such as replacing L-type amino acids with D-type amino acids to resist protease degradation).

[0032] Molecular docking is a computer-based simulation technique used to predict the optimal binding mode of two or more molecules in three-dimensional space. Its core objective is to find the most stable conformation between a ligand (usually a small molecule drug) and a receptor (usually a protein or other macromolecule), and to estimate the binding free energy or affinity using a scoring function, thereby determining the binding strength.

[0033] The docking score is a numerical value obtained from molecular docking simulations, used to quantify the interaction strength and binding stability between ligands (small molecules, peptides, etc.) and target proteins. Its value is typically expressed in kcal·mol⁻¹. -1 The value is expressed in units of , and the more negative (lower) the value, the closer the prediction is and the stronger the affinity.

[0034] Fidgetin (FIGN) is a member of the AAA+ ATPase family and belongs to the "microtubule-severing factor" category. It is an ATP-dependent enzyme involved in the regulation of microtubule dynamics and is crucial for cytoskeleton remodeling, cell division, and development. Its Chinese name is often translated as "Fidgetin protein" or "FIGN protein".

[0035] Myosin heavy chain 2 (MYH2) is a gene that encodes a type of myosin heavy chain in skeletal muscle. It belongs to the myosin heavy chain (MYH) gene family. This gene forms a gene cluster on human chromosome 17 (or chromosome 2 in some literature). It is the gene that encodes the key myosin heavy chain in fast-2A skeletal muscle fibers, which is responsible for converting the chemical energy of ATP into mechanical force to drive muscle contraction. Mutations or dysregulation of its expression can cause a variety of muscle diseases.

[0036] Actinomycin D, also commonly known as "actinomycin," is a mycotoxin produced by Streptomyces spp. (Streptomyces spp.) Streptomyces The polypeptide antibiotics produced by these drugs can intercalate between the GC base pairs of the DNA double helix, inhibiting RNA synthesis and thus blocking cell transcription and proliferation. Therefore, they are widely used in anti-tumor chemotherapy (such as for the treatment of nephroblastoma, choriocarcinoma, etc.) and in the clinical treatment of various solid tumors and hematological malignancies.

[0037] iRGD (internalizing RGD) is a tumor-penetrating peptide with the complete cyclic sequence CRGDKGPDC (also written as c(CRGDKGPDC)). This peptide possesses both the RGD (Arg-Gly-Asp) integrin-binding motif and the CendR (C-terminal rule) penetration motif, enabling it to achieve "dual receptor" localization and deep penetration within tumor tissue. iRGD is a cyclic peptide with both integrin targeting and NRP-1-mediated penetration capabilities. Through a dual-receptor mechanism, it achieves highly efficient drug penetration into the tumor and has been widely used to enhance the efficacy of various tumor treatments and diagnostic techniques, including chemotherapy, gene therapy, and imaging.

[0038] The key residue F321 refers to the phenylalanine residue at position 321 on the polypeptide chain. In many enzymes or receptors, it often acts as a crucial structural or functional fulcrum, influencing the substrate binding posture and catalytic efficiency through its hydrophobic aromatic ring.

[0039] β-catenin is a multifunctional connective protein that plays a key role in human cells, mainly involved in the following two aspects: (1) Cell adhesion: β-catenin binds to E-cadherin, an adhesion molecule on the cell membrane, to form an adhesion complex, which helps cells form tight connections and maintain the integrity of tissue structure; (2) Wnt signaling pathway: In the classic Wnt / β-catenin signaling pathway, β-catenin acts as the core molecule for signal transduction. When Wnt signaling is activated, β-catenin in the cell stabilizes and accumulates, and then enters the cell nucleus, where it binds to TCF / LEF transcription factors to regulate the expression of a series of genes related to cell proliferation, differentiation, migration, and stem cell maintenance. If Wnt signaling is turned off, β-catenin is labeled by complexes (including APC, Axin, GSK-3β, etc.) and degraded through the ubiquitin-proteasome pathway.

[0040] The Transwell assay (also known as the Transwell chamber assay) is the most commonly used in vitro experimental method in cell biology for studying cell migration and invasion. It simulates the process by which tumor cells "break through" the basement membrane from the primary tumor site into surrounding tissues or the bloodstream through physical isolation. The assay uses a polycarbonate membrane to divide a culture plate into two layers: the upper chamber is for seeding the test cells; the lower chamber contains chemokines (such as serum or growth factors). Cells will pass through the membrane pores according to their concentration gradient. If the membrane is covered with Matrigel, invasion is primarily detected; otherwise, migration is primarily detected.

