Peptide with neuroprotective effect and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a peptide with a neuroprotective effect and application thereof. The amino acid sequence of the polypeptide comprises an amino acid sequence P1-Linker-P2, and the polypeptide is a PSD-95 inhibitor, can atypically bind to the PDZ1 and / or PDZ2 structural domain of PSD-95 so as to inhibit the protein-protein interaction of the PDZ1 and / or PDZ2 structural domain with nNOS, and can be used for preparing drugs for treating excitatory neurotoxicity related injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a peptide with neuroprotective effect and application thereof. Background Art

[0002] N-methyl-D-aspartate receptor (NMDAR) plays an important role in the central nervous system, participating in the formation of learning and memory, the formation of synapses in the development of the central nervous system, the formation of plasticity and glutamate-mediated neurotoxicity. NMDA receptor is the main mediator of excitotoxicity (i.e., glutamate-mediated neurotoxicity), which is related to neurodegenerative diseases and acute brain injury. It can be used as a therapeutic target for certain nervous system diseases, such as cerebral infarction, neuropathic pain, epilepsy and schizophrenia.

[0003] Postsynaptic density (PSD) is a super signaling molecule complex of the excitatory postsynaptic membrane and an important substance for the transmission function of synapses. According to the weight of the molecular weight of PSD, it can be divided into four categories: PSD-95, PSD-93, synaptic associated protein 97 (SAP-97) and SAP102.

[0004] PSD-95 is the most abundant and important scaffolding protein, mainly present in mature excitatory glutamatergic synapses. Its relative molecular mass (Mr) is 95,000. It is necessary for receptor activity and stability on the postsynaptic membrane and plays an important role in synaptic plasticity. It is the most important synaptic protein that mediates and integrates synaptic information and is also a member of the guanylate-related kinase family. PSD-95 includes three PDZ domains, one SH3 domain and one guanylate kinase-like (GK) domain, which are connected by a connecting region. PSD-95 is almost entirely located in the postsynaptic density of neurons and is involved in anchoring synaptic proteins. Its direct and indirect binding partners include neuroligins, nNOS, NMDA receptors, AMPA receptors and potassium channels.

[0005] According to the different splicing of mRNA to genes, more than 10 nNOS (neuronal nitric oxide synthase) spliceosomes are known, with a molecular weight of 160.8KD. nNOS contains two structural domains, the C-terminal reduction domain and the N-terminal oxidation domain. The N-terminal has two non-overlapping binding regions: (1) PDZ region: composed of 1 to 99 amino acids, involved in the formation of active nNOS dimers; (2) β-finger structure: composed of 100-300 amino acids, containing a specific amino acid sequence -ETTF-, which can bind to the PDZ of other proteins to exert its function. nNOS can be anchored to the plasma membrane or cytosolic protein through the PDZ-PDZ domain or C-terminal PDZ reaction. PSD-95 can connect nNOS to the NMDA receptor and effectively activate nNOS through the activation of the NMDA receptor.

[0006] Stroke is characterized by the death of neurons in ischemic areas, cerebral hemorrhage areas, and / or traumatic areas. Neuronal death or damage caused by cerebral ischemia is a cascade of injury. After cerebral ischemia, tissue blood perfusion decreases, excitatory neurotransmitters increase, NMDA and AMPA receptors are activated, ion channels open, calcium ions flow in, and a large number of enzymes are activated to trigger signal cascades, resulting in multi-pathway neuronal damage. Although NMDA receptor antagonists effectively reduce excitotoxicity by blocking glutamate-mediated ion flux, they also block some physiologically important processes. Its downstream PSD-95 triggers a series of ischemic injuries by interacting with a variety of proteins. It is a key site of cerebral ischemic injury and a potential target for drug therapy. Therefore, the development of PSD-95 inhibitors has great medicinal significance for nervous system damage caused by various excitotoxic neurotoxicities, including stroke. In contrast, specific inhibition of excitotoxicity can be obtained by using PSD-95 inhibitors to perturb the intracellular nNOS / PSD-95 / NMDA receptor complex.

[0007] In addition, studies have shown that the excitatory neurotransmitter NMDA plays an important role in anxiety, epilepsy and various neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease or Huntington's disease. For example, studies have shown that excessive excitation of the central glutamatergic system can cause anxiety, and NMDA receptors (NMDARs) are responsible for the main part of glutamate excitotoxic neurotoxicity. The onset of epilepsy includes three different and continuous pathophysiological processes: initiation, maintenance and extension of paroxysmal discharges, and inhibition of paroxysmal discharges. In this process, excitatory neurotransmitters such as glutamate and aspartate play an important role. In Alzheimer's disease, PSD-95 participates in the neurotoxic mechanism leading to it through the GluR6-PSD-95-MLK3 pathway. In addition, in Huntington's disease, PSD-95 is a mediator of NMDA receptor and huntingtin mutant neurotoxicity. Therefore, the development of PSD-95 inhibitors is also of great significance for the treatment, improvement and prevention of the above diseases.

