Self-assembled polypeptide, application and medicine

Abstract

The invention belongs to the field of biological medicines, and discloses a self-assembled polypeptide, which comprises a first fragment; the amino acid sequence of the first fragment is LLYpF; wherein Yp represents tyrosine modified by a phosphate group. The self-assembled polypeptide is in a solution state in water based on phosphorylation modification, phosphate groups are removed under the action of alkaline phosphatase, self-assembly is achieved, and nanofibers are formed; meanwhile, the phosphorylation position is a third amino acid in the fragment, the amino acid is a key amino acid for the adiponectin to play a role, and the third amino acid is changed into a non-natural amino acid through phosphorylation modification, so that the third amino acid is prevented from being degraded too early in tissues; the core innovation point of the invention is as follows: the core amino acid is subjected to phosphate group modification, and the triple characteristics of high drug stability, fixed-point release at a target point and self-assembly to form gel after release after modification at the position are utilized, so that the drug effect is ensured to be fully exerted. In addition, the invention also discloses a related application of the polypeptide.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a self-assembling polypeptide, its application and medicine. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a chronic liver injury caused by lipid degeneration independent of alcohol. Initially, lipid accumulation progresses to non-alcoholic steatohepatitis (NAFLD), or further deteriorates to liver fibrosis and even liver cancer. Currently, drugs under investigation for the treatment of NASH and NAFLD include nuclear receptor agonists, such as FXR agonists, PPAR agonists, chemokine receptor inhibitors, thyroid hormone receptor-β agonists, and GLP-1, FGF21, or SGLT2 inhibitors. Among these, GLP-1 analogs and the farnesoid X receptor (FXR) agonist obeticholic acid are the most promising agents for approval in the treatment of NAFLD and NASH. However, obeticholic acid, the first to enter Phase 2 clinical trials, recently had its marketing application rejected again. Consequently, no specific treatment for NAFLD and NASH has been approved worldwide. Therefore, the development of new therapeutic targets and the research of novel therapeutic agents hold significant research and application value for the clinical treatment of NAFLD and NASH.

[0003] According to a large number of studies, adiponectin is an important endogenous bioactive polypeptide or protein secreted by adipose tissue that regulates human energy metabolism. It can specifically bind to adiponectin receptor 1 and adiponectin receptor 2, which are widely distributed in the liver and skeletal muscle. Its main functions include increasing insulin sensitivity, regulating glucose and lipid metabolism balance, and inhibiting oxidative stress and other physiological functions. Some scientific reports have shown that the adiponectin receptor has a highly conserved 15-amino acid polypeptide sequence (GKFHCNIPGLYYFAY) that has the same biological function as adiponectin. Previous studies have further identified a polypeptide GLYYF with only 5 amino acids that has the ability to bind to the AdipoR1 receptor;

[0004] The patent application with application number CN109810176A is a dual agonist peptide of adiponectin receptor-1 and receptor-2 for treating non-alcoholic steatohepatitis and liver fibrosis. It discloses a dual agonist peptide of adiponectin receptor-1 and adiponectin receptor-2, comprising the amino acid sequence: P-X2-LY-X5-F-X7; wherein at least one of X2, X5 and X7 is a non-natural amino acid, P is proline, L is leucine, and Y is tyrosine.

[0005] In this scheme, the purpose of using unnatural amino acids is based on the specific selection of unnatural amino acids due to their high stability.

[0006] However, the effectiveness of a drug is not just related to stability.

[0007] The technical problem to be solved in this case is: how to make adiponectin work efficiently in the liver. Summary of the Invention

[0008] The purpose of the present invention is to provide a self-assembling polypeptide. The self-assembling polypeptide is modified based on phosphorylation so that it is in a solution state in water. Under the action of alkaline phosphatase, the phosphate group is removed to achieve self-assembly and form nanofibers. At the same time, the phosphorylation position is the third amino acid in the fragment. This amino acid is a key amino acid for the efficacy of adiponectin. Through phosphorylation modification, it is converted into a non-natural amino acid, thereby avoiding its premature degradation in tissues.

[0009] At the same time, the present invention also provides applications and medicines based on the polypeptide.

[0010] The specific scheme of the present invention is:

[0011] A self-assembling polypeptide comprising a first segment;

[0012] The amino acid sequence of the first fragment is LLYpF, wherein Yp represents tyrosine modified with a phosphate group.

[0013] In the above self-assembling polypeptide, the first segment is preceded by a natural amino acid or a non-natural amino acid;

[0014] The natural amino acid is glycine; the unnatural amino acid is Nva.