[0041] Western blotting is the gold standard technique in molecular biology for detecting the presence and abundance of specific proteins in a sample. It combines the high resolution of gel electrophoresis with the high specificity of antigen-antibody reactions, enabling the extraction of information about the target protein from complex mixed protein samples. The core logic of Western blotting can be summarized in three key steps: (1) Separation: Proteins of different molecular weights migrate at different rates in an electric field, forming bands on the gel to achieve physical separation; (2) Transfer: The separated proteins are transferred from the gel to a solid support (usually a PVDF or NC membrane) using an electric field; (3) Detection: The target protein is bound to a specific primary antibody, and then amplified and visualized using a secondary antibody (usually a coupling enzyme or fluorescent group). In the Western blot diagram, "Input" refers to "total protein input" or "sample loading," representing the original cell lysate added to the gel system at the start of the experiment. This is a mixed protein solution extracted directly from the sample without any treatment. "IP" (Immunoprecipitation) represents the target protein and its interacting partners that are "fished out" by the antibody.

[0042] Fidgetin (FIGN) is a microtubule-cutting protein that is primarily located in the nucleus of normal cells. However, in various malignant tumor cells (such as breast cancer, liver cancer, and lung adenocarcinoma), FIGN is enriched in the cytoplasm. What causes this imbalance in FIGN's "nucleoplasmic shuttle" in cancer cells? This invention, through proteomics screening, discovered that myosin heavy chain 2 (MYH2) is a key interacting protein with FIGN. Further mechanistic studies revealed that MYH2 acts as a "molecular anchor" by binding to specific domains of the FIGN protein, causing FIGN to remain in the cytoplasm and thus preventing its nuclear localization. Therefore, disrupting the MYH2-FIGN interaction represents a potential therapeutic strategy to "re-enter" oncogenic cytoplasmic FIGN into the nucleus, thereby reversing its tumor-promoting function. Extensive research prior to this invention has confirmed that cytoplasmic FIGN can drive tumor proliferation and metastasis by binding to and stabilizing RNA-binding protein HNRNPA2B1 and β-catenin mRNA. Therefore, this invention proposes a MYH2-FIGN interaction blocking amino acid compound, wherein the MYH2-FIGN interaction blocking amino acid compound is an amino acid sequence (peptide) as described in SEQ ID NO:1 or SEQ ID NO:3 or a peptide derivative thereof.

[0043] Among them, the polypeptide shown in SEQ ID NO: 1 is based on the molecular docking results of MYH2 and FIGN. Through Co-IP experiments, it was verified that the point mutation F321S can indeed significantly weaken the binding of MYH2 and FIGN. Thus, a polypeptide with F321 as the center and mimicking the FIGN sequence was synthesized.

[0044] Among them, the tumor-penetrating peptide iRGD (sequence CRGDKGPDC) is a short peptide that can specifically target and penetrate tumor tissue. Its mechanism of action is as follows: first, it binds to αv integrin, which is highly expressed on the surface of tumor blood vessels or cells, via the RGD motif; subsequently, it is cleaved by a protease, exposing the CendR motif, which binds to neuropilin-1 (NRP-1), thereby activating the tissue penetration pathway. When a therapeutic peptide is fused with iRGD, its tumor targeting and tissue penetration can be significantly improved. Therefore, peptides that bind iRGD, such as the peptide shown in SEQ ID NO: 3, can be synthesized based on the peptide shown in SEQ ID NO: 1.

[0045] Furthermore, the aforementioned polypeptide chains can undergo various other treatments, as long as the amino acid segments capable of blocking the binding of MYH2 and FIGN are retained. For example, the aforementioned polypeptides can be cyclized, with side-chain-side-chain or head-tail cyclization fixing the conformation to improve enzymatic stability; alternatively, N-methylation / non-natural amino acids can be performed to reduce peptidase recognition and improve oral absorption; or, peptide-small molecule fusion (hybrid) can be performed, covalently linking small molecule pharmacophores with short peptide fragments, combining high affinity and good permeability.