[0008] Nerinetide (NA-1, TAT-NR2B9c, sequence: YGRKKRRQRRRKLSSIESDV) is a PSD-95 inhibitor, thereby disrupting the binding of PSD-95 to NMDA receptors and neuronal nitric oxide synthase (nNOS), and reducing excitotoxicity induced by cerebral ischemia. In preclinical ischemic stroke models, it can reduce the infarct size of cerebral ischemia-reperfusion and improve its functional prognosis. Nerinetide has no serious side effects and can be used when stroke or other ischemic or hemorrhagic conditions are suspected but the diagnosis of the absence of hemorrhage has not yet been confirmed according to the standards recognized in the art. However, Nerinetide has a short half-life in vivo, patients need to take large doses of medication every day, patient compliance is poor, clinical costs are high, and this compound may become an ineffective non-selective compound due to its low affinity for PDZ1-2 in PSD-95. WO2010004003A2 describes a dimeric peptide ligand connected by a polyethylene glycol linker (PEG). WO2017185249A1 and WO2017185250A1 disclose peptides for treating central nervous system damage. CN110799547A discloses a compound having a structure shown in R1-S1-YEKL-S2-R2 and the use of the compound. CN115667286A discloses a novel cyclic peptide that can act as an inhibitor of protein-protein interactions, and the polypeptide comprises an amino acid sequence of TX1LETX2X3X4GX5X6X7PX8TIRVX9Q. To meet clinical needs, more PSD-95 inhibitors still need to be developed. Summary of the invention

[0009] In order to solve the above problems, the present invention discloses a peptide with neuroprotective effect and application thereof. The peptide with neuroprotective effect can atypically bind to the PDZ1 and / or PDZ2 domains of PSD-95, thereby inhibiting the protein-protein interaction between PSD-95 and nNOS.

[0010] In one aspect, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, characterized in that the amino acid sequence of the polypeptide comprises an amino acid sequence P1-Linker-P2,

[0011] Wherein, P1 is a cell-penetrating peptide (CPP); P2 is the following general formula (1):

[0012] CX 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 C (1)

[0014] in,

[0015] X 3 ~X 17 Each is independently selected from any amino acid or unnatural amino acid;

[0016] Linker selected from (PEG) m ,

[0017] m is independently selected from integers of 0-10.

[0018] In some embodiments, the P1 is selected from the group consisting of YGRKKRRQRRR, rrrqrrkkrGy, rrrqrrkkr, rrrqrrkkrGy, polyarginine consisting of 2 to 30 residues, GRKKRRQRRRPPQQ, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMKWKK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTSTGR, KLALKLALKALKAALKLA, GALFLAFLAAALSL-MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, and PLIYLRLLRGQF.

[0019] In some embodiments, the P1 is selected from YGRKKRRQRRR, rrrqrrkkrGy.

[0020] In some embodiments, the Linker is selected from m is independently selected from integers of 0-10.

[0021] In some embodiments, m is an integer from 0-4.

[0022] In some embodiments, the linker is selected from AEEA, AEEEA.

[0023] In some embodiments, the P3 is the following general formula (1):

[0024] CX 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 C (1)

[0026] in,

[0027] X 3 Selected from K, R, Y, W, Q, V, F, T, H;

[0028] X4 Selected from M, Q, F, C, W, I, Y, K, R, T;

[0029] X 5 Selected from V, Y, E, I, H, W;

[0030] X 6 Select from Y, M, E, S, T;

[0031] X 7 Selected from T, D, H, W, Q, I, Y, L, M, E, V;

[0032] X 8 Selected from K, R, F, D, M, I;

[0033] X 9 Selected from G, S, E, T, D, V, M, W, I, H, Y, A;

[0034] X 10 Selected from N, A, H, D, E, I, K, R, Q, T, S, L, P, V; X 11 Selected from I, V, T, H, A, Q, E, D, R, W, F, L, Y, N; X 12 Selected from D, T, F, V, I, G, N;

[0035] X 13 Selected from F, W, I, V, Q, S, T, L, R, E, D, N;

[0036] X 14 Selected from C, F, V, S, T;

[0037] X 15 Selected from H, M, I, S, C, D, F, Q, K, R, N, T, Y, E; X 16 Selected from T, E, K, L, M, V, Q, R, I, Y, W;

[0038] X 17 Selected from R, W, V, I, R, S, H, E, Y.

[0039] Preferably, wherein

[0040] X 3 Selected from K, R, Y, W, Q;

[0041] X 4 Selected from M, Q, Y, L, T, R, F;

[0042] X 5 Selected from V, Y, E, I, H;

[0043] X 6 Selected from S, T;

[0044] X 7 Selected from L, E, Q, V, T, D, H;

[0045] X 8 Selected from F, M;

[0046] X 9 Selected from G, S, E, T, D;

[0047] X 10 Selected from I, K, R, T, E, D, Q, S;

[0048] X 11 Selected from D, Q, E, L, Y, R;

[0049] X 12 Selected from V, I;

[0050] X 13 Selected from V, T, R, F, Q, E, I;

[0051] X 14 Selected from S, T;

[0052] X 15 Selected from T, Y, E, D, H;

[0053] X 16 Selected from I, Y, W, T, H;

[0054] X 17 Selected from I, Y, E.

