Polypeptide and use thereof in treatment of nervous system diseases

Abstract

Provided is a polypeptide which contains a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity to amino acid sequence MTYRPGYNPFG (SEQ ID NO: 41) or amino acid sequence AKGYYRPYGVPV (SEQ ID NO: 22), or a polypeptide having one or more amino acid substitutions, deletions and / or additions relative to the amino acid sequence as shown in SEQ ID NO: 41 or 22. The provided polypeptide can interfere with the binding of a Brag2 synaptic protein to the C terminus of an AMPA receptor, thereby inhibiting NMDA-mediated excessive endocytosis of an AMPA receptor and neuronal apoptosis, and playing a role in protecting neuronal cell activity. Therefore, the polypeptide has application prospects and potential development values as a drug for treating nervous system diseases such as strokes, depression, Alzheimer's disease and drug addiction.

Description

A polypeptide and its application in the treatment of nervous system diseases Technical Field

[0001] This disclosure relates to the field of biomedicine, specifically to a polypeptide and its application in the treatment of nervous system diseases. Background Technology

[0002] AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors mediate rapid excitatory synaptic transmission in the central nervous system. Their dynamic expression on the postsynaptic membrane is related to the induction and maintenance of long-term potentiation (LTP) and long-term depression (LTD), and they participate in regulating learning and memory activities.

[0003] Under normal circumstances, the internalization and exoaplasmation of AMPA receptors on the postsynaptic membrane are balanced, ensuring a stable number of receptors on the postsynaptic membrane and maintaining normal nerve signal transmission. In some pathological conditions, overactivation of excitatory glutamate receptors leads to a sustained greater number of AMPA receptors internalized than on the exoaplasm, resulting in a reduction of postsynaptic AMPA receptors. This triggers long-term depression (LTD) of synaptic transmission, ultimately leading to neuronal death. Further research has revealed that this process is related to a specific amino acid sequence (A867-G877) containing multiple tyrosine residues at the C-terminus of the AMPA receptor GluA2 subtype. This amino acid sequence further activates guanosine triphosphatase Arf6 through interaction with Brag2 synaptic protein. Arf6 not only plays a role in actin membrane remodeling but also directly affects clathrin-mediated synaptic vesicle formation, thereby leading to the internalization of postsynaptic AMPA receptors. Throughout the regulatory process, the interaction between the C-terminus of the AMPA receptor and the Brag2 synaptic protein plays a crucial role. By interfering with their interaction, excessive endocytosis of the postsynaptic membrane receptor is greatly reduced, thereby reducing the formation of LTD (Wang Y, J Biol Chem. 2004; 279(40): 41267-41270.).

[0004] It has been confirmed that many neurological diseases (such as stroke, Alzheimer's disease, major depressive disorder, and drug addiction) are closely related to the rapid excitatory synaptic transmission process mediated by AMPA receptors in the central nervous system. Therefore, developing a peptide drug molecule that interferes with the binding of the C-terminus of the AMPA receptor to the Brag2 synaptic protein, and subsequently treating or alleviating these diseases by inhibiting NMDA-mediated excessive internalization of AMPA receptors and regulating neuronal apoptosis, has become a major technical challenge in the development of drugs for neurological diseases.

[0005] Summary of the Invention

[0006] To address the aforementioned technical issues, this disclosure uses the intracellular C-terminal sequence of the GluA2 subtype as a basis and employs peptide chip technology to screen for a batch of peptide fragments that are similar to the intracellular C-terminal sequence of the GluA2 subtype but retain its biological function. Through peptide synthesis and cell experiments, peptides that can effectively protect neuronal cell activity were finally obtained. The protective effect of these peptides on neuronal cell activity is superior to that of positive control drugs. Based on this, this application provides a peptide and its application in the treatment of nervous system diseases.

[0007] On the one hand, this disclosure provides a polypeptide comprising a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence MTYRPGYNPFG (SEQ ID NO: 41) or the amino acid sequence AKGYYRPYGVPV (SEQ ID NO: 22), or a polypeptide having one or more amino acid substitutions, deletions, and / or additions to the amino acid sequence shown in SEQ ID NO: 41 or 22.

[0008] In some embodiments, the N-terminus of the aforementioned polypeptide is further fused with the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 42).

[0009] In some embodiments, the aforementioned polypeptide may also have optional modifications.

[0010] In some embodiments, the aforementioned modifications are chemical group modifications or amino acid modifications.

[0011] In some preferred embodiments, the sites for modification of the aforementioned chemical groups are selected from any one or more of the N-terminus, C-terminus, lysine side chain, and cysteine ​​side chain of the polypeptide.

[0012] In some preferred embodiments, the aforementioned amino acid modification is a hydrophilic amino acid modification or a cysteine ​​modification.

