A short peptide that blocks the binding of vdbp to megalin

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a short peptide for blocking the combination of VDBP and Megalin. The application directly reveals the core molecular basis of the combination of VDBP and Megalin by identifying the key amino acid site sequence of the interaction between VDBP and Megalin. The blocking small molecules or polypeptides designed on the basis can cut off the combination of VDBP and Megalin in a highly specific manner, avoiding the limitations brought by random screening and large molecule antibody intervention in the traditional method. The technical scheme not only significantly improves the research and development efficiency, reduces redundant experiments, and reduces the research and development cost, but also guarantees the repeatability and consistency of the research results. Based on the combination site, the application directly designs a short peptide capable of blocking the combination of VDBP and Megalin, the short peptide does not cause neuron apoptosis or structural damage, can reverse the inhibited signal pathway of the interaction between VDBP and Megalin, and has potential application value in treating depression.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a short peptide that blocks the binding of VDBP to Megalin. Background Technology

[0002] Depression is a common mental illness characterized by persistent low mood, loss of interest, and cognitive impairment. Its pathogenesis is complex, involving multiple factors such as neurotransmitter imbalance, impaired neuroplasticity, abnormal neuroinflammatory responses, and neuro-immune-endocrine system disorders. Currently, the main clinical treatment is still based on small-molecule chemical antidepressants, including selective serotonin reuptake inhibitors (SSRIs), norepinephrine reuptake inhibitors (NRIs), tricyclic antidepressants (TCAs), and monoamine oxidase inhibitors (MAOIs).

[0003] While these medications offer some relief for symptoms, they also have several limitations: slow onset of action and limited efficacy: most antidepressants require 4–8 weeks of continuous use to produce significant effects, and approximately 30%–40% of patients respond poorly to or develop drug resistance to existing medications. Significant side effects: long-term use often causes adverse reactions such as weight gain, sexual dysfunction, gastrointestinal discomfort, and sleep disturbances, affecting patient adherence. Single target: traditional drugs primarily work by regulating monoamine neurotransmitter levels, neglecting key aspects of depression pathogenesis such as neuroinflammation, neuroimmunity, and synaptic plasticity changes. Significant individual variability: differences in genotype, metabolic capacity, and brain region function lead to significant variations in response to the same medication among different patients, making clinical treatment outcomes difficult to predict.

[0004] In recent years, with the interdisciplinary development of neuroscience and immunology, researchers have gradually recognized that depression is not only the result of neurotransmitter imbalance, but also a disease characterized by systemic inflammation and abnormal neuro-immune interactions. Therefore, novel therapeutic strategies targeting inflammatory factors, neurotrophic factors, and neuroimmune pathways have become a research hotspot. Neutralizing antibodies and short peptide drugs, as emerging biologics, can overcome the problems of single-target therapy and significant side effects of traditional drugs by highly specifically regulating key molecular pathways, showing promising application prospects. However, current research on antibody and short peptide treatments for depression is still in the exploratory stage, lacking systematic mechanistic studies and clinical validation.

[0005] Protein molecules play crucial roles in cell signal transduction, substance transport, and pathological mechanisms, and elucidating protein-protein interaction patterns is essential for understanding their molecular functions. While traditional experimental methods (X-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance) can provide high-resolution three-dimensional structures, their limitations—such as experimental conditions, protein complexity, and high cost—make rapid studies of large molecular complexes and their mutagenic effects difficult. Therefore, homology modeling and molecular docking have become important computational tools in structural biology and drug design.

[0006] Against this backdrop, low-density lipoprotein receptor-related protein 2 (LRP2, also known as Megalin), a large transmembrane glycoprotein, participates in protein reabsorption in proximal tubular epithelial cells of the kidney and plays an important role in the nervous and metabolic systems. Vitamin D-binding protein (VDBP) is a plasma protein that primarily binds and transports vitamin D and its metabolites. Previous studies have suggested that the binding between VDBP and Megalin may be a crucial step in regulating vitamin D metabolism and related diseases. However, due to the protein's large size and structural deficiencies, high-resolution structural analysis and systematic docking studies of the Megalin–VDBP complex are currently lacking.

[0007] Therefore, based on existing structural data, constructing models of the possible binding modes of the Megalin–VDBP complex and designing new therapeutic drugs using computational methods such as homology modeling, molecular docking, cluster analysis, and mutation verification has become a key technical problem that urgently needs to be solved. Summary of the Invention

[0008] The first aspect of the present invention is to provide a short peptide or a salt thereof.

[0009] A second aspect of the present invention is to provide a conjugate.

[0010] A third aspect of the present invention is to provide a fusion protein.

[0011] A fourth aspect of the present invention aims to provide biomaterials related to the short peptides or salts thereof of the first aspect of the present invention, the conjugates of the second aspect of the present invention, and the fusion proteins of the third aspect of the present invention.

[0012] The fifth aspect of this invention aims to provide methods for preparing the short peptide or its salt as described in the first aspect of this invention, the conjugate as described in the second aspect of this invention, and the fusion protein as described in the third aspect of this invention.

[0013] The sixth aspect of this invention aims to provide the application of the short peptide or its salt of the first aspect of this invention, the conjugate of the second aspect of this invention, and the fusion protein of the third aspect of this invention.

[0014] A seventh aspect of the present invention is to provide a pharmaceutical composition.

[0015] The object of the eighth aspect of the present invention is to provide a method for blocking the binding of VDBP to Megalin in vitro.

[0016] The ninth aspect of this invention aims to provide the application of the VDBP-Megalin binding site in the preparation of a drug screening platform for screening candidate drugs for depression.

[0017] The tenth aspect of this invention aims to provide a method for treating depression, comprising the following steps:

[0018] Apply an effective dose of the short peptide of the first aspect of the present invention or its salt, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention to the test subject.

[0019] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0020] In a first aspect, the present invention provides a short peptide or a salt thereof, wherein the amino acid sequence of the short peptide is any one of a1) to a2):

[0021] a1) ADYSENTFTEYKKKL (SEQ ID NO: 4);

[0022] a2) and a1) have amino acid sequences that are at least 90% homologous.

[0023] In a first aspect of the invention, a2) is a protein that has the same function as a1).

[0024] In a first aspect of the invention, protein substitutions that have the same function in a2) are generally considered as conservative substitutions, including substitutions between aliphatic amino acids Ala, Val, Leu and Ile, exchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr.

[0025] In a first aspect of the invention, a2) means that the given amino acid sequence shares at least 90% identity with the reference sequence. Alternatively, it means that the given amino acid sequence differs from the reference sequence by one amino acid, which, for proteins, is preferably an amino acid substitution or deletion. This difference in identity can arise from a conserved substitution of amino acids.

[0026] In some embodiments of the present invention, the salt is a pharmaceutically acceptable salt, including acid addition salts and base addition salts.

[0027] "Pharmaceutically acceptable acid addition salts" refer to salts that retain the biological effectiveness and properties of the free base, are not undesirable in biological or other respects, and are formed from an inorganic acid and an organic acid, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and the organic acid including but not limited to acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, fumaric acid, galactopyric acid, gentian acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, etc.

[0028] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acid and are not undesirable in biological or other respects. These salts are prepared by the addition of an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; and basic ion exchange resins, such as various ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, thiazoline, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0029] A second aspect of the present invention provides a conjugate comprising the short peptide or a salt thereof described in the first aspect of the present invention, and a modification portion.

[0030] In some embodiments of the present invention, the modified portion comprises at least one of chemical modification and fluorescent dye.

[0031] In some embodiments of the present invention, the chemical modification includes at least one of amidation, acetylation, amination, methylation, phosphorylation, glycosylation, ubiquitination, and biotinylation.

[0032] In some embodiments of the present invention, the fluorescent dye comprises FITC.

[0033] A third aspect of the invention provides a fusion protein comprising the short peptide or a salt thereof described in the first aspect of the invention, and a coupling portion.

