N-methylation modified cyclic peptide and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to N-methylation modified cyclopeptide and application thereof. On the basis of a GluA2-3Y polypeptide sequence, chemical modification and transformation of amido bond N-methylation and head-tail cyclization are carried out, the N-methylation modified cyclopeptide with stable metabolism and good permeability is obtained, and the endocytosis of a GluA2AMPA receptor is blocked to protect nerve cells, so that the GluA2AMPA receptor can be effectively inhibited, and the GluA2AMPA receptor can be effectively inhibited. The invention also provides application of the cyclic peptide in preparation of drugs for treating cerebral arterial thrombosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to an N-methylated modified cyclic peptide and applications thereof. Background Art

[0002] Glutamate (Glu), the primary excitatory neurotransmitter in the mammalian central nervous system, plays a crucial regulatory role in higher-order neurophysiological processes, including spatial cognitive processing, memory encoding, and storage. Rapid regulation of excitatory synaptic transmission in the central nervous system is primarily achieved through activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor by excitatory glutamate. Overstimulation of AMPA receptors leads to intracellular calcium overload, which in turn triggers cell damage and death. This process is closely associated with numerous acute and chronic neurodegenerative diseases, such as ischemic brain injury, amyotrophic lateral sclerosis, and Parkinson's disease.

[0003] AMPA receptors are mainly distributed in brain areas such as the hippocampus and cerebellum. They are tetrameric ionotropic glutamate receptors composed of GluA1, GluA2, GluA3 and GluA4 subunits. The combination of different subunits gives AMPA receptors different biophysical and pharmacological properties. GluA2 is one of the core subunits of the AMPA receptor. After RNA editing (arginine replaces glutamine) at the Q / R site of the GluA2 subunit, a calcium-impermeable AMPA receptor (CI-AMPAR) is formed, which protects neurons from calcium influx damage. After a stroke occurs, cerebral ischemia causes GluA2 to be endocytosed, and calcium-permeable AMPA receptors (CP-AMPAR) are formed on the postsynaptic membrane, triggering a large amount of calcium ion influx, causing calcium overload, and ultimately leading to neuronal death. The guanine-nucleotide exchange factor 2 (BRAG2)-adenylate ribosylation factor 6 (ARF6) pathway is the regulatory mechanism of GluA2 AMPA receptor endocytosis ( Figure 1 The interplay between GluA2 phosphorylation and dephosphorylation is crucial for controlling AMPA receptor surface expression and endocytosis. After phosphorylation of tyrosine at position 876 in GluA2 by Src family tyrosine kinases, it specifically binds to BRAG2 via a tyrosine-rich motif (YKEGYNVYG, designated as the 3Y motif), activating Arf6 and promoting AMPA receptor endocytosis. Therefore, loss of BRAG2 or inhibition of the GluA2-BRAG2 interaction prevents AMPA receptor endocytosis.

[0004] The active sequence 3Y (YKEGYNVYG) and a membrane-penetrating sequence Tat (YGRKKRRQRRR) were combined to design a peptide drug Tat-GluA2-3Y that can cross the blood-brain barrier. Figure 2 The peptide competitively binds to BRAG2, reducing the excessive endocytosis of AMPA receptors mediated by GluA2. Tat-GluA2-3Y has shown promising therapeutic effects in treating several neurological diseases, such as stroke, Alzheimer's disease, and major depression.

[0005] Cell-penetrating peptides (CPPs) can deliver peptides and even protein carriers into cells via non-invasive pathways and have many applications both in vitro and in vivo. Some CPP-based therapies have entered Phase III clinical trials. However, most of the proteins delivered by cell-penetrating peptides are trapped in endosomes and degraded. At the same time, cell-penetrating peptides are easily degraded or bound to plasma proteins in serum, limiting their efficacy in vivo. In addition, some classic cationic cell-penetrating peptides, including the first discovered TAT, are also limited by their systemic toxicity, which is mainly attributed to their positive charge.

[0006] Cyclosporine A (CsA) is a multi-N-methylated cyclic peptide. This naturally occurring peptide drug exhibits high oral bioavailability. N-methylation is one of the simplest methods to introduce conformational constraints into peptides because it introduces steric constraints, allows cis-peptide bonds, and prevents the formation of hydrogen bonds. N-methylation modification can greatly improve the physicochemical, structural, and biological properties of peptides. In addition, compared with linear peptides, cyclic peptides have better chemical stability and longer biological half-lives. Therefore, the use of cyclization and amino acid N-methylation modification of 3Y may transform 3Y into a biologically active analogue, thereby overcoming the technical shortcomings of CPP. Summary of the Invention

[0007] The purpose of the present invention is to provide an N-methylated modified cyclic peptide. Based on the GluA2-3Y polypeptide sequence, N-methylated and cyclized chemical modifications and transformations are carried out to obtain a metabolically stable and permeable N-methylated modified cyclic peptide, which blocks the endocytosis of the GluA2 AMPA receptor to protect the nervous system. The present invention also provides the use of the cyclic peptide.

[0008] The technical solutions of the present invention are as follows:

[0009] The amino acid N-methylated modified cyclic peptide of the present invention has a structural formula as shown in formula (I) and is named c10c-G1V3.

[0010]

[0011] The present invention provides use of the cyclic peptide in preparing a drug for treating ischemic stroke.

[0012] The present invention also provides a medicine comprising a therapeutically effective amount of acetate, hydrochloride or other pharmaceutically acceptable salt forms of the cyclic peptide.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention uses standard Fmoc solid-phase synthesis technology to synthesize 25 peptides. After surface plasmon resonance experiments, cell protection activity experiments and membrane penetration experiments, the candidate peptide c10c-G1V3 was screened. A series of cell and animal experiments on the candidate peptides revealed that:

[0015] (1) The affinity of c10c-G1V3 for BRAG2 protein is four times that of GluA2-3Y;

[0016] (2) The oxygen-glucose deprivation and glutamate-induced HT22 cell injury models demonstrated that the peptide c10c-G1V3 exerted a significant protective effect at the cellular level;

[0017] (3) Both cyclization and amino acid N-methylation modifications can improve the membrane penetration efficiency of peptides. Compared with the positive drug Tat-GluA2-3Y, peptide c10c-G1V3 can penetrate the cell membrane without relying on the membrane penetration sequence.