[0015] The self-assembling polypeptide of the present invention is also obtained by modifying adiponectin. The main modification position of the present invention is that the third amino acid in the fragment is a tyrosine modified with a phosphate group. The starting point of the modification of the present invention is:

[0016] 1. The present invention selects the last four amino acids in the classic polypeptide sequence GLYYF that has the ability to bind to the AdipoR1 receptor;

[0017] 2. The present invention phosphorylates the second tyrosine, transforming it into a non-natural amino acid, which prevents it from being rapidly degraded in the body before reaching liver tissue. Furthermore, after dephosphorylation, it can self-assemble into gel fibers, further reducing the degradation rate and facilitating the sustained efficacy of the drug.

[0018] 3. The liver is a tissue with high expression of alkaline phosphatase. Under the action of alkaline phosphatase, the second tyrosine-modified phosphate group of the polypeptide of the present invention disappears, which can achieve targeted release of drug efficacy in the target tissue while reducing side effects.

[0019] 4. The second tyrosine is a key amino acid in adiponectin. Modification of this amino acid is the most critical factor in ensuring drug efficacy.

[0020] The core innovation of this invention is to modify the core amino acid with a phosphate group, taking advantage of the triple characteristics of the drug after modification at this position: high drug stability, fixed release at the target site, and self-assembly to form a gel after release, to ensure full efficacy.

[0021] In the above self-assembling polypeptide, the natural amino acid is glycine; and the non-natural amino acid is Nva.

[0022] In the above-mentioned self-assembling polypeptide, the polypeptide is in a non-gel aqueous solution state in an aqueous solution; when the modified phosphate group on the tyrosine in the polypeptide is removed, the polypeptide self-assembles in an aqueous solution or biological tissue.

[0023] In the above-mentioned self-assembling polypeptide, the modified phosphate group on the tyrosine in the polypeptide is removed under the action of alkaline phosphatase.

[0024] At the same time, the present invention also discloses the use of any of the above self-assembling polypeptides to prepare a drug for treating non-alcoholic fatty liver disease.

[0025] At the same time, the present invention also discloses the use of any of the above self-assembling polypeptides to prepare a drug for treating non-alcoholic fatty liver disease.

[0026] Finally, the present invention also discloses a medicine containing any of the above self-assembling polypeptides.

[0027] In the above-mentioned medicine, the medicine is a medicine for treating non-alcoholic fatty liver disease, or a medicine for treating non-alcoholic steatohepatitis.

[0028] In the above-mentioned medicine, the medicine is an injection or an oral preparation.

[0029] The beneficial effects of this application are:

[0030] The polypeptide of the present invention modifies the core amino acid with a phosphate group, utilizing the triple characteristics of high drug stability after modification at this position, being able to be released at the target site, and being able to self-assemble to form a gel after release, thereby ensuring full efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 are the structural formulas of polypeptide 1 and polypeptide 2;

[0032] Figure 2 is the in vitro dephosphorylation kinetics of GLYYpF;

[0033] Figure 3This is the in vitro dephosphorylation kinetics of NvaLYYpF;

[0034] Figure 4 Electron microscopy images of peptide 1 and peptide 2 when assembled and unassembled;

[0035] Figure 5 This is the stability result diagram of GLYYF and GLYYpF dephosphorylation;

[0036] Figure 6 The stability results of NvaLYYF and NvaLYYpF dephosphorylation are shown;

[0037] Figure 7 Figure 2 is the assembly diagram of GLYYpF selectively on cells in response to ALP;

[0038] Figure 8 Figure 2 is the assembly diagram of NvaLYYpF in response to ALP on cells;

[0039] Figure 9 This is a characterization diagram of the binding of peptide 1 and peptide 2 to adiponectin receptor;

[0040] Figure 10 The figure shows the triglyceride content after the action of peptide 1 and peptide 2 on mice fed with high-fat diet;

[0041] Figure 11 The figure shows the cholesterol content after peptide 1 and peptide 2 act on mice fed with high-fat diet;

[0042] Figure 12 Flow chart for animal experimental treatment;

[0043] Figure 13 This is a curve diagram of the weight changes of mice after different drugs acted on the mouse model;

[0044] Figure 14 This is a graph showing changes in blood glucose levels in mice after different drugs acted on the mouse model;

[0045] Figure 15 This is a curve diagram of the changes in mouse liver weight after different drugs acted on the mouse model;

