Dual-targeting polypeptide compound for inhibiting liver cancer cell migration as well as preparation method and application of dual-targeting polypeptide compound

Abstract

The invention discloses a dual-targeting polypeptide compound for inhibiting liver cancer cell migration as well as a preparation method and application of the dual-targeting polypeptide compound. The polypeptide compound protected by the invention is MY6 or MY7, and is a compound obtained by connecting carboxyl ends of polypeptide fragments respectively targeting two extracellular rings of LAPTM4B protein through a PEG arm and coupling through Click reaction. Experiments prove that compared with a single-targeting polypeptide, the binding capacity of MY6 or MY7 to free LAPTM4B protein is improved by an order of magnitude, high-selectivity analysis and detection of the LAPTM4B protein in living cells are successfully realized under low concentration, and meanwhile, MY6 or MY7 can regulate and control positioning of the LAPTM4B protein at microstructures such as cell surface pseudopod and intervene in cell-cell and cell-extracellular matrix interaction, so that high-selectivity analysis and detection of the LAPTM4B protein in the living cells are realized. The migration of cancer cells is further effectively inhibited. The dual-targeting polypeptide compound provided by the invention can be used for detection of malignant tumors and development of targeting drugs.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to a dual-targeting polypeptide compound that inhibits the migration of liver cancer cells, its preparation method, and its application. Background Technology

[0002] Tumor metastasis is one of the fundamental causes of the high mortality rate of cancer. Taking liver cancer as an example, due to its tendency to metastasize and recur, it is one of the most serious malignant diseases threatening human life and health. The abnormal proliferation of tumor cells within diseased tissue, followed by migration and invasion from the primary lesion to other tissues and organs, is a key step in tumor metastasis. Constructing highly specific chemical intervention molecules targeting proteins that promote migration is a powerful strategy for inhibiting cell migration and holds great promise for inhibiting tumor metastasis and thus achieving precise cancer treatment.

[0003] LAPTM4B, a lysosomal four-transmembrane protein, is closely related to the development and progression of various cancers. Overexpression of LAPTM4B promotes the migration of cells in almost all solid tumors, including liver cancer, leading to metastasis and making it a novel target for tumor detection and treatment. Constructing high-affinity and highly selective affinity recognition molecules targeting LAPTM4B to interfere with cell migration at the molecular level holds promise as a novel strategy for efficiently inhibiting malignant tumor metastasis, but such research is currently limited.

[0004] Peptides, as endogenous biomolecules, possess numerous advantages such as small size, abundant building blocks, and the ability to be chemically synthesized and modified, making them a hot topic and frontier in drug development. Globally, pharmaceutical companies are increasing their investment in peptide drug development year by year, and an increasing number of bioactive peptides are being approved for clinical and practical applications, demonstrating unique advantages in the treatment of cancer, diabetes, and other diseases. Therefore, how to rationally design and effectively construct and prepare peptides that stably bind to target proteins and possess functional interventional activities has become a key research focus. Summary of the Invention

[0005] The technical problems to be solved by this invention are how to inhibit the migration of LAPTM4B protein and / or how to prepare a stable binding antibody targeting LAPTM4B protein and / or how to inhibit the migration of tumor cells and / or how to prepare a polypeptide compound that inhibits the migration of tumor cells and / or how to prepare a dual-targeting polypeptide compound that inhibits the migration of tumor cells.

[0006] Addressing the urgent need and existing bottlenecks in peptide drug development, this study utilizes chemical methods to design and construct novel dual-target peptide recognition molecules targeting the extracellular fragment of LAPTM4B protein. These molecules aim to precisely intervene in the migration function of LAPTM4B protein, potentially providing a new and effective method and candidate compounds for the metastasis and treatment of malignant tumors such as liver cancer.

[0007] To address the aforementioned technical problems, this invention first provides compounds, which may be MY6 or MY7. MY6 can be a compound synthesized via a Click reaction by linking the carboxyl terminus of a polypeptide named AP2H and the carboxyl terminus of a polypeptide named AP2 using polyethylene glycol as a linker. MY7 can be a compound synthesized via a Click reaction by linking the carboxyl terminus of a polypeptide named AP2H and the carboxyl terminus of a polypeptide named SR using polyethylene glycol as a linker. The amino acid sequence of AP2H is SEQ ID NO.1 in the sequence listing, the amino acid sequence of AP2 is SEQ ID NO.2 in the sequence listing, and the amino acid sequence of SR is SEQ ID NO.3 in the sequence listing. The polyethylene glycol may contain 3 or more repeating units.

[0008] In the MY6 described above, the length of the linker that mediates the connection between the polypeptide AP2H and AP2 can be greater than or equal to In the MY7 described above, the length of the linker that mediates the connection between the polypeptide AP2H and the SR can be greater than or equal to

[0009] The chemical structural formula of MY6 mentioned above can be represented by the following formula 1:

[0010]

[0011] In Formula 1, IHGHHIISVG is the amino acid sequence from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) of AP2H from left to right; SIRILIVKRRRFQST is the amino acid sequence from the carboxyl terminus (C-terminus) to the amino terminus (N-terminus) of AP2 from left to right.

[0012] The chemical structural formula of MY7 mentioned above can be expressed as Equation 2 below:

[0013]

[0014] In Formula 2, IHGHHIISVG is the amino acid sequence from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) of AP2H from left to right; and SIRILIVKRR is the amino acid sequence from the carboxyl terminus (C-terminus) to the amino terminus (N-terminus) of SR from left to right.

[0015] The structural formula of the above compound MY6 is shown in Formula 1. Its synthesis includes: 1) the carboxyl group of the C-terminal glycine of AP2H with the amino acid sequence SEQ ID NO.1 is linked to the amino group of lysine (obtained by connecting an azide group to the side chain) by forming an amide bond; 2) the carboxyl group of the C-terminal serine of AP2 with the amino group SEQ ID NO.2 is linked to the amino group of NH2-PEG6-CH2CH2COOH by forming an amide bond; 3) the compound obtained in step 2) is linked to the amino group of propargyl glycine (containing an alkynyl group in the side chain) by forming an amide bond through the carboxyl group of the terminal PEG6; 4) the compound obtained in step 1) is linked to the alkynyl group of the compound obtained in step 3) by forming a 1,2,3-triazole ring through the azide group.

[0016] The structural formula of the above compound MY7 is shown in Formula 2. Its synthesis includes: 1) the carboxyl group of the C-terminal glycine of AP2H with the amino acid sequence SEQ ID NO.1 is linked to the amino group of lysine (obtained by connecting an azide group to the side chain) by forming an amide bond; 2) the carboxyl group of the C-terminal serine of SR with the amino acid sequence SEQ ID NO.3 is linked to the amino group of NH2-PEG3-CH2CH2COOH by forming an amide bond; 3) the compound obtained in step 2) is linked to the amino group of propargyl glycine (containing an alkynyl group in the side chain) by forming an amide bond through the carboxyl group of the terminal PEG3; 4) the compound obtained in step 1) is linked to the alkynyl group of the compound obtained in step 3) by forming a 1,2,3-triazole ring through the azide group.

