GluA2 AMPAR endocytosis block transmembrane cyclopeptide and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a GluA2 AMPAR endocytosis block transmembrane cyclopeptide and application thereof. Structural modification and transformation are carried out on nine active amino acid sequences (GluA2-3Y) at the tail end of a GluA2 subunit of an AMPA receptor, a hydrophobic group is connected to the N end of polypeptide, amino acids at different positions in a polypeptide sequence are replaced by two cysteines according to a certain rule, and the amino acids at different positions in the polypeptide sequence are converted into two cysteines according to a certain rule. A chemical connector is used for connecting sulfydryl on cysteine to enable a polypeptide sequence to partially form a ring, so that the aims of stabilizing conformation of the polypeptide sequence, enhancing the transmembrane capability of the modified polypeptide and improving the stability of the polypeptide sequence are fulfilled.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a transmembrane loop peptide for blocking GluA2 AMPAR endocytosis and its application. Background Art

[0002] The AMPA receptor is a tetrameric channel composed of four subunits (GluA1-GluA4), and the GluA2 subunit is the most important functional subunit. Relevant studies have shown that the C-terminus of the GluA2 subunit is extremely vulnerable to phosphorylation. The AMPA receptor directly binds to the synaptic protein Brag-2 through the phosphorylation of tyrosine at the end of the GluA2 subunit. The BRAG2 / GEP100 protein is a guanine nucleotide exchange factor (GEF) of Arf6. In addition, the Brag-2 protein can participate in the invasion of cancer cells by linking the EGF receptor signaling pathway with the activation of Arf6. It has been confirmed that the occurrence of many neurological diseases such as drug addiction, Alzheimer's disease, stroke, severe depression, etc. is closely related to this process. Further research has found that this process is related to a specific amino acid sequence containing multiple tyrosines in the intracellular C-terminus of the GluA2 subunit of the AMPA receptor (369-377). This amino acid sequence further activates the guanosine triphosphatase Arf6 through interaction with the Brag-2 synaptic protein. Arf6 not only plays the function of actin membrane remodeling, but also directly acts on the formation of clathrin-mediated synaptic vesicles, thereby leading to the endocytosis of AMPA receptors on the postsynaptic membrane. Therefore, in the whole regulation process, the binding of the C-terminus of the AMPA receptor to the Brag-2 synaptic protein plays a crucial role. By interfering with the interaction between the two, the excessive endocytosis of receptors on the postsynaptic membrane will be greatly reduced, and thus the formation of LTD will be reduced. Whether to activate the phosphorylation of tyrosine at the C-terminus of the GluA2 subunit becomes the key in this process. It has been confirmed by previous studies that by connecting a transmembrane sequence TAT (RRRYKGRKKKRR) to the N-terminus of the 3Y sequence of AMPA GluA2 (the amino acid sequence is YKEGYNVYG, and the structural formula is shown in formula (Ⅳ)), the designed polypeptide Tat-GluA2-3Y can cross the blood-brain barrier, effectively block the pathological endocytosis of GluA2, play a neuroprotective role, and have a positive therapeutic effect on various neurological diseases such as stroke. Currently, it has entered the clinical stage. However, Tat-GluA2-3Y is a polypeptide composed of natural amino acids, and has disadvantages such as large molecular weight, poor stability, and short half-life.

[0003] (Ⅳ). Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a transmembrane loop peptide that blocks GluA2 AMPAR endocytosis. The loop peptide is chemically modified and transformed on the basis of the GluA2-3Y polypeptide sequence to restrict the flexible conformation of GluA2-3Y, so as to achieve the purpose that the polypeptide still has conformational stability, maintains a certain transmembrane efficiency and biological activity while removing the transmembrane sequence. At the same time, the present invention provides the application of the loop peptide in the preparation of drugs for treating nerve injury and cerebral infarction.

[0005] The technical solution of the present invention is as follows: The GluA2 AMPAR endocytosis-blocking transmembrane loop peptide of the present invention includes at least one of the following loop peptides: loop peptides with amino acid sequences shown in SEQ ID NO: 1-3, named P3LC6LC-M, P3LC7LC-P, P3DC7LC-O respectively, and their corresponding structural formulas are shown in formulas (Ⅰ)-(Ⅲ); The amino acid sequence of P3LC6LC-M: 4-pentynoic acid-YKE[CYNC]YG, as shown in SEQ ID NO: 1; The amino acid sequence of P3LC7LC-P: 4-pentynoic acid-YK[CGYNC]YG, as shown in SEQ ID NO: 2; The amino acid sequence of P3DC7LC-O: 4-pentynoic acid-YK[CGYNc]YG, as shown in SEQ ID NO: 3.