[0046] Figure 16 This is a curve diagram of the changes in serum triglycerides in mice after different drugs acted on the mouse model;

[0047] Figure 17 This is a curve diagram of changes in serum cholesterol in mice after different drugs acted on the mouse model;

[0048] Figure 18 H&E staining and Oil Red staining of mouse liver. DETAILED DESCRIPTION

[0049] The present invention will be described clearly and completely below in conjunction with the examples of the present invention. In the description of the present invention, it should be noted that, where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0050] Example 1 Synthesis of polypeptide

[0051] The designed polypeptide molecule was synthesized by solid phase synthesis by adding non-natural amino acids, and the amino acid sequence was (GLLYpF, NvaLYYpF) (reference Figure 1 Using AM resin, the required amino acids are weighed and placed on a peptide synthesizer. Following the appropriate procedures, the desired molecule is synthesized. Purification by high-performance liquid chromatography and lyophilization yield the corresponding self-assembling peptide powder.

[0052] Hereinafter, the polypeptide having the polypeptide sequence GLLYpF is referred to as polypeptide 1; the polypeptide having the polypeptide sequence NvaLYYpF is referred to as polypeptide 2;

[0053] The molecular formula and structural formula of Nva are: C5H 11 NO2

[0054]

[0055] Example 2 Characterization of polypeptide dephosphorylation kinetics

[0056] The experimental method is as follows: the polypeptide is dissolved in PBS buffer to prepare a working solution (1 mL) with a concentration of 300 μM, and alkaline phosphatase (5 U / mL) is added for reaction treatment at 37°C.

[0057] At different time points (0, 0.5, 1, 2, 4, 8, and 12 hours), 100 μL of sample was taken, mixed with 25 μL of acetonitrile solution containing 0.1% TFA, and stored in a -20°C refrigerator. The resulting samples were analyzed by analytical high-performance liquid chromatography (HPLC) using gradient elution. The chromatographic peak areas of the peptides before and after dephosphorylation were integrated, and the kinetic parameters of the dephosphorylated peptides were calculated based on the area.

[0058] Results Reference Figure 2 and Figure 3 ;

[0059] Figure 2 is the in vitro dephosphorylation kinetics of GLYYpF;

[0060] Figure 3 This is the in vitro dephosphorylation kinetics of NvaLYYpF;

[0061] The experimental results are: Figure 2 and Figure 3 Thus, the in vitro evaluation of peptide responsiveness to ALP by measuring dephosphorylation kinetics showed that both NvaLYYpF and GLYYpF were completely dephosphorylated after 8 hours of incubation with ALP solution, demonstrating the feasibility of enzyme-responsive self-assembly in cells with high ALP expression.

[0062] Example 3 Characterization of nanofiber structure formed by self-assembly of active polypeptides under the action of phosphatase

[0063] The experimental method is as follows: the sample is prepared by diluting 5 μL of a 1.0 wt% peptide working solution into an appropriate amount of water. A drop of this solution is added to a 200-mesh carbon-coated copper grid (EMCN), allowed to stand for 1 minute, and then blotted dry with filter paper. Subsequently, a 2% uranyl acetate solution is added and incubated for 1-2 minutes, blotted again, and allowed to air dry. Images are captured using a Hitachi 7650 transmission electron microscope at 80 kV.

[0064] Results Reference Figure 4 ;

[0065] Figure 4 Electron microscopy images of peptide 1 and peptide 2 when assembled and unassembled;

[0066] The experimental results are: Figure 4 It can be seen that the polypeptide 1 and polypeptide 2 of the present invention can self-assemble to form a nanofiber structure under the action of phosphatase.

[0067] Example 4 Biological stability of phosphorylated active polypeptides

[0068] Experimental Methods: The peptide was dissolved in PBS buffer to a working solution (1 mL) at a concentration of 300 μM. Proteinase K (0.3 U / mL) was added for reaction at 37°C. At different time points (0, 10, 30, 90, 120, 240, and 480 minutes), 100 μL of sample was taken, mixed with 25 μL of acetonitrile containing 0.1% TFA, and stored in a -20°C refrigerator. For phosphorylated peptides, 5 U / mL of alkaline phosphatase (ALP) was first added for overnight assembly, followed by the addition of proteinase K for subsequent treatment. The resulting samples were analyzed by analytical high-performance liquid chromatography (HPLC) using gradient elution. The chromatographic peaks before and after dephosphorylation were integrated, and the kinetic parameters of the dephosphorylated peptide were calculated based on the area.

