Artificial small interfering peptides of DAPK1 phosphorylation substrates and their pharmaceutical applications

Abstract

The present application relates to an artificial small interfering peptide of DAPK1 phosphorylation substrate and its pharmaceutical use, and relates to the field of small polypeptide drug development, and the provided small polypeptide can be used for the manufacture of drugs for preventing and treating ischemic stroke, and for the same mechanism of disease, such as stroke, traumatic brain injury, spinal cord injury, neonatal hypoxic-ischemic encephalopathy, depression and autism, neurodegenerative diseases (e.g., multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease), etc.

Description

[Technical Field]

[0001] The present invention relates to the field of small polypeptide drug development, in particular to artificial small interfering peptides of DAPK1 phosphorylation substrates and their pharmaceutical applications. [Background technology]

[0002] Stroke, also known as "cerebrovascular disease," is a group of acute cerebrovascular diseases characterized by sudden onset and focal neurological damage. It is characterized by high incidence, mortality, disability, and recurrence rates, as well as high associated medical costs and complications, low recognition rates, low treatment rates, and low control rates. According to World Health Organization statistics, stroke is the second leading cause of death and the leading cause of adult disability worldwide. It severely impacts human health and quality of life, causing significant psychological burden and economic losses for patients' families and society. According to the "China Stroke Prevention and Treatment Report 2018," stroke is the leading cause of adult death and disability in China, accounting for approximately one-third of all deaths from cerebrovascular disease worldwide. 1.96 million people die from cerebrovascular disease each year, while 12.42 million survive. Incidence rates are rapidly increasing and tending to be younger.

[0003] Strokes are divided into two types: ischemic and hemorrhagic. Ischemic stroke accounts for approximately 87% of cases. It is a serious neurological disorder caused by the blockage of blood flow due to thrombus formation or embolism, resulting in sudden paralysis, speech impairment, vision loss, or even death. Currently, the most effective treatment is thrombolytic therapy using recombinant tissue plasminogen activator (rtPA). However, the so-called treatment window for stroke is so short that it is difficult to control the time in practice. In addition, blood reperfusion injury is prone to causing intracranial hemorrhage. Therefore, only about 5% of patients currently receive thrombolytic therapy, and most patients receive symptomatic or supportive care. Therefore, using neuroprotective drugs to protect normal neurons and save surviving neural tissue can reduce the area of ​​cerebral infarction and avoid complications associated with thrombolytic or anticoagulant therapy, and allows for early treatment without the need for detailed etiological identification and diagnosis. Furthermore, because neuroprotective drugs can be used for hemorrhagic stroke, their therapeutic effectiveness and future potential have made them a hot topic in stroke treatment research in recent years. However, although laboratories around the world have already developed over 1,000 small molecule compounds as neuroprotective drugs for ischemic stroke, and over 200 clinical trials have been conducted, no generally accepted neuroprotective drug has yet been developed. Therefore, it is extremely important to continue developing new drugs and exploring new treatment methods to effectively treat stroke by reducing neuronal cell death.

[0004] In ischemic stroke, a lack of oxygen and glucose depletes neurons' cellular energy reserves, triggering a complex cascade of reactions involving multiple mechanisms, the interaction of which leads to neuronal damage and death. These reactions include imbalance in ion homeostasis, generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and mitochondrial dysfunction, ultimately leading to cell death through either necrosis or apoptosis. Death-associated protein kinase 1 (DAPK1), a calmodulin-regulated serine / threonine protein kinase, plays a key role in the cell signaling pathways involved in this cascade. Research has shown that the DAPK1-NR2B pathway, DAPK1-DANGER pathway, DAPK1-p53 pathway, and DAPK1-Tau pathway are all involved in cell death induced by oxygen and glucose deprivation, and blocking these cascades in acute treatment of ischemia and oxygen deprivation animal models can effectively reduce neuronal death. However, to date, no effective drugs have been successfully developed for clinical use from this perspective, so more drugs need to be developed for ischemic stroke.

[0005] Polypeptides generally refer to low molecular weight proteins containing less than 100 amino acids, and have advantages not found in high molecular weight drugs such as low molecular weight compounds and proteins. Like low molecular weight compounds, they do not require digestion, so they are quickly absorbed even when injected directly, are absorbed at nearly 100% absorption rate, do not consume energy, do not cause gastrointestinal or metabolic burdens, and can be absorbed as a vector. However, like high molecular weight compounds, they are well recognized as molecules, have clear structure-function relationships, remarkable pharmacodynamic properties, low drug doses, strong physiological activity, low toxicity, few or no side effects, and do not accumulate. Therefore, they have become an important direction in pharmaceutical development in recent years.

[0006] [Prior art document] [Non-patent literature] [Crossref]1]DOI:10.3969 / j.issn.1000-3614.2019.02.001 L Wang,J Liu,Y Yang,B Peng,Y Wang(2019)The prevention and treatment of stroke still faces huge challenges-Brief Report on Stroke Prevention and Treatment in China,2018.Chinese Circulation Journal,34:105-19. [PubMed]DoI:10.3389 / fnmol.2016.00046 P Singh,P Ravanan,P Talwar(2016)Death Associated Protein Kinase 1(DAPK1):A Regulator of Apoptosis and Autophagy.Front.Mol.Neurosci,9:46. [End Page 3]DOI:10.1007 / s12035-016-0008-y S Wang,X Shi,H Li,P Pang,L Pei,H Shen,Y Lu(2017)DAPK1 Signaling Pathways in Stroke:from Mechanisms to Molecular Therapies Neurobiology,54:4716-4722. [ PubMed ] 4]DOI:10.1074 / jbc.M505804200 MShamloo,L Soriano,T Wieloch,K Nikolich,R Urfer,D Oksenberg(2005)Death-associated protein kinase is activated by dephosphorylation in response to cerebral ischemia J Biol Chem,280:42290–9. 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Biochemistry, 45:13614-21. [PubMed 12]DOI:10.1128 / MCB.01595-06 A Craig,J Chrystal,K Fraser,N Sphyris,Y Lin,B Harrion,M Scott,I Dornreiter,T Hupp(2007)The MDMs Ubiquitination Signal in the DNA-Binding Domain of p53 Forms a Docking Site for Calcium Calmodulin Kinase Superfamily Members.MCB,27:3542-55. [End Page 13]DOI:10.1038 / embor.2008.246 EZalckvar, Berissi H, Mizrachy L, Idelchuk Y, Koren I, Eisenstein M, Sabanay H, Pinkas-Kramaiski R, Kimchi A(2009)DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and induction of autophagy.EMBO Rep,10:285-92. [Operations 14]DOI:10.1128 / MCB.24.19.8611-8626.2004 G Shani,L Marash,D Gozuacik,S Bialik,L Teitelbaum,G Shohat,A Kimchi(2004)Death-associated protein kinase phosphorylates ZIP kinase,forming a unique kinase hierarchy to activate its cell death functions.MCB,24:8611-26. 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[Non-Patent Document 22] DOI:10.1161 / 01.str.31.1.193 S Ahmed,Y He,A Nassief,J Xu,X Xu,C Hsu,F Faraci(2000)Effects of lipopolysaccharide priming on acute ischemic brain injury.Stroke,31:193-9. [Non-Patent Document 23] DOI:10.1097 / 01.WCB.0000096063.84070.C1 I Yonekura, N Kawahara, H Nakatomi, K Furuya, T Kirino (2004) A model of global cerebral ischemia in C57BL / 6 mice.J Cereb Blood Flow Metab,24:151-8. [Non-patent Document 24] DOI:10.1155 / 2018 / 6381932 M Ru, H Liu(2018) Association between Y-Maze Acquisition Learning and Major Histocompatibility Complex Class II Polymorphisms in Mice.Biomed Res Int,2018:6381932. [Non-Patent Document 25] DOI:10.1074 / jbc.M104273200 A Velentza, A Schumacher, C Weiss, M Egli, D Watterson (2001) A protein kinase associated with apoptosis and tumor suppression: structure, activity, and discovery of peptide substrates. J Biol Chem, 276:38945-65. Summary of the Invention [Problem to be solved by the invention]