[0013] In some embodiments, the aforementioned polypeptide comprises a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1 or 26, or a polypeptide having one or more amino acid substitutions, deletions, and / or additions with the amino acid sequence shown in SEQ ID NO: 1 or 26.

[0014] On the other hand, this disclosure provides nucleic acid molecules for encoding the aforementioned polypeptides.

[0015] On the other hand, this disclosure provides a pharmaceutical composition comprising the aforementioned polypeptide or nucleic acid molecule.

[0016] In some preferred embodiments, the aforementioned pharmaceutical composition further comprises an optional pharmaceutically acceptable carrier.

[0017] On the other hand, this disclosure provides the use of the aforementioned peptides, nucleic acid molecules and / or pharmaceutical compositions in the preparation of medicaments for treating stroke, Alzheimer's disease, major depression and / or drug addiction, or for reducing the risk of stroke, Alzheimer's disease, major depression and / or drug addiction.

[0018] In some embodiments, the aforementioned drug or health product contains the aforementioned polypeptide at a dose of 0.1-100 mg / kg body weight.

[0019] In some embodiments, the aforementioned medicament comprises a medicament, pharmaceutical composition, or health product compound with the aforementioned polypeptide as an active ingredient, and a pharmaceutically acceptable carrier thereof.

[0020] In some embodiments, the administration of the aforementioned drug in the foregoing uses includes intravenous injection, subarachnoid injection, spinal injection, subcutaneous injection, intramuscular injection, and oral administration.

[0021] The beneficial effects achieved by this disclosure include at least the following:

[0022] In a mouse water maze experiment, a model of cognitive impairment, the iCXNP-01 and iCXNP-18-T peptides disclosed herein effectively reduced escape time and restored the number of times mice crossed the platform. Experimental data show that iCXNP-01 and iCXNP-18-T peptides have a significant neuroprotective effect. iCXNP-01 and iCXNP-18-T peptides show promise for development into novel drugs or health supplements with rapid onset of action for the treatment of various neurological diseases such as stroke, Alzheimer's disease, major depression, and drug addiction.

[0023] The GluR2-CT peptide of the present invention has a rapid onset of action (e.g., within 1 hour) and a long duration of action (e.g., lasting at least about 24 hours after a single dose), compared to traditional antidepressants which usually take several weeks to take effect. It can promptly reduce suicidal tendencies in patients with major depressive disorder and prevent suicidal behavior. Attached Figure Description

[0024] Figure 1 shows the SPR affinity test results of PMS-001 and Brag2.

[0025] Figure 2 shows the SPR affinity test results of iCXNP-01 and Brag2.

[0026] Figure 3 shows the SPR affinity test results of iCXNP-18-T and Brag2.

[0027] Figure 4 shows the test results of the protective effect of the peptide on hippocampal neurons.

[0028] Figure 5 shows the hemolysis test results of peptides PMS-001, iCXNP-01, and iCXNP-18-T.

[0029] Figure 6 shows the results of the cytotoxicity test of peptides PMS-001, iCXNP-01, and iCXNP-18-T on HepG2 cells.

[0030] Figure 7 shows the results of the cytotoxicity test of peptides PMS-001, iCXNP-01, and iCXNP-18-T on 293T cells.

[0031] Figure 8 shows the weight changes of the experimental animals.

[0032] Figure 9 shows the results of the positioning cruise to evade the incubation period.

[0033] Figure 10 shows the neuroprotective effects in areas such as space exploration. Detailed Implementation

[0034] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of this application. For those skilled in the art, other embodiments can be obtained based on these drawings.

[0035] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this application, definitions and explanations of relevant terms are provided below.

[0036] It should also be understood that in some methods described herein that include more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are listed, unless the context otherwise indicates.

[0037] definition

[0038] As used herein, the terms “a” and “an” as well as “the” and similar pronouns indicate singular and plural, unless otherwise specified herein or the context clearly contradicts them.

[0039] As used herein and unless otherwise stated, the terms “about” or “approximately” mean within 10% of a given value or range. Where an integer is required, the term means within 10% of a given value or range, rounded up or down to the nearest integer.

[0040] As used herein, the conjunction term "and / or" between multiple elements is understood to include both individual and combined options. For example, when two elements are joined by "and / or," the first option refers to the applicability of the first element without the second. The second option refers to the applicability of the second element without the first. The third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within the scope of meaning and thus satisfies the requirement of the term "and / or" as used herein. The concurrent applicability of multiple options is also understood to fall within the scope of the term's meaning and thus satisfies the requirement of the term "and / or".

[0041] As used herein, the term "identity" refers to the degree to which two or more amino acid sequences, when aligned, have the same amino acid residues at the same positions. Amino acid identity can be expressed as a percentage and can also be used to assess the similarity and homology between sequences.

