A polypeptide inhibitor targeting sdc4-hs complex and uses thereof

Abstract

This invention relates to a peptide inhibitor targeting the SDC4-HS complex and its application, belonging to the field of biomedical technology. It addresses one of the problems in existing technologies regarding inaccurate early diagnosis of MASLD, poor efficacy and safety of therapeutic drugs, and the lack of specific peptide inhibitors targeting the SDC4-HS complex. This invention provides a peptide inhibitor targeting the SDC4-HS complex, comprising a WYGP sequence, the amino acid sequence of which is shown in SEQ ID NO. 1 of the sequence listing. This invention provides a specific peptide inhibitor targeting the SDC4-HS complex, capable of blocking key pathogenic pathways of MASLD, achieving safe and efficient treatment and early intervention for fatty liver.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a peptide inhibitor targeting the SDC4-HS complex and its application. Background Technology

[0002] Metabolic-associated fatty liver disease (MASLD), a hepatic manifestation of metabolic syndrome, has a global prevalence of nearly 25%. Its pathological progression can gradually advance from simple hepatic steatosis to steatohepatitis (MASH). Liver fibrosis and even liver cancer Currently, liver biopsy remains the gold standard for the clinical diagnosis of MASLD, but its invasiveness carries a 0.3%-0.5% risk of serious complications and has a 20% rate of missed diagnoses of fibrosis; while blood tests (such as ALT) are more reliable. Imaging technology (ultrasound) MRI has a sensitivity of only 60%-70% for early lesions, which is insufficient to meet the needs of early disease screening and dynamic monitoring. In terms of treatment, lifestyle interventions can maintain long-term efficacy in only 30%-40% of patients. Although clinically recommended drugs such as pioglitazone can improve hepatic steatosis, only 15%-20% of patients achieve histological remission, and fractures are also common. Side effects such as heart failure Rezdiffra, the world's first treatment for MASH, has limited efficacy against advanced fibrosis. Phase III clinical trials of drugs such as obeticholic acid have been terminated due to insufficient safety or efficacy. There is an urgent need for novel targeted intervention strategies that address the pathological mechanisms of MASLD.

[0003] Existing research indicates that the SDC4-HS complex, formed by the modification of the extracellular matrix protein SDC4 with heparan sulfate (HS), plays a key regulatory role in the progression of MASLD to MASH, driving disease exacerbation by mediating macrophage recruitment and amplifying inflammatory signals.

[0004] Therefore, how to develop specific inhibitors targeting the SDC4-HS complex, specifically regulate the HS-GAG modification signal of SDC4, and block disease progression is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] Based on the above analysis, the embodiments of the present invention aim to provide a peptide inhibitor targeting the SDC4-HS complex and its application, in order to solve the problems of inaccurate early diagnosis of MASLD, poor efficacy and safety of treatment drugs, and lack of targeting of SDC4 in the prior art. One of the problems is the presence of specific peptide inhibitors for the HS complex.

[0006] The first aspect of the present invention provides a peptide inhibitor targeting the SDC4-HS complex, the peptide inhibitor comprising a WYGP sequence, the amino acid sequence of which is shown in SEQ ID NO.1 of the sequence listing.

[0007] Furthermore, the polypeptide inhibitor includes a WWGP sequence, the amino acid sequence of which is shown in SEQ ID NO. 2 of the sequence listing.

[0008] Furthermore, the polypeptide inhibitor includes a WWHT sequence, the amino acid sequence of which is shown in SEQ ID NO. 3 of the sequence listing.

[0009] Furthermore, the peptide inhibitor can specifically bind to the SDC4 protein and block the interaction between SDC4 and heparan sulfate (HS).

[0010] Furthermore, the polypeptide inhibitor is a linear tetrapeptide.

[0011] Furthermore, the purity of the peptide inhibitor is ≥98%.

[0012] A second aspect of the present invention provides a pharmaceutical composition comprising the above-described polypeptide inhibitor.

[0013] Furthermore, the pharmaceutical composition also contains metformin.

[0014] A third aspect of the present invention also provides the use of the above-mentioned polypeptide inhibitor in the preparation of a medicament for the prevention and / or treatment of metabolic-associated fatty liver disease (MASLD).

[0015] Furthermore, in the aforementioned applications, the metabolic-related fatty liver disease includes simple hepatic steatosis, MASH (muscular steatohepatitis A), and liver fibrosis.