[0018] (4) Peptide c10c-G1V3 significantly reduced the production of reactive oxygen species and the occurrence of cell apoptosis in the glutamate-induced HT22 cell injury model;

[0019] (5) The half-life of c10c-G1V3 was significantly superior to that of Tat-GluA2-3Y, and no significant degradation was observed after 8 h of incubation with rat plasma;

[0020] (6) Peptide c10c-G1V3 significantly reduced cerebral infarction and neurobehavioral deficits caused by cerebral ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 , Regulatory mechanism of AMPA receptor endocytosis: AMPAR-BRAG2-ARF6;

[0022] Figure 2 , the structure of Tat-GluA2-3Y;

[0023] Figure 3 , the synthesis process of c10c-G1V3;

[0024] Figure 4 , RP-HPLC chromatogram of c10c-G1V3;

[0025] Figure 5 , ESI-MS mass spectrum of c10c-G1V3;

[0026] Figure 6 , the interaction between peptides and BRAG2 protein was evaluated by surface plasmon resonance (SPR) technology; A. Structural diagram of c10c-G1Y2 and its binding curve with BRAG2 protein; B. Structural diagram of c10c-G1V3 and its binding curve with BRAG2 protein, with N-methylation sites marked in blue. The peptides were diluted twofold to different concentrations and then flowed through the chip; K d Values ​​were calculated using Biacore T200 analysis software;

[0027] Figure 7 , cytotoxicity of peptides on HT22 cells, all values ​​are expressed as mean ± SD, n = 3;

[0028] Figure 8 , Screening of the neuroprotective activity of peptides on HT22 cells based on the OGD model, all values ​​are expressed as mean ± SD, n = 3, compared with the Tat-GluA2-3Y group, **P < 0.01, ****P < 0.0001; compared with the c10c-G-1 group, ### P < 0.001;

[0029] Figure 9 , Screening of peptides for neuroprotective activity on HT22 cells based on the glutamate model, all values ​​are expressed as mean ± SD, n = 3, compared with the Glu group, ****P < 0.0001;

[0030] Figure 10 , Confocal microscopy images of HEK293T cells after treatment with 5-FAM-labeled peptides (scale bar 25 μm);

[0031] Figure 11 Effects of peptides on the fluorescence intensity of ROS generated in HT22 cells induced by glutamate; A. Fluorescence intensity of HT-22 cells was observed under an inverted fluorescence microscope (×20), scale bar 100 μm; B. Antioxidative stress effects of Tat-GluA2-3Y and c10c-G1V3; All values ​​are expressed as mean ± SD, n = 3, ***P < 0.001 compared with the GLU group;

[0032] Figure 12 , plasma stability of peptides; A. Liquid chromatogram of the remaining amount of Tat-GluA2-3Y; B. Liquid chromatogram of the remaining amount of c10c-G1V3; C. Percentage of the remaining amount of peptides; Data are expressed as mean ± SD, n = 3;

[0033] Figure 13 , protective activity of peptides on MCAO rats; A. Representative images of TTC staining; B. Percentage of infarct area; C. Neurobehavioral scores, data are expressed as mean ± SD, n = 5, **P < 0.01, ***P < 0.001, compared with the model group;

[0034] Figure 14 , a line graph showing the change of peptide RMSD over simulation time;

[0035] Figure 15 , the number of intramolecular hydrogen bonds of c10c-G1V3 in water and chloroform. DETAILED DESCRIPTION

[0036] To further illustrate the present invention, the following will provide a more comprehensive and systematic description of the present invention with reference to the accompanying drawings and application examples. The technical terms used in the present invention are all common terms that can be understood by those skilled in the art. In addition, unless otherwise specified, the materials and reagents used in the present invention can be purchased or prepared by conventional means.

[0037] Example 1 Design and Synthesis of N-Methylated Cyclic Peptides

[0038] To investigate the effects of cyclization and N-methylation on the binding affinity, transmembrane ability, cytoprotective activity, and animal protective activity of 3Y, a series of cyclized and N-methylated 3Y derivatives were synthesized. These derivatives varied in ring size, methylation position, and number of methylations. By replacing the corresponding amino acids in GluA2-3Y with different N-methylated amino acids, cyclization yielded N-methylated cyclic nonapeptides, such as c9c-G-1. By introducing glutamic acid into GluA2-3Y and performing two N-methylation modifications, cyclization yielded double-N-methylated cyclic decapeptides, such as c10c-G1V3. Representative peptides from this example, such as Ac-3Y, have structures shown in (II), c9c-G-1, and c10c-G1V3, respectively.

[0039]

[0040]

[0041] A total of 25 cyclic peptides were synthesized using the Fmoc solid phase synthesis method, as shown in Tables 1 and 2. Figure 3Taking the cyclic peptide c10c-G1V3 as an example, the synthesis method is as follows: the synthesis of c10c-G1V3 begins with the allyl-protected glutamic acid residue fixed on the solid phase support, the N-Fmoc group is removed with piperidine, and then the acylation reaction of the primary amine with the amino acid carboxyl group is completed by the HBTU / HOBT method. The acylation reaction of the secondary amine with the amino acid carboxyl group is completed by the HATU / HOBT method. The peptide resin is treated with Pd(PPh3)4, piperidine and PyBOP to achieve the head-to-tail cyclization of the polypeptide. Finally, c10c-G1V3 is cleaved from the resin with TFA, and the crude peptide is prepared on RP-HPLC. The molecular weight is determined by ESI-MS analysis. The RP-HPLC and ESI-MS images of c10c-G1V3 are shown in Figure 2. Figure 4 and Figure 5 .

[0042] Figure 3 Reagents and conditions: (a) 20% Pip / DMF (v:v), room temperature, 15 min, reaction twice; (b) solid phase peptide synthesis; (c) 3eq Fmoc-AA, 3eq HATU, 3eq HOAT, 6eq DIPEA, DMF, room temperature, 4h; (d) solid phase peptide synthesis; (e) 0.3eq Pd(PPh3)4, 10eq phenylsilane, DCM, room temperature, protected from light, 15 min, reaction twice; (f) 20% Pip / DMF (v:v), room temperature, 15 min, reaction twice; (g) 5eq PyBOP, 5eq HOBT, 5eq DIPEA, DMF, room temperature, 2h, reaction twice; (h) TFA / DTT / phenol / H2O (8.75:0.5:0.25:0.5, v / w / v / v), room temperature, 4h.

[0043] Example 2 Affinity of polypeptide

[0044] Surface plasmon resonance was used to determine the binding affinity of the peptides to the protein, allowing potential active compounds to be screened from the designed peptides. Using Ac-3Y as a positive control, a single N-methylated cyclic peptide was screened for activity at 25 μg / mL. The results are shown in Table 1. Seven cyclic peptides were identified with superior binding affinities to Ac-3Y, with c10c-G-1 (12.68 μM) and c10c-Y-5 (12.32 μM) exhibiting the highest binding affinities. Neither the cyclic nonapeptide c9c nor the N-methylated cyclic nonapeptide series showed binding. However, the cyclic decapeptide c10c, obtained by cyclization after the introduction of glutamic acid, showed similar affinity to Ac-3Y. This result suggests that ring size influences the binding activity of cyclic peptides to the BRAG2 protein. Based on the single N-methylated cyclic peptide, a double N-methylated cyclic peptide was synthesized. However, due to the low yield of c10c-Y-5 at 24%, double methylation was difficult to perform. Seven double-N-methylated cyclic peptides were synthesized based on c10c-G-1. The affinity results are shown in Table 2: c10c-G1Y2 and c10c-G1V3 bind to BRAG2 protein with affinities of 7.45 μM and 7.37 μM, respectively, which are about four times that of the linear peptide Ac-3Y (27.81 μM). The binding pattern of representative cyclic peptides to BRAG2 protein is shown in Table 2. Figure 6 As shown in Figure 3, N-methylation can affect the binding activity of cyclic peptides to the BRAG2 protein by restricting their conformation. The cyclic peptides c10c-G1Y2 and c10c-G1V3 are potential active compounds for further biological activity studies.