[0007] In response to the above-mentioned needs in the art, the present invention aims to develop a drug for ischemic stroke in the direction of a low molecular weight polypeptide drug, and based on the research results, provides a method for developing a drug for preventing and treating ischemic stroke, as well as low molecular weight polypeptides, their intermediate products (gene sequences, expression vectors), and their use in the production of drugs for preventing and treating ischemic stroke. [Means for solving the problem]

[0008] Specifically, the present invention claims the following technical solutions: 1. An artificial small interfering peptide of a DAPK1 phosphorylation substrate, characterized in that the amino acid sequence is as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0009] 2. It has the amino acid motif shown in (A) or (B) below, (A)XXX(R / K)(R / K)(R / K)(R / K)X2(S / T / A)X1XXX (B)XXX(R / K)(R / K)(R / K)X2(R / K)(S / T / A)X1XXX provided that each X is independently selected from any amino acid or no amino acid; X1 is a polar amino acid selected from asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), or threonine (T); X2 is a non-polar amino acid selected from alanine (A), isoleucine (I), leucine (L), methionine (M), and valine (V); where R / K means that either arginine (R) or lysine (K) can be used at that position; where S / T / A means that the position may be any of serine (S), threonine (T), or alanine (A); Preferably, the amino acid sequence of the artificial small interfering peptide of a DAPK1 phosphorylation substrate is different from at least one of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 by two or fewer amino acids, i.e., has at least 85% identity.

[0010] 3. The artificial small interfering peptide according to Example 2, whose amino acid sequence is as set forth in any one of SEQ ID NOs: 6 to 54.

[0011] 4. A mimetic peptide of the artificial small interfering peptide described in Example 2, whose structure is a staple peptide or a cyclic peptide, for example, a ring formed by a chain (amide bond), a ring formed by a side chain, a ring formed by a thioester bond, a ring formed by a lactone bond, a ring formed by oxidation of Se-Cys and Se-Cys, or a cyclic peptide formed by a disulfide bond.

[0012] 5. A retropolypeptide of an artificial small interfering peptide according to any one of Examples 1 to 3, in which the order of amino acids in the peptide chain is changed from the C-terminus to the N-terminus compared to the artificial small interfering peptide.

[0013] 6. A D-retro-inverso peptide of an artificial small interfering peptide described in any one of Examples 1 to 3, in which, compared to the artificial small interfering peptide, each L-amino acid residue in the artificial small interfering peptide is replaced by its corresponding D-amino acid, the amino acid sequence is reversed, and the original spatial orientation and chirality of the side chains are the same as those of the artificial small interfering peptide, i.e., a side chain topology structure similar to that of the artificial small interfering peptide is maintained.

[0014] 7. A derivative peptide of the artificial short interfering peptide described in any one of Examples 1 to 3, characterized in that one or more amino acids in the artificial short interfering peptide described in any one of Examples 1 to 3 are replaced with their corresponding D-amino acids or homoamino acids.

[0015] 8. Two or more small peptides are polymerized in a parallel manner in the same direction, and the C-terminus of each small peptide is free, and the N-terminus of all the small peptides are gathered together to be connected to a delivery vector; The low molecular weight peptide is a polypeptide selected from the group consisting of an artificial low molecular weight interfering peptide described in any one of Examples 1 to 3, a retropolypeptide described in Example 5, a D-type retro-inverso peptide described in Example 6, and a derivative peptide described in Example 7.

[0016] 9. One or more delivery vectors are fused to the N-terminus or C-terminus of a small peptide; A fusion polypeptide, characterized in that the low molecular weight peptide is selected from the artificial low molecular weight interfering peptide described in any one of Examples 1 to 3, the retropolypeptide described in Example 5, the D-type retro-inverso peptide described in Example 6, or the derivative peptide described in Example 7.

[0017] 10. The delivery vector is selected from a membrane-permeable peptide, a ligand, a receptor protein transduction domain (PTD), an antibody, or a high molecular weight polymer; the membrane-permeable peptide is selected from a cationic cell-membrane-permeable peptide, an amphipathic cell-membrane-permeable peptide, a hydrophobic cell-membrane-permeable peptide, or a synthetic cell-membrane-permeable peptide; The polypeptide according to Example 8 or the fusion polypeptide according to Example 9, wherein the polymer is selected from the group consisting of polyethylene glycol (PEG), polylactic acid, poly(lactide-co-glycolide), polyglycolic acid, polycaprolactone, polyethylene oxide, polydioxanone, polypropylene fumarate, trimethylene carbonate, polyesteramide oxirane, esteramide, β-hydroxyphenylpropionate, α-hydroxy acid, polyhydroxyalkanoic acid, polyhydroxybutyric acid, polyimide carbonate, polyurethane, polyanhydride, hyaluronic acid, chitosan, cellulose, gelatin, and collagen.

[0018] 11. The cationic cell membrane-permeable peptide is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 55 to SEQ ID NO: 72; the amphipathic cell membrane-permeable peptide is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 73 to SEQ ID NO: 81, the hydrophobic cell membrane-permeable peptide is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 82 to SEQ ID NO: 85; The fusion polypeptide of Example 10, wherein the synthetic cell membrane-permeable peptide has the amino acid sequence set forth in SEQ ID NO: 86.

[0019] 12. The fusion polypeptide described in Example 11, wherein the artificial small interfering peptide has an amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 and 6 to 54, and the membrane-permeable peptide has an amino acid sequence shown in any one of SEQ ID NOs: 55 to 86.

[0020] 13. A fusion polypeptide according to Example 10, characterized in that the amino acid sequence is as set forth in any one of SEQ ID NOs: 87 to 96.

[0021] 14. An artificially produced nucleic acid molecule encoding a low molecular weight polypeptide, wherein the low molecular weight peptide is selected from the group consisting of an artificial low molecular weight interfering peptide described in any one of Examples 1 to 3 and a retropolypeptide described in Example 5.

[0022] 15. An expression vector, characterized in that it is loaded with the nucleic acid molecule described in Example 14.

[0023] 16. An expression system, characterized in that it is a cell line or a cell-free expression system containing the vector described in Example 15.

[0024] 17. An expression product expressed by the expression system described in Example 14, wherein the main component is a low molecular weight polypeptide, and the low molecular weight peptide is selected from the group consisting of the artificial low molecular weight interfering peptide described in any one of Examples 1 to 3 and the retropolypeptide described in Example 5.

[0025] 18. A composition comprising a polypeptide molecule and pharmaceutically acceptable impurities, additives, solvents, protectants, adjuvants, carriers and / or excipients, wherein the polypeptide molecule is (1) An artificial small interfering peptide described in any one of Examples 1 to 3, a retropolypeptide described in Example 5, a D-type retroinverso peptide described in Example 6, or a derivative peptide described in Example 7, or (2) A modified product of a low molecular weight peptide, wherein the low molecular weight peptide is selected from the group consisting of the artificial low molecular weight interfering peptides described in any one of Examples 1 to 3, the retropolypeptide described in Example 5, the D-type retro-inverso peptide described in Example 6, and the derivative peptide described in Example 7, and the modification is A drug characterized by one or more of the following: N-terminal, C-terminal modified, labeled, cyclized, lipidated, N-methylated, myristoylated and palmitoylated, glycosylated, biotinylated, PEG-modified, fluorescently labeled.

[0026] 19. The drug according to Example 18, wherein the dosage form is an aerosol formulation for inhalation, an oral formulation, or a formulation for intravenous, intraarterial, intracranial, intraperitoneal, intranasal, intramuscular, subcutaneous, intrasynovial, intrasternal, or intraspinal administration.