[0016] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects: 1. The peptide inhibitor targeting the SDC4-HS complex described in this invention specifically binds to the SDC4 protein and effectively blocks the interaction between SDC4 and HS, helping to inhibit the overactivation of key signaling pathways in the MASLD process and providing a new intervention approach to improve hepatic steatosis and inflammation. The peptide acts on the target site with a tetrapeptide structure, has a small molecular size, and is easy to synthesize, which helps to improve the efficiency of action and reduce the preparation cost.

[0017] 2. The peptide inhibitor targeting the SDC4-HS complex described in this invention exhibits good biocompatibility at the cellular level, has no significant adverse effects on hepatocytes within the effective concentration range, and can be effectively taken up by cells and accumulated in liver tissue, which helps to improve the level of local action and reduce the potential risks of systemic exposure, thus providing support for safe in vivo application.

[0018] 3. The peptide inhibitor targeting the SDC4-HS complex described in this invention can improve hepatocellular damage induced by high lipid levels, reduce liver damage-related indicators, alleviate hepatic fat accumulation and inflammatory response, and help delay the progression of MASLD to steatohepatitis and fibrosis. When used in combination with metformin, the improvement effect can be further enhanced, providing a feasible approach for clinical combination therapy.

[0019] 4. The targeted peptide system constructed in this invention provides new candidate therapeutic molecules for MASLD by targeting the SDC4-HS complex, which helps to enrich the targeted treatment strategy for metabolic liver diseases and provides experimental basis and technical support for subsequent new drug development and clinical translation.

[0020] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0021] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts. Figure 1 This is a graph showing the change in root mean square deviation (RMSD) of the SDC4 protein during the simulation process in Example 1. Figure 2 This is a diagram showing the docking boxes and parameters of the SDC4 protein selected using AutoDockTools in Example 2. Figure 3 This is a diagram illustrating the docking and binding pattern between the SDC4 protein (blue) and the Top10 polypeptide compounds (purple) in Example 3. Figure 4 This is a diagram illustrating the docking and binding pattern between the SDC4 protein (blue) and HS (green) in Example 4. Figure 5 This is a diagram illustrating the docking and binding patterns of SDC4 protein (blue), five candidate polypeptide compounds (purple), and HS (green) in Example 4. Figure 6The effect of different concentrations of WYGP polypeptide compound on the viability of HepG2 cells in Example 5; Figure 7 The effect of different concentrations of WWGP polypeptide compound on the viability of HepG2 cells in Example 5; Figure 8 The effect of different concentrations of WWHT polypeptide compound on the viability of HepG2 cells in Example 5; Figure 9 This is a graph showing the changes in HS levels in the supernatant after treating HepG2 cells with the three polypeptide compounds in Example 6. Figure 10 This is a diagram illustrating the protective effect of peptide compounds on HepG2 cells treated with oleic palmitic acid, as analyzed by the CCK-8 assay in Example 7. Figure 11 The graph shows the results of hemolytic reaction analysis of different concentrations of WYGP polypeptide compounds in Example 8; Figure 12 This is a graph showing the results of the enrichment effect analysis of the WYGP polypeptide compound in small animals in Example 8; Figure 13 The results are from the analysis of the absorption efficiency of WYGP polypeptide compound by HepG2 cells in Example 8; Figure 14 This is a diagram illustrating the effect of the peptide inhibitor on delaying the progression of MASLD in HFD (10+3W) mice, as shown in Example 9. Figure 15 This is a diagram illustrating the effect of the peptide inhibitor on delaying the progression of MASLD in HFD (10+6W) mice, as shown in Example 9. Figure 16 This is a violin illustration of the effect of peptide inhibitors on delaying the progression of MASLD in HFD (10+3W) mice in Example 9. Figure 17 This is a violin illustration of the effect of peptide inhibitors on delaying the progression of MASLD in HFD (10+6W) mice in Example 9. Figure 18 This is a diagram illustrating the effect of the peptide inhibitor in delaying the progression of MASLD in MCD (3+1W) mice, as shown in Example 9. Figure 19 This is a diagram illustrating the effect of the peptide inhibitor on delaying the progression of MASLD in MCD (3+2W) mice, as shown in Example 9. Figure 20 This is a violin illustration of the effect of the peptide inhibitor on delaying the progression of MASLD in MCD (3+1W) mice in Example 9. Figure 21 This is a violin illustration of the effect of peptide inhibitors on delaying the progression of MASLD in MCD (3+2W) mice in Example 9. Figure 22 The ALT results of the peptide inhibitor in Example 9 on delaying the progression of MASLD in HFD and MCD mice; Figure 23 The AST results of the peptide inhibitor in Example 9 on delaying the progression of MASLD in HFD and MCD mice; Figure 24 The results of the peptide inhibitor in Example 9 on delaying the progression of MASLD in HFD and MCD mice showed the following effects: Figure 25 The results of cytokine detection in Example 9 show the effect of peptide inhibitors on delaying the progression of MASLD in HFD mice; Figure 26 The results of cytokine detection in Example 9 show the effect of peptide inhibitors on delaying the progression of MASLD disease in MCD mice. Detailed Implementation