[0069] Results Reference Figure 5 and Figure 6 ;

[0070] Figure 5This is the stability result diagram of GLYYF and GLYYpF dephosphorylation;

[0071] Figure 6 The stability results of NvaLYYF and NvaLYYpF dephosphorylation are shown;

[0072] The experimental results showed that the degradation rates of self-assembled and non-assembled peptides were compared using a Proteinase K degradation assay. The results showed that the stability of GLYYpF and NvaLYYpF after ALP-catalyzed self-assembly was significantly improved, with degradation times extending to over 250 minutes, while the non-phosphorylated peptides were degraded in less than 100 minutes. This phenomenon indicates that the self-assembled peptide assemblies significantly slowed the degradation rate of the peptides and improved their stability.

[0073] Example 5 Selective assembly of phosphorylated active polypeptides on the cell surface

[0074] Experimental method: Huh7 cells, a liver cancer cell line, were seeded at a density of 8,000 cells / well in a 35mm confocal culture dish and cultured for 24 hours. The old culture medium was then aspirated, the cells were washed twice with PBS, ALP inhibitors were added or not, and 1 mL of a 200 μM peptide solution was added and incubated for different lengths of time. After the incubation period, the old culture medium was aspirated and the cells were washed twice with PBS. Next, the cells were stained with 2 μg / mL Hoechst33342 dye for 15 minutes. After staining, imaging analysis was performed using a Zeiss LSM980 confocal microscope (60× oil lens). The results were processed using image processing software ZEISS ZEN 3.9.

[0075] Results Reference Figure 7 and Figure 8 ;

[0076] Figure 7 This is a diagram showing the assembly effect of GLYYpF on cells in response to ALP;

[0077] Figure 8 This is a diagram showing the assembly effect of NvaLYYpF on cells in response to ALP;

[0078] The results show that the phosphorylated peptides GLYYpF and NvaLYYpF assembled on the cell surface with and without the addition of an alkaline phosphatase (ALP) inhibitor. In the absence of an ALP inhibitor, GLYYpF and NvaLYYpF assembled on the cell surface (visible green fluorescence), while the addition of an ALP inhibitor resulted in almost no green fluorescence, indicating that the phosphorylated peptides were unable to self-assemble. This experiment demonstrates that phosphorylated peptides can selectively self-assemble in response to the ALP enzyme.

[0079] Example 6 Characterization of active polypeptide binding to adiponectin receptor

[0080] The experimental method is as follows: This example uses immunofluorescence detection, and the specific method is as follows: after digestion, cultured liver cancer cell line Huh7 cells are cultured on a cell slide at a density of 8,000 per well until the cells adhere to the wall.

[0081] After the cells were treated with the peptides, they were washed twice with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 10% goat serum in PBS.

[0082] After washing away the blocking solution, the primary antibody solution containing 5% goat serum (1:500 dilution) was added dropwise to the coverslip containing the cells and incubated on a shaker at 4°C overnight. After washing three times with PBS, the fluorescent-labeled secondary antibody was added and incubated at room temperature for 1 hour. Then, the cells were washed three times with PBS, and the cell nuclei were stained with DAPI for 10 minutes. Finally, the cell slides were removed, washed and sealed, and images were taken using a Zeiss LSM980 laser confocal microscope (60× oil objective). The images were processed using the image processing software ZEISS ZEN 3.9;

[0083] Results Reference Figure 9 ;

[0084] Figure 9 This is a characterization diagram of the binding of peptide 1 and peptide 2 to the adiponectin receptor; the scale bar in the figure is 20 μM;

[0085] The experimental results are: Figure 9 It can be seen that polypeptide 1 and polypeptide 2 of the present invention can interact with the adiponectin receptor AdipoR1 on cells, further demonstrating that they can activate downstream signaling pathways and exert biological functions by targeting the adiponectin receptor.