[0017] To address the aforementioned technical problems, the present invention also provides a fluorescent labeling compound, which may be a compound obtained by coupling the above-described compound with a fluorescent dye.

[0018] In the above-mentioned fluorescent labeling compounds, preferably, the fluorescent dye can be fluorescein isothiocyanate, or other fluorescent groups or fluoresceins.

[0019] To address the aforementioned technical problems, the present invention also provides any of the following applications of the compounds described above and / or the fluorescently labeled compounds described above:

[0020] A1) Applications in the development or preparation of products for detecting LAPTM4B protein;

[0021] A2) Application in the development or preparation of products for detecting cancer cells expressing LAPTM4B protein;

[0022] A3) Application in the development or preparation of products that inhibit the migration and / or invasion of cancer cells expressing LAPTM4B protein;

[0023] A4) Applications in the development or preparation of LAPTM4B protein function inhibitors;

[0024] A5) Applications in the development or preparation of products that regulate the localization of LAPTM4B protein;

[0025] A6) Applications in the development or preparation of products that regulate intercellular and cell-extracellular matrix interactions in cancer cells with high expression of LAPTM4B protein;

[0026] A7) Application in the development or preparation of drugs targeting LAPTM4B protein;

[0027] A8) Application in the development or preparation of drugs that inhibit tumor metastasis;

[0028] A9) Application in the development or preparation of drugs that inhibit cell migration;

[0029] A10) Applications in the development or preparation of tumor detection products;

[0030] A11) Application in the development or preparation of drugs for treating tumors.

[0031] The LAPTM4B mentioned above is a four-transmembrane protein of human lysosomes.

[0032] In the above applications, the tumor may be liver cancer; the cells may be human liver cancer cells HepG2.

[0033] To address the aforementioned technical problems, the present invention also provides a method for preparing the compound described above, wherein the method is C or D;

[0034] The C step may include the following steps: C1) coupling the AP2H described above with an azide compound using the FMOC peptide solid-phase synthesis method to obtain azide-modified AP2H, and coupling the AP2 described in claim 1 with an alkyne compound and PEG using the FMOC peptide solid-phase synthesis method to obtain alkyne-PEG-modified AP2; C2) coupling the azide-modified AP2H with the alkyne-PEG-modified AP2 via a Click reaction to prepare the MY6.

[0035] The D step may include the following steps: D1) coupling the AP2H described above with an azide molecule using the FMOC peptide solid-phase synthesis method to obtain azide-modified AP2H; coupling the SR described above with an alkyne compound and a PEG molecule using the FMOC peptide solid-phase synthesis method to obtain alkyne-PEG-modified SR; D2) coupling the azide-modified AP2H with the alkyne-PEG-modified SR via a Click reaction to prepare the MY7.

[0036] The amino acid sequence of AP2H is SEQ ID NO.1 in the sequence listing, the amino acid sequence of SR is SEQ ID NO.3 in the sequence listing, and the amino acid sequence of AP2 is SEQ ID NO.2 in the sequence listing.

[0037] In method C above, the PEG referred to is PEG6 (a PEG molecule with a degree of polymerization of 6). In method D above, the PEG referred to is PEG3 (a PEG molecule with a degree of polymerization of 3).

[0038] To address the aforementioned technical problems, the present invention also provides a composition, which may contain the compound used for body tattooing and / or the fluorescent marking compound used for body tattooing, and the composition may have at least one of the following uses:

[0039] B1) is used to detect LAPTM4B protein;

[0040] B2) Products used to detect cancer cells expressing the LAPTM4B protein;

[0041] B3) Use in products that inhibit the migration and / or invasion of cancer cells expressing the LAPTM4B protein;

[0042] B4) is used to inhibit the function of LAPTM4B protein;

[0043] B5) is used to regulate the localization of LAPTM4B protein;

[0044] B6) is used to regulate intercellular and cell-extracellular matrix interactions in cancer cells with high expression of LAPTM4B protein;

[0045] B7) is used for tumor detection.

[0046] To address the aforementioned technical problems, the present invention also provides a pharmaceutical composition, which may contain the compounds described above and / or the fluorescently labeled compounds described above, and the pharmaceutical composition may be any of the following drugs:

[0047] E1) Drugs that target the lysosomal four-transmembrane protein LAPTM4B;

[0048] E2) Drugs that inhibit tumor metastasis;

[0049] E3) Drugs that inhibit cell migration;

[0050] E4) Drugs for treating tumors.

[0051] In practical applications, the above-mentioned drugs can be administered directly to patients or animals, or mixed with suitable carriers or excipients before administration to patients or animals, to achieve the purpose of preventing toxoplasmosis infection. The carrier materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. Using these materials, various dosage forms can be formulated, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. To formulate unit-dose dosage forms into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, and ethylcellulose. Various carriers known in the art can be widely used to formulate unit dosage forms into suppositories. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.To formulate unit-dose dosage forms for injection, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Additionally, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injection formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation. The above dosage forms can be administered via injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; via cavities, such as rectal and vaginal; via the respiratory tract, such as nasal; and via mucosal administration. Injection is the preferred route of administration.

[0052] In the above application, the cells expressing the LAPTM4B protein can be human liver cancer cells HepG2.

[0053] LAPTM4B is a 35kD four-transmembrane protein with four hydrophobic transmembrane regions and two extracellular regions. Peptide fragments EL1 (ADPDQYNFSSSELGG) and EL2 (PYRDDVMSVN), located in the two extracellular loops, were selected as target sites for recognizing LAPTM4B.

[0054] The Click reaction mentioned above is a click chemistry reaction.

[0055] Experiments have demonstrated that the dual-targeting peptides and peptide conjugates prepared by Click coupling of the carboxyl ends of two single-targeting peptides using a polyethylene glycol (PEG) chain as the linker, as provided in this invention, can bind to LAPTM4B protein with high selectivity and high affinity. Compared with single-targeting peptides, the binding affinity of the dual-targeting peptides MY6 and MY7 to free LAPTM4B protein is increased by an order of magnitude, successfully achieving highly selective analysis and detection of LAPTM4B protein in living cells at low concentrations. More importantly, the dual-targeting peptides can regulate the localization of LAPTM4B protein at microstructures such as pseudopodia on the cell surface and interfere with cell-cell and cell-extracellular matrix interactions, thereby further effectively inhibiting the migration of cancer cells. Therefore, the dual-targeting peptide compounds provided in this invention can serve as novel functional regulatory tools for LAPTM4B protein, used for the precise inhibition of metastasis in malignant tumors such as liver cancer.

