Therapeutic peptides for excitotoxicity-related injury

Abstract

The present application belongs to the field of biological medicine, and particularly relates to a treatment peptide for excitotoxicity-related injury. The polypeptide of the present application is a PSD-95 inhibitor, and the polypeptide comprises an amino acid sequence represented by (Z1) m (X 0 ) n X 1 ETX 2 F(Z2) q or (Z1) m1 -NH(PEG) u1 C(=O)-(X 0 ) n1 X 1 ETX 2 X 3 (C) n2 (PEG) u2 (Z1) m2 , which can be used for preparing a medicine for treating excitotoxicity-related injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a therapeutic peptide for excitotoxic neurotoxic-related injury. 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 tissue. 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.

[0005] Based on the different splicing of genes by mRNA, more than 10 nNOS (Neuronal nitric oxide synthase) splice variants are currently known, with a molecular weight of 160.8 KD. nNOS contains two structural domains: a C-terminal reduction domain and an N-terminal oxidation domain. The N-terminus has two non-overlapping binding regions: (1) the PDZ region: composed of 1 to 99 amino acids, which participates in the formation of active nNOS dimers; (2) the β-finger structure: composed of 100 to 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 the C-terminal PDZ reaction. PSD-95 can link nNOS to the NMDA receptor, thereby effectively activating nNOS through the activation of the NMDA receptor.

[0006] 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. Following 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. While NMDA receptor antagonists effectively reduce excitotoxicity by blocking glutamate-mediated ion flux, they also inhibit some physiologically important processes. PSD-95, a downstream receptor, interacts with various proteins, inducing a series of ischemic injuries and is a key site of cerebral ischemia-related damage, as well as a potential target for drug therapy. Therefore, the development of PSD-95 inhibitors is of great pharmaceutical significance for neurological damage caused by various excitotoxic neurotoxicities, including stroke. In contrast, specific inhibition of excitotoxicity can be achieved by perturbing the intracellular nNOS / PSD-95 / NMDA receptor complex using PSD-95 inhibitors.

[0007] 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.

[0008] 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 models, it reduces infarct size due to cerebral 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. WO2010004003A2 describes a dimer peptide ligand linked by a polyethylene glycol linker (PEG). WO2017185249A1 and WO2017185250A1 disclose peptides for treating central nervous system injuries. CN110799547A discloses a compound having the structure shown in R1-S1-YEKL-S2-R2 and its uses. CN115667286A discloses a novel cyclic peptide that can act as an inhibitor of protein-protein interactions, the polypeptide containing the amino acid sequence TX1LETX2X3X4GX5X6X7PX8TIRVX9Q. To meet clinical needs, more PSD-95 inhibitors still need to be developed. Summary of the Invention

[0009] To address the aforementioned problem, this invention discloses a therapeutic peptide for excitotoxic neurotoxicity-related injury, which 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, said polypeptide comprising (Z1). m (X 0 ) n X 1 ETX 2 F(Z2) q The amino acid sequence, in which:

[0011] Each X 0 Independently selected from any amino acid;

[0012] X 1 It can be selected from N, Y, Q, I, L, or R;

[0013] X 2 Selectable from T, D, P, F, or G;

[0014] m and q are each independently selected from 0 or 1;

[0015] n is an integer selected from 0 to 6;

[0016] Z1 is a cell-penetrating peptide (CPP);

[0017] Z2 is (X 3 ) p C;

[0018] p is an integer between 0 and 12; each X 3 It can be independently selected from T, D, G, P, K, I, R, or V.

[0019] Preferably, the X 0 Each is independently selected from C, A, G, Y, D, T, H, L, or R; preferably, each X 0 The n is independently selected from C, T, or H; the n is 1, 2, or 3.

[0020] Preferably, Z2 is selected from: TGDGTPKTIRVTC, TGDGTPKTIRVC, TGDGTPKTIRC, TGDGTPKTIC, TGDGTPKTC, TGDGTPKC, TGDGTPC, TGDGTC, TGDGC, TGDC, TGC, TC, C.