[0006] (Ⅰ) (Ⅱ) (Ⅲ) The present invention provides the application of the loop peptide in the preparation of drugs for treating nerve injury and cerebral infarction.

[0007] The present invention also provides a drug, which contains an acetate, hydrochloride or other pharmaceutically acceptable salt form of at least one of the therapeutically effective amounts of the loop peptides.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention is based on the 9 active amino acid sequences (GluA2-3Y) at the end of the AMPAR GluA2 subunit, modifies and transforms its structure, connects a hydrophobic group to the N-terminus of the polypeptide, replaces amino acids at different positions in the polypeptide sequence with two cysteines according to a certain rule, and uses a chemical linker to connect the sulfhydryl groups on the cysteines to form a loop in part of the polypeptide sequence, so as to achieve the purpose of stabilizing the polypeptide sequence conformation, enhancing the transmembrane ability of the modified polypeptide, and improving the stability of the polypeptide sequence.

[0009] The present invention optimizes the polypeptide synthesis method. Through reasonable drug design, three series are designed by selecting the positions of mutant amino acids. A bromobenzyl chemical linker is selected for the connection of the sulfhydryl group of the cysteine side chain, and all the obtained polypeptides are verified by high-resolution mass spectrometry and purified by reverse-phase high-performance liquid chromatography. The obtained target compound polypeptide is systematically evaluated in vitro to test the modification results. Finally, the cyclic peptide described in the present invention is screened, which has high transmembrane efficiency and stability, and has a significant neuroprotective effect. Description of the Drawings

[0010] Figure 1 RP-HPLC chromatogram of cyclic peptide P3LC6LC-M, A is the crude peptide, B is the pure peptide; Figure 2 ESI-MS mass spectrum of cyclic peptide P3LC6LC-M; Figure 3 RP-HPLC chromatogram of cyclic peptide P3LC7LC-P, A is the crude peptide, B is the pure peptide; Figure 4 ESI-MS mass spectrum of cyclic peptide P3LC7LC-P; Figure 5 RP-HPLC chromatogram of cyclic peptide P3DC7LC-O, A is the crude peptide, B is the pure peptide; Figure 6 ESI-MS mass spectrum of cyclic peptide P3DC7LC-O; Figure 7 Polypeptide synthesis process, A is the 36-series polypeptide, B is the 37-series polypeptide, C is the 47-series polypeptide; Figure 8 Polypeptide transmembrane efficiency; Figure 9 A is the binding pose of GluA2-3Y and Brag-2 protein, B is the coincidence result of the energy minimization of P3LC7LC-P and GluA2-3Y; the orange model is the Brag2 protein structure, and the ribbon model is different 3Y conformations; Figure 10 Kinetic characteristics of representative polypeptides and Brag-2 protein; A is GluA2-3Y, B is P3LC6LC-M, C is P3DC7LC-O, D is P3LC7LC-P; Figure 11 Effect of polypeptide on the production of ROS in glutamate-induced HT-22 cells; Figure 12 Anti-apoptosis result of modified polypeptide; Figure 13 Effect of modified polypeptide on glutamate-induced apoptosis of HT-22 cells; Figure 14 、Evaluation of the biological activity of polypeptides in animals. Specific implementation manners

[0011] To further clarify the present invention, the present invention will be described more comprehensively and systematically below in conjunction with the accompanying drawings of the specification and application examples. The professional terms involved 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 methods. Example 1