[0022] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0023] This invention provides a peptide inhibitor targeting the SDC4-HS complex, wherein the peptide inhibitor includes a WYGP sequence, and the amino acid sequence of the WYGP sequence is shown in SEQ ID NO.1.

[0024] Furthermore, the WYGP sequence is tryptophan-tyrosine-glycine-proline.

[0025] The targeted SDC4 provided by this invention A peptide inhibitor of the HS complex, with the WYGP sequence as its active unit, provides a new molecular basis for the intervention of metabolic-related fatty liver disease. This sequence can interact with the SDC4 protein, providing a feasible technical approach for subsequently blocking key pathogenic pathways and improving liver pathology.

[0026] Furthermore, the polypeptide inhibitor includes a WWGP sequence, the amino acid sequence of which is shown in SEQ ID NO. 2 of the sequence listing.

[0027] Furthermore, the WWGP sequence is tryptophan-tryptophan-glycine-proline.

[0028] Furthermore, the polypeptide inhibitor includes a WWHT sequence, the amino acid sequence of which is shown in SEQ ID NO. 3 of the sequence listing.

[0029] Furthermore, the WWHT sequence is tryptophan-tryptophan-histidine-threonine.

[0030] Furthermore, the peptide inhibitor can specifically bind to the SDC4 protein and block the interaction between SDC4 and heparan sulfate (HS).

[0031] The peptide inhibitor of the present invention can bind to the SDC4 protein and block the interaction between SDC4 and heparan sulfate, which helps to regulate the abnormally activated signaling pathways in the MASLD process and provides intervention target support for alleviating liver inflammation and delaying disease progression.

[0032] Furthermore, the polypeptide inhibitor is a linear tetrapeptide.

[0033] The peptide inhibitor of the present invention has a linear tetrapeptide structure, which has a small molecular size and simple structure, making it easy to prepare on a large scale through chemical synthesis. The production process is well controllable, providing favorable conditions for subsequent process scale-up and clinical translation.

[0034] Furthermore, the purity of the peptide inhibitor is ≥98%.

[0035] A second aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide inhibitor.

[0036] Using the peptide inhibitor of this invention as the active ingredient, different dosage forms can be prepared according to actual needs, providing a stable and applicable formulation for the prevention and / or treatment of MASLD, facilitating clinical administration and patient use. Furthermore, the pharmaceutical composition also contains metformin.

[0037] The pharmaceutical composition of the present invention may further contain metformin. By combining active ingredients with different mechanisms of action, it helps to improve the intervention effect on MASLD, providing a new option for clinical combination drug regimens and laying the foundation for subsequent synergistic treatment research.

[0038] A third aspect of the present invention also provides the use of the above-mentioned polypeptide inhibitor in the preparation of a medicament for the prevention and / or treatment of metabolic-associated fatty liver disease (MASLD).

[0039] Furthermore, in the application, the metabolic-related fatty liver disease includes simple hepatic steatosis, MASH (muscular steatohepatitis A), and liver fibrosis.

[0040] Example 1: SDC4 protein structure construction and molecular dynamics simulation optimization 1. Protein sequence acquisition and preliminary construction The SDC4 protein sequence was obtained from the Uniprot database (Uniprot accession number O35988) and derived from mice. The initial protein structure was constructed using the Alpha Fold 3 server, and energy optimization was performed using the Rosetta Relax module to obtain the initial structure for subsequent optimization.