[0034] In some embodiments of the present invention, the coupling portion comprises at least one of a drug, a cytokine, a radionuclide, a fluorescent protein, and a tag protein.

[0035] In some embodiments of the present invention, the fluorescent protein includes GFP and TdTomato.

[0036] In some embodiments of the present invention, the protein tag includes at least one of His, Flag, GST, MBP, HA, and Myc.

[0037] In some embodiments of the present invention, the coupling portion of the fusion protein can lead to changes in protein homology, such as the introduction of a tag protein, a nuclear insertion sequence, or a signal peptide sequence. Adding tags such as His, GFP, or HA to the C-terminus alters the homology, as does adding a signal peptide or a conventional nuclear insertion sequence to the N-terminus.

[0038] A fourth aspect of the present invention provides biomaterials related to the short peptides or salts thereof of the first aspect of the present invention, the conjugates of the second aspect of the present invention, and the fusion proteins of the third aspect of the present invention, including any one of b1) to b12);

[0039] b1) A nucleic acid molecule encoding the short peptide or salt thereof as described in the first aspect of the invention, the conjugate as described in the second aspect of the invention, or the fusion protein as described in any one of the third aspects of the invention;

[0040] b2) An expression cassette containing the nucleic acid molecule described in b1);

[0041] b3) A carrier containing the nucleic acid molecule described in b1);

[0042] b4) A carrier containing the expression box described in b2);

[0043] b5) Transgenic cell lines containing the nucleic acid molecules described in b1);

[0044] b6) Transgenic cell lines containing the expression cassette described in b2);

[0045] b7) A transgenic cell line containing the vector described in b3);

[0046] b8) A transgenic cell line containing the vector described in b4);

[0047] b9) Microorganisms containing the nucleic acid molecules described in b1);

[0048] b10) Microorganisms containing the expression cassette described in b2);

[0049] b11) Microorganisms containing the carrier described in b3);

[0050] b12) contains microorganisms that contain the carrier described in b4).

[0051] In some embodiments of the present invention, the transgenic cell line does not include propagation material.

[0052] In some embodiments of the present invention, the carrier includes a fusion expression tag.

[0053] In some embodiments of the present invention, the fusion expression tag includes, but is not limited to, common fusion expression tags such as GFP, His, and GST.

[0054] In some embodiments of the present invention, the microorganism is a transformant, which is obtained by introducing the recombinant expression vector described above into a host.

[0055] In some embodiments of the present invention, the host is *Escherichia coli*; more preferably, the *Escherichia coli* is *Escherichia coli*. E. coli BL21 (DE3) cells (for protein purification) or DH5α cells (for amplification and preservation of plasmid vectors).

[0056] A fifth aspect of the present invention provides methods for preparing the short peptide or its salt described in the first aspect of the present invention, the conjugate described in the second aspect of the present invention, and the fusion protein described in the third aspect of the present invention.

[0057] It was prepared by culturing the biomaterials described in the fourth aspect of the present invention;

[0058] Alternatively, it can be obtained through peptide chemical synthesis and purified using high-performance liquid chromatography (HPLC) to ensure a purity higher than 95%, and finally identified by mass spectrometry (MS).

[0059] The culture methods described are standard practices in this field. For the section on molecular cloning, please refer to *Molecular Cloning: A Laboratory Manual* (serial coding system: Michael R. Green, Joseph Sambrook, He Fuchu. *Molecular Cloning: A Laboratory Manual* (4th Edition) (Volumes 1, 2, and 3) [M]. Beijing: Science Press, 2017); for the section on cell culture, please refer to *Cell Biology: A Laboratory Manual* (serial coding system: Julio E. Celis [ed.]. *Cell Biology: A Laboratory Manual*. Volume 1: Introduction [M]. Beijing: Science Press, 2008).

[0060] A sixth aspect of the present invention provides the use of the short peptide or its salt described in the first aspect of the present invention, the conjugate described in the second aspect of the present invention, and the fusion protein described in the third aspect of the present invention in c1) to c2):

[0061] c1) Prepare drugs for treating depression;

[0062] c2) Prepare drugs that block the binding of VDBP to Megalin.

[0063] in:

[0064] Vitamin D-binding protein (VDBP) is a glycoprotein synthesized by the liver and secreted into the blood plasma. It plays a crucial role in vitamin D metabolism, immune regulation, and cell signaling. In humans, this protein is encoded by the GC gene, and its reference amino acid sequence can be found in the UniProt database (https: / / www.uniprot.org) with accession number P02774 (GC_HUMAN). The corresponding amino acid sequence is as follows:

[0065] MKRVLVLLLAVAFGHALERGRDYEKNKVCKEFSHLGKEDFTSLSLVLYSRKFPSGTFEQVSQLVKEVVSLTEACCAEGADPDCYDTTRTSALSAKSCESNSPFPVHPGTAECCTKEGLER KLCMAALKHQPQEFPTYVEPTNDEICEAFRKDPKEYANQFMWEYSTNYGQAPLSLLVSYTKSYLSMVGSCCTSASPTVCFLKERLQLKHLSLLTTLSNRVCSQYAAYGEKKSRLSNLIKL AQKVPTADLEDVLPLAEDITNILSKCCESASEDCMAKELPEHTVKLCDNLSTKNSKFEDCCQEKTAMDVFVCTYFMPAAQLPELPDVELPTNKDVCDPGNTKVMDKYTFELSRRTHLPE VFLSKVLEPTLKSLGECCDVEDSTTCFNAKGPLLKKELSSFIDKGQELCADYSENTFTEYKKKLAERLKAKLPDATPTELAKLVNKHSDFASNCCSINSPPLYCDSEIDAELKNIL (SEQ ID NO: 1).

[0066] The sequence contains 474 amino acid residues, with a theoretical molecular weight of approximately 52 kDa. In different populations, the GC gene has natural alleles such as GC1F, GC1S, and GC2. The amino acid differences are mainly concentrated at sites 416 and 420, which belong to the same protein family variants and do not affect the overall structure and function of the sequence.

[0067] Among them, GC1F includes two polymorphic mutation combinations: rs7041 and rs4588;

[0068] GC1S is mutated from Asp (D) to Glu (E) at position 416 of SEQ ID NO: 1.

[0069] GC2 is mutated from Thr(T) to Lys(K) at position 420 of SEQ ID NO:1.

[0070] The VDBPs mentioned in this invention can all refer to sequences that have the above-mentioned sequences or equivalent sequences that have the same function.

[0071] Low-density lipoprotein receptor-related protein 2 (LRP2, also known as Megalin) is a member of the low-density lipoprotein receptor family, belonging to the large transmembrane endocytic receptor group, and is widely expressed in renal proximal tubular epithelial cells, choroid plexus epithelium, and some neurons. In humans, LRP2 is encoded by the LRP2 gene, and its reference amino acid sequence can be found in the UniProt database, accession number P98164 (LRP2_HUMAN). This protein consists of 4655 amino acids, with a theoretical molecular weight of approximately 517 kDa, and possesses a typical LDL receptor domain, EGF repeat sequence, and β-propeller structure. Currently, only one major splice variant of LRP2 is recorded in the database; no other functional variants have been found. Therefore, this sequence can be used as the sole reference sequence for LRP2. The LRP2 described in this invention may also include conserved homologous sequences with the same function.

[0072] The inventors’ previous work has demonstrated (Microglia-Derived Vitamin D Binding Protein Mediates Synaptic Damage and Induces Depression by Binding to the Neuronal Receptor Megalin, DOI: 10.1002 / advs.202410273) that MG-derived VDBP in the PrL region, the core brain region of the emotional circuit, induces depressive-like behavior in mice by binding to the neuronal receptor megalin and regulating the SRC tyrosine kinase signaling pathway, leading to neuronal apoptosis and synaptic damage. This reveals the VDBP-mediated interaction pathway and mechanism between MG and neurons.