[0045] Table 1 Amino acid sequence, molecular weight, retention time and binding affinity of mono-N-methylated cyclic peptides

[0046]

[0047] a The methylated amino acids in the sequence are underlined; b Gradient conditions: Positisil ODS-P C18 analytical column, elution gradient 15%-60% ACN, flow rate 1 mL / min, λ = 220 nm; c represents the equilibrium dissociation constant K of the peptide d .

[0048] Table 2 Sequence, molecular weight, retention time and binding affinity of double N-methylated cyclic peptides

[0049]

[0050]

[0051] a The methylated amino acids in the sequence are underlined; b Gradient conditions: Positisil ODS-P C18 analytical column, elution gradient 15%-60% ACN, flow rate 1 mL / min, λ = 220 nm; c represents the equilibrium dissociation constant K of the peptide d .

[0052] Example 3 Cyclic peptide safety evaluation

[0053] The results of cytotoxicity evaluation of cyclopeptide on HT22 cells are as follows Figure 7 As shown: After all peptides were co-incubated with HT22 cells at 20 μM for 24 hours, the cell survival rate exceeded 95%, which is conducive to subsequent cell activity experiments.

[0054] Example 4 Neuroprotective activity of cyclic peptides on oxygen-glucose deprived HT22 cells

[0055] The cyclic peptide was set up in three dose groups: 0.1μM, 1μM and 10μM, and the drug was administered 24 hours before hypoxia. The hypoxia device containing cells was placed in a 37℃ constant temperature incubator for 3 hours of hypoxia and then reoxygenated for 6 hours. The experimental results are shown in Figure 2. Figure 8 As shown, the survival rate of HT22 cells in the normal cell control group was set at 100%. The survival rate of HT22 cells in the OGD group was (43.58±3.22)%, which was significantly decreased compared to cells in the normal group. At a concentration of 10 μM, the survival rates of cells in the positive drug Tat-GluA2-3Y group and the lead peptide c10c-G-1 group were (59.46±0.79)% and (73.27±1.63)%, respectively.

[0056] The results of the neuroprotective activity experiment of cyclic peptides on HT22 cells showed that c10c-G1V3, c10c-G1N4, and c10c-G1Y5 had better cell protection activities than the lead peptide c10c-G-1 at multiple concentrations. Compared with Tat-GluA2-3Y, at a concentration of 10μM, c10c-G1Y5 showed significantly different cell activity (**P<0.01), and c10c-G-1 and c10c-G1V3 both showed extremely significant differences in cell activity (****P<0.0001); compared with the c10c-G-1 group at a concentration of 10μM, c10c-G1V3 showed highly significant protective activity ( ### P<0.001). Comprehensive analysis of the three dose concentrations revealed that the neuroprotective activities of peptides c10c-G1V3 and c10c-G-1 on HT22 cells were dose-dependent.

[0057] Example 5 Neuroprotective activity of polypeptides on glutamate-induced HT22 cells

[0058] The results of neuroprotection of peptide against Glu-induced HT22 cell death are as follows Figure 9 As shown, the survival rate of HT22 cells in the Glu group was (36.99±0.58)%, which was significantly lower than that in the normal group. At 10 μM, the survival rates of cells in the Tat-GluA2-3Y group and the c10c-G1V3 group were (79.10±5.71)% and (76.34±1.94)%, respectively.

[0059] At 0.1μM and 1μM concentrations of the peptide drug, c10c-G1V3 exhibited highly significant neuroprotective activity (****P<0.0001). At 10μM, both the positive drug Tat-GluA2-3Y and c10c-G1V3 exhibited highly significant neuroprotective activity (****P<0.0001), demonstrating comparable neuroprotective activity. In this model, the neuroprotective activity of c10c-G1V3 on HT22 cells was dose-dependent.

[0060] Example 6 Evaluation of the membrane-penetrating ability of polypeptides

[0061] The cells were observed by confocal microscopy. Figure 10 As shown, the peptide labeled with 5-FAM appears green, while the cell nuclei stained with Hoechst 33342 appear blue. The two images are overlaid to form a merge. The fluorescence intensity of the peptide c10c-G-1-5-FAM, which has undergone cyclization and mono-N-methylation, increases slightly. Furthermore, the fluorescence intensity of the doubly N-methylated cyclic peptide c10c-G1V3-5-FAM increases even more significantly. This result demonstrates that peptide cyclization and methylation can affect its ability to penetrate membranes.

[0062] Example 7 Effect of polypeptides on ROS production in HT22 cells induced by glutamate

[0063] Depend on Figure 11 The results in middle A show that under an inverted fluorescence microscope, the fluorescence intensity of the cells in the Glu group was significantly higher than that in the normal control group. Glutamate treatment of HT22 cells can produce a large amount of ROS, leading to cell death. Figure 11 The results in middle B showed that compared with the glutamate group, the intervention of the positive drug Tat-GluA2-3Y (10 μM) and peptide c10c-G1V3 (10 μM) highly significantly reduced the generation of ROS (***P<0.001).

[0064] Example 8 Evaluation of the Rat Plasma Stability of Cyclic Peptides

[0065] The remaining amount of peptide was analyzed by HPLC, e.g. Figure 12As shown, the modified peptide c10c-G1V3 showed no obvious degradation after incubation with rat plasma for 8 hours, while the half-life of the positive drug Tat-GluA2-3Y was about 1 hour. The stability of c10c-G1V3 was significantly improved compared with Tat-GluA2-3Y.

[0066] Example 9 Evaluation of Animal Protection Activity of Cyclic Peptides

[0067] 1. Effect of cyclic peptide on cerebral infarction area in rats

[0068] The middle cerebral artery occlusion (MCAO) cerebral ischemia-reperfusion model was used to further determine the in vivo neuroprotective activity of c10c-G1V3. Figure 13 As shown in A: After staining brain tissue with TTC, the infarct area appears white and the normal area appears red. Figure 13 As shown in Figure B, the model group rats developed severe cerebral infarction, with an infarct area of ​​29.6%. Compared with the model group, the c10c-G1V3 (8 mg / kg) group showed a 9.4% reduction in infarct area, demonstrating significant neuroprotective activity (**P < 0.01). The positive drug Tat-GluA2-3Y (8 mg / kg) group showed a 13.7% reduction in infarct area, highly significantly reducing damage caused by cerebral ischemia-reperfusion (***P < 0.001). The infarct area percentages in the c10c-G1V3 and Tat-GluA2-3Y groups were similar, with no statistical difference. Therefore, c10c-G1V3 plays a protective role against cerebral infarction caused by cerebral ischemia-reperfusion.

[0069] 2. Effects of cyclic peptides on neurological deficit symptoms in rats

[0070] Effects of peptides on neurological deficit symptoms in rats Figure 13 Figure C: Consistent with the histological staining results, neurological deficit scores were restored in both the 8 mg / kg c10c-G1V3 and Tat-GluA2-3Y groups. Compared with the model group, the c10c-G1V3 (8 mg / kg) group showed statistically significant improvement in neurological deficit symptoms (*P < 0.01), and the Tat-GluA2-3Y (8 mg / kg) group showed a significant improvement in neurological deficit symptoms (**P < 0.01). Neurobehavioral scores were similar between the c10c-G1V3 and Tat-GluA2-3Y groups, with no statistical difference.