[0041] 2. Molecular dynamics simulation optimization A 50 ns molecular dynamics simulation was performed using Gromacs 2023.3 software in a physiological saline system. (1) Force field selection: AMBER14SB protein force field; (2) System processing: Add hydrogen atoms using the pdb2gmx subroutine, add a truncated cube TIP3P solvent box at a distance of 10 Å from the system, add Na+ / Cl- to balance the system charge, and output the topology and parameter files; (3) Energy optimization: The steepest descent method is used to minimize energy, with an initial step size of 0.01 nm and a maximum allowable force of 1000 kJ / mol. nm; (4) Ensemble simulation: First, a 100ps NVT ensemble simulation was performed, with the temperature rising from 0K to 310.15K; then a 100ps NPT ensemble simulation was performed, with the pressure rising to 1 bar; the temperature coupling used a Berendsen temperature controller, and the pressure coupling used a Berendsen constant pressure device. (5) Simulation parameters: Hydrogen bonds were constrained by the LINCS algorithm with an integration step size of 2fs; electrostatic interactions were calculated using the PME method with a cutoff value of 1.2nm; non-bonded interactions had a cutoff value of 10Å, updated every 10 steps. (6) Trajectory Analysis: After eliminating periodicity, RMSD analysis was performed. RMSD analysis is based on protein backbone atoms. The trajectory was preprocessed and fitted using Gromacs, and the data was processed and plotted using Origin. The results are as follows: Figure 1 As shown, the SDC4 protein tends to converge around 30 ns and stabilizes around 0.9 nm. The simulation shows sufficient convergence, which can be used for subsequent screening.

[0042] Example 2: Confirmation of SDC4 protein docking site Schrödinger Sitemap 2021-1 was used to predict protein active sites with default parameters: site score threshold ≥0.5, binding pocket volume 100–500 ų. Site reliability was verified by co-localization of hydrophobic, hydrogen-bonded, and charged regions. The docking box was configured using AutoDock Tools 1.5.7. Figure 2As shown: center coordinates center_x=32.943, center_y=43.226, center_z=48.197, dimensions size_x=16, size_y=22, size_z=18, grid spacing 0.919 Å. The stability and reliability of the box parameters are verified by predicting pockets over the Sitemap, repeat docking binding energy RSD<5%, and ligand conformation overlap RMSD<2.0 Å.

[0043] Example 3: Virtual Screening of Peptides Based on Molecular Docking The peptide database was constructed using Python based on RDKit 2022.09.5. Using 20 natural amino acids as units, a tetrapeptide full combinatorial library and a library of 100,000 pentapeptide random sequences were generated using Python scripts. The resulting library contained approximately 260,000 peptides, which were then screened after energy minimization and conformation optimization.

[0044] The SDC4 protein structure was dehydrated and hydrogenated using PyMOL 2.5.2 software; molecular docking was performed using AutoDock Vina 1.2.3, with the docking box set to cover the active cavity, and the parameters were the same as in Example 2.

[0045] After docking and screening, the top 100 binding energies between ligands and receptors were predicted using the Vina binding energy as an indicator and based on the AMBER force field binding energy prediction algorithm (see Table 1). Protein-peptide interactions were analyzed using PLAP 2.2.0 software, and 3D conformations were visualized using Pymol software. Results are as follows: Figure 3 As shown, the Top 10 peptides (purple) form non-covalent interactions with SDC4 protein (blue), including hydrogen bonds (red), π-cation interactions (green), and salt bridges (yellow). The bond lengths next to the dashed lines are measured in angstroms (Å). Analysis of the overall ligand-protein relationship reveals that the ligands bind within the protein binding cavity with low free energy, resulting in a number of non-covalent interactions. Therefore, the peptides (ligands) and SDC4 protein (receptor) can be considered to form a relatively stable complex with stable binding. The WWGP binding energy is -9.667 kcal / mol, and the WYGP binding energy is -9.658 kcal / mol. The peptide purity was determined by high-performance liquid chromatography (HPLC), with a purity ≥98%. Mass spectrometry (MS) verified that the molecular weight was consistent with the theoretical sequence.

[0046] Table 1. Analysis of the interaction forces between the target compounds of peptides and SDC4 proteins.

[0047] Example 4 Screening for blocking SDC4-HS binding by peptide inhibitors 1. SDC4-HS docking preparation: Heparan sulfate (HS) small molecule was obtained from the Pubchem database, CID number 23696126; using MOEv2019 software, the AMBER10:EHT force field and R-field implicit solvation model were selected to optimize the protonation state and hydrogen orientation of SDC4 protein and minimize energy.