[0056] Meanwhile, the dual-targeting peptide provided by this invention has a short amino acid sequence, making it easy to produce on a large scale and non-toxic to cells. It overcomes the shortcomings of antibodies and other biological agents, such as large molecular weight, weak penetration ability, complicated preparation, high cost, poor stability, and easy to induce immune reactions. It has application prospects in the detection of diseases such as malignant tumors and the development of targeted drugs. Attached Figure Description

[0057] Figure 1 This is a schematic diagram illustrating the targeted recognition of the dual-targeting peptide compounds MY6 and MY7 by the LAPTM4B protein.

[0058] Figure 2 Characterization results for the AP2 and SR peptides targeting and recognizing the extracellular region of LAPTM4B protein. The vertical axis represents the relative fluorescence polarization value.

[0059] Figure 3 The results show the affinity of dual-targeting peptides and their corresponding single-targeting peptides for LAPTM4B protein. The vertical axis represents the relative fluorescence intensity.

[0060] Figure 4 Immunoblotting characterization of LAPTM4B expression levels in HepG2 human liver cancer cells and normal cells HEK293, where LAPTM4B protein is highly expressed.

[0061] Figure 5 The graph shows the statistical results of relative fluorescence intensity after incubation of single-targeting and dual-targeting peptides with HepG2 cells that highly express LAPTM4B protein. The vertical axis of the right graph represents relative fluorescence intensity.

[0062] Figure 6 This study provides in situ imaging analysis of LAPTM4B protein in living cells using single-targeting and dual-targeting peptide compounds.

[0063] Figure 7 Immunofluorescence assays were conducted to demonstrate that dual-targeting peptide compounds regulate the localization of LAPTM4B protein on the membrane of human hepatocellular carcinoma cells.

[0064] Figure 8 This is a wound healing assay demonstrating the inhibitory effect of a dual-targeting peptide compound on the migration of human hepatocellular carcinoma cells by binding to the LAPTM4B protein. The two figures on the left show the wound healing assay of the peptide inhibiting the migration of HepG2 cells; the figure on the right shows the quantitative analysis of the anti-migration efficiency of the peptide after incubation with HepG2 cells.

[0065] Figure 9 Transwell assays demonstrated that the dual-targeting peptide compound effectively inhibited the motility of human liver cancer cells by binding to the LAPTM4B protein. The left image shows a microscopic image of the inhibitory effect of the peptide on HepG2 cell motility; the right image shows the number of HepG2 cells that migrated to the cavity.

[0066] Figure 10 This describes the synthetic route for the dual-targeting peptide compounds MY6 and MY7.

[0067] Figure 11 Synthetic routes for FITC-labeled dual-targeting peptide compounds MY6 and MY7.

[0068] Figure 12 Synthetic routes for single-target peptides AP2H, AP2, and SR.

[0069] Figure 13 Synthetic routes for FITC-labeled single-target peptides AP2H, AP2, and SR. Detailed Implementation

[0070] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0072] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0073] Example 1: Design and preparation of a dual-targeting peptide targeting human LAPTM4B protein

[0074] 1. Selection of extracellular peptide recognition elements for LAPTM4B protein

[0075] Using the tumor marker human lysosomal four-transmembrane protein LAPTM4B as the target, the peptides AP2 and AP2H, which target the two extracellular loop fragments of LAPTM4B protein EL1 (ADPDQYNFSSSELGG) and EL2 (PYRDDVMSVN) respectively, were selected as recognition elements in the laboratory screening (Table 1). Considering that the target peptide AP2 has a long sequence and poor water solubility, the structure of AP2 was optimized to obtain a new peptide SR (Table 1). The interaction between peptides AP2 and SR and the extracellular fragments of LAPTM4B protein was investigated by fluorescence polarization technology. The specific steps are as follows:

[0076] (1) Mix 10 mmol / L phosphate buffer (PBS) (pH 7.4) with the extracellular fragments EL1 and EL2 of LAPTM4B protein to obtain mixture system A; mix FITC-labeled AP2 and SR stock solutions with mixture system A to obtain mixture systems B and C respectively; in mixture systems B and C, the concentration of FITC-labeled AP2 and SR is 40 nmol / L, and the concentration of the extracellular fragment of the protein is 1 μmol / L.

[0077] (2) Take the mixed system obtained in step (1) and incubate it at room temperature in the dark for 1 hour.

[0078] (3) Use an Infinite M1000 pro microplate reader (Tecan, Mennedorf, Switzerland) to scan the fluorescence polarization value of the solution after completing step (2) and record the fluorescence polarization value data at an emission wavelength of 525nm, which is the fluorescence polarization value of the substance to be tested.

[0079] (4) Following steps (1)-(3) above, replace mixture A with mixture D, keeping all other steps unchanged, to obtain the fluorescence polarization value of the blank control. Mixture D consists of PBS buffer.

[0080] (5) Calculate the relative fluorescence polarization value of the analyte. The relative fluorescence polarization value of the analyte = the fluorescence polarization value of the analyte - the fluorescence polarization value of the blank control.

[0081] The results are as follows Figure 2 As shown. The results indicate that the new peptide SR ( Figure 2 The fluorescence polarization values ​​of the extracellular fragment of LAPTM4B protein (represented by FITC-SR) and AP2 ( Figure 2 The FITC-AP2 sequence is consistent with that of the LAPTM4B protein, and the SR sequence of the peptide has a higher proportion of hydrophilic amino acids. Subsequently, AP2H, AP2, and SR were used as targeting recognition elements for the two extracellular regions of the LAPTM4B protein to construct a dual-targeting peptide.

[0082] 2. Design of dual-targeting peptides targeting human LAPTM4B protein

[0083] To achieve simultaneous targeting and recognition of the two extracellular regions of the LAPTM4B protein, a PEG molecule with good hydrophilicity, flexibility, and adjustable length was selected as the linker arm, and covalent assembly of the peptide recognition element was studied. Addressing the challenge of the lack of a crystalline structure for the target protein LAPTM4B, a multi-transmembrane protein, the spatial distance and conformation of the two extracellular target fragments of the target protein were simulated, predicted, and calculated using the deep learning software AlphaFold2. The calculation was based on the theoretical linear distance between the two target sites. PEG molecules with a degree of polymerization of 3 (PEG3) and PEG molecules with a degree of polymerization of 6 (PEG6) were selected as spacer arms. Unlike the traditional solid-phase chemical synthesis of peptides, which involves coupling from the carboxyl terminus to the amino terminus, a click-coupling mechanism was designed based on the spatial conformation of the two extracellular fragments of the target protein, connecting the two target peptide segments from the carboxyl terminus. This resulted in the design of dual-targeting peptides MY6 and MY7, using PEG6 and PEG3 as spacer arms respectively, with two single-targeting peptide segments click-coupled at their carboxyl terms. The lengths of the spacer arms in the structures of the dual-targeting peptides MY6 and MY7 are respectively... and Both are greater than the theoretical straight-line distance between the two target points. The amino acid sequences of AP2H, AP2, SR and the two dual-targeting peptides MY6 and MY7 are shown in Table 1; where each letter represents an amino acid as follows: Q for glutamine, F for phenylalanine, H for histidine, T for threonine, S for serine, L for leucine, G for glycine, I for isoleucine, R for arginine, V for valine, and K for lysine.