[0042] As used herein, the terms “nucleic acid,” “nucleotide,” and “polynucleotide” are used interchangeably to refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and polymers thereof in single-stranded, double-stranded, or multi-stranded form. This term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and / or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic, or derived nucleotide bases. In some embodiments, nucleic acids may include mixtures of DNA, RNA, and the like. The term also covers nucleic acids containing known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. “Nucleic acid” is used interchangeably with “gene,” “DNA,” and “mRNA” encoded by a gene.

[0043] As used in this article, the term "object" refers to mammals, especially primates, and particularly humans.

[0044] As used in this article, the term "stroke" is a general term for a group of diseases caused by sudden local cerebral circulatory disturbances leading to neurological dysfunction. Clinically, it can be diagnosed and graded using a quantitative scale for the degree of neurological deficit. The most widely used rating scale in my country is the Edinburgh and Scandinavian Rating Scale (ERRS). The score ranges from 0 to 45, with 0-15 indicating mild neurological deficit, 16-30 indicating moderate, and 31-45 indicating severe neurological deficit. Stroke patients with severe neurological deficits often present with difficulty walking and dizziness.

[0045] As used herein, the term "depression" refers to a common mental disorder involving prolonged low mood or loss of pleasure or interest in activities, which can be clinically diagnosed and graded using a depressive symptoms rating scale. Appropriate scales include, but are not limited to, the Hamilton Depression Scale (HAMD), the Beck Depression Inventory (BDI), and the Self-rating Depression Scale (SDS). For example, on the HAMD scale, a total score <8 is normal, a total score between 8 and 20 indicates possible depression, a total score between 20 and 35 indicates a diagnosis of depression, and a total score >35 indicates major depressive disorder. The GluR2-CT peptide of this invention is particularly suitable for patients with major depressive disorder who have a total score >35 on the HAMD scale. In the BDI questionnaire, a total score of 10-15 indicates possible depression; a total score greater than 15 indicates existing depression; and a score greater than 25 indicates major depressive disorder. The GluR2-CT peptide of this invention is particularly suitable for patients with major depressive disorder whose total score on the BDI questionnaire is >25. In the SDS scale, the cutoff value for the standard SDS score is 53, with 53-62 indicating mild depression, 63-72 indicating moderate depression, and above 73 indicating severe depression. Severe suicidal tendencies are common in patients with major depressive disorder, causing serious social problems. Suicidal tendencies include, for example, the individual preparing for suicide or attempting suicide but being prevented from doing so.

[0046] As used in this article, the term "Alzheimer's disease," also known as "senile dementia," is a primary degenerative brain disease that occurs in old age and pre-senile stages. It refers to a persistent impairment of higher neurological functions, specifically impairments in memory, thinking, analytical judgment, visuospatial recognition, and emotion, even without loss of consciousness. Clinically, it can be diagnosed using the Mini-Mental State Examination (MMSE). A score of ≤17 for illiteracy, ≤20 for primary school level, ≤22 for secondary school level, or ≤23 for university level indicates cognitive impairment. Based on the degree of cognitive and physical deterioration, it is divided into three stages: mild dementia, moderate dementia, and severe dementia. Individuals in the moderate to severe dementia stage exhibit complete dependence on caregivers and severe memory loss.

[0047] As used in this article, the term "drug addiction" refers to a strong craving or urge for addictive substances and behaviors, and withdrawal symptoms such as general malaise, irritability, restlessness, difficulty concentrating, and sleep disturbances that occur when the addictive substances and behaviors are reduced or stopped.

[0048] As used herein, the term "effective dose" is synonymous with "effective amount," referring to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition. In this disclosure, the "therapeutic effective amount" of iCXNP-01 and iCXNP-18-T peptides is preferably an amount that results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of damage or disability caused by disease-related suffering. For example, applying a therapeutically effective amount of iCXNP-01 or iCXNP-18-T peptides can result in a decrease in the subject's score on a depression scale. Preferably, applying a therapeutically effective amount of iCXNP-01 or iCXNP-18-T peptides results in the subject's remission from major depressive disorder to mild depressive disorder or absence of depressive symptoms. Alternatively, applying a therapeutically effective amount of iCXNP-01 or iCXNP-18-T peptides results in a reduction in the subject's suicide risk. Those skilled in the art can determine the therapeutically effective dose based on factors such as the size of the subject, the severity of the subject's symptoms (e.g., high or low scores), and the specific composition or route of administration chosen. The iCXNP-01, iCXNP-18-T peptides or pharmaceutical compositions of this disclosure can be administered via one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or manner of administration varies depending on the desired outcome. Preferred routes of administration for the iCXNP-01, iCXNP-18-T peptides of this invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. Preferably, the iCXNP-01, iCXNP-18-T peptides of this invention are administered via intravenous injection, subarachnoid injection, spinal injection, subcutaneous, intramuscular injection, and oral administration. For the administration of the iCXNP-01, iCXNP-18-T peptides of this invention, the dose range can be from about 0.0001 to 100 mg / kg, more typically from 0.1 to 100 mg / kg of the recipient's body weight. For example, the dosage can be 0.1 mg / kg body weight, 1 mg / kg body weight, 10 mg / kg body weight, 20 mg / kg body weight, 50 mg / kg body weight, or 100 mg / kg body weight, or within the range of 0.1-100 mg / kg body weight. For the administration of the iCXNP-01 and iCXNP-18-T peptides of the present invention, exemplary treatment regimens may include once daily, every two days, every three days, every four days, every five days, every six days, once weekly, every two weeks, every three weeks, every four weeks, once monthly, every three months, every three to six months, or a slightly shorter initial dosing interval (e.g., once daily to once every three weeks) followed by a longer dosing interval (e.g., once monthly to once every three to six months). The specific administration regimen can be determined by a physician based on the patient's specific symptoms.