[0048] 2. SDC4-HS Interconnection Competitive docking of peptides employed a stepwise substitution docking approach. First, the SDC4 protein and HS molecule were pre-docked, and the optimal complex conformation was preserved. Then, candidate peptides such as WYGP, WWGP, and WWHT were introduced as second ligands into the same docking box, maintaining the receptor and HS positions. The binding free energy was recalculated using Autodock Vina. Docking was performed using the Protein-LigandDock module of MOE, with the receptor set to SDC4 protein and the ligand to HS molecule, Pre-Placement=1000, Placement=30, and the top 10 Refinement models were output. The conformation with the lowest binding free energy was selected. The interaction force analysis between the ligand and receptor was performed using the PLIP online server (https: / / plip-tool.biotec.tu-dresden.de / plip-web), and the 3D conformation of the ligand-receptor was visualized using Pymol software.

[0049] Of all the generated conformations, the PLIF tool clustered the first 10 rotational conformations based on the RMSD between each atom. The representative conformation in each cluster was selected from the conformation with the most neighbors in that cluster. The docking fraction of the SDC4 protein with the HS small molecule was -9.80218315 kcal / mol, as shown in the results. Figure 4 As shown in the figure, the green molecules are HS molecules. The surface plot shows that the docked green HS molecules remain bound within the binding cavity. The protein and compound form a certain number of hydrogen bond interactions (red) and salt bridge interactions (yellow) with different hydrogen bond donors / acceptors. The bond lengths next to the dashed lines are measured in angstroms.

[0050] 3. Verification of peptide blocking effect Quantitative analysis of steric hindrance used ligand binding pocket occupancy, the distance between the center of the HS molecule and the protein binding cavity, and the change in docking binding energy as indicators. The Top 5 peptides (WWGP, WYGP, WRSP, WYFF, WWHT) from Example 3 were selected and added to the SDC4-HS docking system, and the change in the binding free energy of SDC4 and HS was detected and analyzed. Figure 5 As shown: WYGP group: docking fraction -7.17610693 kcal / mol, with the best blocking effect; WWGP group: docking score -7.72010994 kcal / mol; WWHT group: docking score -8.046 kcal / mol; WRSP group: docking fraction -9.2290926 kcal / mol; WYFF group: docking score -10.897 kcal / mol.

[0051] PLIP and Pymol analysis showed that WYGP, WWGP, and WWHT could prevent HS from entering the SDC4 binding cavity through steric hindrance, maintaining their binding to the SDC4 protein surface. In particular, the addition of peptide WYGP resulted in HS being located further out of the pocket, demonstrating a significant blockage in the binding of SDC4 protein to the small HS molecule. Therefore, WYGP, WWGP, and WWHT were subsequently selected as inhibitors for functional validation experiments.

[0052] Example 5: Effect of peptide compounds on HepG2 cell viability 1. Cell Culture HepG2 cells were cultured in high-glucose DMEM medium (Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS, Gibco, 10099141) and 1% penicillin-streptomycin antibiotics (Solarbio, P1400). The cells were purchased from the Cell Bank of the Chinese Academy of Sciences (SCSP-510) and the experiment was limited to passages P10-P25. The culture conditions were 37°C, 5% CO2, and constant temperature culture with saturated humidity (≥95%).

[0053] 2. CCK-8 detection The CCK-8 assay was performed using a Beyotime (Catalog No.: C0038) kit. The experimental design strictly followed the principle of n≥3 independent biological replicates per group, with 3 technical replicates per well to reduce error. Statistical analysis was performed using GraphPad Prism software. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used to analyze differences between two groups. P<0.05 was considered statistically significant.

[0054] Cells were seeded at 5 × 10³ cells / ml in 96-well plates and allowed to adhere overnight. 6.25-200 μM of WYGP, WWGP, and WWHT peptides were added and incubated for 24 h. 10 μL of CCK-8 was added to each well and incubated for another 2 h. The absorbance was measured at 450 nm.

[0055] The CCK8 assay was used to examine the effects of different concentrations of peptide compounds on cell survival and growth. Using the Control group as a reference, cell viability was assessed in HepG2 cells. The measured OD value was directly proportional to cell viability. The results showed that the three peptides at concentrations below 200 μM had no significant toxicity to HepG2 cells. Figure 6 , Figure 7 and Figure 8 As shown.

[0056] Cell viability calculation formula: Viability (%) = [(Ae-Ab) / (Ac-Ab)] × 100% Where Ae represents the absorbance of the treatment group; Ac represents the absorbance of the control group; and Ab represents the absorbance of the blank group.