[0084] The chemical structural formula of MY6 is as follows: Formula 1:

[0085]

[0086] The structural formula of the above compound MY6 is shown in Formula 1. Its synthesis includes: 1) the carboxyl group of the C-terminal glycine of AP2H with the amino acid sequence SEQ ID NO.1 is linked to the amino group of lysine (obtained by connecting an azide group to the side chain) by forming an amide bond; 2) the carboxyl group of the C-terminal serine of AP2 with the amino group SEQ ID NO.2 is linked to the amino group of NH2-PEG6-CH2CH2COOH by forming an amide bond; 3) the compound obtained in step 2) is linked to the amino group of propargyl glycine (containing an alkynyl group in the side chain) by forming an amide bond through the carboxyl group of the terminal PEG6; 4) the compound obtained in step 1) is linked to the alkynyl group of the compound obtained in step 3) by forming a 1,2,3-triazole ring through the azide group.

[0087]

[0088] The structural formula of the above compound MY7 is shown in Formula 2. Its synthesis includes: 1) the carboxyl group of the C-terminal glycine of AP2H with the amino acid sequence SEQ ID NO.1 is linked to the amino group of lysine (obtained by connecting an azide group to the side chain) by forming an amide bond; 2) the carboxyl group of the C-terminal serine of SR with the amino acid sequence SEQ ID NO.3 is linked to the amino group of NH2-PEG3-CH2CH2COOH by forming an amide bond; 3) the compound obtained in step 2) is linked to the amino group of propargyl glycine (containing an alkynyl group in the side chain) by forming an amide bond through the carboxyl group of the terminal PEG3; 4) the compound obtained in step 1) is linked to the alkynyl group of the compound obtained in step 3) by forming a 1,2,3-triazole ring through the azide group.

[0089] Table 1. Peptide sequences and structures of dual-targeting peptide compounds

[0090]

[0091] Note: In Formula 1, IHGHHIISVG, from left to right, is the amino acid sequence of AP2H from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus); SIRILIVKRRRFQST, from left to right, is the amino acid sequence of AP2 from the carboxyl terminus (C-terminus) to the amino terminus (N-terminus); In Formula 2, IHGHHIISVG, from left to right, is the amino acid sequence of AP2H from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus); SIRILIVKRR, from left to right, is the amino acid sequence of SR from the carboxyl terminus (C-terminus) to the amino terminus (N-terminus).

[0092] 3. Preparation of dual-targeting peptides MY6 and MY7

[0093] Two single-target peptides were linked together at their carboxyl ends to synthesize dual-target peptides MY6 and MY7 via a Click reaction: First, the azide-modified single-target peptide AP2H and the alkyne and PEG-modified single-target peptides AP2 and SR were coupled using the FMOC solid-phase synthesis method, and then further prepared by liquid-phase Click reaction.

[0094] First, for the preparation of the azide-modified single-target peptide AP2H: starting with Amide resin, the FMOC solid-phase peptide synthesis method was used to remove the N-terminal FMOC protecting group of the resin using 20% ​​hexahydropyridine at room temperature for 10 min; then, 3 times the amount of Fmoc-Lys(Dde)-OH, 3 times the amount of HATU, and 3 times the amount of HOBt were dissolved in a DMF solution containing 0.4 mol / L N-methylmorpholine, mixed thoroughly, and coupled with the resin at room temperature for 1-2 h; the deprotection and amino acid coupling steps were repeated, and each amino acid on the AP2H peptide was coupled on the resin in the coupling direction from the carboxyl terminus to the amino terminus; the N-terminal FMOC protecting group of the AP2H peptide on the resin was retained, and 1.35 mmol imidazole, 1.80 mmol hydroxylamine hydrochloride, 5 mL N-methylpyrrolidone, and 1 mL The Dde deprotection solution of N,N-dimethylformamide was used to remove the protective layer at room temperature for 4–5 h. Subsequently, a DMF solution containing 3 times the amount of azidoacetic acid, 3 times the amount of HATU, 3 times the amount of HOBt, and 0.4 mol / L N-methylmorpholine was added to the resin, and the reaction was carried out at room temperature for 1–2 h. After successful coupling with azidoacetic acid, the N-terminal FMOC group on the peptide AP2H was removed using a 20% hexahydropyridine solution, finally yielding the azido-modified single-target peptide AP2H (obtained by reacting the amino group of lysine modified with azido groups in the side chain with the carboxyl group of glycine at the C-terminus of AP2H to form an amide bond).

[0095] Based on the Fmoc solid-phase synthesis method, peptides AP2 modified with alkynyl and PEG6 molecules and SR modified with alkynyl and PEG3 molecules were prepared respectively: First, the FMOC protecting group on Amide resin was removed using a 20% hexahydropyridine solution at room temperature for 10 min. Then, a DMF solution containing 3 times the amount of propargyl glycine, 3 times the amount of HATU, 3 times the amount of HOBt, and 0.4 mol / L N-methylmorpholine was added to the resin, and the reaction was carried out at room temperature for 1-2 h. After removing the N-terminal FMOC protecting group of propargyl glycine using a 20% hexahydropyridine solution at room temperature for 10 min, a DMF solution containing 2 times the amount of Fmoc-NH-CH2CH2-PEG3-COOH / Fmoc-NH-CH2CH2-PEG6-COOH (Bomei, China), 2 times the amount of HATU, 2 times the amount of HOBt, and 0.4 mol / L N-methylmorpholine was added. A DMF solution of N-methylmorpholine was added to the resin and reacted at room temperature for 1–2 h. The deprotection and amino acid coupling steps were repeated, and each amino acid on peptides AP2 and SR was coupled onto the resin in a coupling direction from the carboxyl terminus to the amino terminus. After coupling, the N-terminal FMOC protecting groups on peptides AP2 and SR were removed at room temperature using a 20% hexahydropyridine solution, finally yielding peptides AP2 modified with alkynyl and PEG6 molecules (obtained by reacting the amino group of propargyl glycine with the carboxyl group of NH2-CH2CH2-PEG6-COOH molecule to form an amide bond, and simultaneously reacting the amino group of NH2-CH2CH2-PEG6-COOH molecule with the carboxyl group of serine at the C-terminus of AP2 to form an amide bond) and SR modified with alkynyl and PEG3 molecules (obtained by reacting the amino group of propargyl glycine with the carboxyl group of NH2-CH2CH2-PEG3-COOH molecule to form an amide bond, and simultaneously reacting the amino group of NH2-CH2CH2-PEG3-COOH molecule with the carboxyl group of serine at the C-terminus of SR to form an amide bond).