[0055] In another aspect, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, characterized in that the amino acid sequence of the polypeptide comprises an amino acid sequence P1-Linker-P2,

[0056] P1 is a cell penetrating peptide (CPP); P2 is the following general formula (1):

[0057] CX 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 C (1)

[0059] in,

[0060] P1 is selected from YGRKKRRQRRR, rrrqrrkkrGy;

[0061] Linker is selected from m is independently selected from integers of 0-10.

[0062] X 3 Selected from K, R, Y, W, Q;

[0063] X 4 Selected from M, Q, Y, L, T, R, F;

[0064] X 5 Selected from V, Y, E, I, H;

[0065] X 6 Selected from S, T;

[0066] X 7 Selected from L, E, Q, V, T, D, H;

[0067] X 8 Selected from F, M;

[0068] X 9 Selected from G, S, E, T, D;

[0069] X 10 Selected from I, K, R, T, E, D, Q, S;

[0070] X 11 Selected from D, Q, E, L, Y, R;

[0071] X 12 Selected from V, I;

[0072] X 13 Selected from V, T, R, F, Q, E, I;

[0073] X 14 Selected from S, T;

[0074] X 15 Selected from T, Y, E, D, H;

[0075] X 16 Selected from I, Y, W, T, H;

[0076] X 17 Selected from I, Y, E.

[0077] In some embodiments, m is an integer selected from 0-4.

[0078] In some embodiments, the linker is selected from AEEA, AEEEA.

[0079] In some embodiments, the X 3 Selected from K.

[0080] In some embodiments, the X 6 Selected from T.

[0081] In some embodiments, the X 8 Selected from F.

[0082] In some embodiments, the X 9 Selected from G.

[0083] In some embodiments, the X 12 Selected from V.

[0084] In some embodiments, the X 14 Selected from T.

[0085] In some embodiments, the X 17 Selected from I.

[0086] Preferably, P2 is the following general formula (2):

[0087] CKX 4 X 5 TX 7 FGX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C(2).

[0088] Preferably, P2 is the following general formula (3):

[0089] CKX 4 X 5 X 6 X 7 X 8 GX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C(3).

[0090] Further preferably, P2 is the following general formula (4):

[0091] CKX 4 X 5 TX 7 FGX 10 X 11 X 12 X 13TX 15 X 16 X 17 C(4).

[0092] In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence P1-Linker-P2,

[0093] P1 is a cell penetrating peptide (CPP); P2 is the following general formula (2):

[0094] CKX 4 X 5 TX 7 FGX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C(2)

[0095] Wherein, P1 is selected from rrrqrrkkrGy, YGRKKRRQRRR; Linker is selected from AEEA, AEEEA. In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence P1-Linker-P2,

[0096] P1 is a cell penetrating peptide (CPP); P2 is the following general formula (3):

[0097] CKX 4 X 5 X 6 X 7 X 8 GX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C(3)

[0098] Among them, P1 is selected from rrrqrrkkrGy, YGRKKRRQRRR; Linker is selected from AEEEA.

[0099] Preferably, the amino acid sequence of the polypeptide comprises the amino acid sequence P1-Linker-P2,

[0100] P1 is a cell penetrating peptide (CPP); P2 is the following general formula (4):

[0101] CKX 4 X 5 TX 7 FGX 10 X 11 X12 X 13 TX 15 X 16 X 17 C(4)

[0102] Among them, P1 is selected from rrrqrrkkrGy, YGRKKRRQRRR; Linker is selected from AEEEA.

[0103] In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence P1-Linker-P2,

[0104] P1 is a cell penetrating peptide (CPP); P2 is the following general formula (3):

[0105] CKX 4 X 5 X 6 X 7 X 8 GX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C(3)

[0106] Among them, P1 is selected from rrrqrrkkrGy, YGRKKRRQRRR; Linker is selected from AEEA.

[0107] Preferably, the amino acid sequence of the polypeptide comprises the amino acid sequence P1-Linker-P2,

[0108] P1 is a cell penetrating peptide (CPP); P2 is the following general formula (4):

[0109] CKX 4 X 5 TX 7 FGX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C(4)

[0110] Among them, P1 is selected from rrrqrrkkrGy, YGRKKRRQRRR; Linker is selected from AEEA.

[0111] In another aspect, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide is selected from:

[0112]

[0113]

[0114] The polypeptides defined herein may be in the form of a pharmaceutically acceptable salt or a prodrug of the polypeptide.