[0057] Example 6 Effect of peptide compounds on HS levels in cell supernatant The level of heparan sulfate (HS) double antibody sandwich ELISA kit (Catalog No. D711289) in human heparan sulfate was used to detect the HS levels in the supernatant of HepG2 cells treated with three peptides in Example 5. After the reagents were warmed to room temperature, gradient standards and test samples were added to the pre-coated antibody-labeled wells, with three replicates per group. The wells were incubated at 37°C for 1 h and washed 3-4 times. Biotin-labeled antibody was added and incubated at 37°C for 1 h. After washing, HRP streptavidin was added and incubated at 37°C for 0.5 h and washed again. TMB substrate was added and the reaction was developed in the dark for 15 min. The reaction was terminated with stop solution, and the OD value was read at 450 nm. A standard curve was plotted with the OD value of the standard on the ordinate and the corresponding concentration on the abscissa. The curve fit R² > 0.99, indicating a good linear relationship. The HS concentration of the samples was calculated based on the standard curve.

[0058] The results showed that, compared with the control group, the WYGP group had the most significant increase in supernatant HS levels, while the WWGP and WWHT groups showed lower increases, confirming that WYGP had the best effect in blocking SDC4-HS binding. Figure 9 As shown.

[0059] Example 7: Protective effect of peptide compounds on oleic palmitic acid (OPA)-induced HepG2 cell damage. 1. Cell treatment HepG2 cells were seeded at 8 × 10³ cells / well in 96-well plates and treated with oleic acid. High-lipidemia induction was performed using a mixture of palmitic acid (OPA) at a final concentration of 0.6 mM oleic acid and 0.2 mM palmitic acid, prepared in 0.01 M NaOH solvent, for 24 hours. THP-1 macrophages were cultured in RPMI 1640 medium with 10% fetal bovine serum and 1% antibiotics at 37°C, 5% CO2, and saturated humidity. They were then co-cultured with HepG2 cells at a 4:1 ratio (2 × 10³ cells / well) in 96-well plates. 6.25–200 μM (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM) peptide compounds were added, and the cells were cultured for another 24 hours.

[0060] Set up a control group: HepG2 cells were treated with 1640 complete culture medium for 24 h.

[0061] 2. CCK-8 detection Cell viability was detected using the same method as in Example 5. The results are as follows: Figure 10 As shown, different concentrations (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM and 200 μM) of WYGP and WWGP peptide compounds can significantly reduce OPA-induced macrophage damage to HepG2 cells. The cell viability of the WYGP group increased by about 60%, which greatly protected the cells from macrophage attack under high lipid conditions.

[0062] Example 8: Biocompatibility assessment of WYGP peptide compounds 1. Hemolytic test Fresh whole blood from the orbital venous plexus of SPF-grade C57BL / 6 mice was collected, anticoagulated with heparin sodium, centrifuged at 3000 rpm for 10 min, and the supernatant was discarded. The red blood cells were washed three times with sterile physiological saline until the supernatant was clear, and a 5% (v / v) red blood cell suspension was prepared. Distilled water was used as a positive control, and physiological saline as a negative control. Seven concentrations of WYGP peptide groups (0 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM) were set up, with three replicates for each group. 100 μL of red blood cell suspension and 100 μL of the corresponding concentration of peptide solution were added to each well. The mixture was incubated at 37℃ for 1 h, centrifuged at 3000 rpm for 10 min, and the supernatant was collected. The absorbance was measured at 540 nm, and the hemolysis rate was calculated. The results showed that the hemolysis rate of each WYGP concentration group was extremely low, with no significant hemolysis, indicating excellent biocompatibility. Figure 11 As shown.

[0063] 2. In vivo accumulation and biocompatibility The tissue distribution and biocompatibility of WYGP peptide in mice were evaluated using Cy5 fluorescent labeling. WYGP peptide and Cy5 fluorescent dye were mixed at a molar ratio of 1:1 and reacted at room temperature in the dark for 2 hours. Free dye was removed by dialysis to purify WYGP. Cy5 markers were used; a saline control group (Ctrl) and a free Cy5 fluorescent dye control group were also included. Six- to eight-week-old SPF-grade male C57BL / 6 mice, weighing 18–22 g, were selected, with n=5 mice per group. WYGP The Cy5 group and Cy5 control group were administered WYGP via tail vein injection at a volume of 10 mL / kg. Cy5 was administered at a dose of 20 mg / kg; the control group received an equal volume of physiological saline. Thirty minutes after administration, imaging was performed using a small animal in vivo fluorescence imaging system with an excitation wavelength of 646 nm and an emission wavelength of 662 nm to observe fluorescence distribution and liver enrichment.

