GluA2 AMPAR endocytosis-inhibiting transmembrane cyclic peptide and its application

Abstract

The present invention belongs to the field of medicinal chemistry technology, specifically relating to GluA2 AMPAR endocytosis-blocking transmembrane cyclic peptides and their applications. The present invention is based on the nine active amino acid sequence (GluA2-3Y) at the end of the AMPA receptor GluA2 subunit, undergoing structural modification and engineering. A hydrophobic group is attached to the N-terminus of the peptide, and amino acids at different positions in the peptide sequence are replaced with two cysteines according to a specific pattern. The sulfhydryl groups on the cysteines are then linked using a chemical linker to partially cyclicize the peptide sequence, thereby stabilizing the peptide sequence conformation, enhancing the transmembrane ability of the modified peptide, and improving the stability of the peptide sequence.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and in particular relates to a GluA2 AMPAR endocytosis-blocking transmembrane cyclic peptide and an application thereof. Background Art

[0002] AMPA receptors are tetrameric channels composed of four subunits (GluA1-GluA4), of which the GluA2 subunit is the most important functional subunit. Related studies have shown that the C-terminus of the GluA2 subunit is highly susceptible to phosphorylation. AMPA receptors directly bind to the synaptic protein Brag-2 through phosphorylation of the terminal tyrosine of the GluA2 subunit. The BRAG2 / GEP100 protein is a guanine nucleotide exchange factor (GEF) for Arf6. Furthermore, Brag-2 may participate in cancer cell invasion by linking EGF receptor signaling to Arf6 activation. Numerous neurological diseases, such as drug addiction, Alzheimer's disease, stroke, and major depression, have been shown to be closely linked to this process. Further studies have revealed that this process is associated with a specific amino acid sequence (369-377) containing multiple tyrosine residues within the intracellular C-terminus of the AMPA receptor GluA2 subunit. This amino acid sequence further activates the guanosine triphosphatase Arf6 by interacting with the Brag-2 synaptic protein. Arf6 not only plays a role in actin membrane remodeling but also directly affects the formation of synaptic vesicles mediated by clathrin, leading to the endocytosis of AMPA receptors on the postsynaptic membrane. Therefore, the binding between the AMPA receptor C-terminus and the Brag-2 synaptic protein plays a crucial role in the entire regulatory process. By interfering with the interaction between the two, it greatly reduces the excessive endocytosis of postsynaptic receptors, thereby reducing the formation of LTD. Whether or not to activate the phosphorylation of the tyrosine residues on the C-terminus of the GluA2 subunit in this process becomes the key. Studies have demonstrated that by attaching the transmembrane sequence TAT (RRRYKGRKKKRR) to the N-terminus of AMPAGluA2's 3Y (amino acid sequence YKEGYNVYG, structural formula shown in Formula (IV)), the designed peptide Tat-GluA2-3Y can cross the blood-brain barrier, effectively blocking pathological endocytosis of GluA2 and exerting neuroprotective effects. It has a positive therapeutic effect on various neurological diseases, including stroke, and is currently in the clinical stage. However, Tat-GluA2-3Y is a peptide composed of natural amino acids and has drawbacks such as large molecular weight, poor stability, and a short half-life.

[0003] (IV). Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a GluA2 AMPAR endocytosis-blocking transmembrane cyclic peptide. The cyclic peptide is chemically modified and engineered based on the GluA2-3Y polypeptide sequence to restrict the flexible conformation of GluA2-3Y. This achieves the goal of removing the transmembrane sequence while maintaining conformational stability, transmembrane efficiency, and biological activity. The present invention also provides the use of the cyclic peptide in the preparation of a drug for treating nerve damage and cerebral infarction.

[0005] The technical solutions of the present invention are as follows:

[0006] The GluA2 AMPAR endocytosis-blocking transmembrane cyclic peptide of the present invention comprises at least one of the following cyclic peptides: cyclic peptides having amino acid sequences as shown in SEQ ID NOs: 1-3, respectively named P3LC6LC-M, P3LC7LC-P, and P3DC7LC-O, and their corresponding structural formulas are shown in formulas (I) to (III);

[0007] P3LC6LC-M amino acid sequence: 4-pentynoic acid-YKE[CYNC]YG, as shown in SEQ ID NO: 1;

[0008] P3LC7LC-P amino acid sequence: 4-pentynoic acid-YK[CGYNC]YG, as shown in SEQ ID NO: 2;

[0009] P3DC7LC-O amino acid sequence: 4-pentynoic acid-YK[CGYNc]YG, as shown in SEQ ID NO: 3.