[0115] The AEEA structure in the polypeptide structure of the present invention is The structure of AEEEA is

[0116] In some embodiments, the pharmaceutically acceptable salt is selected from trifluoroacetate, acetate, hydrochloride, and phosphate.

[0117] In another aspect, the present invention provides a pharmaceutical composition comprising the above polypeptide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0118] In some embodiments, the polypeptide of the present application can be administered in the form of a pharmaceutical composition. The pharmaceutical composition can be manufactured by conventional mixing, dissolving, granulating, tableting, grinding, emulsifying, encapsulating, capturing or lyophilizing methods. The pharmaceutical composition can be prepared in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that facilitate processing of the compound of the present application into a pharmaceutically acceptable preparation. Appropriate preparation depends on the selected route of administration.

[0119] In some embodiments, administration may be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular.

[0120] In another aspect, the present invention provides the use of the above polypeptide or its pharmaceutically acceptable salt or polynucleotide or pharmaceutical composition in the preparation of a medicament for treating, ameliorating or preventing a disease caused by PSD-95 dysfunction in an individual. The polypeptide of the present invention is a PSD-95 inhibitor and thus can inhibit excitotoxicity.

[0121] In some embodiments, the disease caused by the abnormal function of PSD-95 is selected from at least one of cerebral infarction, neurodegenerative disease, anxiety or epilepsy, and neuropathic pain.

[0122] In some embodiments, the stroke is selected from ischemic stroke or hemorrhagic stroke.

[0123] In some embodiments, the neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

[0124] The term "amino acid" refers to a molecule containing an amino group and a carboxyl group. Suitable amino acids include, but are not limited to, the D- and L-isomers of naturally occurring amino acids, and non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. As used herein, the term amino acid includes, but is not limited to, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

[0125] The term "naturally occurring amino acid" refers to any of the 20 L-amino acids commonly found in peptides synthesized in nature, i.e., the L-isomers of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0126] The meaning of the term "peptide" or "polypeptide" is well known to those skilled in the art. Generally, a peptide or polypeptide is two or more amino acids linked by an amide bond, which is formed by the amino group of one amino acid and the carboxyl group of the adjacent amino acid. The polypeptides described herein may contain naturally occurring amino acids or non-naturally occurring amino acids. They may be modified into analogs, derivatives, functional mimetics, pseudopeptides, and the like containing at least two amino acids. Unless it is specified that the N-terminus or C-terminus has a specific modification, a polypeptide comprising a specific amino acid sequence includes unmodified and modified amino and / or carboxyl termini, which is well known to those skilled in the art. A polypeptide of a specific amino acid sequence may include modified amino acids and / or additional amino acids, unless the N- and / or C-terminus contain modifications that prevent further addition of amino acids. Such modifications include, for example, acetylation of the N-terminus and / or amidation of the C-terminus.

[0127] The polypeptides of the present invention can be modified to form polypeptide derivatives. As is well known to those skilled in the art, various modifications can be made to the polypeptides. Typical modifications include, but are not limited to, N-terminal acetylation, C-terminal amidation, d-amino acid substitution, non-natural amino acid substitution, fatty acid modification, or a combination of the above modifications. The present invention includes any well-known modification of the polypeptide. For example, the polypeptide derivatives may include chemical modifications to the polypeptide, such as alkylation, acylation, carbamylation, iodination, or any other modification that produces a polypeptide derivative. The modification of the polypeptide may include modified amino acids, for example, hydroxyproline or carboxyglutamic acid, and may include amino acids connected by non-peptide bonds.

[0128] For other modifications of the polypeptide of the present invention, non-natural amino acids can be used to replace the natural amino acids in the polypeptide, including but not limited to 2-amino fatty acid (Aad), 3-amino fatty acid (βAad), β-alanine, β-aminopropionic acid (βAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid, piperidine carboxylic acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (βAib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2'- Diaminopimelate (Dpm), 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), isoleucine (aIle), N-methylglycine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle) and ornithine (Orn). Of course, all modified α-amino acids can be replaced by the corresponding β-, γ- or ω-aminocarboxylic acids.

[0129] The polypeptides of the present invention can be prepared using methods well known to those skilled in the art, including well-known chemical synthesis methods. Therefore, when a polypeptide or its derivatives contain one or more non-standard amino acids, it is very likely to be prepared by chemical synthesis. In addition to using chemical synthesis methods to prepare polypeptides or their derivatives, they can also be prepared by expressing encoding nucleic acids. This is particularly applicable to the preparation of polypeptides or their derivatives containing only natural amino acids, in which case well-known methods for preparing nucleic acid encoding polypeptide sequences can be used (see Sambrook et al., Molecular Cloning: A La bora tory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). The polypeptide can be expressed in an organism and purified by well-known purification techniques.

