Polypeptides for treating neuro-related diseases

Abstract

The application belongs to the technical field of biological medicine polypeptide, and particularly relates to a polypeptide for treating nerve-related diseases, wherein the polypeptide can atypically combine with the PDZ1 and / or PDZ2 domain of PSD-95, thereby inhibiting the protein-protein interaction with nNOS, has a relatively small molecular weight, is easy to realize in synthesis, expression and modification, and has a good inhibition efficiency on the combination of PSD95 and its receptor.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a polypeptide for treating neurological diseases. Background Technology

[0002] N-methyl-D-aspartate receptors (NMDARs) play a crucial role in the central nervous system, participating in the formation of learning and memory, synapse formation during central nervous system development, plasticity development, and glutamate-mediated neurotoxicity. NMDA receptors are major mediators of excitotoxicity (i.e., glutamate-mediated neurotoxicity), and are associated with neurodegenerative diseases and acute brain injury. They can serve as therapeutic targets for certain neurological disorders, such as cerebral infarction, neuropathic pain, epilepsy, and schizophrenia.

[0003] Postsynaptic density (PSD) is a supersignaling molecule complex on the excitatory postsynaptic membrane and is an important substance for synaptic transmission. Based on 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 scaffold protein, primarily found in mature excitatory glutamatergic synapses. With a relative molecular mass (Mr) of 95,000, it is essential for receptor activity and stability on the postsynaptic membrane, playing a crucial role in synaptic plasticity. It is the most important synaptic protein mediating and integrating synaptic information and is also a member of the guanylate-associated kinase family. PSD-95 comprises three PDZ domains, one SH3 domain, and one guanylate kinase-like (GK) domain, connected by a connective region. PSD-95 is almost entirely located in the postsynaptic density region of neurons and participates in anchoring synaptic proteins. Its direct and indirect binding partners include neuroconnectins, nNOS, NMDA receptors, AMPA receptors, and potassium channels. Based on different mRNA splicing methods, more than 10 nNOS (Neuronal nitric oxide synthase) splice variants are currently known, with a molecular weight of 160.8 kDa. nNOS contains both a C-terminal reducing domain and an N-terminal oxidizing domain. The N-terminus has two non-overlapping binding regions: (1) PDZ region: composed of 1-99 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 cytosol proteins through the PDZ-PDZ domain or C-terminal PDZ reaction. PSD-95 can link nNOS to NMDA receptors, thereby effectively activating nNOS through the activation of NMDA receptors.

[0005] Stroke is characterized by neuronal death in areas of localized ischemia, hemorrhage, and / or trauma. Neuronal death or damage caused by cerebral ischemia is a cascade of damage. After cerebral ischemia, decreased tissue blood perfusion leads to increased excitatory neurotransmitters, activating NMDA and AMPA receptors, causing ion channel opening, calcium ion influx, and activation of numerous enzymes, triggering a signaling cascade that results in multi-pathway neuronal damage. During cerebral ischemia, NMDA (N-methyl-D-aspartate receptor) receptors are overactivated. Blocking the NMDA / PSD-95 / nNOS pathway can prevent the pathological release of NO. However, studies have shown that blocking the NMDA / PSD-95 coupling may produce unpredictable physiological responses. Although NMDA receptor antagonists effectively reduce excitotoxicity by blocking glutamate-mediated ion flux, they also inhibit some physiologically important processes. PSD-95, a downstream protein, interacts with various proteins to trigger a series of ischemic injuries, making it a key site for ischemic brain injury and a potential target for drug therapy. Blocking the coupling between nNOS and PSD95 is more targeted at preventing the pathological release of NO, thus making it a more ideal target for the prevention and treatment of neuronal damage-related diseases such as ischemic stroke. Therefore, the development of PSD-95 inhibitors has significant pharmaceutical implications for neurological injuries caused by various excitotoxic neurotoxicities, including stroke.

[0006] Furthermore, studies have shown that the excitatory neurotransmitter NMDA plays a crucial role in anxiety, epilepsy, and various neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Huntington's disease. For example, research indicates that excessive excitation of the central glutamatergic system can trigger anxiety, and the NMDA receptor (NMDAR) is responsible for the main part of glutamate excitatory neurotoxicity. Epilepsy involves three distinct but continuous pathophysiological processes: initiation, maintenance and spread of paroxysmal discharges, and inhibition of paroxysmal discharges. Excitatory neurotransmitters such as glutamate and aspartate play important roles in these processes. In Alzheimer's disease, PSD-95 participates in the neurotoxic mechanism through the GluR6-PSD-95-MLK3 pathway. Moreover, 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 these diseases. Nerinetide (NA-1, TAT-NR2B9c, sequence: YGRKKRRQRRRKLSSIESDV) is a PSD-95 inhibitor that disrupts the binding of PSD-95 to NMDA receptors and neuronal nitric oxide synthase (nNOS), and reduces excitotoxicity induced by cerebral ischemia. In preclinical ischemic stroke membranous encephalopathy, it reduces infarct size due to ischemia-reperfusion and improves functional outcomes. Nerinetide has no serious side effects and can be used in suspected stroke or other ischemic or hemorrhagic conditions where a diagnosis of hemorrhage has not yet been confirmed according to established criteria. However, Nerinetide has a short half-life in vivo, requiring high daily doses, leading to poor patient compliance, high clinical costs, and its low affinity for PDZ1-2 in PSD-95 may render it an ineffective, non-selective compound. To meet clinical needs, further development of more PSD-95 inhibitors is required.