[0012] Synthesis of polypeptides by solid-phase synthesis method Swell the resin in 10 mL of DCM and first shake it for 3 hours at 25 °C; deprotect it 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, DMF in sequence, 5 minutes each time (ninhydrin test can be performed); after ensuring that the Fmoc protecting group is removed, activate the amino acid. Weigh the amino acid: HBTU: DIEA at 3eq: 3eq: 5eq respectively and add DMF to dissolve the polypeptide and the condensing agent in the solution. Stir magnetically for 15 minutes at room temperature. After activation, add it to the polypeptide solid-phase synthesis tube, and the polypeptide synthesis tube continues to react in a constant-temperature oscillator with shaking for 1 hour. The reaction conditions are 220 rpm and 25 °C; after the reaction is completed, wash it with DMF, DCM, DMF, DCM, DMF, DMF, DMF, DMF, DMF in sequence, 3 minutes each time (ninhydrin test can be performed). Repeat the Fmoc deprotection and coupling steps until the coupling of the last amino acid is completed; after coupling the last amino acid, deprotect and remove the last Fmoc group. Dry the resin completely with argon. Take out the prepared cleavage solution from the refrigerator, add 10 mL of the cleavage solution under ice bath conditions, and place it in a stirrer to stir for 3 hours. Then wash it three times with ice-cold TFA, mix the washing solution and the cleavage solution, add 100 mL of ice-cold tert-butyl methyl ether, let it stand for precipitation, discard the supernatant after centrifugation, and add water and lyophilize.

[0013] After the polypeptide synthesis is completed, use acetonitrile and water as the solvent conditions (1:1), add 1eq of polypeptide, 3eq of linker and ammonium bicarbonate, stir at room temperature for 1 hour. After the reaction is completed, perform reverse high-performance liquid chromatography detection and mass spectrometry verification. After it is correct, lyophilize it again. Among them, the RP-HPLC chromatogram of the crude peptide P3LC6LC-M is as shown in Figure 1 A in, the RP-HPLC chromatogram of the pure peptide P3LC6LC-M is as shown in Figure 1 B in, the ESI-MS mass spectrum of P3LC6LC-M is as shown in Figure 2 ; the RP-HPLC chromatogram of the crude peptide P3LC7LC-P is as shown inFigure 3 In Figure A, the RP-HPLC chromatogram of the P3LC7LC-P pure peptide is as follows Figure 3 In Figure B, the ESI-MS mass spectrum of P3LC7LC-P is as follows Figure 4 ; the RP-HPLC chromatogram of the P3DC7LC-O crude peptide is as follows Figure 5 In Figure A, the RP-HPLC chromatogram of the P3DC7LC-O pure peptide is as follows Figure 5 In Figure B, the ESI-MS mass spectrum of P3DC7LC-O is as follows Figure 6 . In this example, 36 modified polypeptides were designed, including three series of 36, 37, and 47 (the synthesis process is as shown in Figure 7 ), with 12 modified polypeptides in each series (Table 1). Example 2

[0014] HT-22 cell glutamate injury model To study the in vitro neuroprotective activity of the polypeptides, it is necessary to first study their safety on cells. In this example, the cytotoxicity of the polypeptides at a concentration of 10 μM was tested, and the results are shown in Table 1. Among the 36 series, after treating the cells with the polypeptides P3LC6LC-O, P3LC6LC-M, P3DC6DC-M, P3DC6LC-M, and P3DC6LC-P for 24 hours, the cell viability was lower than 80%, showing a toxic effect on the normal state of the cells; among the 37 series, three polypeptides in the P3DC7LC series showed cytotoxicity; similarly, among the 47 series, the polypeptides P4LC7LC-O, P4LC7DC series, P4DC7DC-M, P4DC7DC-P, P4DC7LC-O, and P4DC7LC-P showed cytotoxicity.

[0015] The experimental results of glutamate-induced excitotoxicity in HT-22 cells are shown in Tables 2 to 4. Among the 36 series, under the drug treatment of 0.2 μM, the polypeptides P3LC6LC-M, P3LC6DC-P, and P3DC6DC-O all showed significant cell protection activity ( P < 0.05); under the drug treatment of 1 μM, most polypeptides showed significant neuroprotective effects, among which P3LC6DC-P and P3DC6DC-O had better protective activities ( P < 0.001); under the drug treatment of 5 μM, most polypeptides showed significant neuroprotective effects, among which P3LC6LC-M and P3DC6DC-O had better protective activities ( P < 0.001). Among the 37 series, under the drug treatment of 0.2 μM, P3LC7DC-M, P3LC7DC-P, and P3DC7DC-O showed significant cell protection activity ( P< 0.05); Under the treatment of 1 μM of the drug, some polypeptides showed significant neuroprotective effects, among which P3DC7DC-O had better protective activity ( P < 0.001); Under the treatment of 5 μM of the drug, most polypeptides showed significant neuroprotective effects, among which the protective activities of P3LC7LC-P, P3LC7DC-O and P3DC7LC-M reached more than 90%, showing significant neuroprotective effects ( P < 0.001). In the 47 series, under the treatment of 0.2 μM of the drug, all polypeptides showed significant neuroprotective effects, and P4LC7LC-P had better protective activity ( P < 0.001); Under the treatment of 1 μM of the drug, all polypeptides also showed significant neuroprotective effects, and P4LC7DC-M had better protective activity ( P < 0.001); Under the treatment of 5 μM of the drug, due to the cytotoxic effects of some polypeptides, only P4DC7DC-P, P4DC7LC-M and P4DC7LC-P showed good protective effects ( P < 0.001).