[0096] The resin was condensed sequentially in dichloromethane and methanol, dried, and then lysed with a lysis buffer containing 95% (v / v) trifluoroacetic acid, 2.5% (v / v) triisopropylsilane, and 2.5% (v / v) water. The single-target peptides modified with different molecules were lysed from the resin and the side-chain protecting groups were successfully removed. The lysis was carried out in an ice bath for 10 min and at room temperature for 2 h. After the lysis was completed, the excess lysis buffer was removed by rotary evaporation and a small amount of diethyl ether was added to precipitate the crude peptide. Finally, single-target peptides modified with azide, alkyne, and PEG molecules were successfully prepared (including the single-target peptide AP2H modified with azide, the peptide AP2 modified with alkyne and PEG6, and the peptide SR modified with alkyne and PEG3). By combining liquid-phase Click reaction, a dual-targeting peptide MY6 was successfully prepared by reacting 1 / 2 amount of azide-modified single-targeting peptide AP2H, 1 / 2 amount of alkynyl and PEG6-modified single-targeting peptide AP2, 5 / 2 amount of cuprous bromide, and 1% triethylamine (v / v) at room temperature for 1 h under nitrogen protection. The dual-targeting peptide compound MY7 was also successfully prepared by combining liquid-phase Click reaction, involving 1 / 2 amount of azide-modified single-targeting peptide AP2H, 1 / 2 amount of alkynyl and PEG3-modified single-targeting peptide SR, 5 / 2 amount of cuprous bromide, and 1% triethylamine (v / v) at room temperature for 1 h under nitrogen protection.

[0097] In the preparation of MY6, the azide group in the lysine side chain of the azide-modified single-target peptide AP2H will form a 1,2,3-triazole ring with the alkynyl group and the glycine side chain of the PEG6-modified single-target peptide AP2 to achieve linkage (Click reaction principle).

[0098] During the preparation of MY7, the azide group in the lysine side chain of the azide-modified single-target peptide AP2H will form a 1,2,3-triazole ring with the alkynyl group in the glycine side chain of the PEG3-modified single-target peptide SR to achieve linkage (Click reaction principle).

[0099] For the synthesis of FITC-labeled dual-targeting peptides MY6 and MY7, based on the above steps, three times the amount of β-alanine, three times the amount of HATU, three times the amount of HOBt, and a 0.4 mol / L DMF solution of N-methylmorpholine were added to the Amide resin of the azide-modified peptide AP2H, and the reaction was carried out at room temperature for 1–2 h. The N-terminal FMOC protecting group of β-alanine was removed using 20% ​​hexahydropyridine solution, and the reaction was carried out at room temperature for 10 min. Three times the amount of FITC and two times the amount of N,N-diisopropylethylamine (DIPEA) were added, and the reaction was carried out at room temperature in the dark for 2 h. After the coupling was completed, a lysis buffer of 95% (v / v) trifluoroacetic acid, 2.5% (v / v) triisopropylsilane, and 2.5% (v / v) water was added, and the lysis was carried out in an ice bath for 10 min and then at room temperature for 2 h. Finally, the azide and FITC-modified peptide AP2H was successfully prepared. Finally, combining liquid-phase Click reaction, 1 part azide and FITC-modified single-target peptide AP2H, 1 part alkyne and PEG6-modified single-target peptide AP2, 5 parts cuprous bromide and 1% triethylamine (v / v) were reacted at room temperature for 1 h under nitrogen protection to successfully prepare FITC-labeled dual-target peptide fluorescent probe MY6; 1 part azide and FITC-modified single-target peptide AP2H, 1 part alkyne and PEG3-modified single-target peptide SR, 5 parts cuprous bromide and 1% triethylamine (v / v) were reacted at room temperature for 1 h under nitrogen protection to successfully prepare FITC-labeled dual-target peptide fluorescent probe MY7.

[0100] The synthesized product was purified using HPLC. The HPLC separation parameters used were as follows: the column was a Diamonsil C18(2) 250×4.6 mm; mobile phase A was an aqueous solution containing 0.1% (v / v) trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid. Elution was performed by increasing the percentage of mobile phase B from 20% to 50% within 25 min. The corresponding target fraction was collected, lyophilized, and the target product with high purity and correct structure was obtained.

[0101] Example 2: High affinity interaction between dual-targeting peptide compounds and target protein LAPTM4B.

[0102] The affinity of single-target peptides AP2H, AP2, and SR, as well as dual-target peptides MY6 and MY7, for LAPTM4B protein was investigated using fluorescence immunosorbent assay. The specific steps are as follows:

[0103] (1) Mix 10 mmol / L phosphate buffer (PBS) (pH 7.4) and LAPTM4B protein (OriGene, catalog number TP307325, China) to obtain mixture A; the concentration of LAPTM4B protein in mixture A is 2 μg / mL.

[0104] (2) Take the mixture obtained in step (1) and add it into a 384 high adsorption plate (PerkinElemer, catalog number 6057260, USA) and incubate overnight at 4°C.

[0105] (3) Remove the protein supernatant from step (2), wash three times with PBS, add 3% bovine serum albumin (BSA) to a 384-well plate, and incubate at room temperature for 1 hour.

[0106] (4) FITC-labeled single-targeting and dual-targeting peptides were obtained through chemical synthesis, as described in the following process: Figure 11 and 13 As shown. FITC-labeled AP2H, AP2, SR, MY6, and MY7 were mixed with PBS to obtain mixed systems B, C, D, E, and F (abbreviated as B to F); the concentrations of FITC-labeled AP2H, AP2, SR, MY6, and MY7 in mixed systems B to F ranged from 20 nmol / L to 600 nmol / L.

[0107] (5) Remove the BSA supernatant from step (3), and add the mixed system obtained in step (4) to the 384 high adsorption plate in step (3) and incubate at room temperature in the dark for 2 hours.

[0108] (6) Remove the mixture from step (5) in the well plate and wash it three times with PBS solution containing 0.05% Tween-20. Use a microplate reader (USA) to scan the fluorescence. The excitation wavelength is 488nm and the fluorescence intensity data at the emission wavelength of 525nm is recorded, which is the fluorescence intensity of the experimental group.