[0027] 20. Pharmaceutical use of a small peptide, The small peptide is selected from the artificial small interfering peptide described in any one of Examples 1 to 3, the retropolypeptide described in Example 5, the D-type retro-inverso peptide described in Example 6, or the derivative peptide described in Example 7; The pharmaceutical use is for the manufacture of a medicament for treating or preventing diseases associated with excitotoxicity mechanisms, including, but not limited to, stroke, traumatic brain injury, spinal cord injury, neonatal hypoxic-ischemic encephalopathy, neurodegenerative diseases, depression, and autism; The neurodegenerative disease is multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

[0028] 21. Pharmaceutical use of a small peptide, The small peptide is selected from the artificial small interfering peptides described in any one of Examples 1 to 3, the retropolypeptides described in Example 5, the D-type retro-inverso peptides described in Example 6, or the derivative peptides described in Example 7, and the pharmaceutical use is to manufacture a drug for treating or preventing physiological disorders associated with the DAPK1-PKD1 pathway, and the diseases include, but are not limited to, stroke, traumatic brain injury, spinal cord injury, neonatal hypoxic-ischemic encephalopathy, neurodegenerative diseases, depression, and autism, The neurodegenerative disease is multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

[0029] 22. A drug according to Example 18 or 19 and an anticoagulant or antiplatelet agent, Preferably, the drug according to Example 18 or 19 and the anticoagulant or antiplatelet drug are packaged together in a single dose, a daily dose, or a course of doses; However, the anticoagulant is not limited to acenocoumarol, 4-hydroxy-3-(1,2,3,4-tetrahydro-1-naphthyl)coumarin (coumatetralyl), dicumarol, dicumarol ethyl, phenprocoumon, warfarin, diphenadione, phenindione, thiochromarol, bemiparin, certoparin sodium, dalteparin, enoxaparin, nadroparin, parnaparin, reviparin, tinzaparin, fondaparinux, idraparinux, danaparoid, sulodexide, dermatan sulfate, apixaban, betrixaban, edoxaban, otamixaban, rivaroxaban, hirudin, bivalirudin, and combinations thereof. recombinant hirudin, desirudin hirudin, argatroban, dabigatran etexilate, melagatran, ximelagatran, defibrotide, antithrombin III, heparin, Coumadin tablets, dabigatran etexilate, apixaban (trade name Eliquis), edoxaban, enoxaparin, fondaparinux, recombinant tissue plasminogen activator, tissue plasminogen activator, alteplase, reteplase, tenecteplase, urokinase, saruplase, streptokinase, anistreplase, monteplase, ancrod, fibrinolysin, brinase, or a composition thereof; The antiplatelet drug is clopidogrel, ticagrelor, prasugrel, dipyridamole, cilostazol, ticlopidine, eptifibatide, aspirin, abciximab, tirofiban, beraprost, prostacyclin, iloprost, treprostinil, aloxipirin, carbaspirin calcium, indobufen, triflusal, picotamide, terutroban, chloricromene, ditazol, or a composition thereof.

[0030] 23. Use of a small peptide for treating a disease, The small peptide is selected from the artificial small interfering peptide described in any one of Examples 1 to 3, the retropolypeptide described in Example 5, the D-type retro-inverso peptide described in Example 6, or the derivative peptide described in Example 7; However, the dosage ranges from 0.001 mg / kg body weight to 50 mg / kg, and the concentration of the small peptides described herein may vary widely and is selected depending on the administration method selected and the subject's body weight, age, sex, etc. A preferred dosage range is a dosage of 0.01 mg / kg body weight to 50 mg / kg; A more preferred dosage range is a dosage of 0.1 mg / kg body weight to 10 mg / kg; or varying the dose range to optimize a treatment regimen in a subject or group of subjects; The diseases include, but are not limited to, stroke, traumatic brain injury, spinal cord injury, neonatal hypoxic-ischemic encephalopathy, neurodegenerative diseases, depression, and autism; The neurodegenerative disease is multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease. [Effects of the Invention]

[0031] After oxidative stress-induced damage in 293T cells and human umbilical vein endothelial cells, DAPK1 is activated, leading to the phosphorylation of its interacting protein kinase D1 (PKD1). PKD1 activation is crucial for the binding of apoptosis signal-regulated kinase 1 (ASK1) and the downstream phosphorylation of c-Jun N-terminal kinase (JNK). Studies have also shown that the ASK1-dependent JNK signaling pathway mediates caspase-dependent apoptosis and caspase-independent necrotic cell death after ischemia. However, no studies have yet concluded whether the DAPK1-PKD1 pathway in neurons is involved in the cascade of events that triggers cell death under ischemia and hypoxia, nor have they investigated whether interference or blockade of the DAPK1-PKD1 pathway can effectively alleviate ischemic neuronal damage or whether it could serve as a novel molecular target for the development of therapeutic drugs for ischemic stroke.

[0032] The inventor hypothesized the influence of the DAPK1-PKD1 pathway on ischemic neuronal injury and based on this, designed a PKD interference peptide, deriving a series of small peptides with the same motif. These small polypeptides were subjected to a series of tests in cells and animal models to obtain the technical solution of the present invention. PKD1 refers to protein kinase D1, a member of the protein kinase D (PKD) family. It is a cytoplasmic serine-threonine kinase widely expressed in cells and possesses unique structure, enzymatic properties, and regulatory characteristics. The PKD family has three members: PKD1, PKD2, and PKD3. PKD1 has been studied more than PKD2 and PKD3. It is generally believed that PKD2 and PKD1 are similar in distribution and function, while PKD3 mainly shuttles between the cytoplasm and the cell nucleus. In terms of domains, the three PKD members share a high degree of homology. PKD has been reported to be involved in many cellular functions, including Golgi self-organization and plasma membrane-directed transport, metastasis, immune response, apoptosis, and cell proliferation (doi:10.1152 / physiol.00037.2010, Progress in Physiological Sciences, 2011, Vol. 42, No. 5).

[0033] The small polypeptide drug developed by the present invention was used in an in vitro oxidative stress injury model and a whole animal stroke model, and the test data demonstrated that the small polypeptide drug developed by the present invention had a significant neuroprotective effect in a glutamate apoptosis model and effectively inhibited oxygen-glucose deprivation-induced damage to primary neurons. That is, it effectively interferes with the neuronal DAPK1-PKD1 pathway, inhibiting the downstream signals of caspase-dependent apoptosis and caspase-independent necrosis of neurons, thereby alleviating brain damage after ischemic stroke. Therefore, the small interfering peptide developed by the present invention can be used to manufacture drugs for treating or preventing diseases related to excitotoxicity mechanisms, including, but not limited to, stroke, traumatic brain injury, spinal cord injury, neonatal hypoxic-ischemic encephalopathy, neurodegenerative diseases (e.g., multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease), depression, and autism.

[0034] Definitions of terms: The term "natural amino acids" refers to the 20 common amino acids that are automatically produced in living organisms, and are generally L-amino acids.

[0035] The term "D-amino acids" refers to amino acids that share the same structural chirality as L-amino acids and are artificially synthesized using D-glyceraldehyde. With the exception of glycine, all other amino acids have stereoisomers (mirror images of the structure).

[0036] The term "homo-amino acids", also called long-chain amino acids, refers to derivatives of naturally occurring amino acids, which are derived by inserting a methylene group (CH) into the naturally occurring amino acid carbon backbone adjacent to the carboxy group, thereby lengthening the carbon chain of the amino acid, thereby improving the physiological activity and biological stability of polypeptides.

[0037] Unless otherwise defined or explained, the terms "polypeptide" and "artificial small polypeptide" have amino acid sequence characteristics generally understood in the art, for example, the amino acid residues are natural L-amino acids, and the left end of the amino acid sequence is an α-NH group, i.e., the left side is the N-terminus, and the right end is an α-COOH group, i.e., the right side is the C-terminus.

[0038] The term "retropolypeptide" refers to a peptide chain in which the order of amino acids in the chain has been changed from the C-terminus to the N-terminus compared to the original peptide chain. For example: N SGVRRRRLSNVSL C The retropolypeptide of N LSVNSLRRRRVGS C is.