[0130] The term "PDZ domain" refers to a modular protein domain of about 90 amino acids characterized by significant (e.g., at least 60%) sequence identity to the brain synaptic protein PSD-95, the Drosophila septate junction protein Discs-Large (DLG), and the epithelial tight junction protein Z01 (Z01). PDZ domains are also known as Discs-Large homology repeats ("DHRs") and GLGF repeats. PDZ domains generally show retention of a core consensus sequence (Doyle, DA, 1996, Cell 85: 1067-76). Exemplary PDZ domain-containing proteins and PDZ domain sequences are disclosed in U.S. Application No. 10 / 714,537. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 This is the HPLC detection chart of compound 1 in Example 1;

[0132] Figure 2 This is the mass spectrum detection diagram of compound 1 in Example 1;

[0133] Figure 3 This is the HPLC detection chart of compound 2 in Example 2;

[0134] Figure 4 This is the mass spectrum detection diagram of compound 2 in Example 2;

[0135] Figure 5 This is the HPLC detection chart of compound 3 in Example 3;

[0136] Figure 6 This is the mass spectrum detection diagram of compound 3 in Example 3. Specific embodiments

[0137] The polypeptide compound and its derivative provided by the present invention adopt a solid phase synthesis method to synthesize its linear precursor, and the crude peptide obtained after cleavage is directly purified to obtain the target compound, or the crude peptide is oxidized with DMSO to form intramolecular disulfide bonds and then purified to obtain the target compound. The synthetic carrier is 2-Chlotrityl Resin resin. During the synthesis process, the 2-Chlotrityl Resin resin is first fully swollen in N, N-dimethylformamide (DMF), and then the solid phase carrier and the activated amino acid derivative are repeatedly condensed → washed → Fmoc protection → washed → the next round of amino acid condensation to achieve the desired length of the synthesized polypeptide chain, and finally a mixed solution of trifluoroacetic acid: water: triisopropylsilane: anisyl thioether (90: 2.5: 2.5: 5, v: v: v: v) is reacted with the resin to cleave the polypeptide from the solid phase carrier, and then the solid crude product of the linear precursor is obtained after precipitation by frozen methyl tert-butyl ether. The crude linear precursor after cleavage is oxidized by disulfide bonds in a neutral solution to obtain the crude target polypeptide. The solid crude product or the oxidized polypeptide crude product is purified and separated by a C18 reverse phase preparative chromatography column in a system of 0.1% trifluoroacetic acid in acetonitrile / water to obtain pure polypeptide and its derivatives.

[0138] Experimental reagents

[0139]

[0140]

[0141]

[0142] Example 1 Synthesis of Compound 1

[0143]

[0144] Step 1: Coupling of the first amino acid Fmoc-Cys(Trt)-OH

[0145] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Cys(Trt)-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed and dissolved in 5 ml of DCM and added to the resin to react at room temperature for 2 h. After the reaction was completed, a blocking solution (10 ml) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0146] Step 2: Synthesis of linear precursor peptide chain

[0147] Linear precursor peptide chain of compound 1

[0148] dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY-AEEA-CKMVTTFGIDVTTTTYIC

[0149] The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second position of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

[0150] · Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0151] • Rinse the resin 6-8 times with DMF until neutral pH.

[0152] Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 h.

[0153] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0154] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0155] Step 3: Cleavage of the linear precursor peptide chain

[0156] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 2 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0157] Step 4: Intramolecular disulfide bond formation

[0158] Add DMSO to the crude product obtained in step 3 to fully dissolve (the volume of DMSO is 20% of the total volume of the reaction system), then slowly add the dissolved polypeptide solution dropwise to 50% acetonitrile aqueous solution to a final concentration of 1 mg / ml, and shake at room temperature for 16 hours. LC-MS monitors the reaction results, and purification is directly performed after the reaction is completed.

[0159] Step 5: Peptide purification

[0160] After filtering through a 0.45um membrane, the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL / min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide.

[0161] Step 6: Detection and characterization methods

[0162] The purified peptide obtained in step 5 was subjected to analytical high performance liquid chromatography and liquid chromatography / mass spectrometry to determine the purity and intramolecular disulfide bond formation of the compound. The results of liquid chromatography and mass spectrometry are as follows: Figure 1 , Figure 2 shown.

[0163] Example 2 Synthesis of Compound 2

[0164]

[0165] Step 1: Coupling of the first amino acid Fmoc-Cys(Trt)-OH

[0166] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Cys(Trt)-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed and dissolved in 5 ml of DCM and added to the resin to react at room temperature for 2 h. After the reaction was completed, a blocking solution (10 ml) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0167] Step 2: Synthesis of linear precursor peptide chain

[0168] Linear precursor peptide chain of compound 2

[0169] dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY-AEEA-CKQYTDFGKQVTTYWIC

[0170] The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second position of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

[0171] · Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0172] • Rinse the resin 6-8 times with DMF until neutral pH.

[0173] Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 h.