[0007] Polypeptides are bioactive substances that constitute various cellular functions in the body. They are characterized by their small relative molecular mass, high specificity, easy absorption, easy synthesis and modification, ability to enhance the body's immunity, and high safety, making them highly valuable in the clinical treatment of tumors. Phage display technology utilizes filamentous bacteriophages to display proteins and peptides, extracting peptides or proteins with desired properties from a large number of variants.

[0008] Phage display technology, a high-throughput screening technique based on directed evolution, has greatly expanded the application of directed evolution technology in peptide modification and screening. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention discloses a polypeptide for treating neurological diseases, wherein the PSD-95 inhibitor can atypically bind to the PDZ1 and / or PDZ2 domains of PSD-95, thereby inhibiting its protein-protein interaction with nNOS.

[0010] On one hand, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence P1-P2-P3.

[0011] Wherein, P1 is cell-penetrating peptide (CPP); P2 is absent or is X. 1 X 2 P3 is given by 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 1 Selected from G, A, V, L, or I;

[0016] X 2 Selected from S, T, C, Y, N, or Q;

[0017] X 3 ~X 17 Each amino acid is independently selected from any amino acid or non-natural amino acid.

[0018] In some embodiments, P1 is selected from YGRKKRRQRRR, yGrkkrrqrrr, rrrqrrkkrGy, polyarginine consisting of 2 to 30 residues, GRKKRRQRRRPPQQ, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMK-WKK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTST-GR, KLALKLALKALKAALKLA, GALFLAFLAAALSL-MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, PLIYLRLLRGQF; preferably, the cell-penetrating peptide (CPP) is selected from YGRKKRRQRRR or yGrkkrrqrrr.

[0019] In some implementations, the X 1 Selected from A; X 2 Selected from S.

[0020] In some implementations, the X 3 Choose from K, R, Y, T, W, or Q.

[0021] In some implementations, the X 4 Selected from M, Q, Y, L, T, F, or R.

[0022] In some implementations, the X 5 Selected from V, E, L, W, K, M, I, H, or Y.

[0023] In some implementations, the X 6 Let T be the value.

[0024] In some implementations, the X 7 Select from T, L, E, D, H, Q, or V.

[0025] In some implementations, the X 8 It is F.

[0026] In some implementations, the X 9 Choose from G, S, E, T, or D.

[0027] In some implementations, the X 10 Selected from I, T, E, D, R, K, Q, or S;

[0028] In some implementations, the X 11Choose from D, L, E, Q, Y, or R.

[0029] In some implementations, the X 12 Selected from V and I.

[0030] In some implementations, the X 13 Choose from T, R, V, I, F, Q, or E.

[0031] In some implementations, the X 14 Choose from T or S.

[0032] In some implementations, the X 15 Choose from T, D, Y, F, H or E.

[0033] In some implementations, the X 16 Choose from I, Y, T, W, or H.

[0034] In some implementations, the X 17 Choose from I, Y, or E.

[0035] In some implementations, P3 is of the following general formula (2):

[0036] CKX 4 X 5 TX 7 FGX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C (2)

[0038] Among them, X 4 Selected from M, Q, or F;

[0039] X 5 Selected from V, Y, or E;

[0040] X 7 Selected from T, D, or H;

[0041] X 10 Selected from I, K, or R;

[0042] X 11 Choose from D, Q, or E;

[0043] X 12 Selected from V or I;

[0044] X 13 Selected from V or T;

[0045] X 15Selected from T, Y, or E;

[0046] X 16 Choose from I, Y, or W;

[0047] X 17 Choose from I or Y.

[0048] On the other hand, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide having an amino acid sequence comprising the amino acid sequence P1-P2-P3.

[0049] Wherein, P1 is cell-penetrating peptide (CPP); P2 is X 1 X 2 P3 is given by the following general formula (1):

[0050] 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)

[0052] in,

[0053] X 1 A;

[0054] X 2 S;

[0055] X 3 ~X 17 Each amino acid is independently selected from any amino acid or non-natural amino acid;

[0056] The cell-penetrating peptide (CPP) is selected from YGRKKRRQRRR, yGrkkrrqrrr, rrrqrrkkrGy, polyarginine consisting of 2 to 30 residues, GRKKRRQRRRPPQQ, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMKWKK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTSTGR, KLALKLALKALKAALKLA, GALFLAFLAAALSL-MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, and PLIYLRLLRGQF.

[0057] In some embodiments, the cell-penetrating peptide (CPP) is selected from YGRKKRRQRRR or yGrkkrrqrrr.

[0058] In some implementations, P3 is of the following general formula (2):

[0059] CKX 4 X 5 TX 7 FGX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C (2)

[0061] in,

[0062] X 4 Selected from M, Q, or F;

[0063] X 5 Selected from V, Y, or E;

[0064] X 7 Selected from T, D, or H;

[0065] X 10 Selected from I, K, or R;

[0066] X 11 Choose from D, Q, or E;

[0067] X 12 Selected from V or I;

[0068] X 13 Selected from V or T;

[0069] X 15 Selected from T, Y, or E;

[0070] X 16 Choose from I, Y, or W;

[0071] X 17 Choose from I or Y.