[0073] This invention designs and verifies a short peptide that blocks the binding of VDBP to Megalin, which can be used to prepare drugs for treating depression.

[0074] A seventh aspect of the present invention provides a pharmaceutical composition.

[0075] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients.

[0076] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, and carriers.

[0077] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the drug. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994).

[0078] In some embodiments of the present invention, the dosage form of the drug includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0079] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet;

[0080] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0081] In some embodiments of the present invention, the dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults.

[0082] In some embodiments of the present invention, the drug is administered to mammals;

[0083] In some embodiments of the invention, the mammal includes humans.

[0084] In some embodiments of the present invention, the pharmaceutical composition further includes other medications for treating depression.

[0085] An eighth aspect of the present invention provides a method for in vitro blocking of VDBP binding to Megalin, comprising the following steps:

[0086] Samples were treated with the short peptides or salts thereof described in the first aspect of the invention, the conjugates described in the second aspect of the invention, and the fusion proteins described in the third aspect of the invention.

[0087] In some embodiments of the present invention, the concentration and time of the treatment can be adjusted according to the specific type of the sample. Those skilled in the art can adjust these conditions using conventional methods, and the treatment conditions are not a limitation of the present invention.

[0088] A ninth aspect of the invention provides the application of the VDBP-Megalin binding site in the preparation of a drug screening platform for screening candidate drugs for depression.

[0089] The VDBP binding sites for Megalin include D82 (aspartic acid) and Y410 (tyrosine). These two sites are crucial for VDBP binding to Megalin. Mutations at these sites can significantly weaken the binding ability between the two molecules. Other mutation sites (R218, K207, F133, K427) have less impact on protein binding and may not be directly involved in the binding interface.

[0090] In some embodiments of the present invention, peptide or small molecule drugs can be screened for the above-mentioned binding sites, and peptides or small molecules that can affect the binding site of VDBP and Megalin can be used as candidate drugs to assist in the prediction and development of candidate drugs for depression.

[0091] A tenth aspect of the present invention provides a method for treating depression, comprising the following steps:

[0092] Apply an effective dose of the short peptide or its salt described in the first aspect of the present invention, the conjugate described in the second aspect of the present invention, the fusion protein described in the third aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention to a test subject.

[0093] In some embodiments of the present invention, the test subject is a mammal;

[0094] In some embodiments of the invention, the mammal includes humans.

[0095] In some embodiments of the invention, the effective dose refers to an amount sufficient to cure, alleviate, or partially prevent the clinical manifestations of a given disease and its complications in a therapeutic intervention including the administration of the drug. An amount sufficient to achieve the above is defined as an "effective dose." The effective dose for each purpose will depend on the severity of the disease or lesion and the subject's weight and general condition. However, it should be recognized that the total dosage of the pharmaceutical compositions of the present invention must be determined by the attending physician within the bounds of reliable medical judgment. For any specific patient, the specific therapeutic effective dose level must be determined based on a variety of factors, including the disorder being treated and its severity; the activity of the specific pharmaceutical composition used; the specific pharmaceutical composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific pharmaceutical composition used; the duration of treatment; other drugs used in combination with or concurrently with the pharmaceutical composition used; and similar factors known in the medical field. It is practiced in the art to start the dosage of the pharmaceutical composition below the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Therefore, the specific dosage of the drug is not a limitation of the invention.

[0096] The beneficial effects of this invention are:

[0097] Existing research often relies on overall structure prediction or molecular docking simulation to infer the binding mode of VDBP and Megalin. However, due to the lack of clear definition of specific amino acid sites, problems such as ambiguous binding regions, high non-specific interference, and difficulty in reproducing verification results often exist, leading to low efficiency and high cost in the development of blocking molecules or drugs. This invention, by clearly identifying the key amino acid sequences of the VDBP-Megalin interaction, directly reveals the core molecular basis of their binding. Based on this, blocking small molecules or peptides designed can cleave the binding of VDBP and Megalin in a highly specific manner, avoiding the limitations of random screening and large-molecule antibody intervention in traditional methods. This technical solution not only significantly improves research efficiency, reduces redundant experiments, and lowers research costs, but also provides a unified and reliable standard for subsequent functional verification, ensuring the reproducibility and consistency of research results.

[0098] Based on this binding site, this invention directly designs a short peptide that can block the binding of VDBP to Megalin. This short peptide does not cause neuronal apoptosis or structural damage and can reverse the signaling pathway inhibited by VDBP–Megalin interaction, thus having potential application value in the treatment of depression. Attached Figure Description

[0099] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0100] Figure 1 The results of immunoprecipitation of HA-VDBP and Megalin in the control group, wild-type (WT) and GC mutant groups.

[0101] Figure 2 Results of proximity connectivity analysis (PLA) to detect the interaction between VDBP and Megalin in neurons.

[0102] Figure 3 The results of immunoblotting analysis show the expression levels of HA-VDBP and Megalin in wild-type (WT) and mutant samples.

[0103] Figure 4 The results show the molecular docking predictions for impaired binding of human VDBP to LRP2 after mutations at key sites, including: A) a schematic diagram of the domain distribution of LRP2 and the binding sites of VDBP; B and C) the active conformation of mouse LRP2 (PDB number: 8EM4) and the human LRP2 model simulated based on the mouse dimer structure, with green and purple representing the two subunits respectively; D) the human VDBP dimer structure, with orange and light orange representing the two subunits respectively; and E) the highest-scoring (Top 1) docking conformation of the human VDBP-LRP2 complex predicted using ZDOCK molecular docking software.

[0104] Figure 5 Results of comparison of amino acid sequences of human and mouse VDBP.

[0105] Figure 6 To present the results of peptide drug design based on human VDBP and LRP2 molecular docking analysis, this paper showcases the secondary structures of seven peptides designed with mutated sites, as well as their three-dimensional binding interface structures.

[0106] Figure 7 The results validate the blocking of VDBP–Megalin interaction by the candidate peptide.

[0107] Figure 8 The results show the effects of peptide 3 on neuronal apoptosis and ultrastructure, including A) immunofluorescence staining results; B) statistical results of cell apoptosis ratio; and C) transmission electron microscopy images.

[0108] Figure 9 Results for peptide 3 reversing the inhibition of VDBP–Megalin interaction on the signaling pathway, including: A) representative Western blot results showing changes in key signaling proteins in neurons after treatment with random sequence control peptide, VDBP + random sequence control peptide, or VDBP + peptide 3, respectively; B) quantitative results of relative protein expression levels, with statistical significance: *p<0.05, **p<0.01, ***p<0.001.

[0109] Figure 10 To investigate the effects of peptide 3 on neuronal biomarkers, peptide 3 was shown to alleviate VDBP-induced loss of neuronal synaptic proteins. A) Representative Western blot images showed the expression of synaptic proteins PSD95, Synapsin 1, SNAP25, Vglut1, and VGAT in primary neurons under different treatment conditions: Con + random sequence control peptide, VDBP + random sequence control peptide, and VDBP + peptide 3; β-actin served as a loading control. B–F) Quantitative analysis of the relative protein levels of B) PSD95, C) Synapsin 1, D) SNAP25, E) Vglut1, and F) VGAT was performed using β-actin as a standard. Data are expressed as mean ± standard error (SEM) (n = 3). Compared with the control group, VDBP exposure significantly reduced the levels of PSD95, Synapsin 1, Vglut1, and VGAT, while peptide 3 treatment partially alleviated these reductions. SNAP25 expression did not change significantly among the groups. Statistical analysis: One-way ANOVA followed by Tukey multiple comparison test; *p<0.05, **p<0.01, ns = not significant.

[0110] Figure 11 The results show the effect of peptide 3 on the expression level of MAP2 in neurons.