[0086] Example 7 Polypeptide Alleviates Cellular Lipid Accumulation

[0087] The experimental method is as follows: Huh7 hepatoma cell line cells are digested into a suspension, counted, and seeded at a density of 1×105 cells / well in a 24-well plate or confocal microplate. The cells are cultured overnight to allow attachment. Subsequently, 400 μM OA is added and incubated for 24 hours to induce lipid accumulation. After a specific period of time following treatment with the corresponding peptide drug, the medium is removed and the cells are washed three times with PBS. Bodipy is added for staining for 10-20 minutes. After washing, DIPA is added to stain the cell nuclei. Subsequently, confocal microplates are used for observation and quantitative analysis. TC and TG quantification kits are used: 100 μL of lipid extraction solution (hexane / isopropanol, 3:2) is added to each well. The plate is tilted and the solution is repeatedly pipetted to ensure that it flows through and covers all cells. The solution is then collected in a 1.5 mL centrifuge tube and heated in a 60°C metal bath to evaporate all organic solvents. Add an appropriate amount of 0.1% Triton X-100, vortex, and let stand for 30 minutes to ensure that the lipids are fully dissolved in the 0.1% Triton X-100 solution. Simultaneously, add 200 μL of 0.2 mM NaOH to each well of the plate, place on a shaker, and shake at room temperature for 3 hours to extract total cellular protein. Finally, measure cholesterol (TC) and triglyceride (TG) levels using the kit instructions and normalize to cellular protein concentration.

[0088] Results Reference Figure 10 and Figure 11 ;

[0089] Figure 10 This is a graph showing the triglyceride content after peptide 1 and peptide 2 acted on high-fat fed mice;

[0090] Figure 11 This is a graph showing the cholesterol content after peptide 1 and peptide 2 acted on high-fat fed mice;

[0091] exist Figure 10 and Figure 11 In the figure, other types of peptides and drugs are also shown; JT003 is a reported positive control;

[0092] Peptides 1 and 2 exhibit comparable pharmacological activity to NvaLYYF and GLYYF, indicating that phosphorylation of Peptides 1 and 2 does not affect their pharmacological activity. In vivo, dephosphorylation by ALP converts them to their active forms (NvaLYYF and GLYYF), activating the adiponectin receptor and exerting lipid-lowering effects. Activation-based drug design may play a role in reducing systemic side effects.

[0093] Example 8: Experiment on the alleviation of non-alcoholic fatty liver disease in mice using polypeptides

[0094] The experimental method is as follows: The 6-week-old C57BL / 6J mice used in this study were purchased from Jicui Yaokang (Jiangsu). After one week of feeding to adapt to the environment, they began to be fed a high-fat diet (XTHF60) containing 60kcal% fat for 8 weeks to induce fatty degeneration of the liver and construct a non-alcoholic fatty liver mouse model. Dosage process: The mice were divided into groups according to their weight, and the mice were intraperitoneally injected once every three days at a dose of 20 mg / kg. Liraglutide was used as a positive control. After eight weeks of continuous injection treatment, the mice were subjected to insulin tolerance test (ITT) and glucose tolerance test (GTT) tests. The mice were further sacrificed and dissected, and the organs were collected for immunohistochemical testing, and the blood was collected for testing of biochemical indicators such as lipids;

[0095] Results Reference Figures 12 to 17 ;

[0096] Figure 12 Flow chart for animal experimental treatment;

[0097] Figure 13 This is a curve diagram of the weight changes of mice after different drugs acted on the mouse model;

[0098] Figure 14 This is a graph showing changes in blood glucose levels in mice after different drugs acted on the mouse model;

[0099] Figure 15 This is a curve diagram of the changes in mouse liver weight after different drugs acted on the mouse model;

[0100] Figure 16 This is a curve diagram of the changes in serum triglycerides in mice after different drugs acted on the mouse model;

[0101] Figure 17 This is a curve diagram of changes in serum cholesterol in mice after different drugs acted on the mouse model;

[0102] Among them, HFD (PBS) refers to the high-fat feeding group and PBS-treated group; Liraglutide refers to the liraglutide-treated group; ND (PBS) refers to the normal feeding PBS-treated group.

[0103] Through the above Figures 12 to 17 It can be seen that polypeptide 1 and polypeptide 2 of the present invention can significantly alleviate non-alcoholic fatty liver disease in mice, control the body weight and blood sugar of mice, and reduce the cholesterol and triglyceride levels in the liver and serum;