[0039] The term "D-retro-inverso peptide" refers to a peptide obtained by replacing each L-amino acid residue in an original L-amino acid peptide with its corresponding D-amino acid, compared to an original L-amino acid peptide composed of natural L-amino acids, in which the amino acid sequence is reversed while the original spatial orientation and chirality of the side chains are the same as in the original L-amino acid peptide, i.e., the side chain topology similar to that of the original L-amino acid peptide is maintained. [Brief explanation of the drawings]

[0040] [Figure 1]Figure 1 shows the identification and mass spectrometry results of the fusion polypeptide RvTAT-PKD-S205 of the present invention. (A) Chromatogram of RP-HPLC analysis of the purified product of RvTAT-PKD-S205: After synthesis and purification, RvTAT-PKD-S205 was synthesized by reverse-phase HPLC and quantified at 210 nm. The peak retention time was 11.797 minutes (arrow indicated), and the purity of the final product was 98.3%. (B) Mass spectrum of RvTAT-PKD-S205: The RvTAT-PKD-S205 peptide was identified by liquid chromatography-mass spectrometry. The molecular weight of the synthetic peptide was 2920.0 daltons (arrow indicated), and the theoretical molecular weight was 2919.43 daltons. [Figure 2] Figure 2 shows the effect of the fusion polypeptide of the present invention on glutamate-induced HT22 cell death. (A) HT22 cells containing five different concentrations of RvTAT-PKD-S205 (0 to 1600 nM) were exposed to 6 mM glutamate (GLUT) for 24 hours at 37°C, and the cytotoxicity of the cells was determined by MTT assay. The 0 nM RvTAT-PKD-S205 group served as the control. Cell death rate (%) = 100% × (control OD - treatment OD) / control OD. Data are mean ± SEM (n = 3). *p < 0.05, **p < 0.01. One-way analysis of variance followed by multiple comparison with the 0 nM RvTAT-PKD-S205 group (Bonferroni t-test). (B) Experimental procedure and sampling plan. [Figure 3]Figure 3 shows the neuroprotective effects of the fusion polypeptides of the present invention in a neuronal oxygen-glucose deprivation / reperfusion (OGD / R) model and a comparison of different fusion polypeptides. (A) Different concentrations of RvTAT-PKD-S205, RvTAT-ZIPK-T299, and RvTAT-opMLC-S20 were added to primary rat cortical neurons (day 7) 30 minutes before OGD treatment. To induce OGD, the neurons were transiently treated with EBSS (i.e., Earle's balanced salt solution (EBSS)) containing 20 mM sodium dithionite (NaSO) at pH 7.2 and the test drug for 1.5 hours. The OGD medium was then replaced with normal NS basal medium to simulate reperfusion. The cultured cells were then incubated for an additional 20 hours, after which cytotoxicity was measured using the MTT assay. Non-OGD-treated cell cultures served as controls because they were 100% viable; cell death (%) due to test drug = 100% × (control OD - test drug OD) / control OD. Data are mean ± SEM (n = 3). ***p < 0.005, one-way ANOVA followed by multiple comparisons with the OGD control group (Bonferroni t-test). (B) Experimental procedure and sampling plan. [Figure 4] Figure 4 compares the effects of different fusion polypeptides on glutamate-induced HT22 cell death. HT22 cells containing 400 nM of each test drug (RvTAT-PKD-S205, RvTAT-ZIPK-T299, RvTAT-rSP6-S235, RvTAT-opMLC-S20, and RvTAT-BECN1-T199) in the culture medium were exposed to 6 mM glutamate at 37°C. After 24 hours, cytotoxicity was determined by MTT assay. HT22 cells without test drug in the culture medium were exposed to 6 mM glutamate alone as a control. Cell death (%) = 100% × (control OD - treatment OD) / control OD. Data are mean ± SEM (n=3), *p<0.05, **p<0.01 by one-way ANOVA followed by multiple comparisons with the glutamate control group (Bonferroni t-test). [Figure 5]Figure 5 shows the neuroprotective effect of the fusion polypeptide of the present invention in a whole-animal transient middle cerebral artery occlusion and reperfusion model. (A) Experimental procedure and sampling plan. Sprague-Dawley rats underwent 90 minutes of middle cerebral artery occlusion followed by reperfusion (tMCAO / R). 4.5 hours later, they received intravenous treatment with 3.5 mg / 2 mL / kg of RvTAT-PKD-S205 or saline as a control. 24 hours after ischemia and reperfusion, some rats underwent TTC staining. The remaining rats underwent rotarod testing on days 3, 5, and 7. (B) Representative images of brain slices. (C) Quantitative comparison of cerebral infarction size between the saline and RvTAT-PKD-S205-treated rats (saline group, n = 13; RvTAT-PKD-S205 group, n = 12). (D) Motor ability was assessed by rotarod testing. The time until the rats fell off the rotarod was recorded. (Sham group n = 8, saline group n = 6, RvTAT-PKD-S205 group n = 10). Data are mean ± SEM, *p < 0.05, **p < 0.01, Student's t-test. Animals were randomly assigned to treatment groups in a blinded manner. [Figure 6]6 shows the neuroprotective function of the fusion polypeptide of the present invention in a transient global cerebral ischemia-reperfusion animal whole-body stroke model. (A) Experimental procedure and sampling plan. C57BL / 6 mice were subjected to bilateral carotid artery occlusion for 20 minutes twice followed by reperfusion (tBCCAO / R). Three hours after tBCCAO, they were treated with 7 mg / 2 mL / kg of RvTAT-PKD-S205 or an equivalent volume of saline via the tail vein. After 24 hours of reperfusion, they were tested in a Y-maze passive avoidance test and MDA and brain water content were measured. (B) Mice that received electric foot shocks and learned to avoid the electric shock zone were tested and evaluated for learning and memory ability in ischemic mice in a Y-maze passive avoidance test. (C) ELISA measurement of MDA content in the brain after 24 hours of reperfusion. (D) Measurement of brain water content after 24 hours of reperfusion. Data are mean ± SEM (n = 11 per group). *p < 0.05, **p < 0.01 compared with the saline control group by Student's t-test. Animals were randomly assigned to treatment groups in a blinded manner. DETAILED DESCRIPTION OF THE INVENTION

[0041] The embodiments described below with reference to the drawings are illustrative and are not intended to limit the present application but to serve to interpret the present application. Any other embodiments that a person skilled in the art can obtain based on the embodiments of the present application without performing any novel work fall within the scope of the claims of the present application. [Example]

[0042] 1. Development of small interfering peptides The present inventors hypothesized the influence of the DAPK1-PKD1 pathway on ischemic neuronal damage and selected amino acids between positions 197 and 210 of protein kinase D to construct a small interfering peptide (PKD-S205), as shown in SEQ ID NO: 1 in Table 1.

[0043] DAPK1 has various phosphorylation-specific substrates, and in addition to its interaction with PKD1, DAPK1 phosphorylates myosin II regulatory light chain (MLC) to activate myosin, leading to plasma membrane vacuolization during stress response-induced signaling cascades. DAPK1 phosphorylates ribosomal protein S6 kinase (rSP6) at serine 235, reducing protein translation. DAPK1 also phosphorylates the autophagy-inducing protein Beclin 1 (BECN1) at threonine 119 in the BH3 domain, promoting the dissociation of Beclin 1 from B-cell lymphoma-extralarge (Bcl-XL), thereby inducing autophagy. DAPK1 phosphorylates Zipper-interacting protein kinase (ZIPK) at threonine 299, thereby altering its intracellular location and promoting cell death.

[0044] The present invention also designs interference peptides shown in SEQ ID NOS: 2 to 6 in Table 1 for the phosphorylation sites of these phosphorylation substrates of DAPK1.

[0045] [Table 1-1]

[0046] [Table 1-2]

[0047] [Table 1-3]

[0048] [Table 1-4]

[0049] 2. Cell membrane-penetrating peptides Cell penetrating peptides (CPPs) are short peptides that can pass through cell membranes or tissue barriers (e.g., the blood-brain barrier) independently, without relying on specific membrane receptors. They generally consist of 30 or fewer amino acids and can transport macromolecules such as proteins, RNA, and DNA into cells by mechanisms such as endocytosis or direct penetration, allowing them to exert their effector functions. They are normally degraded after entering the cells, and therefore have good biocompatibility and low cytotoxicity.

[0050] Membrane-permeable peptides well known in the art (eg, those shown in the table below) can be used in the present invention.

[0051] [Table 2-1]

[0052] [Table 2-2]

[0053] 3. Fusion Polypeptides In an exemplary embodiment of the present invention, the retrosequence TAT(RRRQRRKKRG) of TAT(47-57) is selected as a membrane-permeable peptide and connected to the small interfering peptide developed by the present invention to obtain a fusion molecule. The membrane-permeable peptide is connected to the N-terminus or C-terminus of the small interfering peptide. The amino acid sequences of some fusion polypeptides of the present invention are as shown in the table below.