[0010] (I)

[0011] (II)

[0012] (III)

[0013] The present invention provides the use of the cyclic peptide in preparing medicines for treating nerve damage and cerebral infarction.

[0014] The present invention also provides a drug comprising a therapeutically effective amount of at least one acetate, hydrochloride or other pharmaceutically acceptable salt form of the cyclic peptide.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention is based on the 9 active amino acid sequence (GluA2-3Y) at the end of the AMPAR GluA2 subunit, and structurally modifies and transforms it. A hydrophobic group is connected to the N-terminus of the polypeptide, and the amino acids at different positions in the polypeptide sequence are replaced with two cysteines according to a certain rule. A chemical linker is used to connect the sulfhydryl groups on the cysteine ​​to form a ring in the polypeptide sequence, thereby achieving the purpose of stabilizing the conformation of the polypeptide sequence, enhancing the membrane-penetrating ability of the modified polypeptide, and improving the stability of the polypeptide sequence.

[0017] This study optimized the peptide synthesis method, designing three series of peptides by selecting the amino acid positions for mutation through rational drug design. A brombenzyl-based chemical linker was used to link the cysteine ​​side chain thiol groups. All resulting peptides were validated by high-resolution mass spectrometry and purified by reversed-phase high-performance liquid chromatography. The resulting target peptides were systematically evaluated in vitro to verify the modification results. The resulting cyclic peptides were screened and identified, demonstrating high membrane penetration efficiency and stability, as well as significant neuroprotective effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 , RP-HPLC chromatogram of cyclic peptide P3LC6LC-M, A is crude peptide, B is pure peptide;

[0019] Figure 2 , ESI-MS mass spectrum of cyclic peptide P3LC6LC-M;

[0020] Figure 3 , RP-HPLC chromatogram of cyclic peptide P3LC7LC-P, A is crude peptide, B is pure peptide;

[0021] Figure 4 , ESI-MS mass spectrum of cyclic peptide P3LC7LC-P;

[0022] Figure 5 , RP-HPLC chromatogram of cyclic peptide P3DC7LC-O, A is crude peptide, B is pure peptide;

[0023] Figure 6 , ESI-MS mass spectrum of cyclic peptide P3DC7LC-O;

[0024] Figure 7 , polypeptide synthesis process, A is a 36-series polypeptide, B is a 37-series polypeptide, and C is a 47-series polypeptide;

[0025] Figure 8 , peptide membrane penetration efficiency;

[0026] Figure 9A is the binding posture of GluA2-3Y and Brag-2 protein, and B is the overlap result of P3LC7LC-P energy minimization and GluA2-3Y; the orange model is the Brag2 protein structure, and the ribbon model is different 3Y conformations;

[0027] Figure 10 , Kinetic characteristics of representative peptides and Brag-2 protein; A is GluA2-3Y, B is P3LC6LC-M, C is P3DC7LC-O, and D is P3LC7LC-P;

[0028] Figure 11 , the effect of peptides on ROS production in HT-22 cells induced by glutamate;

[0029] Figure 12 , the anti-apoptotic results of modified peptides;

[0030] Figure 13 , the effect of modified peptides on glutamate-induced apoptosis of HT-22 cells;

[0031] Figure 14 , evaluation of polypeptide animal activity. DETAILED DESCRIPTION

[0032] 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. Example 1

[0033] Solid-phase synthesis of peptides

[0034] Swell the resin in 10 mL of DCM and shake for 3 hours at 25°C. Deprotect the resin with 10 mL of 20% piperidine in DMF for 30 minutes. Wash the resin with 10 mL of DMF, MeOH, DCM, DMF, DCM, DMF, DMF, and DMF, sequentially for 5 minutes each (ninhydrin test is possible). After ensuring removal of the Fmoc protecting group, activate the amino acid. Weigh 3 eq:3 eq:5 eq of amino acid, HBTU, and DIEA, respectively, and add DMF to dissolve the peptide and condensing agent. Stir magnetically at room temperature for 15 minutes. After activation, add the peptide to the solid-phase synthesis tube and continue shaking on a thermostatted shaker at 220 rpm and 25°C for 1 hour. After completion of the reaction, wash the resin with DMF, DCM, DMF, DCM, DMF, DMF, DMF, and DMF, sequentially for 3 minutes each (ninhydrin test is possible). Repeat the Fmoc deprotection and coupling steps until the last amino acid is coupled. After coupling the last amino acid, deprotect and remove the last Fmoc group. Dry the resin completely with argon. Remove the prepared cutting solution from the refrigerator, add 10 mL of cutting solution under ice bath conditions, and stir in a blender for 3 hours. Wash three times with icy TFA. Combine the washing solution and cutting solution, add 100 mL of icy tert-butyl methyl ether, and let it settle. After centrifugation, discard the supernatant and lyophilize.