[0109] (7) Following steps (1)-(6) above, replace mixtures B to F with mixture G, keeping all other steps unchanged, to obtain the fluorescence intensity of the blank control. Mixture G consists of PBS solution.

[0110] (8) Calculate the relative fluorescence intensity of the experimental group (relative fluorescence intensity of the experimental group = fluorescence intensity of the experimental group - fluorescence intensity of the blank control). Use Origin software to calculate the equilibrium dissociation constant K. D The relative fluorescence intensity was used to linearly fit the concentration of FITC-labeled peptides.

[0111] The results are as follows Figure 3As shown. The results indicate that peptides AP2H, AP2, SR, MY6, and MY7 are related to the K of LAPTM4B protein. D The values ​​were 414.6±89.1 nM, 584.4±151.9 nM, 323.7±100.5 nM, 107.05±25.3 nM, and 49.3±12.9 nM, respectively, showing that MY6 and MY7 have high affinity for LAPTM4B protein.

[0112] Example 3: Fluorescently labeled dual-targeting peptides for specific recognition and imaging detection of tumor cells.

[0113] 1. Immunoblotting assay to analyze the expression level of LAPTM4B protein in cells.

[0114] (1) Human liver cancer cells HepG2 and healthy human cells HEK293 (Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, China) were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibiotics (composed of 10,000 Units / mL penicillin and 10,000 μg / mL streptomycin).

[0115] (2) After reaching the required density, remove the DMEM medium, wash three times with 10 mmol / L phosphate buffer (PBS) (pH 7.4), add cell lysis buffer and lyse on ice for 15 min; the cell lysis buffer consists of 98% RIPA, 1% PMSF and 1% protease inhibitor.

[0116] (3) After completing step (2), use a cell scraper to collect the cells into a centrifuge tube, sonicate for 10 seconds, centrifuge at 12000 RPM for 3 minutes, collect the supernatant, and place it in an ice box to wait for loading.

[0117] (4) The protein concentration in the lysis supernatant was measured using the BCA method. 1 μL of cell lysis supernatant was mixed with 19 μL of PBS buffer and 200 μL of BCA working solution. After incubation at 37°C for 30 min using a microplate reader (USA), the absorbance of the mixture at 562 nm was measured. The ratio of solution A to solution B in the BCA working solution was 50:1.

[0118] (5) After completing step (4), mix the cell lysis supernatant with the protein loading buffer at a ratio of 3:1 and heat in boiling water for 10 minutes to denature the protein.

[0119] (6) Based on the absorbance value measured in step (4), mix the solution from step (5) in BeyoGel with the same mass. TM The sample was loaded into the Plus PAGE pre-cast gel and electrophoresis was performed under constant voltage conditions for 1 hour at room temperature.

[0120] (7) After completing step (6), the protein strips on the gel are transferred to the PVDF membrane under constant current conditions and the membrane is transferred for 1 hour under ice water bath conditions.

[0121] (8) After completing step (7), the PVDF membrane is sealed in the sealing solution at room temperature for 1 hour; the sealing solution consists of skim milk powder and TBST (1×), wherein the mass concentration of skim milk powder is 5%.

[0122] (9) Remove the blocking solution from step (8), add LAPTM4B antibody solution, and incubate overnight at 4°C; the LAPTM4B antibody solution consists of LAPTM4B antibody (Abcam, catalog number ab242376, UK), skim milk powder and TBST (1×); the LAPTM4B antibody dilution ratio is 1:1000, and the mass concentration of skim milk powder is 0.5%.

[0123] (10) After completing step (9), discard the solution and wash 5 times with TBST (1×) for 5 min each time. Add secondary antibody solution and incubate at room temperature in the dark for 1 h; the secondary antibody solution consists of HRP-labeled goat anti-mouse IgG antibody (Abcam, catalog number ab205719, UK), skim milk powder and TBST (1×); the secondary antibody dilution ratio is 1:8000 and the mass concentration of skim milk powder is 0.5%.

[0124] (11) After completing step (10), discard the solution and add TBST (1×) to wash 5 times, 5 min each time. Immerse the PVDF membrane in ECL Plus hypersensitive luminescent solution and perform development analysis using a Tanon 5200Muli chemiluminescence imaging system; the ratio of solution A to solution B in ECL Plus hypersensitive luminescent solution is 1:1.

[0125] The results are as follows Figure 4 As shown, human liver cancer cells HepG2 express LAPTM4B protein and are LAPTM4B positive cells; healthy human cells HEK293 do not express LAPTM4B protein and are LAPTM4B negative cells.

[0126] 2. Interaction between fluorescent FITC-labeled MY6 and MY7 and cancer cells with high expression of LAPTM4B protein

[0127] To maintain the native state of the membrane protein LAPTM4B, a flow cytometry-based peptide affinity screening method was established using HepG2 human liver cancer cells overexpressing LAPTM4B as the research subject. To investigate the interaction strength between FITC-labeled dual-targeting peptides MY6 and MY7 and single-targeting peptides and HepG2 cells, the following experiments were conducted:

[0128] (1) Using dimethyl sulfoxide (DMSO) as a solvent, prepare single-target peptide stock solutions (including three single-target peptide stock solutions: AP2H, AP2 and SR) and dual-target peptide compound stock solutions (including two dual-target peptide stock solutions: MY6 and MY7).

[0129] (2) The stock solutions of single-target peptides AP2H, AP2, and SR, and the stock solutions of dual-target peptide compounds MY6 and MY7 were diluted to 0.3 μmol / L with 10 mmol / L phosphate-buffered saline (PBS) (pH 7.4).

[0130] (3) Adherent HepG2 cells were digested with 5 mM ethylenediaminetetraacetic acid solution (pH 8), washed three times with PBS, and then digested with approximately 1 × 10⁻⁶ ppm solution. 6 Cells per tube were resuspended in the dilution buffer of the single-targeting peptide and the dual-targeting peptide from step (2) and incubated at 4°C for 1 hour.

[0131] (4) After completing step (3), wash the cells in the solution three times with PBS, resuspend them in PBS solution, and measure them by flow cytometry (BD, USA) with an excitation wavelength of 488 nm and record the fluorescence intensity data of 20,000 cells.

[0132] (5) Following steps (2)-(4) above, replace the single-target and dual-target peptide stock solutions with PBS solution, while keeping all other steps unchanged, to obtain the fluorescence intensity of the blank control.

[0133] (6) Calculate the relative fluorescence intensity of the candidate peptide. Relative fluorescence intensity of the candidate peptide = Fluorescence intensity of the candidate peptide - Fluorescence intensity of the blank control.

[0134] The results are as follows Figure 5 As shown, compared with the corresponding single-targeting peptides, the dual-targeting peptides MY6 and MY7 ( Figure 5 The binding ability of FITC-MY6 and FITC-MY7 (represented by FITC-MY6 and FITC-MY7) to the LAPTM4B protein in HepG2 cells was increased tenfold.