[0021] Preferably, Z1 is a cell-penetrating peptide (CPP), which is selected from YGRKKRRQRRR, yGrkkrrqrrr, rrrqrrkkr, polyarginine composed of 2 to 30 residues, GRKKRRQRRRPPQQ, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMKWKK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTSTGR, KLALKLALKALKAALKLA, GALFLAFLAAALSL

[0022] -MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, PLIYLRLLRGQF;

[0023] Preferably, the cell-penetrating peptide (CPP) is selected from YGRKKRRQRRR or yGrkkrrqrrr.

[0024] Preferably, the X 0 The n is independently selected from C, T, or H; the n is 1, 2, or 3.

[0025] More preferably, Z2 is selected from TGDGTPKTIRVTC, TGDGTPKTIRVC, TGDGTPKTIRC, TGDGTPKTIC, TGDGTPKTC, TGDGTPKC, TGDGTPC, TGDGTC, TGDGC, TGDC, TGC, TC or C;

[0026] Z1 is a cell-penetrating peptide (CPP), which is selected from YGRKKRRQRRR or yGrkkrrqrrr.

[0027] On the other hand, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising (Z1). m (X 0 ) n LETTF(Z2) q Or (Z1) m (X 0 ) n LETDF(Z2) q The amino acid sequence, in which:

[0028] Each X 0 Independently selected from C, A, G, Y, D, T, H, L, or R;

[0029] m and q are each independently selected from 0 or 1;

[0030] n is an integer selected from 0 to 6;

[0031] Z1 is a cell-penetrating peptide (CPP);

[0032] Z2 is (X 3 ) p C;

[0033] P is an integer between 0 and 12; each X 3 It can be independently selected from T, D, G, P, K, I, R, or V.

[0034] Preferably, the X 0 The n is independently selected from C, T, or H; the n is 1, 2, or 3.

[0035] Preferably, Z2 is selected from TGDGTPKTIRVTC, TGDGTPKTIRVC, TGDGTPKTIRC, TGDGTPKTIC, TGDGTPKTC, TGDGTPKC, TGDGTPC, TGDGTC, TGDGC, TGDC, TGC, TC, or C;

[0036] Z1 is a cell-penetrating peptide (CPP), which is selected from YGRKKRRQRRR or yGrkkrrqrrr.

[0037] On the other hand, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide being selected from one of the following:

[0038] CTHRETTFTGDGTPKTIRVTC(SEQ ID NO:1),

[0039] CTHLETDFTGDGTPKTIRVTC(SEQ ID NO:2),

[0040] CHLETTFTGDGTPKTIRVC(SEQ ID NO:3)、

[0041] CHRETTFTGDGTPKTIRVC(SEQ ID NO:4),

[0042] CHLETDFTGDGTPKTIRVC(SEQ ID NO:5)、

[0043] CLETTFTGDGTPKTIRC(SEQ ID NO:6)、

[0044] CRETTFTGDGTPKTIRC(SEQ ID NO:7),

[0045] CHLETTFTGDGTPKTIRVC(SEQ ID NO:8)ぁ

[0046] CHLETTFTGDGTPKTIRC(SEQ ID NO:9)ぁ

[0047] CHLETTFTGDGTPKTIC(SEQ ID NO:10)、

[0048] CHLETTFTGDGTPKTC(SEQ ID NO:11)、

[0049] CHLETTFTGDGTPKC(SEQ ID NO:12)、

[0050] CHLETTFTGDGTPC(SEQ ID NO:13)、

[0051] CHLETTFTGDGTC(SEQ ID NO:14)、

[0052] CHLETTFTGDGC(SEQ ID NO:15)ぁ

[0053] CHLETTFTGDC(SEQ ID NO:16)ぁ

[0054] CHLETTFTGC(SEQ ID NO:17)ぁ

[0055] CHLETTFTC(SEQ ID NO:18)ぁ

[0056] CHLETTFC (SEQ ID NO:19)