[0049] The term "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, liquid, or gel fillers suitable for human or animal use and must have sufficient purity and sufficiently low toxicity. "Compatibility" refers to the ability of the components in a pharmaceutical composition and the active ingredient of the drug, as well as their intermingling, to not significantly reduce efficacy. In this disclosure, the aforementioned pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, such as salts, buffers, sugars, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants, or emulsifiers, or combinations thereof. The amount of a pharmaceutically acceptable carrier in a pharmaceutical composition can be determined experimentally based on the activity of the carrier and the desired properties of the formulation, such as stability and / or minimal oxidation. Preferably, the carrier is suitable for intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral administration (e.g., by injection or infusion). Depending on the route of administration, active compounds such as iCXNP-01 and iCXNP-18-T polypeptides disclosed herein can be encapsulated in a material to protect the compounds from acids and other natural conditions that can inactivate them.

[0050] Detailed implementation plan

[0051] On the one hand, this disclosure provides a polypeptide comprising a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence MTYRPGYNPFG (SEQ ID NO: 41) or the amino acid sequence AKGYYRPYGVPV (SEQ ID NO: 22), or a polypeptide having one or more amino acid substitutions, deletions, and / or additions to the amino acid sequence shown in SEQ ID NO: 41 or 22.

[0052] In some embodiments, the N-terminus of the aforementioned polypeptide is further fused with the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 42).

[0053] In some embodiments, the aforementioned polypeptide may also have optional modifications.

[0054] In some embodiments, the aforementioned modifications are chemical group modifications or amino acid modifications.

[0055] In some preferred embodiments, the sites for modification of the aforementioned chemical groups are selected from any one or more of the N-terminus, C-terminus, lysine side chain, and cysteine ​​side chain of the polypeptide.

[0056] In some preferred embodiments, the aforementioned amino acid modification is a hydrophilic amino acid modification or a cysteine ​​modification.

[0057] In some embodiments, the aforementioned polypeptide comprises a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1 or 26, or a polypeptide having one or more amino acid substitutions, deletions, and / or additions with the amino acid sequence shown in SEQ ID NO: 1 or 26.

[0058] On the other hand, this disclosure provides nucleic acid molecules for encoding the aforementioned polypeptides.

[0059] On the other hand, this disclosure provides a pharmaceutical composition comprising the aforementioned polypeptide or nucleic acid molecule.

[0060] In some preferred embodiments, the aforementioned pharmaceutical composition further comprises an optional pharmaceutically acceptable carrier.

[0061] On the other hand, this disclosure provides the use of the aforementioned peptides, nucleic acid molecules and / or pharmaceutical compositions in the preparation of medicaments for treating stroke, Alzheimer's disease, major depression and / or drug addiction, or for reducing the risk of stroke, Alzheimer's disease, major depression and / or drug addiction.

[0062] In some embodiments, the aforementioned drug or health product contains the aforementioned polypeptide at a dose of 0.1-100 mg / kg body weight.

[0063] In some embodiments, the aforementioned medicament comprises a medicament, pharmaceutical composition, or health product compound with the aforementioned polypeptide as an active ingredient, and a pharmaceutically acceptable carrier thereof.

[0064] In some embodiments, the administration of the aforementioned drug in the foregoing uses includes intravenous injection, subarachnoid injection, spinal injection, subcutaneous injection, intramuscular injection, and oral administration.

[0065] The embodiments of this disclosure will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. The scope of this disclosure may include some embodiments having combinations of all or some of the described features. Where specific techniques or conditions are not specified in the examples, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0066] Example 1: Constructing a deep learning model using peptide chip data

[0067] (I) Chip Experiment

[0068] Select the intracellular C-terminal sequence of candidate GluA2 isotypes and label it with fluorescence (reagent: Alexa). The 555 Protein Labeling Kit (A30007) was used to detect fluorescently labeled Brag2 protein using a high-throughput short peptide array chip (Healthtell V16 chip).