[0111] Figure 12 The results show the improvement of depressive symptoms by peptide 3 in a mouse model, where A represents the results of the sweet taste preference test, B represents the results of the forced swimming test, C represents the results of the tail suspension test, and D represents the results of the open field test. Detailed Implementation

[0112] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0113] Example 1: VDBP interacts with Megalin

[0114] 1. Experimental Methods

[0115] (1) Immunoprecipitation (IP)

[0116] Objective: To verify whether there is a direct or indirect protein interaction between vitamin D-binding protein (VDBP) and low-density lipoprotein receptor-related protein 2 (LRP2 / Megalin) in cells.

[0117] Experimental materials and reagents:

[0118] Cell line: Hek293 cell line; Antibodies: anti-VDBP antibody (Abcam, ab65636), anti-Megalin antibody (Santa Cruz, sc-515772), IgG isotype control antibody (negative control); Other reagents: immunoprecipitation kit (Thermo Fisher Scientific, Pierce IP Kit), protein lysis buffer (RIPA buffer, containing protease inhibitor), SDS-PAGE and Western blot reagents.

[0119] Experimental steps:

[0120] Protein extraction: Collect cultured cells, lyse with RIPA, incubate on ice for 30 min, then centrifuge (12,000 × g, 15 min, 4℃) and collect the supernatant. Pre-cleaning: Add Protein A / G magnetic beads to 500 μg of total protein, rotate and incubate for 1 h to remove non-specifically bound proteins. Immunoprecipitation: Divide the supernatant into three groups: a. anti-VDBP antibody group; b. anti-Megalin antibody group; c. IgG control group. Each group is incubated with Protein A / G magnetic beads overnight at 4℃. Washing and elution: Wash three times with lysis buffer and collect the immune complexes. Add SDS loading buffer and heat at 95℃ for 5 min to elute. Immunoblot analysis: Transfer to a membrane after SDS-PAGE separation. Primary antibody incubation: Detect the presence of interacting proteins in the precipitate using anti-Megalin or anti-VDBP antibody. Detect by colorimetric detection after secondary antibody incubation.

[0121] Results Interpretation: The detection of a Megalin band in the anti-VDBP antibody immunoprecipitation sample (or the detection of a VDBP band in the anti-Megalin immunoprecipitation sample), while no band was found in the IgG control, indicates an interaction between the two.

[0122] (2) Proximity Ligation Assay (PLA)

[0123] Objective: To further verify whether VDBP and Megalin bind spatially close to each other (<40 nm) at the cellular level.

[0124] Experimental materials and reagents: Cells: primary cortical neurons. Anti-VDBP antibody (rabbit), anti-Megalin antibody (mouse), Duolink® In Situ PLA kit (Sigma-Aldrich).

[0125] Experimental steps:

[0126] Sample preparation: Fixed cultured cells or tissue sections with 4% paraformaldehyde for 15 min, then permeabilized with 0.1% Triton X-100. Antibody incubation: Added anti-VDBP (rabbit) and anti-Megalin (mouse) antibodies, and incubated overnight at 4°C. Probe addition and ligation reaction: Used Duolink® PLA PLUS and MINUS probes to recognize the two antibodies respectively, followed by ligation and rolling circle amplification reactions. Fluorescence detection: Added fluorescently labeled nucleotides (usually a red signal), and stained the nuclei with DAPI. Observed using a confocal microscope.

[0127] 3) Mutant design

[0128] This embodiment designed mutants of different types of VDBP. Therefore, if the substitution of a certain site leads to loss of function or decreased binding, it indicates that the side chain of the original residue is "necessary" for function / binding.

[0129] The specific mutation types are as follows:

[0130] GC1: F415A, K440A, K238A; GC2: Y313A, K419A, T436A; GC3: T414A, N443A, F314A ; GC4: R218A, K207A, F133A; GC5: L200A, N456A, T135A; GC6: K427A, D82A, Y410A.

[0131] Furthermore, the following mutation types were designed for GC4 and GC6:

[0132] GC.4.1: R218A; GC.4.2: K207A; GC.4.3: F133A; GC.6.1: K427A; GC.6.2: D82A; GC.6.3: Y410A.

[0133] 2. Experimental Results

[0134] Immunoprecipitation (IP) test results are as follows Figure 1As shown, the "Input" lane displays the expression levels of HA-VDBP and Megalin in each group, with GAPDH used as an internal reference for sample loading. IP results demonstrate that HA-VDBP and Megalin interact in WT, GC1, GC2, and GC3. This interaction disappears in GC4 and GC6, and weakens in GC5.

[0135] Proximity Linkage Analysis (PLA) Results Figure 2 As shown, wild-type (WT) neurons exhibit strong PLA signaling (red), suggesting significant interaction between VDBP and Megalin, and co-localization with MAP2-positive dendrites (green). In contrast, PLA signaling in GC4 neurons is significantly reduced, while GC5 and GC6 neurons retain moderate levels of interaction. DAPI staining (blue) labels the nuclei, and the merged image shows spatial overlap of MAP2, PLA, and nuclear signals.

[0136] Based on the above results, it can be inferred that the mutation sites in GC4 and GC6 are crucial to the interaction between VDBP and Megalin. Therefore, this embodiment performs more detailed mutations on GC4 and GC6 (including GC.4.1, GC.4.2, GC.4.3, GC.6.1, GC.6.2, and GC.6.3).

[0137] Mutation result analysis such as Figure 3 As shown, HA-VDBP and GAPDH were detected in the "Input" samples (including GC.4.1, GC.4.2, GC.4.3, GC.6.1, GC.6.2, and GC.6.3). GAPDH was used as an internal control to ensure consistent protein loading across samples. Immunoprecipitation (IP) samples showed specific bands for HA-VDBP and Megalin after immunoprecipitation with Megalin antibody. The area marked by the red box represents the detection results of Megalin protein, indicating that the target protein was successfully immunoprecipitated and detected in the IP samples.

[0138] Based on the above results, it can be concluded that samples GC.6.2 and GC.6.3 lost the interaction between VDBP and Megalin. Therefore, D82 and Y410 are key sites on the VDBP protein that interact with the Megalin protein.

[0139] Example 2 Construction of the Megalin–VDBP complex model

[0140] Example 1 analyzed the key binding sites of human VDBP protein with Megalin, but it is still unclear how the key sites of human VDBP protein interact with Megalin protein. Therefore, this example conducted a simulated docking analysis of the specific amino acid binding sites of VDBP and Megalin.

[0141] 1. Homology modeling technique

[0142] 1.1 Protein Download and Processing

[0143] Based on the PDB structure storage URL https: / / www.rcsb.org / , enter PDB ID: 8EM4 or protein name LRP2 to obtain the megalin protein structure; enter PDB ID: 1KW2 or Vitamin D-binding protein to obtain the VDBP protein structure. Both megalin and VDBP proteins are dimers. The megalin protein contains four chains. Chains A and B contain residues from positions 28 to 4413, with residues 104-219, 298-304, 1272-1349, 2779-3033, and 3881-3992 missing. Each chain contains 29 NAG (2-acetamido-2-deoxy-beta-D-glucopyranose) molecules, 16 NGA (2-acetamido-2-deoxy-beta-D-galactopyranose) molecules, and 42 calcium ions. Chains C and D each contain two NAG molecules; only the calcium-bound protein is retained for docking. The VDBP protein contains water molecules, which are removed for docking.

[0144] 1.2 Introduction to Modeling Software (SWISS-MODEL)

[0145] SWISS-MODEL is an online protein homology modeling server developed and maintained by the Swiss Institute of Bioinformatics (SIB). It predicts the three-dimensional structure of unknown proteins based on templates of known protein structures.