[0071] The results showed that c10c-G1V3 had a positive effect on improving neurological function damage caused by cerebral ischemia-reperfusion.

[0072] Example 10 Simulation Analysis of Conformational Stability of Cyclic Peptides

[0073] The conformational stability of the cyclic peptide was simulated by molecular dynamics and root mean square deviation (RMSD) analysis was performed. A stable RMSD value represents a relatively stable conformation, such as Figure 14 As shown, the RMSD values ​​of c10c-G1V3 are stable at The simulation results show that c10c-G1V3 has smaller RMSD fluctuations than Ac-3Y and is more stable.

[0074] Example 11 Simulation analysis of membrane permeability of cyclic peptides

[0075] We used molecular dynamics to predict the membrane permeability of cyclic peptides. We performed molecular dynamics simulations on c10c-G1V3 in two different solvents: water and chloroform (CHCl3). The predicted results of the number of intramolecular hydrogen bonds are as follows: Figure 15 As shown, the average number of hydrogen bonds of c10c-G1V3 in water is 2, and the average number of hydrogen bonds in chloroform is 5. The number of intramolecular hydrogen bonds of c10c-G1V3 in chloroform is more than that in water. c10c-G1V3 undergoes conformational transformation in the two-phase system. The non-polar environment is more conducive to the formation of intramolecular hydrogen bonds of the cyclic peptide, which is beneficial to membrane penetration.

[0076] Based on Examples 1-11, the present invention synthesized 25 peptides using standard Fmoc solid-phase synthesis technology. Surface plasmon resonance, cell protection, and membrane penetration assays were performed to screen the candidate peptide c10c-G1V3. A series of cell and animal experiments on the candidate peptides yielded the following conclusions:

[0077] (1) Affinity experiments identified N-methylated cyclic peptides c10c-G1Y2 and c10c-G1V3 with higher affinity for BRAG2. Their affinity for BRAG2 is four times that of GluA2-3Y. Molecular dynamics simulations showed that c10c-G1V3 exhibited smaller RMSD fluctuations and a more stable conformation than Ac-3Y.

[0078] (2) The oxygen-glucose deprivation and glutamate-induced HT22 cell injury models demonstrated that c10c-G1V3 exerted a significant protective effect at the cellular level.

[0079] (3) Transmembrane penetration experiments demonstrated that both cyclization and amino acid N-methylation modifications can enhance the membrane penetration efficiency of peptides. Compared to the positively charged drug Tat-GluA2-3Y, c10c-G1V3 can penetrate the cell membrane independently of its transmembrane sequence. Molecular dynamics simulations showed that the average number of intramolecular hydrogen bonds of c10c-G1V3 in chloroform is greater than that in water, and that c10c-G1V3 undergoes conformational transformations in the two-phase system, facilitating membrane penetration.

[0080] (4) c10c-G1V3 significantly reduced the production of reactive oxygen species and the occurrence of cell apoptosis in the glutamate-induced HT22 cell injury model.

[0081] (5) The stability of c10c-G1V3 was significantly improved, and no obvious degradation was observed after incubation with rat plasma for 8 h.

[0082] (6) c10c-G1V3 significantly reduced cerebral infarction and neurobehavioral deficits caused by cerebral ischemia-reperfusion injury.

[0083] The experimental part of the embodiment of the present invention is as follows: 1 Reagents and materials

[0084] All materials, reagents, and solvents used were obtained from commercial sources (purity >95%). The molecular weights of all peptides were confirmed by ESI-MS. All peptides were analyzed and purified using reverse-phase HPLC 1220 and 1260 (Agilent Technologies, Inc., China). Positisil ODS-P C18 analytical and semi-preparative columns were purchased from Beijing Yinglai Technology Co., Ltd.

[0085] 2. Synthesis and purification of peptides

[0086] 2.1 Synthesis of peptides

[0087] In this study, peptides were synthesized using standard Fmoc solid-phase synthesis. Amino acids were covalently linked one by one from the C-terminus of the target peptide to Rink Amide MBHA resin to create linear peptides. Glutamic acid (Fmoc-Glu-Oall, with the backbone carboxyl group protected by an allyl group) was used as a linker to cyclize the peptide. The peptide synthesis pathway used was c10c-G1V3.

[0088] (1) Prepare a peptide synthesis table: perform coupling from the C-terminus to the N-terminus of the target peptide sequence, and accurately calculate the amount of amino acids, condensing agents, catalysts, cyclization reagents, cutting solutions, precipitation solutions, etc. in the peptide sequence based on the required amount of resin.

[0089] (2) Selection of peptide synthesis tube specifications and resin substitution degree: Choosing the appropriate peptide synthesis tube based on the amount of target peptide to be synthesized can reduce solvent usage costs, increase the contact area of ​​the reaction, and improve reaction efficiency. In this experiment, a resin with a substitution degree of 0.32 mmol / g was used for the synthesis of two rounds of N-methylated peptides, and a resin with a substitution degree of 0.56 mmol / g was used for the synthesis of other peptides.

[0090] (3) Swelling of Rink Amide MBHA resin: After the resin is taken out of the refrigerator and returned to room temperature, 0.5 g of the resin is weighed into a 25 mL solid phase synthesis tube. 15 mL of DCM is added and the resin is swelled in a thermostatic oscillator at 220 rpm and 25°C for 120-180 min. After the swelling is complete, the DCM is removed by filtration.

[0091] (4) Removal and washing of the Fmoc protecting group: Add 20% Pip / DMF (v:v) at approximately 3 times the volume of the resin bed, react in a thermostatic shaker at 220 rpm and 25°C for 10 min, filter, remove the reaction solution, and add the same volume of 20% Pip / DMF (v:v) again, and react in a thermostatic shaker for 15 min. After the reaction is complete, add DMF, MeOH, DCM, DMF, DCM, DMF, and DMF at approximately 3 times the volume of the resin in sequence, and wash the resin seven times. Shake in a thermostatic shaker for approximately 5 min, then filter. Add MeOH to shrink the resin and fully wash away any residual piperidine in the resin.

[0092] (5) Kaiser test: Place the resin pellets in a centrifuge tube until the bottom of the tube is covered. Two drops each of reagent A (80% phenol-ethanol solution (w:v)) and reagent B (5% ninhydrin-ethanol solution (w:v)) are added and heated in a 100°C metal bath for 3-5 minutes. A positive ninhydrin test indicates that the Fmoc protecting group has been removed. If the result is negative or the blue color is not obvious, a second deprotection step can be performed.