[0135] 3. In situ imaging analysis of LAPTM4B protein in live cells

[0136] (1) with approximately 1×10 6 Human liver cancer cells (HepG2) or normal cells (HEK293) were seeded in confocal dishes (Φ=15mm) at a density of cells / dish and cultured overnight at 37°C and 5% CO2 to allow them to adhere.

[0137] (2) In the confocal dish of adherent cells, discard the original culture medium and then add FITC-labeled peptides AP2H, AP2, SR, MY6 and MY7 solutions (diluted with DMEM medium) to make the final concentration of FITC-labeled peptides 2 μmol / L. Then incubate at 37℃ and 5% CO2 in the dark for 1 h, discard the solution and wash once with 10 mmol / L phosphate buffer (PBS) (pH 7.4).

[0138] (3) The fluorescence distribution in the cells was detected using a laser scanning confocal microscope (Olympus FV1000-IX81, Japan).

[0139] The results are as follows Figure 6 As shown in the figure. For LAPTM4B-positive human hepatocellular carcinoma cells HepG2, the addition of FITC-labeled single-targeting and dual-targeting peptides resulted in significant green fluorescence on the cell membrane. Figure 6 (The five smaller images on the left side of the middle section), and compared to single-target peptides ( Figure 6 The left side of the middle section represents FITC-AP2H, FITC-AP2, and FITC-SR), which are dual-targeting peptides MY6 and MY7 binding to the LAPTM4B protein on the surface of HepG2 cells. Figure 6 The FITC-MY6 and FITC-MY7 cells on the left side of the image (representing the signal-to-noise ratio) have a higher signal-to-noise ratio. For LAPTM4B-negative healthy HEK293 cells, no obvious green fluorescence was observed in the field of view after incubation with FITC-labeled MY6 and MY7. Figure 6 (FITC-MY6 and FITC-MY7 are represented on the right). This demonstrates that FITC markers MY6 and MY7 exhibit high selectivity for recognizing different cells, while specifically recognizing the LAPTM4B protein.

[0140] Example 4: MY6 and MY7 alter the localization of LAPTM4B protein in living tumor cells.

[0141] (1) with approximately 1×10 6 Human liver cancer cells (HepG2) were seeded at a density of cells / dish in confocal dishes (Φ=15mm) and cultured overnight at 37℃ and 5% CO2 to allow them to adhere to the dish.

[0142] (2) In the confocal dish of adherent cells, discard the original culture medium in the confocal dish, wash three times with 10 mmol / L phosphate buffer (PBS) (pH 7.4), and then add peptide AP2H, AP2, SR, MY6 and MY7 solutions (diluted with DMEM medium) (as experimental group) and DMEM medium (as blank group). The final peptide concentration is 2 μmol / L. Then incubate at 37℃ and 5% CO2 for 2 h.

[0143] (3) After completing step (2), aspirate the solution and wash three times with PBS. Fix HepG2 cells with 4% paraformaldehyde PBS for 20 min, and wash three times with PBS. Add blocking solution and block at room temperature for 1 h; the blocking solution consists of bovine serum albumin (BSA) and PBS, wherein the mass concentration of bovine serum albumin is 5%.

[0144] (4) Remove the blocking solution from step (3), add LAPTM4B protein antibody solution, and incubate overnight at 4°C; the LAPTM4B protein antibody solution consists of LAPTM4B antibody (Abcam, catalog number ab242376, UK), BSA and PBS; the LAPTM4B antibody is diluted at 1:500 and the mass concentration of BSA is 0.5%.

[0145] (5) After completing step (4), aspirate the solution and wash five times with PBS solution containing 0.05% Tween 20, 5 min each time. Add secondary antibody solution and incubate at room temperature in the dark for 1 h; the secondary antibody solution consists of Alexa Fluor 647-labeled goat anti-mouse IgG antibody (Abcam, catalog number ab150115, UK), BSA and PBS; the dilution ratio of the secondary antibody is 1:1000, and the mass concentration of BSA is 0.5%.

[0146] (6) After completing step (5), discard the solution and wash five times with PBS solution containing 0.05% Tween 20, 5 min each time. Add DAPI nuclear dye and incubate at room temperature for 10 min.

[0147] (7) After completing step (6), wash three times with PBS and use a laser scanning confocal microscope (Olympus FV1000-IX81, Japan) to detect the fluorescence distribution in the cells.

[0148] The experimental results are shown in Figure 7 (Control group is the blank group). The results showed that compared to the blank group and the single-target peptide group ( Figure 7 (Represented by AP2H, AP2, and SR), the dual-targeting peptides MY6 and MY7 can regulate the localization of LAPTM4B protein on the cell membrane surface, induce LAPTM4B protein to anchor at microstructures such as intercellular pseudopodia, and regulate the interaction between cells and between cells and the extracellular matrix by generating more intercellular structures.

[0149] Example 5: Dual-targeting peptides effectively inhibit tumor cell migration

[0150] (1) In a six-hole plate, 5×10 4Human liver cancer cells (HepG2) were implanted into wound healing inserts (Ibidi, Germany) at a density of cells / well and cultured at 37°C and 5% CO2 until 90%–100% confluence.

[0151] (2) After completing step (1), carefully remove the plug with tweezers, leaving a scratch area with a width of about 500μm.

[0152] (3) After completing step (2), discard the culture medium and carefully wash the detached cells with 10 mmol / L phosphate-buffered saline (PBS) (pH 7.4) buffer. Then add the test solution. The test solution is 10 μmol / L AP2H, AP2, SR, MY6 and MY7 solution (diluted with DMEM medium) (as experimental group) and DMEM medium (as blank group).

[0153] (4) After completing step (3), continue incubation at 37℃ and 5% CO2 for 12 hours. Take pictures of the scratched area at 0h and 12h respectively, and use ImageJ software to calculate the healing area of ​​the scratched area. The cell migration ability is measured by the ratio of the healing area of ​​the experimental group to the healing area of ​​the control group.

[0154] The experimental results are shown in Figure 8 (Control group was the blank group). The results showed that the dual-targeting peptides MY6 and MY7, by targeting and binding to LAPTM4B protein, interfered with the migration function of LAPTM4B protein, thereby inhibiting the migration of human hepatocellular carcinoma HepG2 cells. Meanwhile, compared to a single-targeting peptide (…),… Figure 8 AP2H, AP2, and SR are represented by [a specific peptide name], and the dual-targeting peptides MY6 and MY7 have a higher ability to inhibit tumor cell migration. Figure 8 (See the right image in the middle), the differences are significant.