[0174] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0175] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0176] Step 3: Cleavage of the linear precursor peptide chain

[0177] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 2 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0178] Step 4: Intramolecular disulfide bond formation

[0179] Add DMSO to the crude product obtained in step 3 to fully dissolve (the volume of DMSO is 20% of the total volume of the reaction system), then slowly add the dissolved polypeptide solution dropwise to 50% acetonitrile aqueous solution to a final concentration of 1 mg / ml, and shake at room temperature for 16 hours. LC-MS monitors the reaction results, and purification is directly performed after the reaction is completed.

[0180] Step 5: Peptide purification

[0181] After filtering through a 0.45um membrane, the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL / min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide.

[0182] Step 6: Detection and characterization methods

[0183] The purified peptide obtained in step 5 was subjected to analytical high performance liquid chromatography and liquid chromatography / mass spectrometry to determine the purity and intramolecular disulfide bond formation of the compound. The results of liquid chromatography and mass spectrometry are as follows: Figure 3 , Figure 4 shown.

[0184] Example 3 Synthesis of Compound 3

[0185]

[0186] Step 1: Coupling of the first amino acid Fmoc-Cys(Trt)-OH

[0187] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Cys(Trt)-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed and dissolved in 5 ml of DCM and added to the resin to react at room temperature for 2 h. After the reaction was completed, a blocking solution (10 ml) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0188] Step 2: Synthesis of linear precursor peptide chain

[0189] Linear precursor peptide chain of compound 3

[0190] dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY-AEEA-CKFETHFGREIVTEIYC

[0191] The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second position of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

[0192] · Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0193] • Rinse the resin 6-8 times with DMF until neutral pH.

[0194] Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 h.

[0195] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0196] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0197] Step 3: Cleavage of the linear precursor peptide chain

[0198] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 2 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0199] Step 4: Intramolecular disulfide bond formation

[0200] Add DMSO to the crude product obtained in step 3 to fully dissolve (the volume of DMSO is 20% of the total volume of the reaction system), then slowly add the dissolved polypeptide solution dropwise to 50% acetonitrile aqueous solution to a final concentration of 1 mg / ml, and shake at room temperature for 16 hours. LC-MS monitors the reaction results, and purification is directly performed after the reaction is completed.

[0201] Step 5: Peptide purification

[0202] After filtering through a 0.45um membrane, the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL / min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide.

[0203] Step 6: Detection and characterization methods

[0204] The purified peptide obtained in step 5 was subjected to analytical high performance liquid chromatography and liquid chromatography / mass spectrometry to determine the purity and intramolecular disulfide bond formation of the compound. The results of liquid chromatography and mass spectrometry are as follows: Figure 5 , Figure 6 shown.

[0205] Example 4 Synthesis of Compound 4

[0206]

[0207] Step 1: Coupling of the first amino acid Fmoc-Cys(Trt)-OH

[0208] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Cys(Trt)-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed and dissolved in 5 ml of DCM and added to the resin to react at room temperature for 2 h. After the reaction was completed, a blocking solution (10 ml) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0209] Step 2: Synthesis of linear precursor peptide chain

[0210] Linear precursor peptide chain of compound 4

[0211] dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY-AEEEA-CKFETHFGREIVTEIYC

[0212] The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second position of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

[0213] · Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0214] • Rinse the resin 6-8 times with DMF until neutral pH.

[0215] Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 h.

[0216] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0217] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0218] Step 3: Cleavage of the linear precursor peptide chain

[0219] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 2 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0220] Step 4: Intramolecular disulfide bond formation

[0221] Add DMSO to the crude product obtained in step 3 to fully dissolve (the volume of DMSO is 20% of the total volume of the reaction system), then slowly add the dissolved polypeptide solution dropwise to 50% acetonitrile aqueous solution to a final concentration of 1 mg / ml, and shake at room temperature for 16 hours. LC-MS monitors the reaction results, and purification is directly performed after the reaction is completed.

[0222] Step 5: Peptide purification

[0223] After filtering through a 0.45um membrane, the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL / min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide.

[0224] Step 6: Detection and characterization methods

[0225] The purity of the pure polypeptide obtained in step 5 and the formation of intramolecular disulfide bonds of the compound are determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry.

[0226] Test Example 1 ELISA test peptide sample inhibits the binding of Tat-NR2B9c-B and cMyc-PSD95 alpha 1-392

[0227] (1) Experimental materials:

[0228]

[0229] (2) Experimental steps:

[0230] The ELISA (enzyme-linked immunosorbent assay) method was used to test the inhibitory effect of peptide samples on Tat-NR2B9c-B binding to cMyc-PSD95alpha 1-392. cMyc-PSD95 alpha 1-392 was coated on a 384-well plate (greiner 781097) at 0.37μg / mL, 25μL / well. After blocking the ELSIA plate coated with cMyc-PSD95 alpha 1-392, the peptide samples were diluted to 0-10μM (from 10μM down 3-fold gradient dilution for a total of 8 concentrations). The workstation was used to transfer 12.5μL / well of the peptide sample to the 384-well plate and centrifuged to remove bubbles. Then, an 8-well pipette (10-100μL) was used to transfer 12.5μL / well of 12nM Tat-NR2B9c-B, centrifuged to remove bubbles, and incubated at 37℃ for 1h. Discard the liquid in the wells, add 80μL 1×TBST washing solution with pH=7.4 and wash the plate 3-5 times, 3-5 minutes each time. Drain the test plate and add 25μL / well of 1:10000 diluted Streptavidin HRP, centrifuge to remove bubbles and incubate in a 37℃ incubator for 1h. Wash the plate again and drain the test plate, add 25μl TMB colorimetric solution to each well, and continue to incubate in the incubator at 37℃ for 30min. Finally, add 25μl stop solution (1MHCL) to each well to terminate the reaction. Use the microplate reader Cytation5 to read the absorbance at 450nm.

[0231] The experimental results show that the compounds of the present invention can inhibit the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392 and have a good inhibitory effect. The affinity of the polypeptide of the present invention to cMyc-PSD95 alpha 1-392 is stronger than that of Tat-NR2B9c-B. Test Example 2 ELISA test of polypeptide samples inhibiting the binding of biotin-nNOS1-299 to cMyc-PSD95 alpha 1-392 (1) Experimental materials:

[0232]

[0233] (2) Experimental steps:

[0234] The ELISA (enzyme-linked immunosorbent assay) method was used to test the inhibitory effect of peptide samples on biotin-nNOS1-299 binding to cMyc-PSD95 alpha 1-392. cMyc-PSD95 alpha 1-392 was coated on a 384-well plate (greiner 781097) at 10μg / mL, 25μL / well. After blocking the ELSIA plate coated with cMyc-PSD95 alpha 1-392, the peptide samples were diluted to 0-10μM (from 10μM down 3-fold gradient dilution for a total of 8 concentrations). The peptide sample was transferred to the 384-well plate at 12.5μL / well using the workstation and centrifuged to remove bubbles. Then use an 8-well pipette (10-100μL) to pipette 12.5μL / well of 10μg / mL 743biotin-nNOS1-299, centrifuge to remove bubbles, and incubate at 37℃ for 1h. Discard the liquid in the wells, add 80μl of 1×TBST washing solution with pH=7.4, wash the plate 3-5 times, 3-5min each time. Control the test plate to dry and add 25μL / well of 1:10000 diluted Streptavidin HRP, centrifuge to remove bubbles, and incubate in a 37℃ incubator for 1h. After washing the plate again and controlling the test plate to dry, add 25μL TMB colorimetric solution to each well and continue to incubate in the incubator at 37℃ for 30min. Finally, add 25μL of stop solution (1M HCL) to each well to terminate the reaction. Use the microplate reader Cytation5 to read the absorbance at 450nm.

[0235] The experimental results show that the compound of the present invention can inhibit the binding of biotin-nNOS1-299 and cMyc-PSD95 alpha1-392 and has a good inhibitory effect.

[0236] Test Example 3 FRET Test Peptide Sample Inhibits the Binding of cMyc-PSD95 alpha 1-392 and Tat-NR2B9C-B

[0237] (1) Experimental materials:

[0238]

[0239] (2) Experimental steps:

[0240] The FRET (fluorescence resonance energy transfer) method was used to test the inhibitory effect of peptide samples on the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B. The peptide samples were diluted to 0-10 μM (from 10 μM down 3-fold gradient dilution for a total of 8 concentrations). Use an 8-well gun (1-10 μL) to transfer 4 μL / well of 4nM cMyc-PSD95 alpha 1-392 to a 384-well white plate (Thermo 264706) and centrifuge to remove bubbles. Use a workstation to transfer 4 μL / well of the peptide sample to a 384-well plate and centrifuge to remove bubbles. Use an 8-well gun again to transfer 4 μL / well of 20nM Tat-NR2B9c-B to a 384-well white plate and centrifuge to remove bubbles. Mix the prepared 0.02μg / mL EU-steptavidin with 1ug / mL Mouse Anti-C-MYC IgG SureLight APC in a 1:1 ratio. Use an 8-well pipette to transfer 8μL / well of the EU-APC premix to a 384-well white plate and centrifuge to remove bubbles. Incubate at room temperature in the dark for 2 hours and use a microplate reader cytation5 to read the emission fluorescence values ​​at 320nM excitation 620nm and 665nm.

[0241] The experimental results show that the compound of the present invention can inhibit the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B and has a good inhibitory effect. The affinity of the polypeptide of the present invention to cMyc-PSD95 alpha 1-392 is stronger than that to Tat-NR2B9c-B.