[0072] The present invention also provides a polypeptide or a pharmaceutically acceptable salt thereof, characterized in that the amino acid sequence of the polypeptide comprises amino acid sequences P1-P3, wherein P1 is a cell-penetrating peptide (CPP); and P3 is the following general formula (1):

[0073] 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)

[0075] in,

[0076] X 3 ~X 17 Each amino acid is independently selected from any amino acid or non-natural amino acid;

[0077] The cell-penetrating peptide (CPP) is selected from YGRKKRRQRRR, yGrkkrrqrrr, rrrqrrkkrGy, polyarginine consisting of 2 to 30 residues, GRKKRRQRRRPPQQ, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMKWKK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTSTGR, KLALKLALKALKAALKLA, GALFLAFLAAALSL-MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, and PLIYLRLLRGQF.

[0078] In some embodiments, the cell-penetrating peptide (CPP) is selected from YGRKKRRQRRR or yGrkkrrqrrr.

[0079] In some implementations, P3 is of the following general formula (2):

[0080] CKX 4 X 5 TX 7 FGX 10 X 11 X 12 X 13 TX 15 X 16 X 17 C (2)

[0082] in,

[0083] X 4 Selected from M, Q, or F;

[0084] X 5 Selected from V, Y, or E;

[0085] X 7 Selected from T, D, or H;

[0086] X 10 Selected from I, K, or R;

[0087] X 11 Choose from D, Q, or E;

[0088] X 12 Selected from V or I;

[0089] X 13 Selected from V or T;

[0090] X 15 Selected from T, Y, or E;

[0091] X 16 Choose from I, Y, or W;

[0092] X 17 Choose from I or Y.

[0093] In some embodiments, the amino acid sequence of the polypeptide comprises the amino acid sequence SA-P1-P2-P3.

[0094] Wherein, P1 is cell-penetrating peptide (CPP); P2 is absent or is X. 1 X 2 P3 is given by the following general formula (1):

[0095] CX 3 X 4 X5 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)

[0097] in,

[0098] X 1 Selected from G, A, V, L, or I;

[0099] X 2 Selected from S, T, C, Y, N, or Q;

[0100] X 3 ~X 17 Each amino acid is independently selected from any amino acid or non-natural amino acid.

[0101] In some embodiments, P1 is selected from YGRKKRRQRRR, yGrkkrrqrrr, rrrqrrkkrGy, polyarginine consisting of 2 to 30 residues, GRKKRRQRRRPPQQ, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMK-WKK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTST-GR, KLALKLALKALKAALKLA, GALFLAFLAAALSL-MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, PLIYLRLLRGQF; preferably, the cell-penetrating peptide (CPP) is selected from YGRKKRRQRRR or yGrkkrrqrrr.

[0102] In some implementations, the X 1 Selected from A; X 2 Selected from S.

[0103] In some implementations, the X 3 Choose from K, R, Y, T, W, or Q.

[0104] In some implementations, the X 4 Selected from M, Q, Y, L, T, F, or R.

[0105] In some implementations, the X 5 Selected from V, E, L, W, K, M, I, H, or Y.

[0106] In some implementations, the X 6 Let T be the value.

[0107] In some implementations, the X 7 Select from T, L, E, D, H, Q, or V.

[0108] In some implementations, the X 8 It is F.

[0109] In some implementations, the X 9 Choose from G, S, E, T, or D.

[0110] In some implementations, the X 10 Selected from I, T, E, D, R, K, Q, or S;

[0111] In some implementations, the X 11 Choose from D, L, E, Q, Y, or R.

[0112] In some implementations, the X 12 Selected from V and I.

[0113] In some implementations, the X 13 Choose from T, R, V, I, F, Q, or E.

[0114] In some implementations, the X 14 Choose from T or S.

[0115] In some implementations, the X 15 Choose from T, D, Y, F, H or E.

[0116] In some implementations, the X 16 Choose from I, Y, T, W, or H.

[0117] In some implementations, the X 17 Choose from I, Y, or E.

[0118] In some implementations, P3 is of the following general formula (2):

[0119] CKX 4 X 5 TX 7 FGX 10 X 11 X 12 X 13 TX 15X 16 X 17 C (2)

[0121] Among them, X 4 Selected from M, Q, or F;

[0122] X 5 Selected from V, Y, or E;

[0123] X 7 Selected from T, D, or H;

[0124] X 10 Selected from I, K, or R;

[0125] X 11 Choose from D, Q, or E;

[0126] X 12 Selected from V or I;

[0127] X 13 Selected from V or T;

[0128] X 15 Selected from T, Y, or E;

[0129] X 16 Choose from I, Y, or W;

[0130] X 17 Choose from I or Y.