[0046] Furthermore, the development of small molecule inhibitors that directly target protein-protein interactions (PPIs) is challenging and has a low success rate, while peptide drugs, due to their high specificity and relatively low toxicity, are an ideal choice for targeting PPIs. The aforementioned peptide chains can be formulated into pharmaceutical compositions (peptide drugs) using various excipients or adjuvants in the following ways:

[0047] (1) Polymer-drug covalent or non-covalent conjugation, such as polyethylene glycol (PEG)-drug conjugation, polyvinylpyrrolidone (PVP) carriers, etc., can prolong the cycling half-life and achieve passive targeting (EPR effect); a preferred embodiment is, for example, coupling the modified KAFYMAGQGD with a small molecule drug (payload), such as Bis-Mal-amido-PEG or DBCO (Dibenzocyclooctyne) reagent. It is necessary to control the drug-peptide molar ratio (DAR value) between 2 and 4 to balance stability and toxicity.

[0048] (2) Nanocarriers (liposomes, solid lipid nanoparticles, polymer microspheres) achieve active targeting through surface modification (such as RGD (Arg-Gly-Asp, a tripeptide sequence composed of three amino acids: arginine (Arg), glycine (Gly) and aspartic acid (Asp), folic acid);

[0049] (3) Prodrug strategy: masking key residues of peptides into forms such as esters and amides that can be released by enzymes in vivo, thereby improving oral or transdermal absorption.

[0050] Specifically, suitable excipient combinations can be selected, and formulations can be screened based on solubility, stability, and route of administration (oral, injection, topical).

[0051] The polypeptide drug may be in the following formulation form, for example:

[0052] Oral solid dosage forms (tablets, capsules) require the addition of solubilizers, disintegrants, and preservatives.

[0053] Injectable solutions must use isotonic agents, buffers, etc., and be sterile filtered.

[0054] Nanodelivery systems require the selection of emulsifiers, surfactants, and targeting ligands.

[0055] Therefore, this invention claims protection for a polypeptide drug prepared from an amino acid compound that blocks MYH2-FIGN interaction as described above, and also claims protection for the use of an amino acid compound that blocks MYH2-FIGN interaction as described above in the preparation of a cancer therapeutic drug.

[0056] The present invention will be further illustrated below through specific embodiments. It should be noted that the following embodiments are merely illustrative and not intended to limit the present invention.

[0057] Example 1: Design and Synthesis of Targeted Peptides

[0058] Based on the molecular docking results of MYH2 and FIGN (e.g. Figure 1 As shown in the figure, this invention identifies a key region on the FIGN protein responsible for binding MYH2, which contains the key amino acid residue F321. Figure 2 To verify the effect of the FIGN protein F321 mutation (F321S) on its binding ability to the MYH2 protein using co-immunoprecipitation (Co-IP) and Western blotting techniques, the Co-IP experiment was conducted to confirm the following results: Figure 2 As shown, the point mutation F321S does indeed significantly weaken the binding of MYH2 to FIGN. Accordingly, this invention synthesized a polypeptide (WT) centered on F321 and mimicking the FIGN sequence, with the amino acid sequence: Lys-Ala-Phe-Tyr-Met-Ala-Gly-Gln-Gly-Asp (KAFYMAGQGD), i.e., SEQ ID NO: 1. Simultaneously, as a negative control, a mutant peptide (F321S) with phenylalanine (F) mutated to serine (S) was synthesized, with the sequence: Lys-Ala-Ser-Tyr-Met-Ala-Gly-Gln-Gly-Asp (KASYMAGQGD), i.e., SEQ ID NO: 2.

[0059] To enhance membrane penetration and tumor targeting, the aforementioned core peptide was fused to the iRGD peptide (sequence: Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys) at its C-terminus via a glycine-glycine (GG) linker, yielding the fusion peptide:

[0060] WT-iRGD: KAFYMAGQGDGGCRGDKGPDC (SEQ ID NO: 3);

[0061] F321S-iRGD:KASYMAGQGDGGCRGDKGPDC (SEQ ID NO: 4).

[0062] All peptides were synthesized by the commissioned company using the solid-phase synthesis method described below, and purified by HPLC to a purity >95%, with correct identification by mass spectrometry.

[0063] The above-mentioned peptides were synthesized using a standard 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis strategy on an automated peptide synthesizer. The synthesis followed the principle of extending from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus), and the specific steps are as follows:

[0064] (1) Resin pretreatment and loading of the first amino acid

[0065] The synthesis began with Rink Amide MBHA resin (CAS No.: 431041-83-7) (substitution value: 0.5 mmol / g), which ensured that the C-terminus of the final product was an amide. The resin was first washed and swollen with dimethylformamide (DMF) for 30 minutes. The first Fmoc-protected amino acid (i.e., the C-terminal Asp) was activated in DMF with the activators hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) for 5 minutes, then added to the resin. The reaction was carried out at room temperature for 2 hours, allowing the amino acid to be covalently linked to the resin via ester bonds. After the reaction was complete, the resin was thoroughly washed with DMF, and the coupling was confirmed to be complete using a ninhydrin assay.