[0093] (6) Activation, coupling and washing of amino acids: Weigh 3eq Fmoc-Glu-(OAll)-OH, 3eq HBTU, and 3eq HOBT, dissolve them in DMF (1-2mL / g), slowly add 6eq DIPEA, and activate for 10-15min. Add the activated mixture to the resin and react for 50min. The reaction temperature is controlled at 25-30℃. After the reaction is completed, take the resin balls and add them to the test tube. The bottom of the test tube is covered and tested with ninhydrin. If the result is negative, the coupling is completed. If the result is positive, the material can be re-added for secondary coupling. After the coupling is completed, add DMF, DCM, DMF, DCM, DMF, and DMF in an amount of about 3 times the volume of the resin bed in sequence. Wash the resin 6 times, shake it in a constant temperature oscillator at 220rpm and 25℃ for about 5min, and then filter it.

[0094] (7) Repeat the above steps of deprotection, post-deprotection washing, amino acid activation, coupling, and post-coupling washing until the last amino acid Fmoc-Tyr(tBu)-OH is connected. If the ninhydrin test is negative, the Fmoc protecting group on the amino group is not removed.

[0095] (8) Detection of secondary amines: After deprotection and washing of Fmoc-Sar-OH, take a small amount of resin into a centrifuge tube (it is best to cover the bottom of the test tube with a layer), then add two drops of prepared reagent C (2% tetrachlorobenzoquinone-DMF solution (w:v)) and reagent D (2% acetaldehyde-DMF solution (w:v)), and let it stand at room temperature for 5-10 minutes. If the resin turns blue-green, it indicates the presence of unprotected amino groups.

[0096] (9) For coupling after Fmoc-Sar-OH, replace the HBTU used for amino acid activation with HATU and react in a constant temperature oscillator at 220 rpm and 25°C for 2 h. After the reaction is complete, repeat the above washing and detect with reagents C and D. If the resin is colorless, the coupling is complete. If the resin is blue-green, re-feed the material for secondary coupling. Repeat the above steps until the last amino acid Fmoc-Tyr(tBu)-OH is synthesized and the ninhydrin test is negative, indicating that the Fmoc protecting group on the amino group is not removed.

[0097] (10) Deallylation and cyclization of the peptide: The linear peptide synthesized above was reacted twice with 0.3 eq Pd(PPh3)4 and 10 eq phenylsilane in DCM for 15 min in the dark to remove the allyl group on the glutamic acid backbone. DCM, DMF, DCM, DMF, and DMF were added to the synthesis tube at approximately 3 times the volume of the resin. The resin was washed five times and filtered.

[0098] (11) Cyclization of the polypeptide. Add 20% Pip / DMF (v:v) solution to remove the N-terminal Fmoc protecting group of Fmoc-Tyr(tBu)-OH. Wash the resin seven times with DCM, DMF, DCM, DMF, DCM, DMF, and DMF (approximately 3 times the volume of the resin). Finally, react 5eq PyBOP, 5eq HOBT, and 5eq DIPEA in DMF for 1.5 h twice to complete the cyclization. Add approximately 3 times the volume of the resin to the synthesis tube with DCM, DMF, DCM, DMF, and DMF. Wash the resin five times and filter. The ninhydrin test is negative.

[0099] (12) Transfer and dry the peptide resin: After the reaction is completed, wash the peptide resin three times with approximately 3 volumes of DCM. Use a suction pump to drain the solvent from the resin, transfer it to a drying box, and air dry it for about 2 hours. Then transfer it to an oven and vacuum dry it at room temperature for more than 20 hours until constant weight is achieved (the weight change in 2 hours should not exceed 1.0%). This will yield a fully protected peptide resin.

[0100] (13) Cleavage: Transfer the dried resin to a 50 mL round-bottom flask and add a cleavage solution (0.4 mL phenol, 17.6 mL TFA, 1 mL H2O, 1 g dithiothreitol) that has been previously frozen at -20°C for at least 30 min. Mix the peptide resin at room temperature to achieve peptide cleavage and deprotection. Add 10.0-15.0 mL of cleavage solution per gram of peptide resin and stir at 25-40°C for 180-240 min. Filter the reaction solution and wash the resin twice with a small amount of TFA. Collect and combine the filtrates.

[0101] (14) Precipitation: Prefreeze methyl tert-butyl ether in a refrigerator at -20°C for more than 3 hours. Slowly add the reaction solution to the cold methyl tert-butyl ether and allow to precipitate for more than 2 hours. After the polypeptides are separated, remove the solution from the refrigerator and centrifuge the precipitate at 7500 rpm and 4°C for 15 minutes. Discard the supernatant and collect the precipitate. Wash the precipitate three times with cold methyl tert-butyl ether, making sure to cover the precipitate with methyl tert-butyl ether during washing.

[0102] (15) Vacuum freeze-drying of peptides: Dissolve the resulting precipitate with water and acetonitrile, sonicate to completely dissolve the crude peptide, then carefully transfer the sample to a freeze-drying vial and prefreeze it in a -80°C freezer for at least 6 hours. Then, place the freeze-dried vial in a freeze dryer and dry it for at least 40 hours. After freeze-drying, remove the freeze-dried vial and weigh the crude peptide. Calculate the yield. Store the freeze-dried crude peptide at -20°C.

[0103] (16) Confirmation of molecular weight of peptide: A small amount of crude peptide was dissolved in water and acetonitrile, filtered through a 0.22 μM filter membrane, and purified by RP-HPLC 1220. Fractions were collected based on the absorbance at 280 nm, and the molecular weight was determined by ESI-MS analysis.

[0104] 2.2 Analysis and purification of peptides

[0105] HPLC analysis conditions for peptides: chromatographic column: Positisil ODS-P C18 (analytical type) 5 μM, 4.6 mm × 250 mm; sample solvents: deionized water and acetonitrile; aqueous phase: deionized water containing 0.05% TFA; organic phase: chromatography-grade ACN containing 0.05% TFA; elution time: 1-20 min, gradient: 10%-40% ACN; wavelength: 280 nm; column temperature: 25 ± 2°C; flow rate: 1 mL min -1 ; The injection volume was 20 μL.

[0106] HPLC purification conditions for the peptide were as follows: chromatographic column: Positisil ODS-P C18 (semi-preparative), 5 μM, 10.0 mm × 250 mm; sample solvents: deionized water and acetonitrile; aqueous phase: deionized water containing 0.05% TFA; organic phase: chromatography-grade ACN containing 0.05% TFA; elution time: 1-30 min, gradient: 10%-40% ACN; wavelength: 280 nm; column temperature: 25 ± 2°C; flow rate: 3 mL min -1 ; The injection volume is 2 mL.

[0107] 3 Surface plasmon resonance experiments

[0108] 3.1 Preparation before the experiment

[0109] Preparation of 1× HBS-EP buffer: Accurately weigh 0.15 M NaCl solid, 0.01 M HEPES, and 3 mM ETDA, add 0.05% surfactant P20 to aid solubilization, adjust the solution pH to 7.4, filter with a 0.22 μM filter membrane, degas by ultrasonication, and store at 4°C.

[0110] Preparation of peptide samples: Weigh a certain amount of peptide and dilute it to a 1 mg / mL stock solution with 1× HBS-EP. Then, dilute the peptide to 100 μg / mL as the highest injection concentration with 1× HBS-EP. Repeat this dilution in half for at least five further concentrations. Set a replicate concentration at intervals, including a zero concentration.