[0069] Fluorescently labeled samples were diluted at four concentration gradients: 1:500, 1:1000, 1:5000, and 1:10000. The chips were placed in a chip hydration apparatus, covered with ultrapure water, and hydrated for 20 minutes at 55±5 rpm on a track shaker. The chip surface was then sprayed with isopropanol and centrifuged to dry. The dried chips were assembled into assay cassettes according to the experimental design. 90 μL of diluted sample was added to each well of the assembled chips, and the cassettes were incubated for 1 hour using a constant-temperature shaker. The assay cassettes were then washed in a plate washer. The chips in the assay cassettes were then disassembled, cleaned, dried, and reassembled into an imaging cassette, which was then scanned and imaged using an ImageXpress micro 4 imager from Molecular Devices. Each sample yielded one TIFF image file, representing the raw data.

[0070] (II) Constructing Deep Learning Models

[0071] Using raw data obtained from chip experiments, a deep learning model specifically designed to predict protein-peptide binding affinity for this target protein was trained. The trained model can predict the binding affinity score between any peptide and the target protein.

[0072] The peptide chip used in this application is the HealthTell V16 chip, which has four repeating peptide arrays, each containing more than 3,200,000 peptides. These 3,200,000 peptides are peptide sequences formed by unbiased random combinations of 5-16 amino acids, achieving a diversity coverage of 99.9% for pentamers (5-peptides). Simultaneously, this version of the chip's peptide array also includes some target sequences, such as 1,328,926 target sequences (including potential therapeutic sequences of some drug target proteins, epitope sequences, antibody sequences, and influenza protein therapeutic sequences, etc.).

[0073] Example 2: Screening of candidate peptides

[0074] Using the deep learning model obtained in Example 1, candidate peptides are selected from the peptide database:

[0075] Based on the constructed search space for peptide sequences, a deep learning model built using microarray data is used to predict the binding affinity of peptide sequences, and computationally highly binding peptides are selected for validation. Specific implementation steps:

[0076] (I) Construction of the peptide search space

[0077] 1. The number of polypeptide sequences on the chip is approximately 3,200,000.

[0078] 2. The data records approximately 1,000 peptides that are marketed or have therapeutic effects;

[0079] 3. The data records approximately 25,000,000 endogenous or naturally derived peptides;

[0080] 4. ~20,000 peptides designed based on Brag2 spatial structure calculations.

[0081] (ii) Using the deep learning model obtained in Example 1 to screen candidate peptides in a self-built database.

[0082] (III) Based on the binding strength of each candidate peptide, and taking into account the length of the peptide, the degree of binding strength fluctuation, and the isoelectric point of the peptide, potential functional peptides were selected using relevant knowledge of known sequences to verify their interaction with Brag2 synaptic proteins. A total of 39 peptides were selected (see Table 1), and experiments were conducted to further verify the activity of the peptides.

[0083] Table 1. Information on candidate peptides

[0084] Example 3: Peptide Synthesis

[0085] (I) Peptide Synthesis

[0086] The candidate peptide was synthesized by Jier Biosynthesis, with peptide PMS-001 (patent CN113166721A, Jier Biosynthesis) as a positive reference. The peptide sequence is shown in Table 2.

[0087] Table 2 Information on synthesized peptides

[0088] Example 4: SPR determination of the affinity between the peptide and Brag2 protein

[0089] The affinity of candidate peptides for Brag2 protein was determined using surface plasmon resonance (SPR). Peptides were selected for downstream experimental validation based on the measured KD values. The materials used for SPR determination were as follows: CM5 sensor chip (Cytiva), Amine Coupling Kit (Cytiva), 96-well polystyrene microplate (GREINE), sodium acetate 4.5g (Cytiva), glycine 2.0g (Cytiva), PBS (Gibco), Tween 20 (Aladdin), and DMSO (Sigma). Specific implementation steps:

[0090] Brag2 protein was immobilized using the Amine Coupling Kit. The immobilization process consisted of three steps: activation, coupling, and blocking.

[0091] Activation: The chip surface was activated by reacting an equal volume of EDC and NHS mixture for 7 minutes.

[0092] Coupling: The Brag2 protein was diluted with sodium acetate solution at pH 4.5 to 60 μg / mL and injected into the activation channel at a flow rate of 10 μL / min for 420 s to covalently couple it to the chip surface.

[0093] Blocking: NHS ester groups on the chip activation channel that did not participate in the coupling reaction were blocked with 1M ethanolamine (pH 8.5).