[0066] (2) Fmoc deprotection cycle

[0067] Each round of amino acid extension is performed in the following cycle:

[0068] a. Deprotection: Treat the resin twice (10 minutes each time) with a DMF solution containing 20% ​​piperidine to remove the Fmoc protecting group attached to the N-terminus of the nascent peptide chain, exposing the free α-amino group.

[0069] b. Washing: Wash the resin thoroughly 6 times with DMF to completely remove residual piperidine and byproducts.

[0070] c. Coupling: Dissolve the next Fmoc-protected amino acid (4.0 equivalents), HOBt (4.0 equivalents), and DIC (4.0 equivalents) in DMF, activate for 5 minutes, then add to the resin and bubble at room temperature for 1 hour. For amino acids with large steric hindrance, extend the coupling time to 2 hours.

[0071] d. Washing and detection: After coupling, wash the resin with DMF. Take a small amount of resin for ninhydrin detection. If positive (blue), repeat the coupling steps until the detection is negative (colorless) to ensure complete reaction.

[0072] (3) Chain extension and special treatment

[0073] Repeat step (2) sequentially, following the sequence order of SEQ ID NO: 1-4, to couple all amino acids in turn. All amino acid side chains are protected with acid-sensitive groups to ensure complete coupling.

[0074] (4) Final pyrolysis and side chain deprotection

[0075] After the polypeptide chain synthesis was completed, the resin was washed with dichloromethane (DCM) and dried under vacuum. The polypeptide was cleaved from the resin by stirring at room temperature for 2.5 hours using a cleavage reagent (trifluoroacetic acid:water:triisopropylsilane = 95:2.5:2.5, v / v / v), and the protecting groups of all amino acid side chains were removed simultaneously.

[0076] (5) Precipitation, purification and identification of polypeptides

[0077] The lysis buffer was filtered into cold methyl tert-butyl ether to precipitate the peptides. After centrifugation, the precipitate was collected, washed three times with cold ether, and dried under vacuum to obtain the crude peptide. The crude product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC, C18 column) using an acetonitrile-water (containing 0.1% TFA) gradient elution, and the main peak was collected. Electrospray ionization mass spectrometry (ESI-MS) confirmed that the molecular weight of the obtained product was consistent with the theoretical value, and analytical HPLC analysis showed that the chemical purity was greater than 95%.

[0078] 1. Verification of peptide blocking MYH2-FIGN interaction

[0079] The following experimental groups were set up: blank control group (PBS), F321S-iRGD control group (20 μM), and WT-iRGD treatment group (20 μM). Peptide competition experiments were performed. Biotin-labeled WT peptide (SEQ ID NO: 1) or F321S mutant peptide (SEQ ID NO: 2) was co-incubated with cell lysis buffer and streptavidin magnetic beads. MYH2 and FIGN in the product were detected by Western blotting. The results are as follows: Figure 3 As shown. Figure 3 This is a Western blot analysis of the protein binding assay for peptide competitive binding, in which... Figure 3 In A, IP is MYH2. As shown in the figure, after treatment with WT-iRGD fusion peptide, the binding of MYH2 to FIGN in the immunoprecipitation product is significantly weakened. Figure 3 In figure B, IP stands for FIGN. As shown in the figure, after treatment with the WT-iRGD fusion peptide, the binding of FIGN to MYH2 in the immunoprecipitation product was significantly weakened. In summary, the results indicate that the WT peptide can effectively competitively bind to MYH2 and disrupt the MYH2-FIGN interaction, while the F321S mutant peptide does not have this effect.

[0080] 2. iRGD fusion peptide promotes FIGN nuclear translocation.

[0081] Huh7 (hepatocellular carcinoma) cells were treated with 20 μM WT-iRGD fusion peptide (SEQ ID NO: 3) or F321S-iRGD control peptide (SEQ ID NO: 4) for 24 hours, and the subcellular localization of FIGN was detected by immunofluorescence. Figure 4 Images from immunofluorescence experiments show the subcellular localization of FIGN protein in Huh7 cells in different treatment groups (control, F321S-iRGD, WT-iRGD). Results are as follows: Figure 4 The results showed that after treatment with the WT-iRGD fusion peptide, FIGN signals significantly accumulated in the cell nucleus, while FIGN in the control group and the F321S-iRGD group were mainly distributed in the cytoplasm.