[0104] Example 9 Immunohistochemical Analysis of Lipid Content in Animal Tissues

[0105] The experimental method is as follows: Mouse liver tissue was subjected to histological analysis using paraffin sectioning and frozen sectioning techniques. In the paraffin sectioning process, fresh liver tissue blocks (thickness <0.5 cm) were fixed with 10% neutral formalin for 3 days to stabilize the cell morphology through protein cross-linking. The volume of the fixative was 10-15 times the volume of the tissue to ensure sufficient penetration. Subsequently, the sections were dehydrated with gradient ethanol (75%-100%), transparentized with xylene, and embedded in paraffin (58-65°C). Sections with a thickness of 4-5 μm were prepared. After dewaxing and rehydration, hematoxylin-eosin (H&E) staining was used. After sealing with neutral gum, the morphological characteristics of the cell nucleus (blue) and cytoplasm (pink) were observed under an optical microscope. On the other hand, in the frozen section process for lipid analysis, liver tissue is fixed with 4% paraformaldehyde for 48 hours to maintain lipid structure, then dehydrated in a gradient of 10% and 30% sucrose solutions for 2 days to reduce ice crystal damage. After embedding in OCT embedding medium, 6-8μm sections are cut at -20°C and stained with Oil Red O to specifically label neutral lipids (red). Finally, high-resolution images are obtained using a slice scanner. Both methods require fixation to be initiated within 20 minutes of tissue ex vivo, and the choice of fixative is optimized based on the research objectives.

[0106] Results Reference Figure 18 ;

[0107] Figure 18 H&E staining and Oil Red staining of mouse liver;

[0108] Through the above Figure 18 It can be seen that the degree of fatty degeneration of hepatocytes can be observed by Oil Red O staining of polypeptide 1 and polypeptide 2 of the present invention. The weakening of OA staining indicates that the lipid droplets and total lipid content in hepatocytes are significantly reduced after treatment with adiponectin self-assembling polypeptide drugs polypeptide 1 and polypeptide 2 ( Figure 18 H&E staining showed that after peptide treatment, the number of liver vacuoles caused by fatty degeneration was significantly reduced, indicating that peptide can alleviate cell damage in fatty liver.

[0109] Summarize:

[0110] 1. As shown in Example 2, NvaLYYpF and GLYYpF have a certain feasibility for enzyme-responsive self-assembly in cells with high ALP expression. As shown in Example 3, the polypeptides of the present invention can self-assemble into nanofiber structures under the action of phosphatase. At the same time, the cell experiment in Example 5 further verified that the polypeptides of the present invention have the function of selective self-assembly in response to the ALP enzyme.

[0111] The above three cases demonstrate that the tyrosine phosphorylation modification of the present invention has a positive effect on the efficacy of polypeptides in cells.

[0112] 2. Example 4 demonstrates that the stability of the phosphorylated polypeptide is significantly improved, which can effectively prolong its duration of action and prevent it from being rapidly degraded by the organism.

[0113] 3. Examples 6 and 7 demonstrate that the polypeptide of the present invention can target the adiponectin receptor at the cellular level and exert its lipid-lowering function.

[0114] 4. Examples 8 and 9 demonstrate that the polypeptide of the present invention can significantly alleviate non-alcoholic fatty liver disease in disease model mice, control mouse body weight and blood sugar, reduce cholesterol and triglyceride levels in the liver and serum; and alleviate cell damage in fatty liver.

[0115] The present invention modifies the core amino acid with a phosphate group, utilizing the triple characteristics of the drug after modification at this position: high drug stability, fixed release at the target site, and self-assembly to form a gel after release, thereby ensuring full efficacy.

Claims

1. A self-assembling polypeptide, characterized in that The polypeptide comprises a first segment; The amino acid sequence of the first fragment is LLYpF, wherein Yp represents tyrosine modified with a phosphate group.

2. The self-assembling polypeptide according to claim 1, characterized in that The first segment is preceded by a natural amino acid or an unnatural amino acid; The natural amino acid is glycine; the unnatural amino acid is Nva.

3. The self-assembling polypeptide according to claim 1 or 2, characterized in that The polypeptide is in a non-gel aqueous solution state in an aqueous solution; when the modified phosphate group on the tyrosine in the polypeptide is removed, the polypeptide self-assembles in an aqueous solution or biological tissue.

4. The self-assembling polypeptide according to claim 3, characterized in that The modified phosphate group on the tyrosine in the polypeptide is removed under the action of alkaline phosphatase.

5. Use of the self-assembling polypeptide according to any one of claims 1 to 4 in preparing a medicament for treating non-alcoholic fatty liver disease.

6. Use of the self-assembling polypeptide according to any one of claims 1 to 4 in preparing a medicament for treating non-alcoholic steatohepatitis.

7. A drug, characterized in that Contains the self-assembling polypeptide according to any one of claims 1 to 4.

8. The drug according to claim 7, characterized in that The medicine is a medicine for treating non-alcoholic fatty liver disease, or a medicine for treating non-alcoholic fatty liver disease.

9. The drug according to claim 7, characterized in that The medicine is an injection or an oral preparation.

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