[0057] CRLETTFC(SEQ ID NO:20)ぁ

[0058] HLETTF(SEQ ID NO:21)、

[0059] ANETPF(SEQ ID NO:22)ぁ

[0060] GYETFF(SEQ ID NO:23)ぁ

[0061] YQETPF(SEQ ID NO:24)ぁ

[0062] DYETGF(SEQ ID NO:25)ぁ

[0063] DPETPF(SEQ ID NO:26)、

[0064] YGRKKRRQRRRCHLETTFTGDGTPKTIRVC(SEQ ID NO:27),

[0065] YGRKKRRQRRRCHLETTFTGDGTPKTIRC(SEQ ID NO:28),

[0066] YGRKKRRQRRRCHLETTFTGDGTPKTIC(SEQ ID NO:29),

[0067] YGRKKRRQRRRCHLETTFTGDGTPKTC(SEQ ID NO:30),

[0068] YGRKKRRQRRRCHLETTFTGDGTPKC(SEQ ID NO:31),

[0069] YGRKKRRQRRRCHLETTFTGDGTPC(SEQ ID NO:32)、

[0070] YGRKKRRQRRRCHLETTFTGDGTC(SEQ ID NO:33)、

[0071] YGRKKRRQRRRCHLETTFTGDGC(SEQ ID NO:34)、

[0072] YGRKKRRQRRRCHLETTFTGDC(SEQ ID NO:35)、

[0073] YGRKKRRQRRRCHLETTFTGC(SEQ ID NO:36),

[0074] YGRKKRRQRRRCHLETTTFTC(SEQ ID NO:37)、

[0075] YGRKKRRQRRRCHLETTFC(SEQ ID NO:38)、

[0076] YGRKKRRQRRRCRLETTFC (SEQ ID NO:39).

[0077] On the other hand, the present invention provides a compound or a pharmaceutically acceptable salt thereof, said compound comprising (Z1). m1 -NH(PEG) u1 C(=O)-(X 0 ) n1 X 1 ETX 2 X3 (C) n2 (PEG) u2 (Z1) m2 The amino acid sequence, in which:

[0078] Each X 0 Independently selected from any amino acid;

[0079] X 1 It can be selected from N, Y, Q, I, L, P, or R;

[0080] X 2 Selectable from T, D, P, F, W, or G;

[0081] X 3 Optional from F and V;

[0082] m1 and m2 are each independently selected from 0 or 1, and m1 and m2 are not both 0 at the same time;

[0083] u1 and u2 are each independently selected from integers from 0 to 6, and u1 and u2 are not both 0 at the same time;

[0084] n1 is an integer selected from 0 to 6;

[0085] n2 is selected from 0 or 1;

[0086] Z1 is a cell-penetrating peptide (CPP).

[0087] Preferably, the X 0 Each is independently selected from C, A, G, Y, D, T, H, L, or R; n1 is 1, 2, or 3.

[0088] Preferably, u1 and u2 are each independently selected from 0, 1, 2 or 3, and u1 and u2 are not both 0 at the same time.

[0089] Preferably, u1 and u2 are selected from u1=1, u2=0 or u1=0, u2=1.

[0090] Preferably, Z1 is a cell-penetrating peptide (CPP), which is selected from YGRKKRRQRRR, yGrkkrrqrrr, rrrqrrkkr, polyarginine composed of 2 to 30 residues, GRKKRRQRRRPPQQ, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMKWKK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTSTGR, KLALKLALKALKAALKLA, GALFLAFLAAALSL

[0091] -MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, PLIYLRLLRGQF.

[0092] Preferably, the cell-penetrating peptide (CPP) is selected from YGRKKRRQRRR or yGrkkrrqrrr.

[0093] On the other hand, the present invention provides a compound or a pharmaceutically acceptable salt thereof, said compound being selected from one of the following:

[0094] yGrkkrrqrrr-AEEA-HLETTF (SEQ ID NO:40),

[0095] yGrkkrrqrrr-NH(PEG)3C(=O)-HLETTF (SEQ ID NO:41),

[0096] yGrkkrrqrrr-AEEA-CHLETTFC (SEQ ID NO:42),

[0097] yGrkkrrqrrr-NH(PEG)3C(=O)-CHLETTFC (SEQ ID NO:43),

[0098] YGRKKRRQRRR-AEEA-ANETPF(SEQ ID NO:44),

[0099] YGRKKRRQRRR-AEEA-GYETFF(SEQ ID NO:45),

[0100] YGRKKRRQRRR-AEEA-YQETPF(SEQ ID NO:46),

[0101] YGRKKRRQRRR-AEEA-DYETGF (SEQ ID NO:47),

[0102] YGRKKRRQRRR-AEEA-DPETPF (SEQ ID NO:48),

[0103] YGRKKRRQRRR-AEEA-IETDV(SEQ ID NO:49)、

[0104] YGRKKRRQRRR-AEEA-HLETTF (SEQ ID NO: 50).