[0146] 1.3 The Process of Protein Modeling

[0147] 1.3.1 Modeling of megalin protein

[0148] The megalin protein structure analysis revealed four major deletions (104-219, 1272-1349, 2779-3033, 3881-3992). Based on these four major deletions, the monomeric protein was divided into five parts: (28-103, 220-1271, 1350-2778, 3034-3880, 3993-4413). Using the Modeling > User Template mode on the SWISS-MODEL website, the human megalin protein structure was generated through homology / comparison modeling based on the segmented mouse megalin protein structure (8EM4) and the human megalin protein sequence (the provided sequence covers the length of each protein structure segment, extended by 20-50 amino acids). Subsequently, based on the mouse megalin dimer structure, the complete calcium-bearing human megalin dimer protein was assembled using PyMOL's align, copy to object, and create functions.

[0149] 1.3.2 Modeling of VDBP Protein

[0150] Using the Modeling > User Template mode on the SWISS-MODEL website, mouse VDBP monomer structures and human VDBP monomer structures (without SNPs) were generated through homology / comparison modeling based on the human VDBP protein structure (1KW2), mouse VDBP sequences, and human VDBP sequences (without SNPs). The complete mouse and human (without SNPs) VDBP dimer proteins were obtained by using PyMOL's align, copy to object, and create functions.

[0151] 2. Protein docking, clustering, and mutation site selection

[0152] 2.1 ZDOCK Integration Software

[0153] This embodiment uses ZDOCK software to simulate the potential interaction modes between free-state monomers of megalin and VDBP proteins, generating a large number of predicted conformations. The entire docking process is mainly divided into two parts: preprocessing and formal docking. The preprocessing part includes modifying and cleaning the pre-docked protein structure information, defining protein atom types according to the force field, etc. The formal docking includes calling the ZDOCK program to run the docking command, specifying the output path, etc. During the formal docking process, docking parameters can be set according to docking requirements. The spatial sampling Euler angle density can be adjusted to 15° or 6°, and the software will generate 3600 or 54000 potential docking conformations during the sampling phase. During the docking process, a fixed protein monomer can be specified (the receptor protein is fixed by default), and the number of output conformations can be specified, etc.

[0154] 2.2 Specific Process of Docking and Clustering

[0155] Preprocessing: The calcium-containing megalin protein and VDBP protein PDB files are combined according to the atom types defined in the force field file uniCHARMM. The commands mark_sur receptor.pdb receptor_m.pdb and mark_sur ligand.pdb ligand_m.pdb are used to generate the protein files receptor_m.pdb and ligand_m.pdb with defined force fields.

[0156] Formal docking section: The molecular docking process is executed using the executable file ZDOCK with the command `zdock -R receptor_m.pdb -L ligand_m.pdb -o zdock.out`. `-R` specifies the receptor file after defining the force field, `-L` specifies the ligand file after defining the force field, and `-o` specifies the output file name of the ZDOCK program. The file contains information such as the initial and rotated Euler angle parameters of the receptor and ligand, the number of translation grid points, and the ZDOCK score. An output file containing space configuration information for 2000 predicted conformations is generated according to the default settings of the ZDOCK program (15°, fixed receptor). Then, `create.pl` is used to generate 2000 complex structures based on the output file. (When using `mark_sur`, note that `uniCHARMM` must be in the same directory to define the force field information. When using ZDOCK, `receptor_m.pdb` and `ligand_m.pdb` must be in the same directory to provide the complexes for docking prediction.)

[0157] Clustering: Using the `create_complex_view.pdb` file (receptor-ligand non-contact state) generated by the `create_complex_view` function in `show_ZDOCK_pose.py` as the initial file, two thousand complex conformations were imported using the `view_decoys.tcl` script. In the VMD software, trajectory files for 2001 conformations were generated (the first file (number 0) is `complex_view.pdb`). Subsequently, using the commands `set sel [atomselect 0 "chain CD andbackbone"]` and `measure cluster $sel num 20 distfunc rmsd cutoff 50.0 first 1 last -1 step 1` in VMD, the two thousand predicted conformations were clustered into 20 clusters based on the K-means clustering method, using a 50 Å cutoff value for the protein backbone. The cluster centers and the highest-scoring conformations were then determined based on the clustering results.

[0158] 2.3 Protein docking and selection of mutation sites

[0159] For the highest-scoring conformation in each cluster, conformations that match the potential binding region of VDBP were selected. Detailed interaction analysis was performed using the show_ZDOCK_pose.py script. Six groups of residue mutation combinations, each containing three sites, were designed for human-derived clusters 1 (80 conformations), 3 (120 conformations), and 7 (123 conformations) based on pi-pi interactions, cation-pi interactions, salt bridges, hydrogen bonds, and hydrophobic interactions. Seven groups of residue mutation combinations, each containing three sites, were designed for mouse-derived clusters 1 (89 conformations) and 5 (138 conformations). Further experimental mutations of the VDBP protein binding site were then conducted to explore its binding effectiveness.

[0160] 3. Predictive conformation screening based on experimental data

[0161] Based on human VDBP protein site mutation experiments, it was found that key residues for megalin protein to bind to VDBP protein exist in cluster 1 (80 conformations) combination (D / LYS / 427, D / ASP / 82, D / TYR / 410) and cluster 3 (120 conformations) combination (D / ARG / 218, D / LYS / 207, D / PHE / 133). Based on the above residues, the screening work for predicting conformations was carried out.

[0162] The script `show_ZDOCK_pose.py` generates detailed interaction information files for all conformations or conformations within specific clusters. By filtering key residue information on the ligands and sorting the conformations according to the number of key residue types, the number of interactions, and the ZDOCK score, corresponding files `_filtered_results.txt` (containing conformation information sorted by key residues) and `_keyword_statistics.txt` (containing the probability of each key residue appearing in all or cluster conformations) are obtained. Among the key residues screened across all conformations, the top five conformations are conformations 1, 366, 69, 351, and 355; their key residue types are 6, 5, 4, 4, and 4 respectively; and the number of key residue interactions is 11 for each.

[0163] 4. Experimental Results

[0164] The results of the ZDOCK scoring function are shown in Table 1.

[0165] Table 1. Representative docking conformations of human VDBP with human megalin (top 10 conformations clustered)

[0166]

[0167] The docking conformations were ranked using the ZDOCK scoring function. Subsequently, the K-means algorithm was used to cluster the 2000 docking conformations output by ZDOCK based on the root mean square deviation (RMSD, cutoff value 50 Å) of the docking ligand (VDBP) structure. The table shows the top 10 clusters (out of a total of 20). The last five columns of the table list the non-covalent molecular interactions between the VDBP and megalin structure as represented by each conformation in the first column.

[0168] Predictions of impaired molecular docking between human VDBP and LRP2 following mutations at key sites are as follows: Figure 4 As shown. Figure 4 Figure A shows the domain distribution of LRP2 and the binding site of VDBP; Figures B and C show the active conformation of mouse LRP2 (PDB number: 8EM4) and the human LRP2 model obtained by simulation based on the mouse dimer structure, with green and purple representing the two subunits respectively; Figure D shows the human VDBP dimer structure, with orange and light orange representing the two subunits respectively; Figure E shows the highest score (Top 1) docking conformation of the human VDBP-LRP2 complex predicted by ZDOCK molecular docking software.

[0169] The results of the comparison of amino acid sequences of human and mouse VDBP are as follows: Figure 5 As shown, human VDBP (sequence length: 474) and mouse VDBP (sequence length: 476) share 77.7% sequence identity.

[0170] Example 3: Design of candidate short peptides that can block the binding of VDBP to Megalin

[0171] Based on the simulation docking results of Example 2, this example designed 7 short peptides that may block the binding of VDBP to Megalin, namely:

[0172] SZ_P01: DPDCYDTRT (SEQ ID NO: 2);

[0173] SZ_P02: AEGADPDCYDTRT (SEQ ID NO: 3);

[0174] SZ_P03: ADYSENTFTEYKKKL (SEQ ID NO: 4);

[0175] SZ_P04:ADYSENTFTEYKKKLAERLKAKLPDATPTELAKLVNKH (SEQ ID NO: 5);

[0176] SZ_P05:ADYSENTFTEYKKKLAERLKAKLPDATPTELAKLVNKHSDFASNCCSINSPP LYC (SEQID NO: 6);

[0177] SZ_P06: ATPTELAKLVNKHSDFASNCCSINSPPLYC (SEQ ID NO: 7);

[0178] SZ_P07: PQEFPTYVEP (SEQ ID NO: 8).