[0064] The results showed that WYGP-Cy5 was enriched in the liver of mice without any abnormal physiological response or tissue damage. Figure 12 As shown, this indicates that the WYGP peptide not only exhibits low hemolytic activity in vitro but also maintains good biocompatibility in vivo, with no significant toxicity or adverse reactions, providing safety support for its subsequent research and application.

[0065] 3. Cell absorption efficiency HepG2 cells were treated with FITC-labeled WYGP peptide using the same method. Cell uptake efficiency was measured at 0h, 1h, and 4h using flow cytometry via the FITC channel. Figure 13 As shown, the uptake efficiency of WYGP peptides in HepG2 cells was 0.49% at 0 h, 53.8% at 1 h, and 76.5% at 4 h, indicating good cellular uptake. This demonstrates that the uptake capacity of WYGP peptides by HepG2 cells continuously increases over time, without any absorption barriers caused by cellular rejection. This cellular uptake confirms that WYGP peptides can be readily taken up by cells without significant cytotoxicity or rejection, further providing experimental evidence at the cellular level for its excellent biocompatibility.

[0066] Example 9: WYGP peptide alone and in combination with metformin intervention in MASLD model mice 1. Model Building Male C57BL / 6 mice, aged 6-8 weeks and weighing 18-22g, were selected, with n=5 mice per group. The mice were housed in an environment with a temperature of 22-25℃, humidity of 50%-60%, 12h light / 12h dark, and free access to food and water. MASLD mouse models were established by continuous feeding with either a high-fat diet (HFD, D12492, 60% kcal fat) or a methionine-choline-deficient diet (MCD, Research Diets, SFD024).

[0067] 2. Animal grouping and treatment Mice that successfully developed the model were randomly divided into three groups: normal control group, WYGP peptide alone group, and WYGP peptide combined with metformin group. MCD model group: Mice were first fed MCD diet for 3 consecutive weeks to establish the MASLD model, and then drug administration was started: One week of drug administration is recorded as MCD3+1 week. Two weeks of drug administration is recorded as MCD3+2 weeks.

[0068] HFD model group: Mice were first fed HFD diet for 10 consecutive weeks to establish the MASLD model, and then drug administration was started: The drug was administered for 3 weeks, which was recorded as HFD10+3 weeks. The drug was administered for 6 weeks, denoted as HFD10+6 weeks.

[0069] WYGP peptides were administered via tail vein injection at a dose of 20 mg / kg every 2 days, using sterile saline as a solvent. Metformin was dissolved in sterile drinking water at a concentration of 5 mg / mL and administered to mice freely, at a dose of approximately 200 mg / kg / day. The normal control group received an equal volume of saline via tail vein injection plus ordinary drinking water.

[0070] 3. Statistical methods All data are expressed as mean ± standard deviation. GraphPad Prism 8 software was used for analysis. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0071] 4. Detection of the degree of fat degeneration in the sample Mouse livers were collected at different time points for formalin fixation, paraffin embedding, sectioning, and HE staining. Fat vesicles were quantified using the Halo digital pathology system.

[0072] The results showed that, Figure 14 , Figure 15 , Figure 16 , Figure 17 , Figure 18 , Figure 19 , Figure 20 and Figure 21 As shown, in both the HFD and MCD model groups, after a short treatment period (HFD 10+3 weeks; MCD 3+1 weeks), the number of liver fat vesicles in mice treated with WYGP peptide alone and in the WYGP peptide combined with metformin group showed a decreasing trend; the liver-to-kidney ratio also decreased. After a longer treatment period (HFD 10+6 weeks; MCD 3+2 weeks), the number, area, and area ratio of liver fat vesicles in mice treated with WYGP peptide alone and in the WYGP peptide combined with metformin group all decreased significantly, with the decrease being more significant in the WYGP peptide combined with metformin group. These results confirm that small molecule peptide inhibitors can effectively alleviate the progression of MASLD, and that combining them with metformin shows a more prominent effect in delaying the progression of MASLD.

[0073] 5. Detection of liver function indicators in samples To evaluate the protective effect of WYGP peptide on liver function in MASLD model mice, we used the Shenzhen Mindray BS-240VET fully automated veterinary biochemical analyzer to detect changes in the activity of three liver injury markers, ALT, AST, and LDH, in serum.