[0016] 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, P4DC7DC-P polypeptides had better cytoprotective activities, and among them, P3LC7LC-P had the best neuroprotective activity.

[0017] Table 1 Cytotoxicity results of polypeptides at a concentration of 10 μM

[0018] Table 2 Experimental results of excitotoxicity of 36 series polypeptides on glutamate-induced HT-22 cells

[0019] Note: The data of the three experiments were all expressed as mean ± standard deviation (μM). Control was the control group, and Model was the model group. P <0.05, ** P <0.01, *** P <0.001, compared with the model group at each concentration.

[0020] Table 3 Experimental results of excitotoxicity of 37 series polypeptides on glutamate-induced HT-22 cells

[0021] Note: The data of the three experiments are all expressed as mean ± standard deviation (μM). Control is the control group, and Model is the model group. P <0.05, ** P <0.01, *** P <0.001, compared with the model group at each concentration.

[0022] Table 4 Experimental results of excitotoxicity induced by 47 series polypeptides glutamate in HT-22 cells

[0023] Note: The data of the three experiments are all expressed as mean ± standard deviation (μM). Control is the control group, and Model is the model group. P <0.05, ** P <0.01, *** P <0.001, compared with the model group at each concentration.

[0024] Example 3 Determination of drug permeability and prediction of drug absorption In this example, 8 polypeptides such as P3LC6LC-M, P3LC7LC-P, and P3DC7LC-O were selected to determine the cell membrane penetration efficiency. The results are as Figure 8 , It is proved by the MDCK-MDR1 cell membrane penetration model that they all have a certain membrane penetration ability. The membrane penetration efficiency of P3LC6LC-M, P3LC7LC-P, and P3DC7DC-P has no obvious difference from that of Tat-GluA2-3Y, and the membrane penetration efficiency of P3DC7LC-O is about 4 times that of Tat-GluA2-3Y. Example 4

[0025] Molecular docking of polypeptides with Brag-2 protein By comparing the binding pose of computer-simulated GluA2 with Brag-2 protein and the pose with the minimum energy of P3LC7LC-P, the results are as Figure 9 shown: The first Tyr in GluA2-3Y forms hydrogen bonds with the amino acid residues Val 603 and Lys 602, the second Tyr forms a hydrogen bond with the amino acid residue Lys 403; the methyl group of Val forms hydrophobic interactions with Tyr 406 and Arg 407; the third Tyr forms a hydrogen bond with the amino acid residue Gly 428 and at the same time forms a π-π conjugation with Phe429. Example 5

[0026] Conformation comparison between the best docking conformation of GluA2-3Y and the designed cyclic peptide The results in Table 5 show that, in comparison, P3DC7LC-O has a lower average strain energy and a higher similarity measure, suggesting a higher affinity.

[0027] Table 5 Comparison of the best docking conformations of GluA2-3Y and the designed cyclic peptides

[0028] Note: U a represents 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 configuration. F is the negative value of the P density overlap function as described in the method section. A lower value indicates a greater similarity; S c represents the large alignment score, which is simply the sum of the U column and the F column. A lower value is intended to indicate a better alignment. Example 6

[0029] Determination of the affinity of the polypeptides for the Brag-2 protein In this example, the affinity of the polypeptides for the Brag-2 protein was determined by surface plasmon resonance technology, with GluA2-3Y as the positive control, and the polypeptides were ranked according to the priority of their binding levels. The binding modes of the representative polypeptides to the Brag-2 protein are as Figure 10 shown. The binding of the polypeptides to the Brag-2 protein is in a state of slow binding and slow dissociation. Therefore, in this example, a kinetic fitting method was used to evaluate 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 bioactivity studies. The amino acid sequences of the 36 series of polypeptides are shown in SEQ ID NO: 1; the amino acid sequences of the 37 series of polypeptides are shown in SEQ ID NO: 2; the amino acid sequences of the 47 series of polypeptides are shown in SEQ ID NO: 4.