[0105] YGRKKRRQRRR-AEEA-IETDIW(SEQ ID NO:51)、

[0106] YGRKKRRQRRR-AEEA-YIETDV(SEQ ID NO:52)、

[0107] YGRKKRRQRRR-AEEA-NIETDV(SEQ ID NO:53),

[0108] YGRKKRRQRRR-AEEA-YIETWV(SEQ ID NO:54)、

[0109] YGRKKRRQRRR-AEEA-NIETWV(SEQ ID NO:55)、

[0110] HLETTF-AEEA-yGrkkrrqrrr-NH2 (SEQ ID NO:56),

[0111] CHLETTFC-AEEA-yGrkkrrqrrr-NH2 (SEQ ID NO:57),

[0112] CHLETTFC-NH(PEG)3C(=O)-yGrkkrrqrrr-NH2 (SEQ ID NO:58).

[0113] The polypeptides defined herein may be in the form of a pharmaceutically acceptable salt or prodrug of the polypeptide.

[0114] In some embodiments, the pharmaceutically acceptable salt may be selected from trifluoroacetate, acetate, hydrochloride and phosphate.

[0115] On the other hand, the present invention provides a polynucleotide that encodes the aforementioned polypeptide.

[0116] On the other hand, the present invention provides a pharmaceutical composition comprising the above-described polypeptide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, excipient and / or diluent.

[0117] In some embodiments, the polypeptides of this application can be administered in the form of a pharmaceutical composition. The pharmaceutical composition can be manufactured using conventional methods of mixing, dissolving, granulating, tableting, grinding, emulsifying, encapsulating, capturing, or lyophilizing. The pharmaceutical composition can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or excipients that facilitate the processing of the compounds of this application into pharmaceutically acceptable formulations. Appropriate formulation depends on the chosen route of administration.

[0118] In some implementations, administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, local, intranasal, or intramuscular.

[0119] On the other hand, the present invention provides the use of the above-described polypeptide or a 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. The polypeptide of the present invention is a PSD-95 inhibitor and therefore can inhibit excitotoxicity.

[0120] In some implementations, the disease caused by the PSD-95 dysfunction can be selected from at least one of stroke, neurodegenerative disease, anxiety or epilepsy, and neuropathic pain.

[0121] In some embodiments, the stroke is selected from ischemic stroke or hemorrhagic stroke.

[0122] In some implementations, the neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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).

[0128] 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.

[0129] 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. Attached Figure Description

[0130] Figure 1 This is the HPLC detection chromatogram of compound 55 from Example 1;

[0131] Figure 2 This is the mass spectrometry result of compound 55 from Example 1;

[0132] Figure 3 This is the HPLC detection chromatogram of compound 3 in Example 2;

[0133] Figure 4 This is the mass spectrometry detection chromatogram of compound 3 in Example 2;

[0134] Figure 5 This is the HPLC detection chromatogram of compound 6 in Example 3;

[0135] Figure 6 This is the mass spectrometry result of compound 6 in Example 3;

[0136] Figure 7 This is the HPLC detection chromatogram of compound 27 in Example 4;

[0137] Figure 8 This is the mass spectrometry result of compound 27 in Example 4;

[0138] Figure 9 This is the HPLC detection chromatogram of compound 52 in Example 5;

[0139] Figure 10 This is a mass spectrometry result of compound 52 from Example 5. Specific Implementation

[0140] The polypeptide compounds and their derivatives provided in this method are synthesized using a solid-phase synthesis method to obtain their linear precursors. After cleavage, the crude peptides are directly purified to obtain the target compounds, or the crude peptides are oxidized with DMSO to form intramolecular disulfide bonds and then purified to obtain the target compounds. The synthesis support is 2-Chlotrityl Resin resin. In the synthesis process, the 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 derivatives → washing → deprotection of 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 with a mixed solution of trifluoroacetic acid:water:triisopropylsilane:aniline sulfide (90:2.5:2.5:5, v:v:v:v) with the resin. After precipitation with frozen methyl tert-butyl ether, the solid crude linear precursor is obtained. The cleaved crude linear precursor is then subjected to disulfide bond oxidation in a neutral solution to obtain the crude target polypeptide. The crude solid product or the crude oxidized peptide product was purified and separated by a C18 reversed-phase preparative chromatography column in a system of 0.1% trifluoroacetic acid in acetonitrile / water to obtain pure peptides and their derivatives.