[0179] The secondary structure of the aforementioned short peptides, and the simulation of their three-dimensional binding interface structure, are as follows: Figure 6 As shown.

[0180] Example 4: Validation of candidate short peptides that block the binding of VDBP to Megalin

[0181] 1. Experimental Methods

[0182] The seven candidate short peptides from Example 3 were tested at treatment concentrations of 0.1 nM, 1 nM, and 10 nM.

[0183] To verify whether the seven candidate short peptides screened can effectively block the binding of vitamin D-binding protein (VDBP) to low-density lipoprotein receptor-associated protein 2 (Megalin), a co-immunoprecipitation (Co-IP) assay was performed. The experimental steps are as follows:

[0184] Seven candidate short peptides, synthesized and purified chemically, were prepared into 1 μM stock solutions using sterile deionized water or PBS before the experiment. These solutions were further diluted to working concentrations of 0.1 nM, 1 nM, and 10 nM before use. Recombinant human VDBP protein (purity >95%) and recombinant Megalin-Fc fusion protein (purity >90%) were prepared at a final concentration of 100 nM in binding buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 2 mM CaCl2, 0.05% Tween-20].

[0185] First, VDBP and each candidate short peptide were gently incubated at 4°C for 30 min by rotation to ensure sufficient binding of the short peptides to VDBP. Then, an equimolar concentration (100 nM) of Megalin protein (500 μL) was added to the reaction system, and incubation was continued at 4°C for 1 h by rotation to promote the formation of a VDBP-Megalin complex. The control group included a negative control group without the short peptide and a specific control group with an equal concentration of a random sequence control peptide (scrambled peptide, YDYKESTENTKLAFK, SEQ ID NO: 9). After incubation, approximately 20 μL of Protein A / G magnetic beads pre-bound with anti-Megalin antibody was added, and the mixture was gently rotated at 4°C for 1 h to capture the Megalin-Fc complex. The mixture was then washed three times with binding buffer (500 μL each time) and then washed once more with a high-salt buffer containing 300 mM NaCl to remove non-specific binding. After washing, the sample was eluted with 2× SDS loading buffer containing reducing agent, heated at 95°C for 5 min, and the supernatant was collected for SDS-PAGE electrophoresis and Western blotting. The signal intensity of the co-precipitated VDBP was detected with anti-VDBP antibody, and the precipitation efficiency was verified with anti-Megalin antibody.

[0186] 2. Experimental Results

[0187] The results were obtained by analyzing the VDBP band intensities of different short peptides and different concentration groups. Figure 7 As shown, the short peptide 3 significantly reduced the co-precipitation of VDBP and Megalin at concentrations of 1 nM and 10 nM, with the binding ratio decreasing by more than 50% compared to the negative control group, suggesting that the short peptide can specifically block the interaction between VDBP and Megalin.

[0188] In summary, some of the candidate short peptides (hereinafter referred to as peptide 3) have significant blocking effects and can serve as VDBP–Megalin interaction inhibitors, providing a candidate molecular basis for the development of drugs to intervene in VDBP-mediated signaling pathways.

[0189] Example 5: SZ_P03 has no neurotoxicity.

[0190] 1. Experimental Methods

[0191] TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining methodology (for neuronal apoptosis detection). In this example, the experiment was conducted using Thermo Fisher Click-iT TUNEL Alexa Fluor Imaging Assay, and the relevant procedures were the same as those in the reagent instructions.

[0192] Other materials: Fixative: 4% paraformaldehyde (PFA) or 10% neutral buffered formalin. Proteinase K (20 μg / mL). Osmosis buffer: 0.1% Triton X-100 in PBS. DAPI (4',6-diamidinyl-2-phenylindole, 1 μg / mL). Neuron-labeling antibody (anti-NeuN, diluted 1:500). Positive control: DNase I (treatment of samples produces DNA breaks). Negative control: TdT-free samples.

[0193] Sample preparation steps: Fix cultured neurons or brain tissue slices in 4% PFA at room temperature for 4-10 minutes (optimize the time to preserve cell morphology and avoid over-fixation that could weaken the signal). Wash three times with PBS.

[0194] Osmosis and protease digestion: Treat with 0.1% Triton X-100 in PBS for 10 minutes to promote reagent osmosis. Add proteinase K (20 μg / mL) and digest at 37°C for 5-15 minutes (optimize to enhance the signal, but avoid over-digestion to prevent false positives).

[0195] Blocking: Non-specific sites were blocked with PBS containing 5% BSA at room temperature for 30 minutes.

[0196] Neuron labeling: Incubate with anti-NeuN antibody (4°C overnight), followed by incubation with fluorescently labeled secondary antibody (Alexa Fluor594) for 1 hour at room temperature in the dark.

[0197] TUNEL reaction: Following the kit instructions, incubate the sample in the TdT reaction mixture (TdT enzyme + fluorescent dUTP) at 37°C for 60 minutes. The TdT enzyme catalyzes the attachment of fluorescent dUTP to the DNA break ends, achieving green fluorescent labeling (excitation / emission: 495 / 519 nm). Stain and mount using standard immunofluorescence assays.

[0198] Quantitative analysis: The proportion of TUNEL-positive neurons (TUNEL+ / NeuN+ cells) was calculated using ImageJ software. A threshold was set to exclude background fluorescence. At least three independent experiments were counted, with n≥3 for each group.

[0199] Electron microscopy experimental methodology (for neuronal apoptosis detection): Transmission electron microscopy (TEM) is used to observe the ultrastructural features of apoptosis, nuclear chromatin condensation, cytoplasmic shrinkage, apoptotic body formation, and mitochondrial changes.

[0200] Materials used: Fixative: 2.5% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.4). Post-fixative: 1% osmium tetroxide. Dehydrating agents: ethanol gradient (30%-100%). Embedding agent: epoxy resin (Epon 812). Staining agents: urethane acetate and lead citrate. Ultramicrotome, TEM microscope (Hitachi H-7650). Positive control: neuronal samples known to have induced apoptosis (treated with dexamethasone). Experimental tests were performed using standard electron microscopy sample preparation methods.

[0201] TEM observation: Observation was performed at an accelerating voltage of 80-100 kV. At least 200 neurons were scanned to record apoptotic features (intact nuclear membrane, condensed chromatin, and vesicled organelles).

[0202] Image analysis: The apoptosis index (apoptotic cells / total cell count) was quantified using DigitalMicrograph software. 3D reconstruction (in conjunction with SBF-SEM) is optional for observing mitochondrial division.

[0203] 2. Experimental Results

[0204] The effects of peptide 3 (SZ_PO3) on neuronal apoptosis and ultrastructure are as follows: Figure 8 As shown. No significant increase in TUNEL-positive cells was observed in the control group, the random sequence control peptide group, or the peptide 3 treatment group. Figure 8 (A). Quantitative analysis of the apoptosis rate (%) showed no significant difference among the groups. Figure 8 Transmission electron microscopy images showed that the ultrastructure of neurons treated with peptide 3 was intact, comparable to that of the random sequence control peptide group and the control group, suggesting that peptide 3 did not induce neuronal apoptosis or structural damage. Figure 8 (C)

[0205] Example 6: VDBP peptide 3 reverses the signaling pathway inhibited by VDBP–Megalin interaction.

[0206] 1. Experimental Methods

[0207] Cell lines: Primary neurons, Neurobasal-A medium, supplemented with 2% B-27 rehydration solution, 0.5 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0208] Peptide treatment: VDBP peptide 3, purity >95%; VDBP + random sequence control peptide (a mixed control of VDBP protein and random sequence peptide, the sequence being a randomly shuffled VDBP peptide 3 sequence, used as a negative control); Scramble peptide (random sequence control peptide, biologically inactive, YDYKESTENTKLAFK). All peptides were dissolved in sterile PBS at a concentration of 10 μM.