[0035] After the peptide synthesis is completed, acetonitrile and water are used as solvent conditions (1:1), 1eq of peptide is added with 3eq of linker and ammonium bicarbonate, and stirred at room temperature for 1 hour. After the reaction is completed, reverse HPLC detection and mass spectrometry verification are performed, and then lyophilization is performed after it is correct. The RP-HPLC chromatogram of the crude peptide P3LC6LC-M is shown in the figure. Figure 1 A, RP-HPLC chromatogram of pure P3LC6LC-M peptide Figure 1 The ESI-MS mass spectrum of P3LC6LC-M is shown in Figure B. Figure 2 ; RP-HPLC chromatogram of P3LC7LC-P crude peptide is as follows Figure 3 A, RP-HPLC chromatogram of pure P3LC7LC-P peptide Figure 3 The ESI-MS mass spectrum of P3LC7LC-P is shown in Figure B. Figure 4 ; RP-HPLC chromatogram of P3DC7LC-O crude peptide is as follows Figure 5 A, RP-HPLC chromatogram of pure P3DC7LC-O peptide Figure 5 In Figure B, the ESI-MS mass spectrum of P3DC7LC-O is shown in Figure Figure 6 This embodiment designed and obtained 36 modified peptides, including three series: 36, 37, and 47 (the synthesis process is as follows Figure 7), 12 modified peptides in each series (Table 1). Example 2

[0036] HT-22 cell glutamate injury model

[0037] In order to study the neuroprotective activity of polypeptides in vitro, their safety to cells must first be studied. In this example, cytotoxicity tests were performed on polypeptides at a concentration of 10 μM. The results are shown in Table 1. In series 36, after treating cells with polypeptides P3LC6LC-O, P3LC6LC-M, P3DC6DC-M, P3DC6LC-M, and P3DC6LC-P for 24 hours, the cell viability was less than 80%, showing a toxic effect on the normal state of the cells; in series 37, three polypeptides of the P3DC7LC series showed cytotoxicity; similarly, in series 47, polypeptides P4LC7LC-O, P4LC7DC series, P4DC7DC-M, P4DC7DC-P, P4DC7LC-O, and P4DC7LC-P showed cytotoxicity.

[0038] The results of the glutamate-induced excitotoxicity experiment on HT-22 cells are shown in Tables 2 to 4. In the 36 series, under the treatment of 0.2 μM drug, the peptides P3LC6LC-M, P3LC6DC-P and P3DC6DC-O all showed significant cell protection activity ( P <0.05); Under 1μM drug treatment, most peptides showed significant neuroprotective effects, among which P3LC6DC-P and P3DC6DC-O had better protective activities ( P <0.001); Under 5μM drug treatment, most peptides showed significant neuroprotective effects, among which P3LC6LC-M and P3DC6DC-O had better protective activities ( P <0.001). In the 37 series, P3LC7DC-M, P3LC7DC-P and P3DC7DC-O showed significant cytoprotective activity under 0.2μM drug treatment ( P <0.05); Under 1μM drug treatment, some peptides showed significant neuroprotective effects, among which P3DC7DC-O had the best protective activity ( P <0.001); Under 5μM drug treatment, most peptides showed significant neuroprotective effects, among which P3LC7LC-P, P3LC7DC-O and P3DC7LC-M had protective activities exceeding 90%, showing significant neuroprotective effects ( P <0.001). In the 47 series, all peptides showed significant neuroprotective effects under 0.2μM drug treatment, and P4LC7LC-P had the best protective activity ( P<0.001); Under 1μM drug treatment, all peptides showed significant neuroprotective effects, and P4LC7DC-M had better protective activity ( P <0.001); Under 5μM drug treatment, due to the cytotoxicity of some peptides, only P4DC7DC-P, P4DC7LC-M and P4DC7LC-P showed good protective effects ( P <0.001).