[0141] Experimental reagents

[0142]

[0143]

[0144]

[0145] The structural formula of Fmoc-AEEA-OH is as follows: Example 1: Synthesis of Compound 55 (SEQ ID NO: 55)

[0146]

[0147] Step 1: Couple the first amino acid Fmoc-Val-OH

[0148] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-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 then washed 5 times with DCM and 5 times with DMF.

[0149] Step 2: Synthesis of linear precursor peptide chains

[0150] YGRKKRRQRRR-AEEA-NIETWV

[0151] 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 W position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0152] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0153] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0154] • 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.

[0155] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0156] 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.

[0157] Step 3: Cleavage of the linear precursor peptide chain

[0158] 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.

[0159] Step 4: Preparation of Peptides

[0160] 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 C-18 (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 to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0161] Step 5: Detection and Characterization Methods

[0162] The purity and molecular weight of the peptide obtained in step 4 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 1 , Figure 2 .

[0163] Example 2: Synthesis of Compound 3 (SEQ ID NO:3)

[0164]

[0165] Step 1: Couple the first amino acid Fmoc-Val-OH

[0166] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-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 then washed 5 times with DCM and 5 times with DMF.

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

[0168] CHLETTFTGDGTPKTIRVC

[0169] 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 W position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0170] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0171] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0172] • 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.

[0173] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0174] 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.

[0175] Step 3: Cleavage of the linear precursor peptide chain

[0176] 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.

[0177] Step 4: Preparation of Peptides

[0178] 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 C-18 (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 to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0179] Step 5: Detection and Characterization Methods

[0180] The purity and molecular weight of the peptide obtained in step 4 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 3 , Figure 4 .

[0181] Example 3 Synthesis of Compound 6 (SEQ ID NO: 6)

[0182]

[0183] Step 1: Couple the first amino acid Fmoc-Val-OH

[0184] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-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 then washed 5 times with DCM and 5 times with DMF.

[0185] Step 2: Synthesis of linear precursor peptide chains

[0186] CLETTFTGDGTPKTIRC

[0187] 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 W position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0188] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0189] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0190] • 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.

[0191] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0192] 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.

[0193] Step 3: Cleavage of the linear precursor peptide chain

[0194] 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.

[0195] Step 4: Preparation of Peptides

[0196] 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 C-18 (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 to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0197] Step 5: Detection and Characterization Methods

[0198] The purity and molecular weight of the peptide obtained in step 4 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 5 , Figure 6 .

[0199] Example 4 Synthesis of compound 27 (SEQ ID NO:27)

[0200]

[0201] Step 1: Couple the first amino acid Fmoc-Val-OH

[0202] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-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 then washed 5 times with DCM and 5 times with DMF.

[0203] Step 2: Synthesis of linear precursor peptide chains

[0204] YGRKKRRQRRRCHLETTFTGD-GTPKTIRVC

[0205] 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 W position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0206] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0207] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0208] • 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.

[0209] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0210] 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.

[0211] Step 3: Cleavage of the linear precursor peptide chain

[0212] 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.

[0213] Step 4: Preparation of Peptides

[0214] 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 C-18 (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 to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0215] Step 5: Detection and Characterization Methods

[0216] The purity and molecular weight of the peptide obtained in step 4 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 7 , Figure 8 .

[0217] Example 5 Synthesis of compound 52 (SEQ ID NO:52)

[0218]

[0219] Step 1: Couple the first amino acid Fmoc-Val-OH

[0220] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-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 then washed 5 times with DCM and 5 times with DMF.

[0221] Step 2: Synthesis of linear precursor peptide chains

[0222] YGRKKRRQRRR-AEEA-YIETDV

[0223] 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 W position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0224] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0225] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0226] • 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.

[0227] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0228] 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.

[0229] Step 3: Cleavage of the linear precursor peptide chain

[0230] 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.