[0131] In some embodiments, the polypeptide amino acid sequence comprises the amino acid sequence SAYGRKKRRQRRRASCX 3 X 4 X 5 TX 7 FX 9 X 10 X 11 X 12 TTX 15 X 16 X 17 C, where

[0132] X 3 Selected from K, R, Y, T, W, or Q;

[0133] X 4 Selected from M, Q, Y, L, T, F, or R;

[0134] X 5 Selected from V, E, L, W, K, M, I, H, or Y;

[0135] X 7 Selected from T, L, E, D, H, Q, or V;

[0136] X 9 Selected from G, S, E, T, or D;

[0137] X 10 Selected from I, T, E, D, R, K, Q, or S;

[0138] X 11 Selected from D, L, E, Q, Y, or R;

[0139] X 12 Selected from V and I;

[0140] X 15 Selected from T, D, Y, F, H, or E;

[0141] X 16 Selected from I, Y, T, W, or H;

[0142] X 17 Choose from I, Y, or E.

[0143] In some embodiments, the polypeptide amino acid sequence comprises the amino acid sequence SAYGRKKRRQRRRASCKX 4 X 5 TX 7 FX 9 X 10 X 11 X 12 TTX 15 X 16 X 17 C.

[0144] In some embodiments, the polypeptide amino acid sequence comprises the amino acid sequence SAYGRKKRRQRRRASCKQX 5 TX 7 FX 9 X 10 X 11 X 12 TTX 15 X 16 X 17 C.

[0145] In some embodiments, the polypeptide amino acid sequence comprises the amino acid sequence SAYGRKKRRQRRRASCKX 4 X 5 TX 7 FX 9 X 10 X 11 X 12 TTTX 16 X 17 C.

[0146] In some embodiments, the polypeptide amino acid sequence comprises the amino acid sequence SAYGRKKRRQRRRASCKX 4 X 5 TX 7 FX 9 X 10 X 11 X 12 TTX 15 X 16 IC.

[0147] In some embodiments, the polypeptide amino acid sequence comprises the amino acid sequence SAYGRKKRRQRRRCX 3 X 4 X 5 TX 7 FX 9 X 10 X 11 X 12 TTX 15 X 16 X 17 C, where

[0148] X 3 Selected from K, R, Y, T, W, or Q;

[0149] X 4 Selected from M, Q, Y, L, T, F, or R;

[0150] X 5 Selected from V, E, L, W, K, M, I, H, or Y;

[0151] X 7 Selected from T, L, E, D, H, Q, or V;

[0152] X 9 Selected from G, S, E, T, or D;

[0153] X 10 Selected from I, T, E, D, R, K, Q, or S;

[0154] X 11 Selected from D, L, E, Q, Y, or R;

[0155] X 12 Selected from V and I;

[0156] X 15 Selected from T, D, Y, F, H, or E;

[0157] X 16 Selected from I, Y, T, W, or H;

[0158] X 17 Choose from I, Y, or E.

[0159] In some embodiments, the polypeptide amino acid sequence comprises the amino acid sequence SAYGRKKRRQRRRCKX 4 X 5 TX 7 FX 9 X 10 X 11 X 12 TTX 15 X 16 X 17 C.

[0160] In some embodiments, the polypeptide amino acid sequence comprises the amino acid sequence SAYGRKKRRQRRRCKQX 5 TX 7 FX 9 X 10 X 11 X 12 TTX 15 X 16 X 17 C.

[0161] In some embodiments, the polypeptide amino acid sequence comprises the amino acid sequence SAYGRKKRRQRRRCKX 4 X 5 TX 7 FX 9 X 10 X 11 X 12 TTTX 16 X 17 C.

[0162] In some embodiments, the polypeptide amino acid sequence comprises the amino acid sequence SAYGRKKRRQRRRCKX 4 X 5 TX 7 FX 9 X 10 X 11 X 12 TTX 15 X 16 IC.

[0163] In some implementations, P3 is CKMVTTFGIDVTTTYIC, CKQYTDFGKQVTTYWIC, CKFETHFGREIVTEIYC, CKQETLFSTDVTTYYIC, CKQVTEFSEDVTTYYIC, CRYVTTFSDLITTDYYC, CKQITLFERDVTTYIIC, CRMITEFTDEVRTFTIC, or CKMETTFESDIITEIIC.