[0054] [Table 3]

[0055] 4. Preparation of fusion polypeptides The fusion polypeptide may be expressed by an expression system and then purified, which is a mature technique in the art and therefore will not be described here.

[0056] The fusion polypeptides provided by the present invention are small polypeptides, and are preferably chemically synthesized.

[0057] The fusion polypeptide used in the subsequent tests of the present invention was synthesized by GenScript Biotechnology Co., Ltd. (GenScript, China) using Fmoc-based chemical solid phase peptide synthesis (SPPS).

[0058] The SPPS method involves sequentially adding amino acids to the resin to construct a peptide chain. After synthesis, the Fmoc group at the N-terminus was deprotected, followed by deprotection of the side chain protecting groups. The peptide was then cleaved from the resin and purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The purification solvent was acetonitrile + deionized water, each buffered with 0.1% trifluoroacetic acid (TFA), using a gradient elution. Analytical RP-HPLC confirmed the purity to be greater than 95%, and the components were determined by measuring their molecular weights using electrospray ionization mass spectrometry (ESI-MS) (Figure 1). The polypeptide content was measured by nitrogen quantification using a Vario Micro Element Analyzer. The actual polypeptide amount was calculated by weight x purity x content.

[0059] All fusion polypeptides herein are white powders, stored in the dark at −20°C, completely soluble in water, prepared as 200 μM stock solutions in sterile water or saline, and evaluated in injury models at concentrations ranging from 0.1 to 2 μM.

[0060] 5. In vitro oxidative stress damage model 1. Glutamate loading model of the HT22 mouse hippocampal neuron line reagent: Neurobasal Medium: Thermo Fisher Scientific Gibco 10888022 DMEM medium (Dulbecco's Modified Eagle Medium): Thermo Fisher Scientific 10569077 B-27 商標 Plus Supplement (50x): Thermo Fisher Scientific Gibco A3582801 1.1 Glutamate tolerance test HT22 mouse hippocampal neuron line cells were cultured and maintained in DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were maintained at 37°C in a 5% CO2 / 95% air atmosphere. For drug testing, 6 × 10 3 HT22 cells were seeded into a 96-well plate at 100 cells / well and cultured at 37°C in a CO2 incubator for 24 hours. After that, the medium was replaced with 2% B27 (B-27 商標 Neurobasal medium supplemented with NS Plus Supplement (50x) and different concentrations of the test drug were then replaced, and HT22 cells were exposed to 6 mM glutamate and continuously cultured at 37°C in a CO2 incubator for another 24 hours. Cell viability was measured by MTT colorimetric assay.

[0061] 1.2 Analysis and evaluation of cell viability by MTT assay Cell viability was quantitatively measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Thiazolyl Blue Tetrazolium Bromide, MTT) assay.

[0062] Water-soluble yellow dye MTT reagent (final concentration 0.5 mg / mL) was added to each well and incubated at 37°C. After 4 hours, the incubation medium was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added. After dissolution at room temperature for 30 minutes, the absorbance of the formazan solution was measured spectrophotometrically at 570 nm.

[0063] MTT absorbance data are transformed to show the ratio of cell viability relative to untreated and treated controls, with the untreated control considered to have 100% viability.

[0064] 1.3 Test Results HT22 cells were exposed to 6 mM glutamate while adding different concentrations of the RvTAT-PKD-S205 polypeptide. Cell viability was measured by MTT colorimetric assay 24 hours later. The RvTAT-PKD-S205 peptide at 100 nM, 200 nM, 400 nM, and 1600 nM reduced cell death by 11%, 22%, 32%, and 24%, respectively. The 400 nM and 1600 nM concentrations showed significant neuroprotective effects, but the 1600 nM concentration showed a lower neuroprotective effect.

[0065] The test results revealed that the RvTAT-PKD-S205 chimeric peptide provided by the present invention had a significant concentration-dependent neuroprotective effect in the glutamate-induced apoptosis model, and showed that 400 nM was the optimal administration concentration of the RvTAT-PKD-S205 polypeptide (Figure 2).

[0066] 2. Oxygen-glucose deprivation (OGD) reperfusion model of rat primary cortical neuron cultures reagent: Dulbecco's Modified Eagle Medium (DMEM): Thermo Fisher Scientific 30030 Earle's Balanced Salt Solution (EBSS): Thermo Fisher Scientific 14155063 2.1 Oxygen-glucose deprivation reperfusion test Primary cultures of cortical neurons were prepared from Sprague-Dawley (SD) rats on embryonic days 15-18 (Wenxiang Fan, Xiang Li, Liangliang Huang, Shucheng He, Zhicheng Xie, Yuxin Fu, Weirong Fang, Yunman Li). S-oxiracetam ameliorates ischemic stroke induced neuronal apoptosis through up-regulating α7 nAChR and PI3K / Akt / GSK3β signal pathway in rats. Neurochemistry International Volume 115, May 2018, Pages 50-60 https: / / doi.org / 10.1016 / j.neuint.2018.01.008). Specifically, the following procedures were performed: Cerebral cortices were dissociated in Dulbecco's modified Eagle's medium (DMEM) and digested with 0.25% trypsin for 5 minutes at 37°C. The digestion was stopped by adding fetal bovine serum (10% final concentration). The cells were then centrifuged at 500g for 5 minutes and then resuspended by repeated pipetting with a Pasteur pipette. The cells were dissociated in Neurobasal medium supplemented with 2% B27 (v / v), 1 mM glutamine, 50 U / mL penicillin, and 50 U / mL streptomycin. The cells were seeded into 24-well plates (coated with 0.1 mg / mL poly-D-lysine) at a density of 1.5 x 10 cells per well. 5 The cells were cultured in a humidified incubator at 37°C with 5% CO2 / 95% air, with the medium changed once every three days, and kept for 7 days before use.

[0067] The oxygen-glucose deprivation test was based on a previously established method (J. Huang, N.D. Kodithuwakku, W. He, Y. Zhou, W. Fan, W. Fang, G. He, Q. Wu, S. Chu, Y. Li. The neuroprotective effect of a novel agent N2 on rat cerebral ischemia associated with the activation of PI3K / Akt signaling pathway. Neuropharmacology, 95 (2015), pp. 12-21) with some modifications to simulate in vivo ischemia / reperfusion (I / R) injury, specifically as follows: Prior to OGD exposure, primary cultures in the drug-testing groups were incubated at 37°C for 30 minutes in a CO2 incubator with different concentrations of the test drug in NS basal medium. The medium was then removed from all primary cultures and replaced with glucose-free Earle's balanced salt solution (EBSS) (20 mM sodium dithionite (NaSO4, pH 7.2) with or without the test drug) in the OGD group, or with EBSS containing 6 mM glucose in the control group. The OGD challenge was terminated by replacing the medium with normal NS basal medium, followed by an additional 20 hours of incubation at 37°C. Finally, cell viability was measured using the MTT colorimetric assay (same as in 1.2).

[0068] 2.2 Test results Two concentrations of RvTAT-PKD-S205 polypeptide were added to primary neurons 30 minutes before OGD, and then sodium dithionite was added to induce oxygen and glucose deprivation in the neurons for 60 minutes. After this period, the neurons were restored to normal glucose and oxygen culture conditions, and cell viability was measured 20 hours later using the MTT colorimetric assay.

[0069] As shown in Figure 3, the protective effect of RvTAT-PKD-S205 on oxygen-glucose-deprived neurons was concentration-dependent, with 300 and 1000 nM RvTAT-PKD-S205 significantly reducing neuronal death by 12% and 22% (Figure 3). The data also demonstrated that RvTAT-PKD-S205 reduced neuronal death in a concentration-dependent manner, and 300 nM of the RvTAT-PKD-S205 polypeptide could effectively inhibit oxygen-glucose deprivation-induced injury to primary neurons.