[0030] Table 6 Binding ability of the 36 series of modified polypeptides to the Brag-2 protein and their retention times on HPLC

[0031] Table 7 Binding ability of the 37 series of modified polypeptides to the Brag-2 protein and their retention times on HPLC

[0032] Table 8 Retention times of the 47 series of modified polypeptides on HPLC

[0033] Example 7 Effect of Modified Polypeptides on Glutamate-Induced ROS Generation in HT-22 Cells Based on the aforementioned results of transmembrane evaluation and in vitro neuroprotective activity of cells, the effects of P3DC7LC-O and P3LC7LC-P on glutamate-induced ROS generation in HT-22 cells were further investigated. In this example, HT-22 cells were stained with DCFH-DA, and the fluorescence intensity of the cells was observed through an inverted fluorescence microscope (×20). The antioxidant stress effects of the polypeptides were statistically analyzed. All values were expressed as mean ± standard deviation (n = 3). Compared with the Glu group, *** P <0.001, ** P <0.01, * P <0.05; as Figure 11 shown, compared with the normal group, the Glu group induced a large amount of ROS production in HT-22 cells. When the cells were intervened with 5 μM polypeptides, the positive control drug Tat-GluA2-3Y and the polypeptides P3DC7LC-O and P3LC7LC-P all improved the production of ROS in nerve cells to a certain extent. This result indicates that the polypeptides P3DC7LC-O and P3LC7LC-P can reduce glutamate-induced ROS production in nerve cells to a certain extent. Example 8

[0034] Anti-Apoptotic Effect of Polypeptides The anti-apoptotic results of the modified polypeptides P3LC6LC-M, P3LC7LC-P, and P3DC7LC-O are as Figure 12 shown. The modified polypeptides reduced the activation of caspase-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 a microtubule protein in Example 9

[0035] Effect of Modified Polypeptides on Glutamate-Induced Apoptosis of HT-22 Cells Based on the aforementioned experimental results, the effect of P3LC7LC-P on glutamate-induced apoptosis of HT-22 cells was further investigated. In this example, 1 hour before Glu induction, HT-22 cells were treated with Tat-GluA2-3Y or modified polypeptides (5 μM). HT-22 cells were stained with Hoechst, and the apoptotic morphology of HT-22 cells was observed under an inverted fluorescence microscope to obtain the apoptosis rates of the Tat-GluA2-3Y and modified peptide groups, * P <0.001. Compared with the Glu group, all values were expressed as mean ± SD, n = 3 for each group. One-way ANOVA was performed, followed by Dunnett's multiple comparison; as Figure 13As shown, compared with the normal group, the Glu group induced apoptosis in HT-22 cells. Intervention with 5 μM polypeptide on the cells showed that the positive control drug Tat-GluA2-3Y and the polypeptide P3LC7LC-P both improved neuronal apoptosis to a certain extent. The results indicate that the polypeptide P3LC7LC-P has a protective effect on glutamate-induced neuronal apoptosis. Example 10

[0036] Effect of Modified Polypeptide on Infarct Area and Neurological Function Given that the modified polypeptide has certain neuroprotective ability, blood-brain barrier permeability and water solubility in vitro, this example further evaluated its in vivo neuroprotective effect through a widely used ischemic stroke model. As Figure 14 shown, MCAO injury induced obvious infarction, which was shown as the white area in brain sections. Tat-GluA2-3Y and P3LC7LC-P at a dose of 8 mg / kg significantly reduced the infarct area in mice compared with the vehicle-treated MCAO group ( P <0.001), and the infarct area decreased from 324.047 mm 2 to 216.242 mm 2 . In vitro experiments showed that P3LC7LC-P had an obvious protective effect on cerebral I / R injury and could reduce neurological deficits after cerebral I / R injury.

[0037] The materials and methods of the examples of the present invention are as follows: 1. Chemicals and Reagents All chemicals and solvents used were from commercial sources. The reaction process 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 column (5 μm, 4.6 mm × 250 mm) and Positisil OSD-P C18 semi-preparative column (5 μm, 10 mm × 250 mm) were purchased from Beijing Yinglai Technology Co., Ltd.