[0164] In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1 to SEQ ID NO: 36, SAYGRKKRRQRRRCKMVTTFGIDVTTTYIC (SEQ ID NO: 1), SAYGRKKRRQRRRASCKMVTTFGIDVTTTYIC (SEQ ID NO: 2), SAYGRKKRRQRRRCKQETLFSTDVTTYYIC (SEQ ID NO: 3), SAYGRKKRRQRRRASCKQETLFSTDVTTYYIC (SEQ ID NO: 4), SAYGRKKRRQRRRCKQVTEFSEDVTTYYIC (SEQ ID NO: 5), SAYGRKKRRQRRRASCKQVTEFSEDVTTYYIC (SEQ ID NO: 6), SAYGRKKRRQRRRCRYVTTFSDLITTDYYC (SEQ ID NO: 7), SAYGRKKRRQRRRASCRYVTTFSDLITTDYYC (SEQ ID NO: 8), SAYGRKKRRQRRRCKQITLFERDVTTYIIC (SEQ ID NO: 9), SAYGRKKRRQRRRASCKQITLFERDVTTYIIC (SEQ ID NO: 10), SAYGRKKRRQRRRASCRMITEFTDEVRTFTIC (SEQ ID NO: 11), SAYGRKKRRQRRRASCYQVTDFSTEIVSYYIC (SEQ ID NO: 12), SAYGRKKRRQRRRASCRQITTFGEEVITFWEC (SEQ ID NO: 13), SAYGRKKRRQRRRASCKLHTHFGEEIFTTWYC (SEQ ID NO: 14), SAYGRKKRRQRRRASCKQYTDFGKQVTTYWIC (SEQ ID NO: 15), SAYGRKKRRQRRRASCYTETTFGEEVTTTHIC (SEQ ID NO: 16), SAYGRKKRRQRRRASCKQVTTFGTYVTTHYEC (SEQ ID NO: 17), SAYGRKKRRQRRRASCKFETHFGREIVTEIYC (SEQ ID NO: 18), SAYGRKKRRQRRRASCWYETQFSEDVVTTYIC (SEQ ID NO: 19), SAYGRKKRRQRRRASCQTHTVFSTEIQTYIIC (SEQ IDNO:20)、SAYGRKKRRQRRRASCKQVTEFGQRIITHIYC(SEQ ID NO:21)、SAYGRKKRRQRRRASCWQETTFTTEIETYIIC(SEQ ID NO:22)、SAYGRKKRRQRRRCQMETTFGEQVVTTWEC(SEQ ID NO:23)、SAYGRKKRRQRRRCKMETTFESDIITEIIC(SEQ ID NO:24)、SAYGRKKRRQRRRASCKMETTFESDIITEIIC(SEQ ID NO:25)、SAYGRKKRRQRRRCRMHTVFDEDVQTYWYC(SEQ ID NO:26)、SAYGRKKRRQRRRASCRMHTVFDEDVQTYWYC(SEQ ID NO:27)、SAYGRKKRRQRRRCRRVTDFGQEIRTYYIC(SEQ ID NO:28)、YGRKKRRQRRRCKMVTTFGIDVTTTYIC(SEQ ID NO:29)、YGRKKRRQRRRASCKMVTTFGIDVTTTYIC(SEQ ID NO:30)、YGRKKRRQRRRASCKQYTDFGKQVTTYWIC(SEQ ID NO:31)、YGRKKRRQRRRASCKFETHFGREIVTEIYC(SEQ ID NO:32)、yGrkkrrqrrrCKMVTTFGIDVTTTYIC(SEQ ID NO:33)、

[0165] yGrkkrrqrrrASCKMVTTFGIDVTTTYIC(SEQ ID NO:34)、

[0166] yGrkkrrqrrrASCKQYTDFGKQVTTYWIC(SEQ ID NO:35)、yGrkkrrqrrrASCKFETHFGREIVTEIYC(SEQ ID NO:36)、

[0167] rrrqrrkkrGyCKMVTTFGIDVTTTYIC(SEQ ID NO:37)ぁ

[0168] rrrqrrkkrGyASCKMVTTFGIDVTTTYIC(SEQ ID NO:38)ぁ

[0169] rrrqrrkkrGyASCKQYTDFGKQVTTYWIC (SEQ ID NO:39), rrrqrrkkrGyASCKFETHFGREIVTEIYC (SEQ ID NO:40).

[0170] On the other hand, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide being at least one of (I) to (II):

[0171] (I) The amino acid sequence contains an amino acid sequence that has at least 90% sequence identity with the above-mentioned polypeptide and has the same or similar function;

[0172] (II) A polypeptide with the same or similar function obtained by substitution, deletion or addition of one or more amino acids in the polypeptides described above.

[0173] In some embodiments, the polypeptide amino acid sequence has at least 95% sequence identity and is a polypeptide with the same or similar function.

[0174] In some embodiments, the polypeptide is a polypeptide with the same or similar function obtained by substituting, deleting, or adding one, two, or three amino acids to the polypeptide described above.

[0175] In some embodiments, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide being at least one of (I) to (II):

[0176] (I) The amino acid sequence contains a polypeptide with at least 90% sequence identity with SEQ ID NO:1 to SEQ ID NO:36 and has the same or similar function;

[0177] (II) A polypeptide with the same or similar function obtained by substitution, deletion or addition of one or more amino acids in the polypeptides described in SEQ ID NO:1 to SEQ ID NO:36.

[0178] In some embodiments, the polypeptide amino acid sequence comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:1 to SEQ ID NO:36.

[0179] In some embodiments, the polypeptide is a polypeptide with the same or similar function obtained by substituting, deleting, or adding one, two, or three amino acids to the polypeptides described in SEQ ID NO:1 to SEQ ID NO:36.

[0180] On the other hand, the present invention provides a polynucleotide comprising the nucleotide sequence encoding the above.

[0181] On the other hand, the present invention provides a pharmaceutical composition comprising the above-described polypeptide or a pharmaceutically acceptable salt or polynucleotide thereof, and a pharmaceutically acceptable carrier, excipient, and / or diluent.

[0182] On the other hand, the present invention provides the use of the above-mentioned polypeptide or pharmaceutically acceptable salt or polynucleotide or pharmaceutical composition thereof in the preparation of a medicament for treating, improving or preventing diseases caused by PSD-95 dysfunction in an individual.