[0074] Biochemical test results showed that, Figure 22 , Figure 23 and Figure 24 As shown, serum ALT, AST, and LDH activities were significantly elevated in both HFD and MCD model control mice, indicating significant hepatocellular damage. After treatment with WYGP or WYGP+Met, the activities of these three liver injury markers decreased in a time-dependent manner, gradually approaching steatosis levels after long-term treatment (HFD 6 weeks, MCD 2 weeks). The WYGP+Met combined treatment group showed a more significant decrease in ALT, AST, and LDH activities, superior to the WYGP alone group in both models, with some indicators approaching normal physiological levels, demonstrating a stronger protective effect on liver function. In conclusion, WYGP peptides can effectively improve liver function damage in MASLD model mice, and the protective effect is even better when used in combination with metformin, providing key functional evidence for its potential as a candidate therapeutic agent for MASLD.

[0075] 6. Detection of inflammatory factors in samples To elucidate the molecular mechanism by which WYGP peptide improves MASLD, we used OLINK high-throughput cytokine detection technology to systematically analyze the expression changes of different inflammation-related factors in serum in two mouse models, HFD and MCD.

[0076] Assays were performed using an Olink Target 48 mouse cytokine plate. 1 µL of plasma was extracted from freshly aliquoted plasma stored at -80°C, thawed, and incubated for 16 hours at 4°C in an incubation mixture containing cytokine-specific antibody pairs, with each antibody conjugated to both forward and reverse probes. Extension of the complementary probes occurred on a SimpliAmp thermal cycler. For assays, real-time PCR was performed on a BioMark HD system (StandardBioTools) using an MX controller primer IFC 48.48 microfluidic chip loaded with samples and probes. PCR data analysis was performed using BioMark HD Real-Time PCR analysis software with the following parameters set for automatic (global) Ct threshold determination: quality threshold = 0.5, linear baseline correction, and a missing freq value set below 20%. Data processing, quality control, and absolute concentration determination were performed using Olink® NPX Signature software (v1.13.0).

[0077] The heatmap results of cytokine expression profile characteristics show that, for example Figure 25 and Figure 26 As shown, compared with the control group, the expression of pro-inflammatory chemokines (such as Cxcl1, Cxcl2, Ccl11), interleukins (such as Il1a, Il17a), and inflammation-related cytokines were significantly downregulated in the WYGP group and the WYGP+Met group, while the expression of anti-inflammatory factors did not increase significantly, showing an overall phenotype of reduced inflammation. In summary, WYGP peptides can alleviate the inflammatory microenvironment of MASLD liver by inhibiting the expression of chemokines and pro-inflammatory interleukins, blocking the recruitment and activation of inflammatory cells, and the anti-inflammatory effect is more prominent when used in combination with metformin.

[0078] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide inhibitor targeting the SDC4-HS complex, characterized in that, The peptide inhibitor includes a WYGP sequence, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The polypeptide inhibitor of claim 1, wherein, The peptide inhibitor includes a WWGP sequence, the amino acid sequence of which is shown in SEQ ID NO. 2 of the sequence listing.

3. The polypeptide inhibitor of claim 1, wherein, The polypeptide inhibitor includes a WWHT sequence, the amino acid sequence of which is shown in SEQ ID NO. 3 of the sequence listing.

4. The polypeptide inhibitor of any one of claims 1-3, wherein, The peptide inhibitor can specifically bind to the SDC4 protein and block the interaction between SDC4 and heparan sulfate (HS).

5. The polypeptide inhibitor of any one of claims 1-3, wherein, The polypeptide inhibitor is a linear tetrapeptide.

6. The polypeptide inhibitor of any one of claims 1-3, wherein, The purity of the peptide inhibitor is ≥98%.

7. A pharmaceutical composition, characterized by, A polypeptide inhibitor comprising 6. The polypeptide inhibitor of any one of the preceding claims.

8. The pharmaceutical composition of claim 7, wherein, The pharmaceutical composition also contains metformin.

9. The use of a peptide inhibitor according to any one of claims 1-6 in the preparation of a medicament for the prevention and / or treatment of metabolic-associated fatty liver disease (MASLD).

10. Use according to claim 9, characterized in that, The metabolic-related fatty liver diseases include simple hepatic steatosis, MASH (muscular steatohepatitis A), and liver fibrosis.

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