[0231] Step 4: Preparation of Peptides

[0232] 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 C-18 (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 to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0233] Step 5: Detection and Characterization Methods

[0234] The purity and molecular weight of the peptide obtained in step 4 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 9 , Figure 10 .

[0235] Example 6 Synthesis of compound 42 (SEQ ID NO:42)

[0236]

[0237] Step 1: Couple the first amino acid Fmoc-Val-OH

[0238] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-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 then washed 5 times with DCM and 5 times with DMF.

[0239] Step 2: Synthesis of linear precursor peptide chains

[0240] dY-G-dR-dK-dK-dR-dR-dQ-dR-dR-dR-AEEA-CHLETTFC

[0241] 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 W position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0242] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0243] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0244] • 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.

[0245] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0246] 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.

[0247] Step 3: Cleavage of the linear precursor peptide chain

[0248] 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.

[0249] Step 4: Intramolecular disulfide bond formation

[0250] 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.

[0251] Step 5: Preparation of Peptides

[0252] 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.

[0253] Step 6: Detection and Characterization Methods

[0254] 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.

[0255] Example 7 Synthesis of compound 43 (SEQ ID NO:43)

[0256]

[0257] Step 1: Couple the first amino acid Fmoc-Val-OH

[0258] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-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 then washed 5 times with DCM and 5 times with DMF.

[0259] Step 2: Synthesis of linear precursor peptide chains

[0260] dY-G-dR-dK-dK-dR-dR-dQ-dR-dR-dR-NH(PEG)3C(=O)-CHLETTFC

[0261] 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 W position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0262] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0263] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0264] • 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.

[0265] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0266] 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.

[0267] Step 3: Cleavage of the linear precursor peptide chain

[0268] 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.

[0269] Step 4: Intramolecular disulfide bond formation

[0270] 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.

[0271] Step 5: Preparation of Peptides

[0272] 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.

[0273] Step 6: Detection and Characterization Methods

[0274] 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.

[0275] Example 8 Synthesis of compound 56 (SEQ ID NO:56)

[0276]

[0277] Step 1: Couple the first amino acid Fmoc-Val-OH

[0278] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-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 then washed 5 times with DCM and 5 times with DMF.

[0279] Step 2: Synthesis of linear precursor peptide chains

[0280] HLETTF-AEEA-dY-G-dR-dK-dK-dR-dR-dQ-dR-dR-dR--NH2

[0281] 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 W position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0282] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0283] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0284] • 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.

[0285] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0286] 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.

[0287] Step 3: Cleavage of the linear precursor peptide chain

[0288] 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.

[0289] Step 4: Preparation of Peptides

[0290] 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 C-18 (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 to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0291] Step 5: Detection and Characterization Methods

[0292] The purity and molecular weight of the peptide obtained in step 4 were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry.

[0293] Example 9 Synthesis of compound 57 (SEQ ID NO:57)

[0294]

[0295] Step 1: Synthesis of linear precursor peptide chains

[0296] CHLETTFC-AEEA-dY-G-dR-dK-dK-dR-dR-dQ-dR-dR-dR

[0297] 294 mg (0.2 mmol) of RinkAmide-AM Resin resin was fully swollen in DMF for 1 h, followed by synthesis in the order of the linear precursor sequence from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0298] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0299] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0300] • 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.

[0301] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0302] 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.

[0303] Step 3: Cleavage of the linear precursor peptide chain

[0304] 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.

[0305] Step 4: Intramolecular disulfide bond formation

[0306] 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.

[0307] Step 5: Preparation of Peptides

[0308] 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.

[0309] Step 6: Detection and Characterization Methods

[0310] 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.

[0311] Other compounds of the present invention can be synthesized by referring to the synthesis method of the above embodiments.

[0312] Example 1: ELISA test of peptide samples to inhibit the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392.

[0313] (1) Experimental materials:

[0314]

[0315] (2) Experimental steps:

[0316] The inhibitory effect of peptide samples on the binding of Tat-NR2B9c-B to cMyc-PSD95alpha 1-392 was tested using ELISA (enzyme-linked immunosorbent assay). cMyc-PSD95 alpha 1-392 was coated at 0.37 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). 12.5 μL / well of peptide sample was transferred to the 384-well plate using a workstation, and the plate was briefly centrifuged to remove air bubbles. Then, 12.5 μL / well of 12 nM Tat-NR2B9c-B was pipetted using an 8-well pipette (10–100 μL), briefly centrifuged to remove air bubbles, and incubated at 37°C for 1 h. Discard the liquid in the wells, add 80 μL of 1×TBST washing buffer (pH 7.4) and 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 it completely. 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 (1 MHCL) to each well to terminate the reaction. Read the absorbance at 450 nm using a Cytation 5 microplate reader.