[0155] Example 6: Dual-targeting peptides effectively inhibit tumor cell motility

[0156] (1) HepG2 cells were incubated for 12 h in DMEM medium containing 0.1% fetal bovine serum and 1% penicillin and streptomycin (composed of 10,000 units / mL penicillin and 10,000 μg / mL streptomycin).

[0157] (2) HepG2 cells were digested with trypsin, washed once with 10 mmol / L phosphate-buffered saline (PBS) (pH 7.4), and then the HepG2 cells were digested at 5 × 10⁻⁶ cells / mL. 4 The density was resuspended in solution A; solution A was a 10 μmol / L solution of AP2H, AP2, SR, MY6 or MY7 (diluted with DMEM medium) (as experimental group) and DMEM medium (as blank group).

[0158] (3) Place the Transwell insert (Labselect, Cat#14341) into a 24-well plate (Costar, Cat#3524). Add HepG2 cells and solution A to the upper chamber, and add DMEM medium containing 10% FBS and solution A to the lower chamber. Incubate at 37°C and 5% CO2 for 10 h.

[0159] (4) After completing step (3), fix the cells that have migrated to the bottom of the upper chamber with 4% paraformaldehyde for 20 min. After washing three times with PBS, stain the number of cells that have migrated to the bottom of the upper chamber with crystal violet dye (Beyotime, China) at room temperature for 30 min. After washing three times with PBS, quantitatively observe the cells at the bottom of the upper chamber using a microscope.

[0160] The experimental results are shown in Figure 9 (Control group was the blank group). The results also showed that, compared to single-target peptides ( Figure 9 AP2H, AP2, and SR are representative of dual-targeting peptide compounds. Figure 9 MY6 and MY7 showed a stronger ability to inhibit tumor cell motility, with significant differences.

[0161] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A compound, characterized in that: The compound is MY6 or MY7. MY6 is a compound synthesized via a Click reaction by linking the carboxyl terminus of a polypeptide named AP2H and the carboxyl terminus of a polypeptide named AP2 using polyethylene glycol as an arm. MY7 is a compound synthesized via a Click reaction by linking the carboxyl terminus of a polypeptide named AP2H and the carboxyl terminus of a polypeptide named SR using polyethylene glycol as an arm. The amino acid sequence of AP2H is SEQ ID NO.1 in the sequence listing, the amino acid sequence of AP2 is SEQ ID NO.2 in the sequence listing, and the amino acid sequence of SR is SEQ ID NO.3 in the sequence listing. The polyethylene glycol contains at least three ethylene glycol repeating units.

2. The compound according to claim 1, characterized in that: The chemical structural formula of MY6 is as follows: Formula 1: In Formula 1, IHGHHIISVG is the amino acid sequence from the amino terminus to the carboxyl terminus of AP2H from left to right; SIRILIVKRRRFQST is the amino acid sequence from the carboxyl terminus to the amino terminus of AP2 from left to right. The chemical structural formula of MY7 is as follows: Formula 2: In Formula 2, IHGHHIISVG is the amino acid sequence from the amino terminus to the carboxyl terminus of AP2H from left to right; SIRILIVKRR is the amino acid sequence from the carboxyl terminus to the amino terminus of SR from left to right.

3. A fluorescently labeled compound, characterized in that: The fluorescent labeling compound is a compound obtained by coupling the compound of claim 1 or 2 with a fluorescent dye.

4. The fluorescently labeled compound according to claim 3, characterized in that: The fluorescent dye is fluorescein isothiocyanate.

5. Any of the following applications of the compound of claim 1 or 2 and / or the fluorescently labeled compound of claim 3 or 4: A1) Applications in the development or preparation of products for detecting LAPTM4B protein; A2) Application in the development or preparation of products for detecting cancer cells expressing LAPTM4B protein; A3) Application in the development or preparation of products that inhibit the migration and / or invasion of cancer cells expressing LAPTM4B protein; A4) Applications in the development or preparation of LAPTM4B protein function inhibitors; A5) Applications in the development or preparation of products that regulate the localization of LAPTM4B protein; A6) Applications in the development or preparation of products that regulate intercellular and cell-extracellular matrix interactions in cancer cells with high expression of LAPTM4B protein; A7) Application in the development or preparation of drugs targeting LAPTM4B protein; A8) Application in the development or preparation of drugs that inhibit tumor metastasis; A9) Application in the development or preparation of drugs that inhibit cell migration; A10) Applications in the development or preparation of tumor detection products; A11) Application in the development or preparation of drugs for treating tumors.

6. The application according to claim 5, characterized in that: The tumor is liver cancer; the cells are HepG2 human liver cancer cells.

7. A method for preparing the compound according to claim 1 or 2, characterized in that: The method is C or D; The C step includes the following steps: C1) coupling the AP2H described in claim 1 or 2 with an azide compound using the FMOC peptide solid-phase synthesis method to obtain azide-modified AP2H, and coupling the AP2 described in claim 1 with an alkyne compound and PEG using the FMOC peptide solid-phase synthesis method to obtain alkyne-PEG-modified AP2; C2) coupling the azide-modified AP2H with the alkyne-PEG-modified AP2 via a Click reaction to prepare the MY6. The D step includes the following steps: D1) coupling the AP2H described in claim 1 or 2 with an azide molecule using the FMOC peptide solid-phase synthesis method to obtain azide-modified AP2H; coupling the SR described in claim 1 with an alkyne compound and a PEG molecule using the FMOC peptide solid-phase synthesis method to obtain alkyne-PEG-modified SR; D2) coupling the azide-modified AP2H with the alkyne-PEG-modified SR via a Click reaction to prepare the MY7. The amino acid sequence of AP2H is SEQ ID NO.1 in the sequence listing, the amino acid sequence of SR is SEQ ID NO.3 in the sequence listing, and the amino acid sequence of AP2 is SEQ ID NO.2 in the sequence listing.

8. A composition, characterized in that: The composition contains the compound of claim 1 or 2 and / or the fluorescent labeling compound of claim 3 or 4, and the composition has at least one of the following uses: B1) is used to detect LAPTM4B protein; B2) Products used to detect cancer cells expressing the LAPTM4B protein; B3) Use in products that inhibit the migration and / or invasion of cancer cells expressing the LAPTM4B protein; B4) is used to inhibit the function of LAPTM4B protein; B5) is used to regulate the localization of LAPTM4B protein; B6) is used to regulate intercellular and cell-extracellular matrix interactions in cancer cells with high expression of LAPTM4B protein; B7) is used for tumor detection.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the compound of claim 1 or 2 and / or the fluorescently labeled compound of claim 3 or 4, wherein the pharmaceutical composition is any of the following drugs: E1) Drugs that target the lysosomal four-transmembrane protein LAPTM4B; E2) Drugs that inhibit tumor metastasis; E3) Drugs that inhibit cell migration; E4) Drugs for treating tumors.

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