[0209] Antibodies: Primary antibodies included antiphosphorylated SRC (p-SRC, Cat. No. Cell Signaling #6943), antiphosphorylated AKT (p-AKT, Cat. No. [#4060]), antiphosphorylated ERK1 / 2 (p-ERK1 / 2, Cat. No. [#4370]), antiphosphorylated CREB (p-CREB, Cat. No. [#9198]), anti-BCL2 (Cat. No. [#2876]), and anti-BAX (Cat. No. [#2772]); secondary antibodies were HRP-conjugated goat anti-rabbit IgG (Cat. No. [#7074]). A GAPDH antibody (Cat. No. [#2118]) was used as an internal control. All antibodies were diluted 1:1000 (primary antibody) and 1:5000 (secondary antibody).

[0210] The proteins detected include:

[0211] p-SRC: Phosphorylated SRC kinase (Src family kinase). SRC is a non-receptor tyrosine kinase, and its phosphorylated activated form (p-SRC) is involved in cell adhesion, migration, and proliferation.

[0212] p-AKT: Phosphorylated AKT (also known as PKB, protein kinase B). AKT is an effector of the PI3K / AKT signaling pathway. Phosphorylation promotes cell survival and anti-apoptosis.

[0213] p-ERK1 / 2: Phosphorylated extracellular signal-regulated kinase 1 / 2 (ERK1 / 2), a downstream pathway of the MAPK pathway, regulates cell division and differentiation upon phosphorylation activation.

[0214] p-CREB: Phosphorylated cAMP response element-binding protein (CREB), a transcription factor. Phosphorylation activates gene transcription and promotes the expression of genes related to cell survival.

[0215] BCL2: B-cell lymphoma 2 protein, an anti-apoptotic protein that maintains mitochondrial membrane integrity and inhibits programmed cell death.

[0216] BAX: BCL2-associated X protein, a pro-apoptotic protein that promotes the permeability of the outer mitochondrial membrane, leading to apoptosis.

[0217] Other reagents: RIPA lysis buffer (containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, a mixture of protease inhibitors and phosphatase inhibitors); BCA protein quantification kit; 10-12% SDS-PAGE precast gel; transfer buffer (25mM Tris, 192mM glycine, 20% methanol); ECL chemiluminescence kit; ImageJ software for density analysis.

[0218] Experimental procedure: Cell treatment: Seed cells in logarithmic growth phase into 6-well plates (density 5 × 10⁶). 5 Cells were cultured at 24 h until 70-80% confluence. Cells were starved for 12 h (using serum-free DMEM), followed by the addition of VDBP peptide 3 (experimental group, concentration 10 μM) and VDBP + random sequence control peptide (control group 1) and incubated at 37 °C for 1-2 h. Each treatment was performed in triplicate.

[0219] After protein extraction, immunoblotting experiments were performed.

[0220] Data analysis: ImageJ software was used to quantify the gray values ​​of the bands and calculate the relative expression levels of the target protein (normalized with GAPDH, with the control group random sequence peptide set to 1). GraphPad Prism software was used to generate bar charts, and t-tests were performed for statistical analysis (p < 0.05 was considered statistically significant). Results are presented as mean ± standard deviation (n = 3).

[0221] 2. Experimental Results

[0222] Representative immunoblotting results as follows Figure 9 As shown in Figure A, the statistical results are as follows: Figure 9 As shown in Figure B, peptide 3 treatment significantly reduced the expression levels of p-SRC, p-AKT, p-ERK1 / 2, p-CREB, BCL2, and BAX, indicating that VDBP peptide 3 can specifically inhibit the activation of downstream signaling pathways.

[0223] Example 7: Effects of peptide 3 on mature neurons

[0224] 1. Experimental Methods

[0225] Cell Culture and Processing: Primary cortical neurons were isolated from C57BL / 6 mouse embryos at day 18 (E18) and cultured in Neurobasal medium supplemented with B27 and GlutaMAX (Thermo Fisher Scientific) at 37°C in a humidified incubator with 5% CO2. Neurons were maintained in vitro until day 14 (DIV14) to ensure the formation of mature synapses before treatment. Cells were divided into three experimental groups (n = 3 biological replicates per group): (1) control group + random sequence control peptide (vector + 10 nM random sequence control peptide), (2) VDBP + random sequence control peptide (200 ng·mL -1 (3) Recombinant VDBP + 10 nM random sequence control peptide), and (4) VDBP + peptide 3 (200 ng·mL) -1 Recombinant VDBP + 10 nM peptide 3). Recombinant human VDBP (R&D Systems) was prepared as a 1000× stock solution in sterile PBS. Peptide 3 and a random sequence control peptide (GenScript) were dissolved in sterile water to prepare a 100× stock solution, which was then diluted in culture medium to a final concentration of 10 nM (final DMSO ≤ 0.1% where applicable). For treatment, neurons were pre-incubated with either peptide 3 or the random sequence control peptide (10 nM) at 37°C for 1 hour. Subsequently, recombinant VDBP (200 ng / mL) was added. -1Cells were added to the designated groups with either a carrier (PBS) or a buffer, and then co-incubated at 37°C in 5% CO2 for 12 hours. After treatment, cells were harvested for downstream analysis. For protein extraction and Western blot analysis, cells were lysed in RIPA buffer (Sigma-Aldrich) containing protease and phosphatase inhibitors (Roche). Protein concentrations were determined using the BCA assay (Pierce). An equal volume of protein (20 µg) was separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad). The membrane was probed using antibodies against PSD95 (1:1000, Abcam), Synapsin1 (1:1000, Cell Signaling Technology), SNAP25 (1:1000, Sigma-Aldrich), Vglut1 (1:500, Synaptic Systems), VGAT (1:500, Synaptic Systems), and β-actin (1:5000, Sigma-Aldrich) as loading controls. Bands were visualized using enhanced chemiluminescence (ECL, ThermoFisher Scientific), and quantitative analysis was performed using ImageJ software. Technical replicates were performed for each sample. Statistical analysis data are presented as mean ± SEM from three independent experiments. Statistical significance was determined using one-way ANOVA, followed by Tukey post-hoc tests for multiple comparisons (GraphPad Prism 9). A p-value <0.05 was considered statistically significant.

[0226] 2. Experimental Results

[0227] After treating neurons with peptide 3, the expression of neuronal-related markers was detected, and the results are as follows: Figure 10 As shown, A represents the results of Western blot analysis, and B-F represent the statistical results.

[0228] The results showed that VDBP exposure significantly altered the expression of synaptic proteins in primary neurons. Specifically, compared with the control group, the levels of the postsynaptic marker PSD95 and the presynaptic vesicle protein Synapsin 1 were significantly reduced in the VDBP + random sequence control peptide group, indicating impaired synaptic structure. The SNARE complex component SNAP25 showed no significant changes among the groups, indicating that the vesicle fusion mechanism remained largely intact. Conversely, Vglut1 (a marker of excitatory glutamatergic termination) and VGAT (a marker of inhibitory GABAergic termination) were significantly upregulated after VDBP treatment, reflecting a disruption of the excitatory-inhibitory synaptic balance. Importantly, administration of peptide 3 effectively reversed these changes, partially restoring the expression of PSD95 and Synapsin 1, and returning Vglut1 and VGAT levels to normal. These findings suggest that peptide 3 can alleviate VDBP-induced synaptic dysfunction and contribute to maintaining neuronal homeostasis.