[0039] Among all the modified polypeptides, P3LC6LC-O, P3LC6LC-M, P3LC6LC-P, P3LC6DC-O, P3LC7LC-O, P3LC7LC-P, P3LC7DC-M, P3LC7DC-P, P3DC7LC-O, P4DC7LC-M, P4DC7LC-P, and P4DC7DC-P polypeptides had better cytoprotective activity, among which P3LC7LC-P had the best neuroprotective activity.

[0040] Table 1 Cytotoxicity results of peptides at 10 μM concentration

[0041]

[0042] Table 2 Results of glutamate-induced excitotoxicity experiments on HT-22 cells by 36 series peptides

[0043]

[0044] Note: The data of three experiments are expressed as mean ± standard deviation (μM), Control is the control group, Model is the model group, P <0.05,** P <0.01,*** P <0.001, compared with the model group at each concentration.

[0045] Table 3 Results of glutamate-induced excitotoxicity experiments on HT-22 cells by 37 series peptides

[0046]

[0047] Note: The data of three experiments are expressed as mean ± standard deviation (μM), Control is the control group, Model is the model group, P <0.05,** P <0.01,*** P <0.001, compared with the model group at each concentration.

[0048] Table 4 Results of glutamate-induced excitotoxicity experiments on HT-22 cells by 47 series peptides

[0049]

[0050] Note: The data of three experiments are expressed as mean ± standard deviation (μM), Control is the control group, Model is the model group, P <0.05,** P <0.01,*** P <0.001, compared with the model group at each concentration.

[0051] Example 3

[0052] Determination of drug permeability and prediction of drug absorption

[0053] In this example, eight peptides including P3LC6LC-M, P3LC7LC-P, and P3DC7LC-O were selected to measure cell membrane penetration efficiency. Figure 8 The MDCK-MDR1 cell transmembrane model demonstrated that they all had certain transmembrane abilities. The transmembrane efficiency of P3LC6LC-M, P3LC7LC-P, and P3DC7DC-P had no significant difference from that of Tat-GluA2-3Y, while the transmembrane efficiency of P3DC7LC-O was about 4 times that of Tat-GluA2-3Y. Example 4

[0054] Molecular docking of peptides and Brag-2 protein

[0055] By comparing the computer simulation of the binding posture of GluA2 and Brag-2 protein with the energy-minimized posture of P3LC7LC-P, the results are as follows Figure 9 As shown: the first Tyr in GluA2-3Y forms a hydrogen bond with Val 603 amino acid residue and Lys 602 amino acid residue, the second Tyr forms a hydrogen bond with Lys 403 amino acid residue; the methyl group of Val forms a hydrophobic interaction with Tyr 406 and Arg 407; the third Tyr forms a hydrogen bond with Gly 428 amino acid residue and forms a π-π conjugation with Phe429. Example 5

[0056] Comparison of the optimal docking conformation of GluA2-3Y and the conformation of the designed cyclic peptide

[0057] The results in Table 5 show that P3DC7LC-O has a lower average strain energy and a higher similarity metric, which suggests that it has a higher affinity.

[0058] Table 5 Comparison of the optimal docking conformation of GluA2-3Y and the conformation of the designed cyclic peptide

[0059]

[0060] Note:U arepresents the average strain energy of the molecules in the orientation, in kcal / mol, calculated as the sum of the individual force field potentials (possibly using a solvent model) divided by the number of molecules; F b represents the similarity measure of the configurations, F is the negative value of the P density overlap function, and the lower the value, the greater the similarity; S c stands for Grand Alignment Score, which is simply the sum of columns U and F, with lower values ​​intended to indicate better alignment. Example 6

[0061] Determination of affinity between peptides and Brag-2 protein

[0062] In this example, the affinity of peptides to Brag-2 protein was determined by surface plasmon resonance technology, with GluA2-3Y as a positive control, and the peptides were prioritized according to their binding level. The binding patterns of representative peptides to Brag-2 protein are shown in Figure 2. Figure 10 As shown, the binding of the polypeptide to the Brag-2 protein is a slow association and dissociation state. Therefore, this example used kinetic fitting to estimate the dissociation constant, KD. The results are shown in Tables 6 to 8. P3DC7DC-O, P3DC7LC-O, P3LC7LC-M, P3LC7LC-P, and P3LC7DC-P can be used as potential active compounds for further biological activity studies. The amino acid sequence of the 36 series polypeptide is shown in SEQ ID NO: 1; the amino acid sequence of the 37 series polypeptide is shown in SEQ ID NO: 2; and the amino acid sequence of the 47 series polypeptide is shown in SEQ ID NO: 4.