[0111] 3.2 Ligand preconcentration and ligand immobilization

[0112] Before coupling the ligand protein to the chip, the protein was dissolved in a solution below its isoelectric point. At this point, the protein's net surface charge was positive, allowing it to bind to carboxyl groups on the chip surface through electrostatic adsorption as it passed over the chip. However, excessively low pH can affect protein activity, so we used preconcentration experiments to identify optimal pH conditions. First, BRAG2 protein was diluted to 15 μg / mL using sodium acetate at pH 4.0, pH 4.5, pH 5.0, and pH 5.5. Setting the flow rate to 10 μL / min, ligands diluted with various sodium acetate buffer concentrations and a regeneration solution (5 mM NaOH) were placed in the sample holder. The Injection command was used to set the Contact time to 60 s, and the Regeneration command icon was used to set the Contact time to 30 s. The optimal coupling buffer was selected by comprehensively considering the relative signal changes after diluting the ligand with different coupling buffers and the effects of the buffer on protein activity and coupling efficiency.

[0113] When immobilizing the ligand, covalent coupling was used to connect the BRAG2 protein to the CM5 chip, and the optimal pH was determined based on the pre-enrichment experiment for coupling. First, according to formula (1): Calculate the ligand coupling amount (RL): RL = 3917 RU, actual coupling amount = 1.5 RU = 5875 RU. In this experiment, the molecular weight of the ligand BRAG2 is 47 kDa, the molecular weight of the analyte is 1200 daltons, and the stoichiometric ratio (Sm) is 1. The actual coupling amount is 1.5 RL. To account for subsequent kinetic testing, Rmax is set to 100 RU. Then, place a 1:1 mixture of EDC and NHS condensing agents and ethanolamine blocking reagent in the sample rack. Remove the caps from all sample tubes and secure the sample stoppers. First, inject the EDC / NHS mixture with a contact time of 430 s. Then, inject the ligand molecule using a multi-injection method to achieve a coupling amount slightly above the target coupling amount. Finally, inject ethanolamine for blocking with a contact time of 420 s. After all commands are completed, calculate the actual coupling amount.

[0114] 3.3 Preliminary screening of peptide drug activity

[0115] Selection of initial screening concentrations for affinity experiments: Set the flow rate to 30 μL / min, the ligand-protein contact time to 60 s, and the ligand regeneration time to 30 s. Prepare the control drug GluA2-3Y at different concentrations: 1.0625 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. Manually inject the sample at zero concentration and the control drug concentration, and determine the screening concentration for control drug binding to the BRAG2 protein based on the corresponding sensorgram intensity (RU).

[0116] The other peptides were prepared into the initial screening concentration of the control drug GluA2-3Y. The same sampling mode as above was used to detect the binding strength of the peptides to the BRAG2 protein. Compounds with RU values ​​greater than 5 were selected for kinetic testing and KD values ​​were calculated.

[0117] 3.4 Determination of affinity and kinetics of peptide drugs

[0118] Based on the initial screening results of peptide activity, select compounds with active potential for the next step of fitting to calculate the KD value. Set the contact time of the analyte to 60s, the dissociation time to 120s, and the regeneration time to 30s. Set the flow rate to 30μL / min. Let the configured concentration gradient drugs flow through the chip in sequence. After binding, dissociation, and regeneration, collect and draw a curve showing the change in the response value of each compound over time. According to the binding mode of the peptide and BRAG2 protein, select kinetic analysis or affinity analysis. Select different fitting models according to different binding mode diagrams, and fit and calculate the binding constant K on and dissociation constant K off , find its KD value.

[0119] Toxicity experiment of 4 peptides on HT22 cells

[0120] The toxicity experiment of HT22 cells (mouse hippocampal neuronal cell line) was carried out using the method reported in the literature. The complete culture medium was prepared by adding 10% FBS and 1% double antibody to DMEM basic culture medium, mixing well, and storing in a refrigerator at 4°C. The cell viability was determined by CCK-8 colorimetry. HT22 cells were cultured in a culture dish containing 4 mL of complete culture medium. After the cells entered the logarithmic growth phase, HT22 cells were plated at 4×10 3 The cells were plated into 96-well plates at a density of 1000 cells / well and cultured in a 37°C, 5% CO2 environment for 24 hours. When the cells grew to 80%, the culture medium in the well plate was aspirated and replaced with 20 μM polypeptides prepared with basic culture medium. The cells were incubated with the cells for 24 hours. A blank group (containing only basic culture medium) and a normal cell group (cells and basic culture medium) were also set up. After the incubation, 10 μL of CCK8 was added. The culture plate was incubated at 37°C in the dark for 2 hours, and the absorbance was detected at a wavelength of 450 nm.

[0121] Neuroprotective experiment of 5 peptides on oxygen-glucose deprived HT22 cells

[0122] 5.1 Cell recovery, passaging, and cryopreservation

[0123] Resuscitation: Quickly remove HT22 cells from the -80°C freezer, lyse in a 37°C water bath, and quickly transfer to a 15 mL centrifuge tube containing 3 mL of complete medium. Centrifuge at 1000 rpm / min for 5 min. After centrifugation, remove the supernatant and resuspend in 1 mL of complete medium. Inoculate the cell suspension into a 6 cm dish containing 4 mL of culture medium and culture in a 37°C cell culture incubator.

[0124] Passaging: Subculture cells when they have grown to over 90%. Discard the complete medium in the culture dish and wash the cells with PBS buffer to prevent the effect of serum in the culture medium on trypsin. Add 2 mL of trypsin preheated at 37°C for digestion. Observe the digestion status of HT22 cells under a microscope. When the intercellular spaces are clear and the cells appear to be round and fall off, add 6 mL of complete medium to stop digestion. After pipetting and mixing, transfer and centrifuge, remove the supernatant, add 2 mL of complete medium to resuspend, and inoculate into culture dishes at a ratio of 1:4 to continue culturing.

[0125] Cryopreservation: Prepare sufficient cell freezing solution in advance. Add 3 mL of trypsin to digest adherent cells. Once cells are fully suspended, add 5 mL of complete culture medium to terminate digestion. Transfer the mixture to a centrifuge tube and centrifuge at 1500 rpm for 3 minutes at room temperature. Discard the supernatant and resuspend the cells in 2 mL of cell freezing solution. Gently mix thoroughly, transfer the tube to a cryovial, and gradually cool the tube until it is transferred to liquid nitrogen for long-term storage.

[0126] 5.2 Preparation of peptide drugs

[0127] Accurately weigh a certain mass of peptide and dispense it into a 1.5 mL EP tube. Calculate the required amount of cell-grade DMSO based on the mass and molecular weight of the peptide to prepare a stock solution with a concentration of 10 mM. Before starting the experiment, use the basic culture medium DMEM to prepare each group of drugs at concentrations of 0.1, 1, and 10 μM. After vortexing, store in a -20°C refrigerator for later use.