[0038] 2. Peptide Synthesis, Cleavage and Purification The polypeptide synthesis reagent and rink amide resin (100 - 200 mesh, 0.5 mmol / g) were purchased from Beijing Euro-United Technology Co., Ltd. Acetonitrile and dichloromethane were purchased from Guangzhou Bixiluo Technology Co., Ltd. Trans fatty acid was purchased from Aladdin Reagent (Shanghai) Co., Ltd. The peptide was synthesized by the standard solid-phase peptide synthesis method on the rink amide resin. After swelling the resin in DCM, the first C-terminal residue was coupled by mixing 3 eq of Fmoc-protected amino acid and 3 eq of diisopropylcarbodiimide in DMF at RT for 1 hour. In all subsequent coupling steps, 20% piperidine in DMF was used and stirred at RT for 30 minutes to remove the Fmoc protecting group. For peptide coupling, 3 eq of Fmoc-protected amino acid, 3 eq of HBTU / 3 eq of HATU and 6 eq of DIEA were used and mixed in DMF at RT for 1 hour. After completing the linear sequence, the peptide was acetylated at the N-terminus by mixing 8 eq of Ac 2 O and 6 eq of DIEA in DCM at RT for 1 hour.

[0039] The resin was treated with the prepared cleavage solution (9.4 mL of trans fatty acid, 0.5 g of dithiothreitol and 0.5 mL of deionized water) by stirring at RT for 3 hours to complete peptide cleavage and deprotection. The crude peptide solution was extracted with cooled methyl ether and the precipitate was separated by centrifugation. The precipitate was resuspended in cold methyl ether and centrifuged (repeated twice). The crude peptide was lyophilized, dissolved in the minimum volume of dimethyl sulfoxide, and then purified by reverse-phase high performance liquid chromatography equipped with a Positisil OSD-P C18 analytical column. The linear gradient of the chromatographic column was 10 - 90% acetonitrile dissolved in ddH 2 O containing 0.05% TFA. The fractions were collected according to the absorption peak at 280 nm wavelength, analyzed by ESI-MS, and then lyophilized.

[0040] After confirming the molecular weight by mass spectrometry, the crude peptide was dissolved in dimethyl sulfoxide and diluted at a ratio of 1:10 (v / v) in 10×H 2 O (final concentration 10% dimethyl sulfoxide). Using a Positisil OSD-P C18 semi-preparative column, the mixture was fractionated by RP-HPLC with a linear gradient of 10 - 70% acetonitrile and ddH 2 O containing 0.05% TFA to separate the linear peptide and the disulfide cyclized peptide. All peptides used in this study were determined to have a purity ≥90% by analytical HPLC.

[0041] 3. BRAG2 Expression and Purification For the Biacore experiment, a part of the human IQ motif and the sequence of protein 1 containing the SEC7 domain (Uniprot ID: Q6DN90) 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 strain BL21. The bacilli were induced to express overnight at 16 °C with 0.3 mM IPTG when the OD 600 was 0.6 - 0.8. 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 ultrasonic generator model. The lysate was clarified by centrifugation at 12,000 rpm and 4 °C for 30 min, and then loaded onto a NiSO 4 column. Using elution buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol), with a flow rate of 1 mL / min, elution was performed using a 0 - 100% gradient over 40 min. The eluate was collected, digested with TEV protease, purified by a secondary nickel column and Superdex 75, and 9.52 mg of BRAG2 protein with a purity > 95% was obtained.

[0042] 4. Surface Plasmon Resonance The SPR experiment was carried out at 25 °C using a BIAcore CM5 series S sensor chip (BIAcore T200 from GE Healthcare Life Sciences). Polyclonal anti-GST antibody (Abcam, Cambridge) was immobilized on all flow cells by amine coupling, and the capture level was between 2280 RU and 6700 RU (GE Healthcare GST Capture Kit). As a control, the reference flow cell (between 520 RU and 1280 RU) only captured GST, while the active flow cell (between 690 RU and 1990 RU) immobilized the GST-tagged protein. The lyophilized peptides (in triplicate) were resuspended in the assay buffer and injected into all four flow cells at a rate of 30 μL-min -1 for 60 - 80 s. +PO 4 3- The buffer consisted of 50 mM Na 3 PO 4 , 150 - 300 mM NaCl and 1 mM DTT (pH 7.4), while the -PO 4 3- buffer consisted of 1 mM Na 3 PO 4Composed of 150 mM NaCl, 20 mM Tris, and 1 mM DTT (pH 7.4) (reflecting physiological concentration). Peptide 3 needs to be regenerated with 2 M NaCl before injecting the next peptide cycle. Data was analyzed using Scrubber 2.0 (BioLogic Software, Campbell, ACT, Australia) and Prism 6.0 (GraphPad Software, California, USA).