[0063] Table 6 Binding ability of 36 series modified peptides to Brag-2 protein and their retention time on HPLC

[0064]

[0065] Table 7 Binding ability of 37 series modified peptides to Brag-2 protein and their retention time on HPLC

[0066]

[0067] Table 8 Retention time of 47 series modified peptides on HPLC

[0068]

[0069] Example 7

[0070] Effects of modified peptides on glutamate-induced ROS production in HT-22 cells

[0071] Based on the above-mentioned transmembrane evaluation and in vitro neuroprotective activity results, the effects of P3DC7LC-O and P3LC7LC-P on glutamate-induced ROS production in HT-22 cells were further studied. In this example, HT-22 cells were stained with DCFH-DA, and the fluorescence intensity of the cells was observed under an inverted fluorescence microscope (×20). The anti-oxidative stress effect of the peptides was statistically analyzed. All values ​​are expressed as mean ± standard deviation (n=3). Compared with the Glu group, *** P <0.001,** P <0.01,* P <0.05; e.g. Figure 11 As shown in the results, compared with the normal group, the Glu group induced HT-22 cells to produce a large amount of ROS. When the cells were intervened with 5 μM peptide, the positive control drug Tat-GluA2-3Y and the peptides P3DC7LC-O and P3LC7LC-P all improved the production of ROS in neurons to a certain extent. The results showed that the peptides P3DC7LC-O and P3LC7LC-P can reduce the production of ROS in neurons induced by glutamate to a certain extent. Example 8

[0072] Anti-apoptotic effect of peptides

[0073] The anti-apoptotic results of the modified peptides P3LC6LC-M, P3LC7LC-P, and P3DC7LC-O, e.g. Figure 12 As shown, the modified peptides reduced the activation of cysteine ​​protease 3 (Caspases-3) to varying degrees, decreased the expression level of the pro-apoptotic protein Bax, and increased the expression level of the anti-apoptotic protein Bcl-2. Figure 12 Tubulin is microtubule protein. Example 9

[0074] Effects of modified peptides on glutamate-induced apoptosis in HT-22 cells

[0075] Based on the above experimental results, the effect of P3LC7LC-P on glutamate-induced apoptosis of HT-22 cells was further studied. In this example, HT-22 cells were treated with Tat-GluA2-3Y or modified peptide (5 μM) 1 hour before Glu induction. The HT-22 cells were stained with Hoechest and the apoptotic morphology of HT-22 cells was observed under an inverted fluorescence microscope. The apoptotic rates of the Tat-GluA2-3Y and modified peptide groups were obtained. P <0.001, compared with the Glu group, all values ​​are expressed as mean ± SD, n = 3 per group, one-way ANOVA followed by Dunnett's multiple comparison; e.g. Figure 13As shown in the results, compared with the normal group, the Glu group induced apoptosis in HT-22 cells. When the cells were intervened with 5 μM peptide, the positive control drug Tat-GluA2-3Y and the peptide P3LC7LC-P both improved the apoptosis of neurons to a certain extent. The results showed that the peptide P3LC7LC-P had a protective effect on glutamate-induced apoptosis of neurons. Example 10

[0076] Effects of modified peptides on cerebral infarction area and neurological function

[0077] Given that the modified polypeptide has certain neuroprotective ability, blood-brain barrier permeability and water solubility in vitro, this example further evaluated its neuroprotective effect in vivo by studying the widely used ischemic stroke model. Figure 14 As shown, MCAO injury induced significant infarction, which is shown as the white area in brain sections. Tat-GluA2-3Y and P3LC7LC-P at a dose of 8 mg / kg significantly reduced the brain infarct size of mice compared with the vehicle-treated MCAO group ( P <0.001), the infarct area increased from 324.047 mm 2 Reduced to 216.242mm 2 In vitro experiments showed that P3LC7LC-P has a significant protective effect against cerebral I / R injury and can alleviate neurological deficits after cerebral I / R injury.

[0078] The materials and methods of the embodiments of the present invention are as follows:

[0079] 1. Chemicals and reagents

[0080] All chemicals and solvents used were obtained from commercial sources. Reaction progress was monitored by thin-layer chromatography (silica gel GF254, Qingdao Haiyang Chemical Co., Ltd.), and the crude product was purified by silica gel column chromatography (silica gel 200-300 mesh, Shanghai Sanpeng Co., Ltd.). Reverse-phase high-performance liquid chromatographs 1220 and 1260 were purchased from Agilent Technologies. Positisil OSD-PC18 analytical columns (5 μm, 4.6 mm × 250 mm) and Positisil OSD-P C18 semi-preparative columns (5 μm, 10 mm × 250 mm) were purchased from Beijing Yinglai Technology Co., Ltd.