[0094] Then, 1×PBS-P buffer containing 0.5% DMSO was used as the buffer system for concentration gradient analysis. During sample analysis, the flow rate was set to 30 μL / min, the binding time to 120 s, and the dissociation time to 120 s. After each sample analysis, the sample was regenerated with glycine 2.0 for 30 s to maintain binding activity. Response data from the test channels were collected using Biacore 8K Control software (Cytiva), and the data from the fitting analysis were obtained after subtracting the response values ​​from the reference channels. The data were fitted using a 1:1 binding model using Biacore 8K Evaluation software (Cytiva) to obtain the equilibrium dissociation constant KD value. The SPR affinity detection results are shown in Table 3; the SPR affinity detection results for PMS-001, iCXNP-01, and iCXNP-18-T are shown in Figures 1-3.

[0095] Table 3. Affinity results of SPR assay

[0096] Example 5: Validation of Peptide Biological Functions

[0097] Based on the isoelectric point and SPR affinity results from Examples 2 and 4, a subset of peptides were selected to test their protective effect on hippocampal neuronal cell activity. Specific implementation steps:

[0098] (I) Culture of hippocampal neurons (self-isolated in the laboratory)

[0099] Chemicals and reagents: maintenance medium (98% Neurobasal medium, 2% B27, glutamine 0.5 mmol / L, penicillin 50 U / ml, streptomycin 50 U / ml), inoculation medium (90% high glucose DMEM medium, 10% PBS, penicillin 100 U / ml, streptomycin 100 U / ml), cytidine solution (Beyotime).

[0100] 1. Coating: Add 50 μL of 0.1 mg / mL poly-L-lysine solution to a 96-well plate and incubate at 37°C with 5% carbon dioxide for 2 hours. After the predetermined time, remove the 96-well plate and aspirate any excess poly-L-lysine solution. Wash the wells coated with poly-L-lysine solution three times with PBS. Cover the 96-well plate and incubate at 37°C for at least 4-24 hours, ensuring the bottom of the plate is dry and free of liquid.

[0101] 2. Primary Culture of Hippocampal Neurons: Newborn SD rats (within 24 hours of birth) were sterilized with 75% ethanol. Under aseptic conditions, the rats were rapidly decapitated to remove the brain. The cranial suture was cut open with scissors, and the cranial suture was also cut at 45° and 135° along the neck. The brain was removed and placed in ice-cold brain preservation solution, gently shaken and rinsed, then placed in clean ice-cold brain preservation solution. The cerebellum and brainstem were gently removed, and the brain was cut in half along the midline. The olfactory bulb, septum, thalamus, and hypothalamus were removed. The meninges and choroid plexus were dissected, and the cortex was harvested and placed in a culture dish. It was gently rinsed with preservation solution, and the cortex was removed and placed in a second culture dish. The cortex was cut into 0.5mm × 0.5mm pieces, the supernatant was discarded, and 5ml of digestion solution containing 0.125% trypsin + 0.02% EDTA was added for digestion (37℃, 15min). Shaking was performed during digestion. An equal volume of inoculation culture medium was added to stop digestion. The tissue was dispersed using a Pasteur tube and passed through a 70-mesh cell sieve. Centrifuge at 1000 rpm for 8 min and discard the supernatant. Add inoculation medium and gently pipette to prepare a single-cell suspension. Seed 100 μL per well in 96-well plates at a density of 4 × 10^4 / ml and incubate at 37℃ with 5% CO2. After 4-6 h of inoculation, remove the inoculation medium and replace it with maintenance medium; thereafter, change the medium twice a week, replacing half the medium each time. On the second day after inoculation, depending on the cell growth status, add the cell division inhibitor cytarabine (final concentration 1 μmol / L, acting for 24-36 h) to inhibit the growth of glial cells. Select hippocampal neurons cultured for 10-12 days for experiments.

[0102] (II) In vitro detection of peptide bioactivity

[0103] Experiments were conducted based on the state of hippocampal neurons cultured in 96-well plates. Culture medium was aspirated from each well, and 10 μM of a peptide precisely dissolved in DMSO (Sigma) was added. 100 μL of neuronal culture medium was added to each well, and the plates were incubated at 37°C and 5% CO2 for 1 hour. After the predetermined time, NMDA solution was added to bring the final concentration in the wells to 200 nM, and the mixture was gently shaken and incubated at 37°C and 5% CO2 for 1 hour. Cell death was detected using the LDH assay (LDH kit purchased from Beyotime (C0016)).

[0104] LDH experimental procedures:

[0105] One hour before the scheduled detection time, remove the cell culture plate from the cell culture incubator. Add the LDH release reagent provided in the kit to the untreated wells (sample maximum enzyme activity control wells) used for subsequent cell lysis, at a volume of 10% of the original culture medium. After adding the LDH release reagent, mix thoroughly by pipetting several times, and then continue incubation in a cell culture incubator at 37°C, 5% CO2, and 95% humidity.