[0183] In some implementations, the diseases caused by the PSD-95 dysfunction are selected from stroke, neurodegenerative diseases, anxiety or epilepsy, and neuropathic pain.

[0184] In some embodiments, the stroke is selected from ischemic stroke or hemorrhagic stroke.

[0185] In some embodiments, the neurodegenerative disease is selected from at least one of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

[0186] As used in this article, the term "phage display" was proposed by Smith et al. in 1985 and successfully implemented in 1989. Phage display is a selection technique that fuses peptides or proteins with phage capsid proteins and displays them on the surface of viral particles. Phage display involves genetically engineering the insertion of DNA sequences encoding exogenous peptides or proteins into the phage gene, thereby displaying the peptides or proteins on the surface of the phage capsid proteins. A large number of phages displaying different random peptides can form phage libraries for screening specific targets to explore the interactions between peptides and targets. In the field of biology, this technique is widely used in the study of molecular interactions such as protein-protein, protein-peptide, and protein-DNA interactions.

[0187] The principle of phage display technology is to insert a foreign gene into an appropriate position in the structural gene of the phage coat protein. Under normal reading frame conditions and without affecting the normal function of the coat protein, the foreign gene will be expressed along with the coat protein, thus displaying the polypeptide or protein as a fusion protein on the phage surface. The displayed protein can maintain a relatively independent spatial structure and biological activity, which is conducive to the binding of the target protein. Therefore, the target protein can be used for rapid screening of phage display libraries. After the display library is constructed, the target protein is used as a stationary phase and co-incubated with the display library for a period of time. Unbound phages are washed away, and then the adsorbed phages are eluted using a competitive receptor. The eluted phages infect the host bacteria to multiply and amplify, and then the next round of elution is performed. After 3-5 rounds of "adsorption-elution-amplification" (more rounds are required for some antibodies with weak affinity), a high enrichment of phages that specifically bind to the target protein can be obtained. A significant feature of this technology is the establishment of a correspondence between genotype and phenotype.

[0188] The term "amino acid" refers to a molecule containing both an amino and a carboxyl group. Suitable amino acids include, but are not limited to, D- and L-isomers of naturally occurring amino acids, as well as non-natural amino acids prepared through 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.

[0189] The term "naturally occurring amino acid" refers to any one of the 20 L-amino acids commonly found in peptides synthesized in nature, namely, 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).

[0190] The meaning of the terms "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 an adjacent amino acid. Polypeptides described herein may contain naturally occurring or non-naturally occurring amino acids. They can be modified into analogs, derivatives, functional mimics, pseudopeptides, and other compounds containing at least two amino acids. Unless a specific modification is specified at the N-terminus or C-terminus, a polypeptide containing a particular amino acid sequence includes both unmodified and modified amino and / or carboxyl terminals, as is well known to those skilled in the art. A polypeptide with a particular amino acid sequence may include modified amino acids and / or additional amino acids unless the N- and / or C-terminus contain modifications that prevent the further addition of amino acids. Such modifications include, for example, acetylation of the N-terminus and / or amidation of the C-terminus.

[0191] The peptides of this invention can be modified to form peptide derivatives. As is well known to those skilled in the art, various modifications can be made to the peptides. 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 combinations of the above modifications. This invention includes any well-known modification of peptides. For example, peptide derivatives may include chemical modifications to the peptide, such as alkylation, acylation, carbamylation, iodination, or any other modification that produces peptide derivatives. The modification of the peptide may include modified amino acids, such as hydroxyproline or carboxyglutamic acid, and may include amino acids linked by non-peptide bonds.

[0192] For other modifications of the polypeptides of the present invention, non-natural amino acids can be used to replace the natural amino acids in the polypeptides. Non-natural amino acids include, but are not limited to, 2-amino fatty acids (Aad), 3-amino fatty acids (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), desmokinin (Des), 2,2'- Diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesin (Ide), isoleucine (aIle), N-methylglycine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), n-valine (Nva), n-leucine (Nle), and ornithine (Orn). Of course, all modified α-amino acids can be replaced by the corresponding β-, γ-, or ω-aminocarboxylic acids.

[0193] 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 derivative contains one or more non-standard amino acids, it is highly likely that it was prepared by chemical synthesis. Besides preparing polypeptides or their derivatives using chemical synthesis, they can also be prepared by expression encoding nucleic acids. This is particularly suitable for preparing polypeptides or their derivatives containing only natural amino acids, in which case well-known methods for preparing nucleic acid-encoded polypeptide sequences can be used (see Sambrook et al., Molecular Cloning: A. Alabatory 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 polypeptides can be expressed in organisms and purified using known purification techniques.

[0194] The term "PDZ domain" refers to a modular protein domain of approximately 90 amino acids, characterized by significant (e.g., at least 60%) sequence identity with the brain synaptic protein PSD-95, the Drosophila septal connexin Discs-Large (DLG), and the epithelial tight junction protein Z01 (Z01). PDZ domains are also referred to as Discs-Large homologous repeats ("DHRs") and GLGF repeats. PDZ domains typically exhibit a preserved core concordance sequence (Doyle, DA, 1996, Cell 85:1067-76). Exemplary proteins containing PDZ domains and PDZ domain sequences are disclosed in U.S. Patent Application No. 10 / 714,537.