[0082] 3. iRGD fusion peptide downregulates the β-catenin pathway.

[0083] Downstream signal detection: Figure 5 A-5F is a set of experimental results showing the effects of the WT-iRGD fusion peptide on the β-catenin signaling pathway; among them, Figure 5 A is a map showing the detection of β-catenin protein levels using Western blot; Figure 5 B is a bar chart showing the qPCR detection of β-catenin mRNA levels; Figure 5 C is a line graph showing the stability of β-catenin mRNA under Actinomycin D treatment; Figure 5 D, 5E and Figure 5 F is a bar chart showing the mRNA levels of downstream target genes c-MYC, Cyclin D1, and MMP7 detected by qPCR; where... Figure 5 The qPCR and Western blot results of A, 5B, 5D, 5E and 5F showed that treatment with the WT-iRGD fusion peptide significantly reduced the expression levels of β-catenin and its downstream target genes (c-MYC, Cyclin D1, MMP7). Figure 5 The C mRNA stability experiment further demonstrated that treatment with the WT-iRGD fusion peptide reduced the stability of β-catenin mRNA.

[0084] 4. iRGD fusion peptide inhibits tumor cell proliferation

[0085] A blank control group was set up, and Huh7 cells were treated with either WT-iRGD fusion peptide or F321S-iRGD control peptide for CCK-8 assays, cell counting, and colony formation experiments. Figure 6 A-6D is a set of experimental results showing the effect of the WT-iRGD fusion peptide on the proliferation ability of Huh7 cells; among them, Figure 6 A is a line graph showing cell viability as detected by the CCK-8 assay; Figure 6 B is a line graph of the direct cell counting experiment; Figure 6 C is a representative photograph from a cell clonogenic experiment; Figure 6 D is the correct answer. Figure 6 A bar chart showing the number of clones formed in C. (Example:) Figure 6 As shown in A-6D, the WT-iRGD fusion peptide significantly inhibited the proliferation of cancer cells.

[0086] 5. iRGD fusion peptide inhibits tumor cell migration and invasion.

[0087] A blank control group and Huh7 cells treated with either WT-iRGD fusion peptide or F321S-iRGD control peptide were set up for scratch healing experiments: Figure 7 A shows representative photographs of the cell scratch healing experiment at different time points; Figure 7 B is the correct answer. Figure 7 A statistical bar chart of the healed area of ​​a scratch; such as Figure 7 As shown in A-7B, the cell migration ability of the WT-iRGD fusion peptide treatment group was significantly impaired.

[0088] Transwell cell invasion assay: Figure 7 C is a representative photograph of the Transwell cell invasion assay (stained with crystal violet); Figure 7 D and 7E are opposites Figure 7 A bar chart showing the number of C-cells that have penetrated the membrane. (See also:) Figure 7 As shown in C-7E, the WT-iRGD fusion peptide significantly inhibits the migration and invasion of cancer cells.

[0089] 6. iRGD fusion peptide inhibits tumor growth in vivo.

[0090] In the BALB / c nude mouse hepatocellular carcinoma orthotopic model, the WT-iRGD fusion peptide or F321S-iRGD control peptide (10 mg / kg) was administered every other day.

[0091] Figure 8 A-8F is a collection of in vivo experimental results showing the inhibitory effect of the WT-iRGD fusion peptide on tumor growth in a BALB / c nude mouse hepatocellular carcinoma orthotopic model; among them... Figure 8 A represents the gross liver photographs of representative mice in each group at the experimental endpoint, high-power field images of HE-stained liver tissue sections, and immunohistochemical (IHC) staining images of Ki-67, β-catenin, and FIGN in tumor tissues. Figure 8 B is a violin plot showing the ratio of mouse liver weight to body weight (liver-to-body ratio); Figure 8 C is a statistical bar chart showing the number of tumor nodules on the liver surface; Figure 8 D is a statistical bar chart of the Ki-67 positive cell index in tumor tissue; Figure 8 E is a scatter plot of the β-catenin IHC staining score in tumor tissue; Figure 8 F is a scatter plot of the ratio of FIGN protein nucleoplasmic staining intensity in tumor cells. After 6 weeks of treatment, as... Figure 8 As shown in A-8F, compared with the F321S-iRGD control group, mice in the WT-iRGD fusion peptide treatment group:

[0092] like Figure 8 As shown in A-8C, there were fewer in situ liver tumors and a significantly lower liver-to-body ratio.