[0229] Example 8: Effects of peptide 3 on MAP2 expression and cell-cell interactions

[0230] This embodiment evaluated the effects of peptide 3 on neuronal differentiation and synaptic network formation, selecting MAP2 as the detection indicator. MAP2 is a neuron-specific microtubule-binding protein, mainly located in dendrites and the cell body, stabilizing microtubule structure and promoting neuronal differentiation and dendritic growth. MAP2 regulates cytoskeleton dynamics through interaction with microtubules and also participates in axonal transport and synapse formation.

[0231] 1. Experimental Methods

[0232] Cell origin: primary cortical neurons

[0233] Culture medium: Neurobasal-A medium supplemented with 2% B-27 rehydration solution, 0.5 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin, cultured at 37℃ and 5% CO2.

[0234] Peptide treatment: VDBP peptide 3 (purity >95%, prepared by solid-phase synthesis); VDBP + random sequence control peptide (mixed control of VDBP protein and random sequence peptide); random sequence control peptide (YDYKESTENTKLAFK). All peptides were dissolved in sterile PBS at a concentration of 10 μM.

[0235] Antibodies: Anti-MAP2 monoclonal antibody (mouse-derived, Cat. No. Abcam ab11267, dilution 1:500); DAPI (4', 6-diamidinyl-2-phenylindole, nuclear dye, 1 μg / mL); Alexa Fluor 488-labeled goat anti-mouse secondary antibody (Cat. No. Invitrogen A-11001, dilution 1:1000).

[0236] Experimental Procedure: Cell Culture and Treatment: Primary neurons were isolated according to standard protocols (refer to the aforementioned neuron culture conditions) and seeded on coverslips pre-coated with poly-D-lysine (density approximately 5 × 10⁻⁶). 4 cells / cm 2 ).

[0237] Culture for 3-7 days until neuronal differentiation occurs, then add 5 μM cytarabine to inhibit glial cell proliferation.

[0238] On day 4 of culture, cells were treated with a random sequence control peptide (control group), VDBP + random sequence control peptide (mixed control group), and VDBP peptide 3 (experimental group) at a concentration of 10 μM and incubated at 37°C for 24 hours. Triple replicates were set up.

[0239] Collect samples for immunofluorescence experiments.

[0240] Imaging and Analysis: Observe under a fluorescence microscope (488 nm to excite the green MAP2 signal, 405 nm to excite the blue DAPI signal), and acquire multiple fields of view (at least 5 per group).

[0241] ImageJ software was used to quantify the length of MAP2-positive neurons and the number of crossovers. Crossover was defined as the intersection point between MAP2-labeled neurons. The mean number of crossovers (n=50 neurons / group) was calculated, and a line graph was plotted. The differences were analyzed using a t-test (p<0.05 was considered significant).

[0242] 2. Experimental Results

[0243] The results are as follows Figure 11 As shown, the MAP2 expression level and synaptic interleaving number in the VDBP peptide 3 group were significantly higher than those in the random sequence control peptide group and the VDBP + random sequence control peptide group, indicating that VDBP peptide 3 promotes neuronal differentiation and synaptic network formation. This method can be used to evaluate the application of VDBP-derived peptides in neuroprotection or repair.

[0244] Example 9: Efficacy test of peptide 3 in treating depression in animal models

[0245] 1. Experimental Methods

[0246] 1) Sweetness Preference Test (SPT)

[0247] First, allow the mice to freely access two bottles of water: one with tap water and the other with a sugar solution of a certain concentration (e.g., 1–2% sucrose) for two days. Place the two bottles side by side and record the intake from each bottle over a certain period of time, usually 24 or 48 hours.

[0248] Calculate the sugar water preference rate: P = sugar water intake / (sugar water intake + water intake) × 100%.

[0249] Compared to the control group, a decrease in preference indicates enhanced depressive-like behavior; an increase in preference indicates improved mood or no depressive-like manifestations.

[0250] 2) Forced Swim Test (FST)

[0251] When mice are placed in water, their initial active escape behaviors gradually decrease, and they exhibit restlessness. The longer the restlessness, the more it reflects the negative emotions and helplessness of the depressive-like sample.

[0252] Pre-training phase: Observe and record behavior upon initial placement in water, typically divided into two phases: early escape behavior and later rigidity. Testing usually lasts 6–15 minutes, recording the total immobility time. Perform an FST test after drug administration to assess efficacy. Compared to the control group, prolonged immobility time suggests significant depressive-like behavior, while shortened immobility time suggests an antidepressant effect.

[0253] 3) Tail Suspension Test (TST)

[0254] The mouse's tail is fixed, and it is suspended head down. When the mouse stops struggling, its body remains vertically upside down and motionless. This indicator can be seen as the animal's behavior of giving up struggling after experiencing learned helplessness. The longer the animal remains still in the tail suspension experiment, the more severe its depression.

[0255] 4) Open Field Test (OFT)

[0256] Mice were placed in an open, typically square, area, and their movement and behavior in the new environment were recorded. More centering time indicated lower anxiety or higher exploratory behavior; less centering time may suggest higher anxiety.

[0257] 5) Experimental Grouping

[0258] The experiment was divided into four groups: negative control group, VDBP overexpressing mice (OE-VDBP), VDBP overexpressing mouse peptide 3 treatment group, and VDBP overexpressing mouse random sequence control peptide treatment group.

[0259] In the OE-VDBP treatment, AAV virus was injected into the nucleus accumbens region of the mouse brain, causing overexpression of VDBP protein in inhibitory neurons of the nucleus accumbens region, resulting in depressive-like symptoms. Following this, the peptide 3 and random sequence control peptide treatment groups received intraperitoneal injections of either peptide 3 or random sequence control peptide at a dose of 1 mg / kg once daily for one week, after which the mouse phenotype was observed.

[0260] 2. Experimental Results

[0261] Experimental results are as follows Figure 12 As shown, treatment with peptide 3 in depressed mice effectively alleviated depressive-like behaviors.

[0262] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1.A short peptide or a salt thereof, characterized in that: an amino acid sequence of the short peptide is ADYSENTFTEYKKKL. 2.A conjugate, characterized in that: the conjugate comprises the short peptide or the salt thereof according to claim 1, and a modification moiety; the modification moiety comprises at least one of a chemical modification, a fluorescent dye. 3.A fusion protein, characterized in that: the fusion protein comprises the short peptide according to claim 1, and a coupling moiety; the coupling moiety comprises at least one of a drug, a cytokine, a radionuclide, a fluorescent protein, a tag protein. 4.Biological materials related to the short peptide or the salt thereof according to claim 1 and the fusion protein according to claim 3, comprising any one of b1) to b12): b1) a nucleic acid molecule encoding the short peptide according to claim 1 or the fusion protein according to claim 3; b2) an expression cassette comprising the nucleic acid molecule according to b1); b3) a vector comprising the nucleic acid molecule according to b1); b4) a vector comprising the expression cassette according to b2); b5) a transgenic cell line comprising the nucleic acid molecule according to b1); b6) a transgenic cell line comprising the expression cassette according to b2); b7) a transgenic cell line comprising the vector according to b3); b8) a transgenic cell line comprising the vector according to b4); b9) a microorganism comprising the nucleic acid molecule according to b1); b10) a microorganism comprising the expression cassette according to b2); b11) a microorganism comprising the vector according to b3); b12) a microorganism comprising the vector according to b4). 5.A method for preparing the short peptide or the salt thereof according to claim 1, the conjugate according to claim 2, or the fusion protein according to claim 3, comprising the following steps: obtained by using the biological materials according to claim 4; or, obtained by a method of chemical synthesis of polypeptides. 6.The short peptide or the salt thereof according to claim 1, the conjugate according to claim 2, or the fusion protein according to claim 3 for use in the preparation of a medicament for treating depression. 7.A pharmaceutical composition, characterized in that: the pharmaceutical composition comprises the short peptide or the salt thereof according to claim 1, the conjugate according to claim 2, or the fusion protein according to claim 3. 8.The pharmaceutical composition according to claim 7, characterized in that: the pharmaceutical composition comprises a pharmaceutically acceptable excipient. ​

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