[0081] 2. Synthesis, cleavage and purification of peptides

[0082] Peptide synthesis reagents and lincosamide resin (100-200 mesh, 0.5 mmol / g) were purchased from Beijing Ouhe Technology Co., Ltd. Acetonitrile and dichloromethane were purchased from Guangzhou Bishilo Technology Co., Ltd. Trans-fatty acids were purchased from Aladdin Reagents (Shanghai) Co., Ltd. Peptides were synthesized using standard solid-phase peptide synthesis on lincosamide resin. After swelling the resin in DCM, the first C-terminal residue was coupled using 3 eq of an Fmoc-protected amino acid and 3 eq of diisopropylcarbodiimide in DMF for 1 hour at room temperature. In all subsequent coupling steps, the Fmoc protecting group was removed using 20% ​​piperidine in DMF with stirring for 30 minutes at room temperature. Peptide couplings were performed using 3 eq of an Fmoc-protected amino acid, 3 eq of HBTU / 3 eq of HATU, and 6 eq of DIEA in DMF for 1 hour at room temperature. After completion of the linear sequence, the peptide was acetylated at the N-terminus using 8 eq of Ac2O and 6 eq of DIEA in DCM mixed for 1 hour at RT.

[0083] Cleavage and deprotection of the peptide were completed by treating the resin with the prepared cleavage solution (9.4 mL of trans-fatty acid, 0.5 g of dithiothreitol, and 0.5 mL of deionized water) at room temperature for 3 hours with stirring. The crude peptide solution was extracted with cold dimethyl ether, and the precipitate was isolated by centrifugation. The precipitate was resuspended in cold dimethyl ether and centrifuged twice. The crude peptide was freeze-dried, dissolved in a minimum volume of dimethyl sulfoxide, and purified by reversed-phase high-performance liquid chromatography equipped with a Positisil OSD-P C18 analytical column using a linear gradient of 10–90% acetonitrile in ddH2O containing 0.05% TFA. Fractions were collected based on the absorbance peak at 280 nm, analyzed by ESI-MS, and then lyophilized.

[0084] After molecular weight confirmation by mass spectrometry, the crude peptide was dissolved in dimethyl sulfoxide (DMSO) and diluted 1:10 (v / v) in 10× H₂O (final concentration: 10% DMSO). The mixture was fractionated by reversed-phase HPLC using a Positisil OSD-P C₁ₒ semi-resolving column with a linear gradient of 10–70% acetonitrile and ddH₂O containing 0.05% TFA to separate the linear peptide from the disulfide-cyclized peptide. All peptides used in this study were determined to have a purity of ≥90% by analytical HPLC.

[0085] 3. BRAG2 expression and purification

[0086] For biacore experiments, a portion of the human IQ motif and the SEC7 domain-containing protein 1 sequence (Uniprot ID: Q6 DN 90) were used. The sequence encoding amino acids 512−885 was cloned into the pET-28a vector (with an N-6 His-TEV tag). This plasmid was transformed into Escherichia coli BL21. The bacterium was expressed at OD 600 When the pH value was 0.6-0.8, expression was induced overnight at 16°C with 0.3 mM IPTG. The bacterial culture was centrifuged, and the resulting pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100) and lysed using an ultrasonicator. The lysate was clarified by centrifugation at 12,000 rpm and 4°C for 30 minutes and then applied to a NiSO4 column. Elution was performed using a 0-100% gradient over 40 minutes at a flow rate of 1 mL / min using elution buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol). The eluate was collected, digested with TEV, purified using a secondary nickel column, and purified using Superdex 75 to yield 9.52 mg of BRAG2 protein with a purity >95%.