[0195] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0196] Compared with traditional monoclonal antibodies, phage-displayed peptides selected from a random phage-displayed peptide library that bind to the target can specifically bind to the PDZ1 and / or PDZ2 domains of PSD-95. The peptides screened in this invention have small relative molecular mass, and their synthesis, expression, and modification are easy to achieve. Attached Figure Description

[0197] Figure 1 HPLC chromatogram of the compound corresponding to the amino acid sequence of Seq ID No.1

[0198] Figure 2 Ms spectrum of the compound corresponding to the amino acid sequence of Seq ID No.1 Specific Implementation

[0199] Synthesis Examples

[0200] The polypeptide compounds and their derivatives disclosed herein are synthesized using a solid-phase synthesis method. The synthesis support is Fmoc-Cys(Trt)-2-Chlotrityl Resin resin. During the synthesis process, the Fmoc-Cys(Trt)-2-Chlotrityl Resin resin is first fully swollen in N,N-dimethylformamide (DMF). Then, the solid support is repeatedly condensed with the activated amino acid derivative → washing → deprotecting the Fmoc → washing → the next round of amino acid condensation to achieve the desired polypeptide chain length. Finally, the polypeptide is cleaved from the solid support by reacting the resin with a mixed solution of trifluoroacetic acid:water:triisopropylsilane:aniline sulfide (90:2.5:2.5:5, v:v:v:v). After precipitation with frozen methyl tert-butyl ether, a solid crude linear precursor is obtained. The cleaved crude linear precursor is then subjected to disulfide bond oxidation in an alkaline solution to obtain the target crude polypeptide. The crude polypeptide was purified and separated by a C-18 reversed-phase preparative chromatography column in a system of 0.1% trifluoroacetic acid in acetonitrile / water to obtain pure polypeptides and their derivatives.

[0201] Experimental reagents

[0202]

[0203]

[0204] The following only lists the synthesis method of Seq ID No.1 as an example. The other sequences can be synthesized by referring to the method in Example 1. The specific synthesized amino acid sequences are shown in Table 1.

[0205] Example 1: Synthesis of the compound corresponding to the amino acid sequence of Seq ID No.1

[0206]

[0207] Step 1: Couple 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, dissolved in 5 ml of DCM, and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 ml) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was washed 5 times with DCM and 5 times with DMF.

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

[0210] SAYGRKKRRQRRRCKMVTTFG-IDVTTTYIC

[0211] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the straight-chain precursor sequence from the second I position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0212] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0213] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0214] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0215] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0216] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0217] Step 3: Cleavage of the linear precursor peptide chain

[0218] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 2, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washings with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product three times with MTBE and dry it under vacuum.

[0219] Step 4: Intramolecular disulfide bond formation

[0220] The crude product obtained in step 3 was dissolved in DMSO (DMSO volume was 20% of the total reaction volume). Then, the dissolved peptide solution was slowly added dropwise to a 50% acetonitrile aqueous solution to a final concentration of 1 mg / ml. The mixture was shaken at room temperature for 16 hours. The reaction results were monitored by LC-MS. After the reaction was completed, the product was directly purified.

[0221] Step 5: Preparation of Peptides

[0222] The crude peptide was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient was 20-50% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0223] Step 6: Detection and Characterization Methods

[0224] The purity and molecular weight of the peptide obtained in step 5 were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry.

[0225] Table 1. Amino acid sequences synthesized by the method described in Example 1

[0226]

[0227] Biological test examples

[0228] Example 2: FP test peptide samples inhibit the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2.

[0229] 1) Main experimental materials

[0230]

[0231]

[0232] 2) Experimental steps:

[0233] 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 was tested using the FP (fluorescence polarization immunoassay) method. Peptide samples were serially diluted to 0–10 μM (8 concentrations in 3-fold serial dilutions from 10 μM downwards). Using an 8-well pipette (1–10 μL), 5 μL / well of 5 nM 6H-PSD95 alpha 61-249 was transferred to a 384-well plate (Corning 3575), and the sample was briefly centrifuged to remove air bubbles. Finally, using a workstation, 5 μL / well of peptide sample was transferred to the 384-well plate, and the sample was briefly centrifuged to remove air 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 into a 384-well plate, and briefly centrifuge to remove air bubbles. Incubate at room temperature in the dark for 2 hours, and read the values ​​using a Cytation 5 FP microplate reader.

[0234] 3) Experimental Results

[0235] The FP method was used to test the inhibition of the binding of the peptide of the present invention to 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2. The results were obtained by analyzing the IC50. 50 The values ​​are shown in Table 2; the results show that the peptides of the present invention have a higher affinity for 6H-PSD95 alpha 61-249 compared to 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2.