[0070] 6. Whole Animal Stroke Model 1. Suture-based middle cerebral artery occlusion and reperfusion model in rats For acute cerebral ischemia, we established a rat transient middle cerebral artery occlusion / reperfusion (tMCAO / R) stroke model based on a previous method (Wenxiang Fan, Xiang Li, Liangliang Huang, Shucheng He, Zhicheng Xie, Yuxin Fu, Weirong Fang, Yunman Li. S-oxiracetam ameliorates ischemic stroke induced neuronal apoptosis through up-regulating α7 nAChR and PI3K / Akt / GSK3β signal pathway in rats. Neurochemistry International Volume 115, May 2018, Pages 50-60 https: / / doi.org / 10.1016 / j.neuint.2018.01.008). Specifically, we performed the following steps: Male Sprague-Dawley rats weighing 220–250 g were used. They were allowed free access to food and water and were housed under constant environmental conditions (12 / 12 h light / dark cycle). The night before surgery, rats were fasted but allowed free access to water. For surgery, rats were anesthetized with chloral hydrate (300 mg / kg, intraperitoneal injection) and then secured supine on an electric pad on the operating table. Body temperature was continuously monitored using a rectal probe during the surgical procedure, and maintained at 36.5–37.0°C. The surgical area was disinfected with povidone-iodine or 70% alcohol. A midline neck incision was made, and the soft tissue above the trachea was gently separated using a retractor. The common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully isolated from the vagus nerve. The ECA was ligated at its distal end, and the pterygoid artery was closely connected to the ICA. Then, to prevent backflow of blood, a microvascular clip was placed on the ICA and another on the CCA. The ligated ECA was cauterized to form a stump, and a 4-0 nylon suture with a rounded end was inserted from the ECA stump to the connection with the CCA. The microvascular clip placed in the ICA was removed to allow for suture insertion.The suture was carefully inserted 18–20 mm from the CCA connection into the MCA and then secured at two locations (at the base of the ECA stump and on the ICA). After 90 minutes of occlusion, the suture was carefully withdrawn, the ECA incision was closed, the microvascular clips placed in the CCA were removed, and after confirming reperfusion, the neck skin was sutured. To alleviate postoperative pain and discomfort, topical lidocaine gel was applied to the surgical incision area, and 1.0 mL of saline was injected subcutaneously after surgery to provide hydration. Sham-operated animals underwent the same procedure, except that no suture was inserted into the artery to occlude blood flow. 4.5 hours after tMCAO, the test drug or sterile saline was intravenously injected at a dose of 3.5 mg / 2 mL / kg within 3 minutes. The body temperature of the mice was maintained at 37°C using a heat lamp during the recovery period.

[0071] 2. Brain Tissue Processing and Infarct Volume Measurement Twenty-four hours after reperfusion, the rats were sacrificed under deep anesthesia, and the entire brain tissue was removed. The brains were coronally sliced ​​to obtain 2-mm-thick slices, which were then immediately stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37°C for 15 minutes. The area of ​​the left hemisphere and the unstained area of ​​the right hemisphere (non-infarcted area) were calculated using ImageJ image processing software, and the percentage of infarct area was calculated using the formula: percentage of cerebral infarct area = (area of ​​left hemisphere - non-infarcted area of ​​right hemisphere) / area of ​​left hemisphere × 100%.

[0072] Results: As shown in Figure 5 (A, B, C), a therapeutic study was conducted in a 90-minute transient middle cerebral artery embolization and reperfusion (tMCAO / R) model to assess the therapeutic effects of the RvTAT-PKD-S205 peptide on whole animals. SD rats were divided into two groups and treated with saline alone (control) or saline and RvTAT-PKD-S205 (3.5 mg / kg). 4.5 hours after focal ischemia, a single dose of RvTAT-PKD-S205 (n = 13) or saline (n = 12) was administered via tail vein injection. Approximately 24 hours after model creation, infarct volume was assessed by TTC staining (Figure 5A). After treatment with RvTAT-PKD-S205, total cerebral infarction volume was statistically significantly reduced (approximately 60%) compared to the control group (Figure 5B, C). The results demonstrated that RvTAT-PKD-S205 could exert its activity in vivo and reduce brain damage caused by ischemic stroke.

[0073] 3. Evaluation of motor function using the rotarod device Motor function tests were performed on SD rats using a rotarod apparatus within 7 days before and after focal acute cerebral ischemia. Three days before tMCAO / R surgery, rats were trained on the rotarod apparatus for three consecutive days. The initial ramp was set at 4 rpm, allowing the animals to maintain their hold on the rotarod for 1 min. After a 10-min rest period, the rpm was steadily increased to 20 rpm within 180 s, and the rats were trained to maintain their hold on the rotarod for at least 150 s. For animals unable to maintain their hold on the rotarod for at least 150 s, new criterion tests were set for each of the following two days until they met the criterion. The duration of the rat's ability to maintain its hold on the rotarod within 180 s was measured. The test was terminated if the animal fell off the rotarod or grasped and rotated the rotarod twice without attempting to climb back up. The average duration of the rotarod test was recorded three times on the day before tMCAO / R surgery. The animals were tested using the rotarod apparatus on days 3, 5, and 7 after tMCAO / R (or sham surgery). Three consecutive measurements were taken each day, and the average of the three measurements was calculated. The animals were allowed to rest for 15 minutes between each test.

[0074] Results: To further examine the recovery effect of the RvTAT-PKD-S205 peptide on motor neuron dysfunction in the brain, a 90-minute transient middle cerebral artery occlusion and reperfusion (tMCAO / R) model was used for post-treatment studies. SD rats were divided into three groups: a sham group (without tMCAO / R) (n = 8), a saline-only (control) group (n = 6), or a saline and PKD-interfering peptide (3.5 mg / kg) treatment group (n = 6). For the RvTAT-PKD-S205 and saline treatment groups, a single dose of RvTAT-PKD-S205 and saline was administered via tail vein injection 4.5 hours after focal ischemia. Neuronal dysfunction was assessed using the rotarod test on days 3, 5, and 7 after tMCAO / R.

[0075] After treatment with the RvTAT-PKD1-S205 peptide, the motor performance of rats subjected to tMCAO / R was significantly improved. Compared to the untreated control group, the rod-holding time on days 5 and 7 after tMCAO / R was increased by 70% and 75%, respectively. There was no significant difference in the mean rod-holding time on day 3 after tMCAO / R between the treated and untreated groups (Figure 5D; sham-operated group, n = 8; saline group, n = 6; RvTAT-PKD-S205 group, n = 10). These results demonstrated that RvTAT-PKD-S205 significantly improved the neurobehavioral symptoms induced by ischemic stroke in rats.

[0076] 4. Transient Bilateral Common Carotid Artery Occlusion and Reperfusion (tBCCAO / R)-Induced Cerebral Ischemia in Mice Model Creation: Male C57BL / 6 mice weighing 20–30 g (6–7 weeks old) were used. They had free access to food and water and were housed under a constant environmental condition (12 / 12 h light / dark cycle). Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (350 mg / kg). Then, they were placed on a heating pad to maintain a rectal temperature of 37°C. BCCAO surgery was performed based on previously established methods. The ventral neck area of ​​shaved mice was cleaned with povidone-iodine and then 70% ethanol. A small skin incision was made in the midline of the neck. The medial sternocleidomastoid muscle was dissected and carefully separated from the vagus nerve and connective tissue to expose the common carotid arteries. Atraumatic vascular clips were placed to occlude each artery for 20 min. The clips were then removed, and blood flow was allowed to recover for 10 min. Next, both common carotid arteries were occluded for an additional 20 min, allowing perfusion to recover for 24 h. Three hours after tBCCAO, the test drug was intravenously injected at 7 mg / 2 mL / kg within 3 minutes. During the recovery period, the body temperature of the mice was maintained at 37°C using a heat lamp. The sham-operated control group underwent all surgical procedures except for bilateral common carotid artery occlusion.

[0077] Test: Detection of learning and memory ability of tBCCAO / R mice by passive avoidance test The Y-maze apparatus consisted of three arms (A, B, and C) at 120° angles to each other, connected via a central zone (CZ). An electrical grid (made of stainless steel) was placed under each arm, and a light bulb was located at the outer end of each arm to provide a light source for the safe zone. One of the three arms was designated as the starting zone by the computer, and after the start of the test, it was defined as the non-safe zone. The remaining two arms were randomly divided into a safe zone without foot shock (electrical stimulation) and a non-safe zone with foot shock by the Y-maze video tracking and analysis system.