[0087] 4. Surface Plasmon Resonance

[0088] SPR experiments were performed at 25°C using a BIAcore CM5 Series S sensor chip (BIAcore T200, GE Life Sciences). Polyclonal anti-GST antibodies (Abcam, Cambridge) were immobilized on all flow cells via amine coupling, with capture levels ranging from 2280 RU to 6700 RU (GE Life Sciences GST Capture Kit). As controls, reference flow cells (between 520 RU and 1280 RU) captured only GST, while active flow cells (between 690 RU and 1990 RU) immobilized GST-tagged proteins. Lyophilized peptides (triplicates) were resuspended in assay buffer and eluted at 30 μL / min. -1 +PO4 was injected into all four flow cells at a rate of 100 μg / min for 60-80 seconds. 3- The buffer consisted of 50 mM Na3PO4, 150-300 mM NaCl, and 1 mM DTT (pH 7.4), while -PO4 3-The buffer consisted of 1 mM Na3PO4 (reflecting physiological concentrations), 150 mM NaCl, 20 mM Tris, and 1 mM DTT (pH 7.4). Peptide 3 required regeneration with 2 M NaCl before injection into the next peptide cycle. Data were analyzed using Scrubber 2.0 (BioLogic Software, Campbell, ACT, Australia) and Prism 6.0 (GraphPad Software, California, USA).

[0089] 5. Cell culture and drug preparation

[0090] HT-22 cells were placed in a 37°C, 5% CO2 incubator and cultured with DMEM complete medium containing 10% FBS and 5% penicillin-streptomycin mixture. When the cells entered the logarithmic growth phase, 4×10 4 The cells were seeded at a density of 100 / mL in a 96-well plate and cultured in an incubator.

[0091] Glutamate was dissolved in FBS-free DMEM medium and adjusted to a pH of 7.2-7.4. Test peptides were prepared as 10 mM stock solutions in 100% DMSO and freshly diluted in cell culture medium to the specified concentrations (0.2, 1, and 5 μM) before use. Twenty-four hours after plating, the medium was discarded and replaced with the test drug for a pre-incubation of 1 hour, using 100 μL per well. The glutamate solution was then replaced. Experiments were divided into blank, control, model, and compound groups.

[0092] 6. Cytotoxicity test

[0093] HT-22 cells were cultured at 4 × 10 4 Cells were plated at a density of 1 μg / mL in a 96-well plate and incubated in DMEM supplemented with 10% FBS for 24 hours at 37°C in a 5% CO2 incubator. Tat-GluA2-3Y and peptides with enhanced cell viability were prepared at a 10 μM concentration and co-cultured with the cells for 24 hours. After 24 hours, the cells were rinsed three times with PBS and incubated with CCK-8 for 2 hours.

[0094] 7. Cell survival assay

[0095] After the above treatments, the cells to be tested were removed and 10% CCK-8 was added directly to the cell culture medium. Mix thoroughly, and then incubate the cells in a 37°C incubator for another 2 hours. The absorbance (OD) at 450 nm was measured using a microplate reader and compared with that of untreated cells for standardized analysis. The experiment was repeated three times, and the average value was used to compare cell viability among the groups.

[0096] 8. Transwell assay

[0097] MDCK cells were obtained from the American Type Culture Collection and cultured in an atmosphere of 90% air and 10% CO2. 5 Cells were seeded in 96-well cell culture plates (Beaver) and allowed to grow and differentiate for 7 days before the cell monolayers were used for transport experiments.

[0098] 9. Transportation Research

[0099] The markers were dissolved in Hank's balanced salt solution, pH 7.4, containing 1 mM Hepes (HBSS pH 7.4). The final concentration of the different peptides was 100 μM, and the detection limit of the HPLC analysis, the presence of saturable active transport mechanisms, and the effects on monolayer integrity are described below. The monolayers were placed in HBSS, pH 7.4, and incubated at 37°C in a humidified atmosphere for 20 minutes before the transport experiments were started. The drug solutions were added to the donor side of the cell monolayer and maintained at 37°C throughout the experiment. To examine the possible effects of active transport or efflux mechanisms, we measured the permeability of all compounds in both the apical to basolateral (ab) and basolateral to apical (ba) directions. Under "permeable" conditions, the apparent permeability coefficient (Papp) was calculated by the following formula:

[0100]

[0101] in, is the steady-state flux (mol / s), C0 is the initial concentration in the donor compartment at each time interval (mol / mL), and A is the filter surface area (cm 2 ).

[0102] The analysis method used a Hichrom Partisil ODS3 analytical column (100 × 4.6 mm) with an average particle size of 5 µm. The mobile phase consisted of water (0.1% formic acid) and acetonitrile (0.1% formic acid). The acetonitrile concentration ranged from 2% to 60%, the flow rate was 1 mL / min, and the retention time was 1 to 4 minutes. The injection volume ranged from 10 to 200 µL.