[0043] 5. Cell culture and drug preparation HT-22 cells were placed in an incubator at 37 °C and 5% CO 2 and cultured in complete DMEM medium containing 10% FBS and 5% penicillin-streptomycin mixture. When the cells entered the logarithmic growth phase, they were seeded into 96-well plates at a density of 4×10 4 cells / mL and cultured in the incubator.

[0044] Glutamic acid was dissolved in DMEM medium without FBS and the pH was adjusted to 7.2 - 7.4. All the polypeptides to be tested were prepared as 10 mM stock solutions in 100% DMSO and freshly diluted to specific concentrations (0.2, 1, 5 μM) with cell medium before use. After plating for 24 hours, the medium in the original plate was discarded, and the cells were pre-incubated with the drug to be tested for 1 hour, with a system of 100 μL per well. Then, it was replaced with the glutamic acid solution. The experiments were divided into blank group, control group, model group, and compound group.

[0045] 6. Cytotoxicity assay HT-22 cells were seeded into 96-well plates at a density of 4×10 4 cells / mL, and DMEM containing 10% FBS was added. They were cultured in an incubator at 37 °C and 5% CO 2 for 24 hours. Tat-GluA2-3Y and the polypeptide with better cell activity were prepared at a concentration of 10 μM and co-cultured with the cells for 24 hours. After 24 hours, the cells were washed three times with PBS, and CCK-8 was added and incubated for 2 hours.

[0046] 7. Cell survival assay After the above treatment, the cells to be detected were taken out, 10% CCK-8 was directly added to the cell culture medium, and they were mixed well. Then, the cells were further incubated in the incubator at 37 °C for 2 hours. The absorbance (OD value) at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the results were compared with those of untreated cells for normalization analysis. The experiment was repeated three times, and the average values were taken to compare the cell viability of each group.

[0047] 8. Transwell assay The MDCK cells were obtained from the American Type Culture Collection and cultured in an environment of 90% air and 10% CO 2 2. In the transportation experiment, 2×10 5 cells were seeded on a 96-well cell culture plate (Beaver), allowed to grow and differentiate for 7 days, and then the cell monolayer was used for the transportation experiment.

[0048] 9. Transport Studies The marker was dissolved in Hank's balanced salt solution (HBSS pH 7.4) with a pH of 7.4 and containing 1 mM Hepes. The final concentration of different polypeptides was 100 μM. The detection limit of HPLC analysis, the presence of a saturable active transport mechanism, and the effect on monolayer integrity are described below. The monolayer cells were placed in HBSS solution with a pH of 7.4 and cultured in a humidified environment at 37 °C for 20 minutes, and then the transportation experiment was started. The drug solution was added to the donor side of the cell monolayer and maintained at 37 °C throughout the experiment. To detect the possible effects of active transport or efflux mechanisms, we measured the permeability of all compounds in two directions: apical to basolateral (a - b) and basolateral to apical (b - a). Under the "permeation" condition, the apparent permeability coefficient (Papp) was calculated by the following formula:

[0049] where, is the steady-state flux (mol / s), C 0 is the initial concentration in the donor chamber at each time interval (mol / mL), and A is the filter area (cm 2 ).

[0050] Analytical method: A Hich-rom Partisil ODS3 analytical column (100×4.6 mm) with an average particle size of 5 µm was used. The mobile phase consisted of water (0.1% formic acid) and acetonitrile (0.1% formic acid); the acetonitrile concentration was 2 - 60%, the flow rate was 1 mL / min, and the retention time was 1 - 4 min. The injection volume was 10 - 200 µL.