[0093] like Figure 8 As shown in D-8F, IHC staining of tumor tissue revealed downregulated expression of Ki-67 (a proliferation marker) and β-catenin, and FIGN showed a nuclear localization pattern.

[0094] 7. iRGD fusion peptide inhibits tumor metastasis in vivo.

[0095] In the BALB / c nude mouse tail vein lung metastasis model, WT-iRGD fusion peptide or F321S-iRGD control peptide (10 mg / kg) were injected every other day. Figure 9 Figures A and 9B present the in vivo experimental results of the WT-iRGD fusion peptide inhibiting tumor metastasis in a BALB / c nude mouse tail vein lung metastasis model; among them... Figure 9 A represents the lungs of representative mice in each group at the experimental endpoint, a panoramic HE-stained lung tissue section, and a high-power HE-stained image of metastatic lesions. Figure 9 B is a bar chart showing the number of lung metastases. After 6 weeks of treatment, if... Figure 9 As shown in A-9B, compared with the F321S-iRGD control group, the number of lung metastatic nodules was significantly reduced in mice treated with the WT-iRGD fusion peptide.

[0096] The above results fully demonstrate that the WT-iRGD fusion peptide of the present invention can effectively inhibit tumor growth and metastasis in vivo.

[0097] Example 2: Polypeptide Drug 1

[0098] Peptide drugs were prepared by bioorthogonal reactions via click chemistry coupling using either azido-alkyne cycloaddition (CuAAC) or strain-promoted alkyne-azido cycloaddition (SPAAC) reactions.

[0099] The specific steps include:

[0100] 1. Modify the C-terminus or N-terminus of the polypeptide (KAFYMAGQGD) shown in SEQ ID NO: 1 with an azide group (-N3).

[0101] 2. Modify small molecule toxins (such as DOX (doxorubicin) and MMAE (monomethyl olprestatin E)) with alkyne groups;

[0102] 3. Mixing under mild conditions forms a stable triazole ring linker.

[0103] The specific reaction conditions and parameters are set according to conventional reaction conditions in this field. The above reaction conditions are relatively mild, produce few byproducts, and are suitable for the in vivo environment.

[0104] One option is to design a linker that can be either cleavable or non-cleavable. Cleavable linkers include GFLG (enzymatic cleavage) and pH-sensitive bonds (cracked under the acidic conditions of the tumor microenvironment). Non-cleavable linkers include polyethylene glycol (PEG) chains, which are primarily metabolized and cleared by the kidneys.

[0105] Example 3: Polypeptide Drug 2

[0106] The maleimide coupling method is used to perform site-specific modification by using a thiol-maleimide reaction, for example, by reacting a cysteine ​​(Cys) residue on the side chain of the polypeptide or an introduced thiol (-SH) group with a maleimide group.

[0107] The specific steps include:

[0108] 1. Introduce thiol groups by site-directed mutagenesis or chemical modification of the polypeptide (KAFYMAGQGD) shown in SEQ ID NO: 1;

[0109] 2. Modify the end of the drug linker with maleimide;

[0110] 3. React in a redox buffer solution.

[0111] The specific reaction conditions and parameters were set according to the conventional maleimide coupling reaction conditions in this field. The above reaction is very rapid and highly specific.

[0112] Industrial applicability

[0113] The polypeptides and fusion peptides provided by this invention can be prepared on a large scale using the mature and stable solid-phase synthesis method described above, and have promising industrialization prospects. They exhibit significant antitumor activity both in vivo and in vitro, and the iRGD modification improves targeting and delivery efficiency, showing good drug development potential and can be used to develop novel anticancer drugs.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound that blocks MYH2-FIGN interactions with amino acids, characterized in that, The MYH2-FIGN interaction blocking amino acid compound is a polypeptide with the amino acid sequence shown in SEQ ID NO:

3.

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the MYH2-FIGN interaction blocking amino acid compound of claim 1, and the route of administration of the pharmaceutical composition includes local injection.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition may be formulated as an injection or a nanodelivery system.

4. A method for preparing a pharmaceutical composition according to any one of claims 2-3, characterized in that, Includes the following steps: The MYH2-FIGN interaction-blocking amino acid compound of claim 1 or 2 is mixed with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

5. The use of the MYH2-FIGN interaction-blocking amino acid compound as described in claim 1 in the preparation of a liver cancer therapeutic agent.

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