[0078] To investigate whether the RvTAT-PKD-S205 peptide could improve cognitive and memory impairment in whole animals after stroke, we conducted a therapeutic study of the interfering peptide in a mouse model of global cerebral ischemia induced by bilateral transient common carotid artery occlusion and reperfusion (tBCCAO / R). In this model, bilateral transient common carotid artery occlusion and reperfusion (tBCCAO / R) primarily damages hippocampal neurons, affecting the animals' cognitive and memory functions.

[0079] The day before surgery, researchers trained the mice to avoid the electric shock triggered by the light source, which could maximally reduce the effect of odor. C57BL / 6 mice were divided into three groups (n = 11 per group): a sham model group, a single-dose intravenous injection of saline alone (control), and a saline and RvTAT-PKD-S205 (7 mg / kg) treatment group. The RvTAT-PKD-S205 peptide and saline were injected via the tail vein 3 hours after model creation. Twenty-four hours after tBCCAO / R model creation, learning and memory abilities were assessed using a Y-maze passive avoidance task (Figure 6A).

[0080] After 3 min, the test began by placing the mouse in the start area. A current stimulus of a predetermined intensity (0.05–0.8 mA) was administered, and the animal had to escape to the adjacent safe area and remain there for 30 s to avoid the footshock. The animal was then placed in the start position for the subsequent test. The number of escapes into the safe area was recorded, and any escapes into the non-safe area were considered incorrect. The average escape percentage was calculated over 10 trials. Between animal changes, the apparatus was thoroughly cleaned with disinfectant and / or alcohol spray.

[0081] Results: The activity status of each mouse was determined by the percentage of successful escape attempts. Observation results showed that the percentage of successful escape attempts in the RvTAT-PKD-S205-treated group was significantly higher than in the saline-treated group. The RvTAT-PKD-S205 peptide ameliorated the decline in memory indices caused by tBCCAO / R, with the treated group showing a 30% improvement in memory indices compared to the untreated group (Figure 6B). These results confirmed that RvTAT-PKD1-S205 treatment could significantly alleviate hippocampal neuronal damage after global ischemia.

[0082] Measurement of malondialdehyde (MDA) in the brain of tBCCAO / R mice MDA is one of the important products of lipid peroxidation, and the degree of lipid oxidation can be determined by measuring MDA levels. MDA levels in tissues were estimated using the thiobarbituric acid (TBA) method. After the behavioral test, mice were decapitated and brain tissue was collected. The brain tissue was homogenized in ice-cold saline. The lysate was centrifuged at 3500 rpm for 10 minutes at 4°C, and the supernatant was collected. The MDA content was measured using an MDA TBA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. Results were evaluated using a standard curve and calculated as nM / mg protein.

[0083] Measurement of brain edema in tBCCAO / R mice Twenty-four hours after tBCCAO / R in mice, stroke-induced brain edema was assessed by the wet-dry method. The wet weight was determined immediately after brain dissection, and the tissue was dried at 55°C until a constant weight was reached within 24 hours to determine the dry weight. The formula for the percentage of brain water content was (wet weight - dry weight) / wet weight × 100%.

[0084] Measurement results: MDA concentration in brain tissue is used to assess the degree of neuronal ischemic injury. Compared to saline control tBCCAO / R mice, MDA concentration in the brain of RvTAT-PKD-S205-treated tBCCAO / R mice was reduced by 55%, significantly inhibiting the increase in MDA after neuronal ischemic injury (Figure 6C). Brain water content was measured to assess cerebral edema. After RvTAT-PKD-S205 treatment, brain water content was significantly reduced by 8.5% compared to the saline control group (Figure 6D). Results demonstrate that RvTAT-PKD-S205 can reduce cerebral edema and oxidative stress-induced damage in the experimental tBCCAO / R model.

[0085] Statistical Methods: In whole-animal studies, researchers created stroke models and evaluated stroke identification and test parameters in a blinded manner for the test drug and control (sterile saline) groups. All data are presented as mean ± SEM. Data from two or more groups were analyzed using one-way analysis of variance (ANOVA), followed by multiple comparisons with the control group using post-hoc tests or unpaired two-tailed t-tests to assess mean differences between treatment and untreated control groups. If data did not meet the above one-way ANOVA specifications, multiple comparisons with the control group (Dunn's test) were performed using a nonparametric one-way ANOVA (Kruskal-Wallis test). When comparing, *p<0.05, **p<0.01, and ***p<0.005 were considered statistically significant.

[0086] 7. Comparison of different fusion polypeptides In this study, fusion polypeptides containing other interfering peptides shown in Table 1 were synthesized and evaluated in a glutamate loading model of the HT22 cell line and an OGD model of primary neuronal cultures.

[0087] The OGD model revealed the following: RvTAT-PKD-S205 (membrane-permeable peptide connected to the N-terminus of SEQ ID NO: 1) and its homologous interfering peptides (membrane-permeable peptides connected to the N- or C-terminus of SEQ ID NO: 23 to SEQ ID NO: 39 in Table 1) provided by the present invention provide significant neuroprotection, reducing cortical neuron death by 12% and 18% in a concentration-dependent manner at 300 and 1000 nM, respectively. RvTAT-ZIPK-T299 (a membrane-permeable peptide connected to the N-terminus of SEQ ID NO: 2) and its homologous interfering peptides (a membrane-permeable peptide connected to the N- or C-terminus of SEQ ID NO: 8 to SEQ ID NO: 22 in Table 1) can effectively reduce cortical neuron death by 12% at 1000 nM. The RvTAT-opMLC-S20 peptide (a membrane-permeable peptide attached to the N-terminus of SEQ ID NO: 4) did not significantly improve neuronal survival, and the results of a comparative neuroprotection study using RvTAT-PKD-S205, RvTAT-ZIPK-T299, and RvTAT-opMLC-S20 as examples are shown in Figure 3.

[0088] In a glutamate loading model, we found that RvTAT-PKD-S205 (a membrane-permeable peptide attached to the N-terminus of SEQ ID NO: 1) and its homologous interfering peptides (a membrane-permeable peptide attached to the N- or C-terminus of SEQ ID NO: 23 to SEQ ID NO: 39 in Table 1) and RvTAT-ZIPK-T299 (a membrane-permeable peptide attached to the N- or C-terminus of SEQ ID NO: 2 to SEQ ID NO: 22 in Table 1) are promising neuroprotective peptides, and both peptides can reduce HT22 cell death by an average of approximately 33%. Next, RvTAT-rSP6-S235 (a membrane-permeable peptide attached to the N-terminus of SEQ ID NO: 3) and its homologous interfering peptides (a membrane-permeable peptide attached to the N- or C-terminus of SEQ ID NO: 3) are promising neuroprotective peptides. RvTAT-PKD-S205 (membrane-permeable peptides attached to the N- or C-terminus of SEQ ID NO:40 to SEQ ID NO:54 in Table 1), RvTAT-opMLC-S20 (membrane-permeable peptides attached to the N-terminus of SEQ ID NO:4), RvTAT-BECN1-T199 (membrane-permeable peptides attached to the N-terminus of SEQ ID NO:5) and their homologous interfering peptides (membrane-permeable peptides attached to the N- or C-terminus of SEQ ID NO:6 and SEQ ID NO:7 in Table 1) reduced HT22 cell death by an average of approximately 29%, 25%, and 21%, respectively, at 400 nM. The comparative results using RvTAT-PKD-S205, RvTAT-rSP6-S235, RvTAT-opMLC-S20, and RvTAT-BECN1-T199 as examples are shown in Figure 4.

[0089] The above results also revealed that although all of the interfering peptides interact with the phosphorylation domain of DAPK1, their neuroprotective effects differ depending on the DAPK1 substrate, with RvTAT-PKD-S205 and its homologous interfering peptides being the most effective.

Claims

1. An artificial small interfering peptide of a DAPK1 phosphorylation substrate, characterized in that the amino acid sequence is as shown in any one of SEQ ID NO: 1, SEQ ID NOs: 23-24, and SEQ ID NOs: 26-38.