[0106] After the predetermined time has elapsed, the cell culture plate is removed from the cell culture incubator and centrifuged at 400g for 5 minutes using a multi-well plate centrifuge. 120 μL of the supernatant from each well is then added to the corresponding well of a new 96-well plate, and the samples are immediately analyzed.

[0107] According to the required amount of INT solution (1X), take an appropriate amount of INT solution (10X) and dilute it to 1X with INT diluent. According to the number of samples to be tested (including controls), prepare an appropriate amount of detection working solution fresh just before the test (total volume 60μL / test, including 20μL lactate solution, 20μL INT solution (1X) and 20μL enzyme solution).

[0108] Add 60 μL of LDH detection working solution to each well. Mix well and incubate at room temperature (approximately 25°C) in the dark for 30 min (you can wrap the sample in aluminum foil and place it on a horizontal shaker or a side-swing shaker with gentle shaking). Measure the absorbance at 490 nm using an ELISA reader.

[0109] A bar chart was plotted with concentration on the x-axis and cell viability on the y-axis, and the results are shown in Figure 4. The results show that iCXNP-01 and iCXNP-18-T have a significant protective effect on hippocampal neurons, and the effect is better than that of the positive control PMS-001.

[0110] Example 6: Hemolytic activity test of peptides

[0111] Dilute 20% rabbit red blood cells with PBS to 4% rabbit red blood cells, and gently invert to mix. Prepare the test drug and positive control PMS-001 with PBS at the concentrations set below, and add 100 μL to each well of a 96-well plate, setting up two replicates.

[0112] The drug concentration settings are as follows: 256 μg / ml, 128 μg / ml, 64 μg / ml, 32 μg / ml, 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0 μg / ml.

[0113] Positive reference concentrations were set as follows: 1%, 0.5%, 0.25%, 0.125%, 0.06%, 0.03%, 0.015%, 0.008%, 0.004%, 0.002%, 0.001%, 0%.

[0114] Add 100 μL of diluted rabbit red blood cell suspension to each well, gently tap to mix, and incubate at 37°C for 1 hour. Observe the hemolysis results after the time is up. If the liquid in the well is clear red, hemolysis is present; if it is colorless and transparent, and the red blood cells settle at the bottom, there is no hemolysis. Transfer the supernatant from the wells to a new 96-well plate and measure the absorbance at 450 nm using an ELISA reader.

[0115] As shown in Figure 5, neither iCXNP-01 nor iCXNP-18-T showed hemolysis.

[0116] Example 7: Cytotoxicity test of peptides

[0117] The toxicity of the peptides to 293T cells (human embryonic kidney cells) and HepG2 cells (human liver cancer cells) was tested using a CCK8 assay (kit purchased from Shanghai Sangon Biotech (E606335-0250)). Specific steps were as follows:

[0118] 293T and HepG2 cells were maintained in their respective culture media and cultured at 37°C with a carbon dioxide (CO2) content of 5%.

[0119] The cytotoxicity of the peptides was detected in vitro. 293T cells and HepG2 cells were seeded in 96-well plates, with 1 × 10⁶ cells per well. 4Cells were divided into 100 μL wells containing 10% FBS, with 8 wells left empty as a blank control. Incubation was performed overnight (37°C, 5% CO2) to allow cell adhesion. Different concentrations (30 μM, 300 μM, 500 μM) of iCXNP-01, iCXNP-18-T, and PMS-001 precisely dissolved in DMSO were added to each well, along with medium containing 10% FBS. Incubation was performed at 37°C, 5% CO2 for 48 hours to allow peptide activation. Cell proliferation was assessed using the CCK8 assay.

[0120] Add 10 μL of CCK-8 Solution to each well. After gentle mixing, incubate in a 5% CO2 incubator at 37°C for 0.5–4 hours. Read the optical density at 450 nm and record the results. Plot a bar graph with concentration on the x-axis and cell viability on the y-axis. The HepG2 cytotoxicity test results are shown in Figure 6, and the 293T cytotoxicity test results are shown in Figure 7. The results show that iCXNP-01 and iCXNP-18-T have no significant cytotoxicity to cells.

[0121] Example 8: Animal Experiments with Polypeptides

[0122] Male mice were divided into groups of 10 mice each, according to experimental requirements. Specific grouping information is shown in the table below:

[0123] Table 4. Mouse grouping information

[0124] Note: The first digit of the animal number represents the group (1, 2, 3, 4, 5 represent the normal control group, the model control group, the iCXNP-01 group, and the iCXNP-18-T group, respectively). (Group and positive control group). The second letter represents the sex (M for male), and the last three digits represent the animal serial number, with "-" indicating that it is not applicable.