[0051] 10. Determination of Cellular ROS Formation: The HT-22 cells were seeded at 3×10 4Inoculate cells with a uniform density of holes into a 24-well plate, and set up a normal control group, a model glutamate group, and a drug administration group. After the cells grow for 24 hours, discard the culture medium in the cells. In the drug administration group, add polypeptides Tat-GluA2-3Y, P3DC7LC-O, and P3LC7LC-P with a concentration of 5 μM. Replace the culture medium of the cells in the normal control group and the model group with basal medium and continue culturing. After 1 hour, expose the cells in the drug administration group and the model group to 6 mM glutamate for culturing. After 24 hours, place 10 μM 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) in the dark environment of the cell culture incubator for staining for 30 minutes, then wash 3 times with PBS. After thoroughly washing the unbound DCFH-DA from the cells, use an inverted fluorescence microscope to observe and record the intensity of dichlorofluorescein (DCF) to estimate the production of ROS.

[0052] 11. Apoptosis detection The modeling method is the same as that for the HT22 cell ROS experiment. After the cells are treated with glutamate for 24 hours, wash the cells 3 times with PBS, and stain with Hoechst 33342 (50 μg / mL) for 8 minutes in the dark environment, then wash 3 times with PBS. Observe and record the morphological changes of the cells under an inverted fluorescence microscope.

[0053] 12. tMCAO model Prepare a middle cerebral artery occlusion (MCAO) cerebral ischemia-reperfusion model using the internal carotid artery wire embolization method. After the animals are anesthetized with isoflurane (a mixture of 2.0 - 2.5% isoflurane and oxygen), fix them in the supine position on the operating table, disinfect the skin, make a midline incision in the neck, separate the left common carotid artery, external carotid artery, and internal carotid artery, gently dissect the vagus nerve, ligate and cut the external carotid artery. Clamp the proximal end of the common carotid artery, make an incision distal to the ligation line of the external carotid artery, insert a nylon wire with an outer diameter of 0.26 ± 0.01 mm, evenly apply silicone rubber at the front end 5 - 6 mm away, insert it into the internal carotid artery through the bifurcation of the common carotid artery until there is slight resistance (about 20 mm from the bifurcation), blocking all blood supply to the middle cerebral artery. After 1.5 hours of cerebral ischemia, gently pull out the nylon wire. After 1.5 hours of cerebral ischemia, gently pull out the nylon wire, restore blood supply for reperfusion, suture the neck skin, disinfect, and then put it back into the cage for feeding.

[0054] 13. Experimental groups and drug administration The rats are randomly divided into the following four groups: Sham group (saline), vehicle group (saline), Tat-GluA2-3Y group (2, 4, and 8 mg / kg), and design group (2, 4, and 8 mg / kg). The drugs are administered by intravenous injection 2 hours after the start of reperfusion.

[0055] 14. Infarct volume analysis Animals were anesthetized with isoflurane, and the brain was removed from the head. The olfactory bulbs, cerebellum, and lower brainstem were removed. The surface of the brain was rinsed with saline to remove bloodstains, and the remaining water stains on the surface were blotted off. The brain was stored at -80°C for 7 minutes. After the animal was removed, a coronal section was immediately cut vertically downward in the plane of the optic chiasm, and a slice was cut every 2 mm backward. The brain slices were placed in freshly prepared TTC (20 g / L) staining solution and incubated at 37°C for 20 minutes. This process used the method reported in the literature. Animals were anesthetized with isoflurane, and the brain was removed from the head. The olfactory bulbs, cerebellum, and lower brainstem were removed. The surface of the brain was rinsed with saline to remove bloodstains, and the remaining water stains on the surface were blotted off. The brain was stored at -80°C for 7 minutes. After the animal was removed, a coronal section was immediately cut vertically downward in the plane of the optic chiasm, and a slice was cut every 2 mm backward. The brain slices were placed in freshly prepared TTC (20 g / L) staining solution and incubated at 37°C for 20 minutes.

[0056] 15. Statistical analysis Quantitative information was expressed as mean ± standard error. One-way analysis of variance (ANOVA) and Scheffe's test were used to determine the significance of differences between the two groups for the infarct area and neurological deficit symptom scores, and ANOVA test was used for the mortality rate. P A difference was considered significant when < 0.05.

Claims

1. A GluA2 AMPAR endocytosis blocking transmembrane cyclic peptide, characterized in that: It comprises at least one of the following cyclic peptides: a cyclic peptide having an amino acid sequence as shown in SEQ ID NO: 1-3, and a corresponding structural formula as shown in formula (I)-(III); （Ⅰ） （Ⅱ） （Ⅲ）。 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: A therapeutically effective amount of at least one acetate, hydrochloride or other pharmaceutically acceptable salt of the cyclic peptide according to claim 1.

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