2. The mimetic peptide of the artificial small interfering peptide of claim 1, wherein the structure is a staple peptide or a cyclic peptide, and the cyclic peptide is a cyclic peptide formed by a ring formed by a chain, a side chain, a thioester bond, a lactone bond, or a disulfide bond.

3. 2. The artificial small interfering peptide retropolypeptide of claim 1, wherein the order of amino acids in the peptide chain from the C-terminus to the N-terminus is changed compared to the artificial small interfering peptide.

4. Compared with the artificial small interfering peptide, each L-amino acid residue in the artificial small interfering peptide is replaced by its corresponding D-amino acid, the amino acid sequence is reversed, and the original spatial orientation and chirality of the side chains are the same as those of the artificial small interfering peptide, i.e., a side chain topology structure similar to that of the artificial small interfering peptide is maintained. A D-retro-inverso peptide of the artificial small interfering peptide described in claim 1.

5. A derivative peptide of the artificial small interfering peptide described in claim 1, characterized in that it is obtained by replacing one or more amino acids in the artificial small interfering peptide described in claim 1 with their corresponding D-amino acids or homoamino acids.

6. The polymer is composed of two or more small peptides polymerized in a parallel manner in the same direction, wherein the C-terminus of each small peptide is free and the N-terminus of all the small peptides are gathered together to be connected to a delivery vector; The polypeptide is characterized in that the low molecular weight peptide is selected from the artificial low molecular weight interfering peptide described in claim 1, the retropolypeptide described in claim 3, the D-type retro-inverso peptide described in claim 4, or the derivative peptide described in claim 5.

7. One or more delivery vectors are fused to the N-terminus or C-terminus of a small peptide; A fusion polypeptide characterized in that the low molecular weight peptide is selected from the artificial low molecular weight interfering peptide described in claim 1, the retropolypeptide described in claim 3, the D-type retroinverso peptide described in claim 4, or the derivative peptide described in claim 5.

8. the delivery vector is selected from a membrane-permeable peptide, a ligand, a receptor protein transduction domain (PTD), an antibody, or a high molecular weight polymer; the membrane-permeable peptide is selected from a cationic cell-membrane-permeable peptide, an amphipathic cell-membrane-permeable peptide, a hydrophobic cell-membrane-permeable peptide, or a synthetic cell-membrane-permeable peptide; 8. The polypeptide according to claim 6 or the fusion polypeptide according to claim 7, wherein the high molecular weight polymer is selected from the group consisting of polyethylene glycol (PEG), polylactic acid, poly(lactide-co-glycolide), polyglycolic acid, polycaprolactone, polyethylene oxide, polydioxanone, polypropylene fumarate, trimethylene carbonate, polyesteramide oxirane, esteramide, β-hydroxyphenylpropionate, α-hydroxy acid, polyhydroxyalkanoic acid, polyhydroxybutyric acid, polyimide carbonate, polyurethane, polyanhydride, hyaluronic acid, chitosan, cellulose, gelatin, and collagen.

9. the cationic cell membrane-permeable peptide is selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 55 to 72; the amphipathic cell membrane-penetrating peptide is selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 73 to 81; the hydrophobic cell membrane-permeable peptide is selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 82 to 85; The fusion polypeptide described in claim 8, characterized in that the synthetic cell membrane-permeable peptide has the amino acid sequence shown in SEQ ID NO:

86.

10. The artificial small interfering peptide has an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 23-24 and SEQ ID NO: 26-38, and the membrane-permeable peptide has an amino acid sequence shown in any one of SEQ ID NO: 55 to SEQ ID NO:

86. The fusion polypeptide according to claim 9.

11. The fusion polypeptide described in claim 8, characterized in that the amino acid sequence is as shown in SEQ ID NO: 87 or SEQ ID NO:

92.

12. The artificial small interfering peptide according to claim 1 is encoded as a small peptide. and a retropolypeptide according to claim 3. An artificially produced nucleic acid molecule.

13. An expression vector comprising the nucleic acid molecule of claim 12.

14. 14. An expression system characterized in that it is a cell-based expression system containing the vector of claim 13 or a cell-free expression system containing the vector of claim 13.

15. The artificial small interfering peptide of claim 1 is expressed by the expression system of claim 14, and the main component is a small peptide. and an expression product selected from the retropolypeptides of claim 3.

16. A method for producing a pharmaceutical composition comprising a peptide molecule and pharmaceutically acceptable impurities, additives, solvents, protectants, adjuvants, carriers and / or excipients, wherein the peptide molecule is (1) The artificial small interfering peptide according to claim 1, the retropolypeptide according to claim 3, the D-type retroinverso peptide according to claim 4, or the derivative peptide according to claim 5, or (2) A drug characterized in that it is a modified product of a small peptide, the small peptide being selected from the artificial small interfering peptide of claim 1, the retropolypeptide of claim 3, the D-type retroinverso peptide of claim 4, or the derivative peptide of claim 5, and the modification is one or more of N-terminal modification, C-terminal modification, labeling, cyclization, lipidation, N-methylation, myristoylation and palmitoylation, glycosylation, biotinylation, PEG modification, and fluorescent labeling.

17. 17. The drug of claim 16, wherein the dosage form is an aerosol formulation for inhalation; an oral formulation; or a formulation for intravenous, intraarterial, intracranial, intraperitoneal, intranasal, intramuscular, subcutaneous, intrasynovial, intrasternal, or intraspinal administration.

18. A medicament for use in the treatment or prevention of a disease associated with an excitotoxic mechanism, comprising a small peptide, The small peptide is selected from the group consisting of the artificial small interfering peptide of claim 1, the retropolypeptide of claim 3, the D-type retro-inverso peptide of claim 4, and the derivative peptide of claim 5; the disease is selected from stroke, traumatic brain injury, spinal cord injury, neonatal hypoxic-ischemic encephalopathy, neurodegenerative disease, depression, and autism; The neurodegenerative disease is multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

19. A pharmaceutical for use in treating or preventing a physiological disorder associated with the DAPK1-PKD1 pathway, comprising a small peptide, The small peptide is selected from the group consisting of the artificial small interfering peptide of claim 1, the retropolypeptide of claim 3, the D-type retro-inverso peptide of claim 4, and the derivative peptide of claim 5; the physiological abnormality is selected from stroke, traumatic brain injury, spinal cord injury, neonatal hypoxic-ischemic encephalopathy, neurodegenerative disease, depression, and autism; The neurodegenerative disease is multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

20. 18. A method for treating a pulmonary artery disease comprising administering to a subject a drug according to claim 16 or 17 and an anticoagulant or antiplatelet drug, The drug and the anticoagulant or antiplatelet drug are packaged together in a single dose, a daily dose, or a course of doses; However, the anticoagulant is not limited to acenocoumarol, 4-hydroxy-3-(1,2,3,4-tetrahydro-1-naphthyl)coumarin (coumatetralyl), dicumarol, dicumarol ethyl, phenprocoumon, warfarin, diphenadione, phenindione, thiochromarol, bemiparin, certoparin sodium, dalteparin, enoxaparin, nadroparin, parnaparin, reviparin, tinzaparin, fondaparinux, idraparinux, danaparoid, sulodexide, dermatan sulfate, apixaban, betrixaban, edoxaban, otamixaban, rivaroxaban, hirudin, bilirubin, Valirudin, recombinant hirudin, desirudin hirudin, argatroban, dabigatran etexilate, melagatran, ximelagatran, defibrotide, antithrombin III, heparin, Coumadin tablets, apixaban (trade name Eliquis), edoxaban, enoxaparin, fondaparinux, recombinant tissue plasminogen activator, tissue plasminogen activator, alteplase, reteplase, tenecteplase, urokinase, saruplase, streptokinase, anistreplase, monteplase, ancrod, fibrinolysin, brinase, or a composition thereof; The antiplatelet drug is clopidogrel, ticagrelor, prasugrel, dipyridamole, cilostazol, ticlopidine, eptifibatide, aspirin, abciximab, tirofiban, beraprost, prostacyclin, iloprost, treprostinil, aloxipirin, carbaspirin calcium, indobufen, triflusal, picotamide, terutroban, chloricromene, ditazol, or a composition thereof.

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