[0125] Mice in the model control group and each drug-treated group were intraperitoneally injected with 3 mg / kg scopolamine at a volume of 10 mL / kg. Mice in the normal control group were intraperitoneally injected with 10 mL / kg of physiological saline. The administration was via tail vein injection for 14 consecutive days. During the water maze test, the scopolamine was administered 30 minutes before the modeling injection. The first day of administration was defined as day 1 of the experiment. The animals were observed daily, including but not limited to the administration site (whether there was redness, swelling, bleeding, etc.), behavioral status, and symptoms in the eyes, mouth, nose, ears, hair, feces, urine, and genitals. Any abnormalities were recorded in detail. The mice were weighed twice a week during the administration period, and the results are shown in Figure 8. One hour before each water maze behavioral experiment, mice in each group were given the corresponding drug. Thirty minutes before the behavioral experiment, mice in the model group and each drug-treated group were intraperitoneally injected with scopolamine solution, while mice in the normal control group were intraperitoneally injected with physiological saline.

[0126] On day 9, mice underwent water maze swimming adaptation training. From day 10 to day 13, they underwent a navigation experiment, lasting 4 days. Each mouse participated in 4 experiments per day, entering the water from a different quadrant each time. The specific entry quadrant order is shown in the table below. When placed in the pool, the mice were required to face the pool wall. The time taken for the mouse to find the underwater hidden platform within 60 seconds was recorded, i.e., the escape latency. If the mouse found the platform within 60 seconds, it stayed on the platform for 15 seconds, after which the system automatically stopped the timer and recorded the escape latency. If the mouse did not find the platform within 60 seconds, it was guided to stand on the platform and stay for 15 seconds, and the escape latency was recorded as 60 seconds. The day after the navigation experiment (day 14), a spatial exploration experiment was conducted. The platform was removed, and the entry point for the spatial exploration experiment was the quadrant opposite to the original platform quadrant. The number of times each group of mice crossed the original platform position within 60 seconds, the specific time spent in the quadrant where the original platform was located, the average swimming speed, and the swimming speed were recorded. The results of the escape latency of the navigation experiment are shown in Figure 9, and the neuroprotective effects in spatial exploration are shown in Figure 10.

[0127] The iCXNP-01 and iCXNP-18-T groups showed significant neuroprotective effects, effectively reducing escape time and restoring the number of times mice crossed the platform in the water maze animal model.

Claims

1. A polypeptide, characterized in that, The polypeptide comprises a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence MTYRPGYNPFG (SEQ ID NO: 41) or the amino acid sequence AKGYYRPYGVPV (SEQ ID NO: 22), or a polypeptide having one or more amino acid substitutions, deletions, and / or additions to the amino acid sequence shown in SEQ ID NO: 41 or 22.

2. The polypeptide according to claim 1, characterized in that, The N-terminus of the polypeptide is also fused with the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 42).

3. The polypeptide according to claim 1 or 2, characterized in that, The polypeptide may also have optional modifications; the modifications may be chemical group modifications or amino acid modifications. Preferably, the sites of the chemical group modification are selected from any one or more of the N-terminus, C-terminus, lysine side chain, and cysteine ​​side chain of the polypeptide; Preferably, the amino acid modification is a substituted hydrophilic amino acid modification or a cysteine ​​modification.

4. The polypeptide according to any one of claims 1-3, characterized in that, The polypeptide comprises a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1 or 26, or a polypeptide having one or more amino acid substitutions, deletions, and / or additions with the amino acid sequence shown in SEQ ID NO: 1 or 26.

5. A nucleic acid molecule for encoding the polypeptide of any one of claims 1-4.

6. A pharmaceutical composition, wherein, The pharmaceutical composition comprises a polypeptide as described in any one of claims 1-4 or a nucleic acid molecule as described in claim 5; Preferably, the pharmaceutical composition further comprises an optional pharmaceutically acceptable carrier.

7. Use of the polypeptide according to any one of claims 1-4, the nucleic acid molecule according to claim 5, and / or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating stroke, Alzheimer's disease, major depression, and / or drug addiction, or for reducing the risk of stroke, Alzheimer's disease, major depression, and / or drug addiction.

8. The use according to claim 7, wherein the drug or health product comprises a polypeptide according to any one of claims 1-4 at a dose of 0.1-100 mg / kg body weight.

9. The use according to claim 7 or 8, wherein the medicament comprises a medicament, pharmaceutical composition or health product compound with a polypeptide as an active ingredient as described in any one of claims 1-4, and a pharmaceutically acceptable carrier thereof.

10. The use according to any one of claims 7-9, wherein the method of administration of the drug includes intravenous injection, subarachnoid injection, spinal injection, subcutaneous injection, intramuscular injection, and oral administration.

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