[0128] 5.3 Activity Evaluation of Peptide Drugs

[0129] The protective effect of peptides on cerebral ischemia reperfusion injury was explored by performing an in vitro experiment on HT22 cells to simulate a cerebral ischemia model. The oxygen-glucose deprivation HT22 cell injury model was constructed using the method reported in the literature. The cells were divided into 4 groups, including a blank group, a normal cell control group, an OGD / R treatment group, and a peptide drug treatment group (peptide + OGD / R). Cells in good growth condition and in the logarithmic growth phase were selected and plated with complete culture medium at 3×10 3 Cells were seeded at a density of 100 cells / well in a 96-well plate and incubated at 37°C for 12 hours. After 12 hours, a pre-incubation period with drug administration was performed. The normal culture medium was discarded and the cells were replaced with basal culture medium for the OGD / R-treated, blank, and normal cell control groups. The drug-treated groups were treated with basal DMEM medium containing varying concentrations of peptides. Following this procedure, the cells were incubated in a constant-temperature incubator for 24 hours.

[0130] After incubation, the culture medium in the OGD / R-treated and drug-treated groups was replaced with sugar-free medium and placed in a hypoxia device for hypoxia. The control group of normal cells continued to be cultured in a 37°C, 5% CO2 cell culture incubator. After 3 hours of hypoxia, the culture medium in the OGD / R-treated and drug-treated groups was replaced with basal medium and reoxygenated in a normal culture environment for 6 hours. After reoxygenation, the culture plates were removed and 10 μL of CCK8 was added to each well of cells from all groups. After addition, the plates were wrapped in tinfoil and placed in a 37°C incubator for another 2 hours. After incubation, the OD value of the cells in each well was measured at a wavelength of 450 nm using an automated microplate reader.

[0131] Neuroprotective experiment of 6 peptides on HT22 cells induced by glutamate

[0132] 6.1 Culture of HT22 cells

[0133] The cell culture steps and procedures are the same as those in Experimental Section 5.1.

[0134] 6.2 Construction of glutamate model

[0135] The glutamate-induced HT22 cell injury model was established using the method reported in the literature. Cells with a growth rate of more than 90% were taken and 4×10 3 Cells were seeded onto 96-well plates at a density of 100 μL / well and incubated in a 37°C incubator for 24 hours. The complete medium in the plates was then aspirated and 100 μL of glutamate solution at different concentrations was added. A normal cell group and a blank control group were also set up. After 24 hours of incubation, 10 μL of CCK8 was added to each well and shaken. The cells were then placed in a 37°C cell incubator and incubated for another 2 hours. The absorbance was measured at 450 nm to determine cell viability. Cell viability was calculated using formula (2): Cell viability (%) = [(absorbance of glutamate-treated group - absorbance of blank group) / (absorbance of control cell group - absorbance of blank group)] × 100%.

[0136] 6.3 Activity Evaluation of Peptide Drugs

[0137] Take cells that have grown more than 90% and use 4×10 3 Cells were seeded onto 96-well plates at a density of 100 μL / well and incubated in a 37°C incubator for 24 hours. The complete medium in the plate was then aspirated and the different drugs diluted in minimal medium were added. A normal cell group, a glutamate group, and a blank group were also set up. After a 30-minute incubation, the drug-treated group was incubated with a certain concentration of glutamate for 24 hours. After the treatment, 10 μL of CCK8 was added and the cells were incubated for another 2 hours. Cell viability was then measured at 450 nm. Cell viability was calculated according to formula (3): Cell viability (%) = [(absorbance of experimental group / model group - absorbance of blank group) / (absorbance of control group - absorbance of blank group)] × 100%.

[0138] Experimental wells: culture medium containing cells, CCK-8, glutamate, and the drug to be tested; model wells: culture medium containing cells, CCK-8, and glutamate; control wells: culture medium containing cells and CCK-8; blank wells: culture medium without cells or the drug to be tested, and CCK-8.

[0139] 7. Fluorescent peptide membrane penetration experiment

[0140] The fluorescent peptide membrane penetration experiment was carried out using the method reported in the literature. 4 HEK293T cells were seeded in 35 mm glass-bottom microwell dishes containing 2 mL of culture medium and cultured for 24 hours. The HEK293T cells were washed and incubated with 5-FAM-labeled peptides (50 μM) in DMEM supplemented with 10% serum and 1% penicillin-streptomycin for 4 hours at 37°C in the presence of 5% CO2. The peptide-containing medium was removed, and the cells were stained with 1 mL of Hoechst 33342 for 10 minutes at room temperature. Finally, the cells were washed three times with PBS and imaged on a confocal laser scanning microscope.

[0141] Experiment on the effects of 8 peptides on ROS production in HT22 cells induced by glutamate

[0142] 8.1 Cell pretreatment: HT22 cells in good growth condition were cultured at a rate of 1×10 5 The cells were uniformly seeded into a 24-well plate at a density of 100 μM / mL. After the cells grew for 24 h, the culture medium was replaced with 10 μM peptides Tat-GluA2-3Y and c10c-G1V3 and incubated with the cells for 30 min. Based on the glutamate-induced HT22 cell injury model, the cells were exposed to 5 mM glutamate and cultured for 24 h.

[0143] 8.2 Intracellular ROS Assay: ROS production in HT22 cells induced by glutamate was investigated using a reported method. After washing twice with ice-cold PBS, cells were stained with 10 μM 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37°C in the dark for 20 min and then washed three times with PBS. After washing, ROS production was estimated by observing the DCF intensity using an inverted fluorescence microscope.

[0144] 9 Animal protection experiments of peptides

[0145] 9.1 Experimental Animals

[0146] All experimental animals were adult male Sprague-Dawley (SD) rats weighing 240-270g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. After all rats arrived at the laboratory, a health check was performed to determine their health status. The rat breeding environment was controlled: the ambient temperature was stable at (22±2)℃, and the relative humidity was 60%. The experimental rats were fed freely before and after surgery with a 12-h light and dark cycle from 9:00AM to 21:00PM. If some rats were found to be in poor condition after surgery, they were given 1% glucose water to drink. All operating instruments and materials that came into contact with animals were sterilized by ultraviolet disinfection. All experimental protocols followed the animal experiment principles of the International Association for Study of Pain and met the requirements of the Ethical Use Committee of Experimental Animals of Qingdao University.

[0147] 9.2 Construction of MCAO Model

[0148] A rat model of middle cerebral artery occlusion (MCAO) cerebral ischemia-reperfusion was established using a previously reported internal carotid artery suture method. Rats were housed for 3-5 days upon arrival at the laboratory and, once they reached the required weight, were randomly divided into groups for subsequent experiments. Animal handlers were specially trained. All rats were sacrificed under anesthesia.

[0149] (1) Rat anesthesia: Rats were anesthetized with 4.0%-5.0% isoflurane mixed with oxygen. The rats were fixed on the operating table in a supine position and anesthesia was maintained with 2.0%-2.5% isoflurane mixed with oxygen.

[0150] (2) Anatomical positioning: The rat neck was depilated and the skin was disinfected. The inner side of the neck was opened with surgical scissors, and the neck muscle tissue was bluntly separated using fine forceps to expose the left common carotid artery (CCA). The vagus nerve was gently peeled off, and the external carotid artery (ECA) and internal carotid artery (ICA) were separated upward along the common carotid artery.