[0242] Test Example 4 FP Test Peptide Sample Inhibits the Binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2

[0243] (1) Experimental materials:

[0244]

[0245] (2) Experimental steps:

[0246] The FP (fluorescence polarization immunoassay) method was used to test the inhibitory effect of peptide samples on the binding of 6H-PSD95 alpha 61-249 to 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2. The peptide samples were diluted to 0-10μM (from 10μM down 3-fold gradient dilution for a total of 8 concentrations). Use an 8-well pipette (1-10μl) to transfer 5μL / well of 5nM 6H-PSD95 alpha 61-249 to a 384-well plate (Corning 3575) and centrifuge to remove bubbles. Use a workstation to transfer 5μL / well of the peptide sample to a 384-well plate and centrifuge to remove bubbles. Use an 8-well pipette to transfer 5 μL / well of 0.5 nM 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2 to a 384-well plate, centrifuge briefly to remove bubbles, incubate at room temperature in the dark for 2 h, and read the values ​​using an ELISA reader, Cytation5 FP module.

[0247] The experimental results show that the compounds of the present invention can inhibit the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2, and have a good inhibitory effect. The affinity of the polypeptide of the present invention to 6H-PSD95 alpha 61-249 is stronger than that of FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2.

[0248] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the polypeptide comprises the amino acid sequence P1-Linker-P2, Wherein, P1 is a cell-penetrating peptide; P2 is the following general formula (1): CX 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 C (1) in, X 3 ~X 17 Each is independently selected from any amino acid or unnatural amino acid; Linker selected from (PEG) m , m is independently selected from integers of 0-10.

2. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The P1 is selected from YGRKKRRQRRR, rrrqrrkkrGy, rrrqrrkkr, rrrqrrkkrGy, polyarginine composed of 2 to 30 residues, GRKKRRQRRRPPQQ, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMKWKK, LLIILRRRIRKQAHAHSK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTSTGR, KLALKLALKALKAALKLA, GALFLAFLAAALSL-MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, PLIYLRLLRGQF, preferably, the P1 is selected from YGRKKRRQRRR, rrrqrrkkrGy.

3. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The Linker is selected from m is independently selected from integers of 0-10.

4. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The P3 is the following general formula (1): CX 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 C (1) in, X 3 Selected from K, R, Y, W, Q, V, F, T, H; X 4 Selected from M, Q, F, C, W, I, Y, K, R, T; X 5 Selected from V, Y, E, I, H, W; X 6 Select from Y, M, E, S, T; X 7 Selected from T, D, H, W, Q, I, Y, L, M, E, V; X 8 Selected from K, R, F, D, M, I; X 9 Selected from G, S, E, T, D, V, M, W, I, H, Y, A; X 10 Selected from N, A, H, D, E, I, K, R, Q, T, S, L, P, V; X 11 Selected from I, V, T, H, A, Q, E, D, R, W, F, L, Y, N; X 12 Selected from D, T, F, V, I, G, N; X 13 Selected from F, W, I, V, Q, S, T, L, R, E, D, N; X 14 Selected from C, F, V, S, T; X 15 Selected from H, M, I, S, C, D, F, Q, K, R, N, T, Y, E; X 16 Selected from T, E, K, L, M, V, Q, R, I, Y, W; X 17 Selected from R, W, V, I, R, S, H, E, Y.

5. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the polypeptide comprises the amino acid sequence P1-Linker-P2, Wherein, P1 is a cell-penetrating peptide; P2 is the following general formula (1): CX 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 C (1) in, P1 is selected from YGRKKRRQRRR, rrrqrrkkrGy; Linker is selected from m is independently selected from integers of 0 to 10; X 3 Selected from K, R, Y, W, Q; X 4 Selected from M, Q, Y, L, T, R, F; X 5 Selected from V, Y, E, I, H; X 6 Selected from S, T; X 7 Selected from L, E, Q, V, T, D, H; X 8 Selected from F, M; X 9 Selected from G, S, E, T, D; X 10 Selected from I, K, R, T, E, D, Q, S; X 11 Selected from D, Q, E, L, Y, R; X 12 Selected from V, I; X 13 Selected from V, T, R, F, Q, E, I; X 14 Selected from S, T; X 15 Selected from T, Y, E, D, H; X 16 Selected from I, Y, W, T, H; X 17 Selected from I, Y, E.

6. The polypeptide or pharmaceutically acceptable salt thereof according to claim 5, characterized in that: The P2 is the following general formula (2): CKX 4 X 5 TX 7 FGX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C(2); Preferably, P2 is the following general formula (3): CKX 4 X 5 X 6 X 7 X 8 GX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C(3); Further preferably, the P2 is the following general formula (4): CKX 4 X 5 TX 7 FGX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C(4).

7. The polypeptide or pharmaceutically acceptable salt thereof according to claim 5, characterized in that: The linker is selected from AEEA and AEEEA.

8. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide is selected from:

9. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

10. Use of the polypeptide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating, ameliorating or preventing a disease caused by PSD-95 dysfunction in an individual, wherein the disease caused by PSD-95 dysfunction is selected from at least one of cerebral stroke, apoplexy, neurodegenerative disease, anxiety or epilepsy, and neuropathic pain; preferably, the cerebral stroke is selected from ischemic stroke or hemorrhagic stroke; the neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease or Huntington's disease.

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