[0103] 10. Determination of Cellular ROS Formation HT-22 cells were cultured at 3×10 4Cells were seeded evenly onto 24-well plates at a density of 10 μM / well. A normal control group, a model glutamate group, and a drug-treated group were set up. After 24 hours of growth, the culture medium was discarded. The drug-treated group was treated with 5 μM peptides Tat-GluA2-3Y, P3DC7LC-O, and P3LC7LC-P. The normal control and model groups were cultured with minimal culture medium. One hour later, the drug-treated and model groups were exposed to 6 mM glutamate. Twenty-four hours later, cells were stained with 10 μM 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 minutes in a dark-protected cell culture incubator. The cells were then washed three times with PBS to thoroughly remove unbound DCFH-DA. The DCFH-DA was then observed using an inverted fluorescence microscope to measure the intensity of dichlorofluorescein (DCF) and estimate ROS production.

[0104] 11. Cell apoptosis detection

[0105] The modeling method was the same as that used in the HT22 cell ROS assay. After 24 hours of glutamate treatment, cells were washed three times with PBS, stained with Hoechst 33342 (50 μg / mL) for 8 minutes in the dark, and washed three times with PBS. Cell morphology was observed and recorded under an inverted fluorescence microscope.

[0106] 12. tMCAO Model

[0107] A middle cerebral artery occlusion (MCAO) model of cerebral ischemia-reperfusion was established using an internal carotid artery guidewire embolization. After anesthesia with isoflurane (2.0-2.5% isoflurane mixed with oxygen), the animals were immobilized in the supine position on the operating table. After skin disinfection, a midline neck incision was made, and the left common, external, and internal carotid arteries were isolated. The vagus nerve was gently dissected, and the external carotid artery was ligated and clipped. The proximal common carotid artery was clamped, and an incision was made distal to the external carotid artery ligature. A nylon filament with an outer diameter of 0.26 ± 0.01 mm was inserted, coated with silicone rubber evenly within 5-6 mm of the distal end. The filament was inserted through the common carotid bifurcation into the internal carotid artery until slight resistance was felt (approximately 20 mm from the bifurcation), thereby completely occluding the blood supply to the middle cerebral artery. After 1.5 hours of cerebral ischemia, the nylon filament was gently withdrawn, blood supply was restored, and reperfusion was allowed. The neck skin was sutured, and the animals were disinfected and returned to their cages for maintenance.

[0108] 13. Experimental Groups and Drug Administration

[0109] Rats were randomly divided into four groups: sham group (normal saline), vehicle group (normal saline), Tat-GluA2-3Y group (2, 4, and 8 mg / kg), and design group (2, 4, and 8 mg / kg). Drugs were administered intravenously 2 hours after the start of reperfusion.

[0110] 14. Infarct volume analysis

[0111] The animals were anesthetized with isoflurane, and the brains were removed from the head. The olfactory bulbs, cerebellum, and lower brainstem were removed. The brain surface was rinsed with saline to remove blood, and any remaining water was aspirated. The brains were stored at -80°C for 7 minutes. Immediately after removal, the coronal section was cut vertically downward in the plane of vision, and then cut 2 mm apart posteriorly. The brain slices were placed in freshly prepared TTC (20 g / L) staining solution and incubated at 37°C for 20 minutes. This procedure was based on a method previously reported in the literature. The animals were anesthetized with isoflurane, and the brains were removed from the head. The olfactory bulbs, cerebellum, and lower brainstem were removed. The brain surface was rinsed with saline to remove blood, and any remaining water was aspirated. The brains were stored at -80°C for 7 minutes. Immediately after removal, the coronal section was cut vertically downward in the plane of vision, and then cut 2 mm apart posteriorly. The brain slices were placed in freshly prepared TTC (20 g / L) staining solution and incubated at 37°C for 20 minutes.

[0112] 15. Statistical analysis

[0113] Quantitative information is presented as mean ± standard error. One-way analysis of variance (ANOVA) and Scheffe's test were used to determine the significance of the differences between the two groups for cerebral infarction area and neurological deficit symptom score. ANOVA test was used to determine the significance of the mortality rate. P The difference was significant when the value was <0.05.

Claims

1. A GluA2 AMPAR endocytosis-blocking transmembrane cyclic peptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO: 2, and its structural formula is shown in formula (II); （Ⅱ）。 2. Use of the cyclic peptide according to claim 1 in the preparation of drugs for treating nerve damage and cerebral infarction.

3. A drug, characterized in that: Comprising a therapeutically effective amount of acetate, hydrochloride or other pharmaceutically acceptable salt form of the cyclic peptide according to claim 1.

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