[0236] Table 2: FP test results for the binding IC50 of peptides to 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2. 50 value

[0237]

[0238]

[0239] Example 3: FRET test of peptide sample inhibiting the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B

[0240] 1) Main experimental materials

[0241]

[0242] 2) Experimental steps:

[0243] The inhibitory effect of peptide samples on the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B was tested using the FRET (fluorescence resonance energy transfer) method. Peptide samples were serially diluted to 0–10 μM (8 concentrations in total, with 3-fold serial dilutions from 10 μM downwards). Using an 8-well pipette (1–10 μL), 4 μL / well of 4 nM cMyc-PSD95 alpha 1-392 was pipetted into a 384-well white plate (Thermo 264706), and the sample was briefly centrifuged to remove air bubbles. Then, using a workstation, 4 μL / well of peptide sample was transferred into the 384-well plate, and the sample was briefly centrifuged to remove air bubbles. Finally, using an 8-well pipette, 4 μL / well of 20 nM Tat-NR2B9c-B was pipetted into the 384-well white plate, and the sample was briefly centrifuged to remove air bubbles. Mix 0.02 μg / ml EU-steptavidin with 1 μg / ml Mouse Anti-C-MYC IgG SureLight APC at a 1:1 ratio. Pipe 8 μl / well of the EU-APC premix into a 384-well white plate using an 8-well multipipe pipette, and briefly centrifuge to remove air bubbles. Incubate at room temperature in the dark for 2 hours. Use a Cytation 5 microplate reader to read the emission fluorescence values ​​at 620 nm and 665 nm after excitation at 320 nM.

[0244] 3) Experimental Results

[0245] The FRET method was used to test the inhibition of the binding of the peptide of the present invention to Tat-NR2B9c-B and PSD95-related protein. The results were obtained by analyzing the IC50. 50 The values ​​are shown in Table 3; the results show that the peptide of the present invention has good inhibitory activity against the binding of cMyc-PSD95alpha1-392 and Tat-NR2B9C-B.

[0246] Table 3: FRET test results for the binding IC50 of peptides to cMyc-PSD95 alpha 1-392 and Tat-NR2B9C-B 50 value

[0247]

[0248]

[0249] Example 4: ELISA test of peptide sample inhibition of binding between biotin-nNOS1-299 and cMyc-PSD95 alpha 1-392 1) Main experimental materials

[0250]

[0251] 2) Experimental steps:

[0252] The inhibitory effect of peptide samples on the binding of biotin-nNOS1-299 to cMyc-PSD95 alpha 1-392 was tested using ELISA (enzyme-linked immunosorbent assay). cMyc-PSD95 alpha 1-392 was coated at 10 μg / ml, 25 μL / well onto 384-well plates (Greiner 781097). After blocking the cMyc-PSD95 alpha 1-392-coated ELISA plates, the peptide samples were serially diluted to 0–10 μM (8 concentrations in 3-fold serial dilutions from 10 μM downwards). Transfer 12.5 μl / well of peptide sample to a 384-well plate using a workstation, and briefly centrifuge to remove air bubbles. Then, pipe 12.5 μl / well of 10 μg / ml 743biotin-nNOS1-299 using an 8-well multipipe (10-100 μl), briefly centrifuge to remove air bubbles, and incubate at 37°C for 1 h. Discard the liquid in the wells, add 80 μl of 1×TBST washing buffer (pH 7.4) to wash the plate 3-5 times, 3-5 min each time. After drying the plate, add 25 μl / well of Streptavidin HRP diluted 1:10000, briefly centrifuge to remove air bubbles, and incubate at 37°C for 1 h. Wash the plate again and dry the plate, add 25 μl of TMB chromogenic solution to each well, and continue incubating at 37°C for 30 min. Finally, add 25 μl of stop solution (1M HCl) to each well to terminate the reaction. The absorbance at 450 nm was read using a Cytation5 microplate reader.

[0253] 3) Experimental Results

[0254] The ELISA method was used to test the inhibitory effect of the peptide of the present invention on the binding of biotin-nNOS1-299 to PSD95-related protein. The results were obtained by analyzing the IC50. 50 The values ​​are shown in Table 4; the results show that the peptide of the present invention has good inhibitory activity against the binding of biotin-nNOS 1-299 and PSD95-related proteins.

[0255] Table 4: IC50 of ELISA assay for peptide inhibition of biotin-nNOS1-299 and cMyc-PSD95 alpha 1-392-related protein binding. 50 value

[0256] 1 0.019 15 0.018 2 0.011 16 0.052 3 0.018 17 0.028 4 0.029 18 0.021 5 0.03 19 0.038 6 0.017 20 0.039 7 0.032 21 0.033 8 0.012 22 0.018 9 0.021 23 0.038 10 0.01 24 0.056 11 0.033 25 0.05 12 0.026 26 0.032 13 0.021 27 0.026 14 0.016 28 0.042

[0257] This invention provides a postsynaptic density protein-95 inhibitor peptide, which can be implemented by those skilled in the art with appropriate modifications to the process parameters, based on the content of this document. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:1~SEQ ID NO:

2. SAYGRKKRRQRRRCKMVTTFGIDVTTTTYIC (SEQ ID NO:1), SAYGRKKRRQRRRASCKMVTTFGIDVTTTYIC (SEQ ID NO: 2).

2. A polynucleotide, characterized in that, The polynucleotide encodes the nucleotide sequence of the polypeptide of claim 1.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, or the polynucleotide of claim 2 and a pharmaceutically acceptable carrier.

4. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, or the polynucleotide of claim 2, or the pharmaceutical composition of claim 3, in the preparation of a medicament for treating, improving, or preventing diseases caused by PSD-95 dysfunction in an individual, wherein the disease caused by PSD-95 dysfunction is stroke.

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