[0317] Table 1 Results of the compounds inhibiting the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392

[0318] 27 0.005 28 0.22 29 1.09 30 8.79 31 14.43 32 14.47 33 27.02 34 3.57 35 15.63 36 3.48 37 4.63 38 4.19 39 0.65 40 0.5 41 0.32 43 4.08 56 1.8 57 4.72 58 0.61

[0319] The experimental results are shown in Table 1. The compounds of the present invention can inhibit the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha1-392 and have good affinity for the target protein. The peptides of the present invention have a stronger affinity for cMyc-PSD95 alpha1-392 than for Tat-NR2B9c-B.

[0320] Test Example 2 ELISA test peptide sample inhibits the binding of biotin-nNOS1-299 and cMyc-PSD95 alpha 1-392 (1) Experimental materials:

[0321]

[0322] (2) Experimental steps:

[0323] 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). 12.5 μL / well of peptide sample was transferred to each 384-well plate using a workstation, and the plates were briefly centrifuged to remove air bubbles. Using an 8-well multipipe (10-100 μL), pipette 12.5 μL / well of 10 μg / mL 743biotin-nNOS1-299, 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. Read the absorbance at 450 nm using a Cytation 5 microplate reader.

[0324] The experimental results are shown in Table 2. The compounds of the present invention can inhibit the binding of biotin-nNOS1-299 and cMyc-PSD95alpha1-392 and have a good inhibitory effect.

[0325] Table 2 Results of the compounds inhibiting the binding of biotin-nNOS1-299 and cMyc-PSD95 alpha 1-392

[0326] 1 92.75 2 103.8 3 16.95 4 49.24 5 42.05 6 29.83 7 133.7 27 0.113 49 0.372 50 6.144

[0327] Test Example 3: FRET test of peptide samples inhibited the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B.

[0328] (1) Experimental materials:

[0329]

[0330]

[0331] (2) Experimental steps:

[0332] 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 (eight concentrations in total, three-fold serial dilutions from 10 μM downwards). Using an 8-well multipipe (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 multipipe, 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 pipette and briefly centrifuge to remove air bubbles. Incubate at room temperature in the dark for 2 hours. Read the emission fluorescence values ​​at 620 nm and 665 nm using a Cytation 5 microplate reader after excitation at 320 nM.

[0333] Table 3 shows the results of the compounds inhibiting the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B.

[0334] 27 0.275 40 3.67 42 3.58 51 46.69 52 2.91 53 9.24 54 2.78 55 3.73 57 26.85 58 52.6

[0335] The experimental results are shown in Table 3. 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 peptide of the present invention has a stronger affinity for cMyc-PSD95 alpha 1-392 than for Tat-NR2B9c-B.

[0336] Test Example 4FP tested the peptide sample to inhibit the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2.

[0337] (1) Experimental materials:

[0338]

[0339]

[0340] (2) Experimental steps:

[0341] 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 pipetted into a 384-well plate (Corning 3575), and the sample was briefly centrifuged to remove air bubbles. Finally, 5 μL / well of the peptide sample was transferred to the 384-well plate using a workstation, 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.

[0342] Table 4 shows the results of the compounds inhibiting the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2.

[0343] 27 1.64 49 14.5 50 194.7

[0344] The experimental results are shown in Table 4. The compounds of this invention can inhibit the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2 with good inhibitory effects. The peptides of this invention have a stronger affinity for 6H-PSD95 alpha 61-249 than for FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2.

[0345] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within 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 is shown in SEQ ID NO:27: YGRKKRRQRRRCHLETTFTGDGTPKTIRVC (SEQ ID NO: 27).

2. A polynucleotide, characterized in that, The polynucleotide encodes 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, the polynucleotide of claim 2, and a pharmaceutically acceptable carrier.

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

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