[0151] (3) Ligation of blood vessels: Use an artery clamp to clamp the proximal end of the common carotid artery, use 4-0 suture to ligate the distal end of the external carotid artery, leave the suture at the proximal end of the external carotid artery for future use, and use an artery clamp to clamp the internal carotid artery.

[0152] (4) Inserting a thrombus: Use ophthalmic scissors to make a small beveled cut at the proximal end of the external carotid artery ligature, and insert a nylon suture plug with a uniform silicone rubber coating on the front end (for rats weighing less than 255g, use a suture plug with a head diameter of 0.33±0.02mm; for rats weighing more than 255g, use a suture plug with a head diameter of 0.36±0.02mm). Tie the spare suture (slipknot) to fix the suture plug and prevent it from slipping. Use microtweezers to gently lift the suture at the distal end of the external carotid artery, use a multifunctional surgical instrument to electrocoagulate the distal end of the external carotid artery, and use ophthalmic scissors to cut the external carotid artery from the proximal end of the suture. Adjust the direction of the suture plug to be consistent with the direction of the internal carotid artery, and remove the artery clamp on the internal carotid artery. A suture was inserted from the external carotid artery through the common carotid bifurcation into the internal carotid artery until slight resistance was felt (approximately 20 mm from the bifurcation). At this point, the suture reached the origin of the middle cerebral artery, completely blocking the artery's blood supply. Sutures previously reserved for use at the proximal end of the external carotid artery were ligated, and the exposed portion of the suture was cut with surgical scissors. The artery clamp on the common carotid artery and the suture at the distal end of the external carotid artery were removed, and the skin was sutured. Rats were transferred to a 37°C intensive care unit, where their body temperature was maintained at 37.0 ± 0.5°C.

[0153] (5) Reperfusion blood supply: After 1.5 hours of cerebral ischemia, the rats were anesthetized and the common carotid artery was clamped with an artery clamp. The suture was gently removed. The internal carotid artery was clamped with an artery clamp and the suture was completely removed. The blood vessels were ligated with sutures. If blood still flowed out, the ligature was electrocoagulated with a multifunctional surgical instrument. The artery clamps on the common carotid artery and internal carotid artery were removed, and reperfusion blood supply was restored.

[0154] (6) Postoperative treatment: The rat's neck skin was sutured and after alcohol disinfection, the rat was transferred to the intensive care unit at a temperature of 37°C until it fully woke up. During the recovery process, general observations were also required, including: death, coma, respiration, urine and stool characteristics, coat color, mental state, vomiting and vomitus, bleeding, convulsions, etc. Animals with abnormalities caused by unexpected factors were eliminated.

[0155] 9.3 Experimental Grouping

[0156] Rats were randomly divided into a sham operation group (Sham group), a model group (Model group), a positive drug group (Tat-GluA2-3Y), and an experimental drug group (c10c-G1V3). Two hours after blood supply was restored after cerebral ischemia-reperfusion, the drug was administered via intraperitoneal injection at a volume of 4.0 mL / kg. Neurological deficits were observed 24 hours after administration, and cerebral infarction area was measured. Each drug-dosing group was prepared by a blinded person. The experimental surgeon, drug administration personnel, and indicator test personnel were unaware of whether the injection was saline or the peptide drug. After the evaluation of each indicator, the blinded person was unblinded.

[0157] Sham operation group: performed steps (1), (2), (3), and (6) of the experimental section 9.2, only isolated the blood vessels without inserting the suture plug, and injected an equal volume of blank excipients intraperitoneally; model group: injected an equal volume of blank excipients intraperitoneally; positive drug group: injected the positive drug Tat-GluA2-3Y intraperitoneally at a dose of 8 mg / kg; experimental drug group: injected the experimental drug c10c-G1V3 intraperitoneally at a dose of 8 mg / kg.

[0158] 9.4 Analysis of Cerebral Infarction Volume

[0159] 24 hours after administration, rats were anesthetized with 4.0%-5.0% isoflurane mixed with oxygen, and the brains were removed by decapitation. The brain tissue was dissected, the olfactory bulb, cerebellum, and lower brainstem were removed, and the pia mater was carefully peeled off. The brain surface was rinsed with normal saline (sodium chloride injection) to remove blood stains, and residual water stains on the surface were aspirated. The brain was placed in a -80°C environment for 10 minutes and then taken out. A coronal section was immediately made downward at the intersection of the visual field, and one slice was cut every 2 mm backward. The brain slices were transferred to freshly prepared 2,3,5-triphenyltetrazolium chloride (TTC, 20 g / L) staining solution with a wet brush and incubated for 20 minutes at 37°C in the dark for staining. During the incubation process, the brain slices were gently turned over once with a brush.

[0160] After staining, normal brain tissue appears dark red, and ischemic brain tissue appears pale white. The TTC staining solution was aspirated and the brain slices were immersed in 4% paraformaldehyde for 24 hours.

[0161] Rinse with saline, quickly arrange the brain slices from front to back, wipe off any residual surface moisture, and photograph them. Statistical analysis of the photographs was performed using image analysis software (ImageJ). The non-ischemic area (red area) and the total area were circled, and the percentage of infarct area was calculated. The percentage of infarct area was calculated according to formula (4): percentage of infarct area (%) = (normal brain area - non-ischemic brain area) / normal brain area × 100%.

[0162] 9.5 Neurological Deficit Symptom Score

[0163] Twenty-four hours after administration, the neurological deficits in rats following cerebral ischemia-reperfusion injury were evaluated using the modified Bederson 5-point scale.

[0164] 0 points: When the rats were suspended by their tails, their forelimbs were naturally extended and pointed toward the ground, and no abnormal motor function was detected.

[0165] 1 point: In the tail-suspension state, the forelimb on the side opposite to the MCAO surgery shows characteristic movement disorders, manifested as complex signs of wrist and elbow flexion, accompanied by shoulder internal rotation and elbow abduction, and the limb is in a state of continuous contact with the lateral wall of the chest cavity.

[0166] 2 points: When the experimental rats were placed on a high-smoothness surface, a lateral thrust was applied to the shoulder on the operated side, and a significant decrease in the resistance exercise ability was observed.

[0167] 3 points: During the spontaneous movement observation, the experimental rats showed a continuous circumferential or circular movement pattern centered on the contralateral side of the surgery.

[0168] 4 points: The affected limb of the experimental rat showed complete flaccid paralysis, with complete loss of motor neuron innervation function and no signs of autonomous movement.

[0169] 10 Statistical processing

[0170] GraphPad Prism 7 software was used for data analysis, and the experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA), and subsequent multiple comparisons between groups were performed using the Dunnett test or Tukey test. *P < 0.05 indicates statistical significance, **P < 0.01 indicates a significant difference, ### or ***P < 0.001 indicates a highly significant difference, and ****P < 0.0001 indicates an extremely significant difference.

Claims

1. An N-methylated cyclic peptide, characterized in that: The structural formula is shown in formula (Ⅰ); 2. Use of the cyclic peptide according to claim 1 in the preparation of a drug for treating ischemic stroke.

3. A drug, characterized in that: The invention also comprises a therapeutically effective amount of acetate, hydrochloride or other pharmaceutically acceptable salt forms of the cyclic peptide.

Images