GALR2 selective polypeptide agonist as well as preparation method and application thereof

Abstract

The invention relates to the technical field of polypeptides, in particular to a GALR2 selective polypeptide agonist as well as a preparation method and application thereof. The polypeptide agonist comprises one of polypeptide of an amino acid sequence in a formula I, polypeptide salt of the amino acid sequence in the formula I, polypeptide of an amino acid sequence in a formula II and polypeptide salt of the amino acid sequence in the formula II. Asn-Trp-Thr-X3-X4-Ala-Met-Leu-X1-Leu-X2-Gly-Ala-Gln-NH2, which is represented by a formula I in the specification, is represented by a formula II in the specification, y < 5 >-Trp-Thr-Leu-Asn-Y < 4 >-Al -Leu-Y < 1 >-Leu-Y < 3 >-Gly-Y < 2 >-NH2, which is shown in a formula II; the GALR2 selective polypeptide agonist molecule provided by the invention has high affinity with GALR2 and strong agonist activity, and can be applied to preparation of ischemic brain injury drugs.

Description

Technical Field

[0001] This invention relates to the field of peptide technology, specifically to a GALR2 selective peptide agonist, its preparation method, and its application. Background Technology

[0002] Ischemic stroke, a major chronic non-infectious disease posing a serious threat to public health, is the leading cause of death and disability in adults. Due to its complex pathogenesis and narrow treatment window, current clinical treatment outcomes remain unsatisfactory. Galanin receptor 2 (GALR2) is widely distributed in the mammalian central nervous system. After binding to endogenous ligands, it participates in regulating multiple biological functions, including endocrine regulation, neuroprotection, and learning and memory. Its function is closely related to the occurrence and development of various neurological diseases, including ischemic stroke. However, endogenous ligands exhibit low selectivity and unstable pharmacokinetic properties during GALR2 activation, requiring further optimization and in-depth research. Currently, no specific compounds targeting GALR2 have been found in clinical practice.

[0003] The literature H. Liu, P. Brumovsky, R. Schmidt, W. Brown, K. Payza, L. Hodzic, C. Pou, C. Godbout, & T. Hökfelt, Receptor subtype-specific pronociceptive and galaninin the spinal cord: Selective actions via GalR1 and GalR2 receptors, Proc. Natl. Acad. Sci. USA 98(17) 9960-9964, discloses the role of GALR2 specific agonists in the field of neuropathic pain. It focuses on the function of galaninin and its receptors GALR1 and GALR2 in the spinal cord, and focuses on the specific role of receptor subtypes, aiming to explore the mechanism of galaninin in pain modulation. The study only mentions the name of the GALR2 agonist AR-M1896, does not cover the treatment scenario of ischemic brain injury, lacks specific peptide molecular structure design and functional validation data related to brain injury, and cannot provide technical support for the development of GALR2 targeted drugs for ischemic brain injury. Summary of the Invention

[0004] The first aspect of the present invention provides a GALR2 selective peptide agonist, the peptide agonist comprising one of the following: a peptide with an amino acid sequence of Formula I, a peptide salt with an amino acid sequence of Formula I, a peptide with an amino acid sequence of Formula II, or a peptide salt with an amino acid sequence of Formula II. Asn-Trp-Thr-X3-X4-Ala-Met-Leu-X1-Leu-X2-Gly-Ala-Gln-NH2, formula I; Y5-Trp-Thr-Leu-Asn-Y4-Ala-Leu-Y1-Leu-Y3-Gly-Y2-NH2, formula II; X1 includes one of the following: Tyr, Tyr(3-Cl), Tyr(3-F), Tyr(3-I), Tyr(3-Br), Tyr(3,5-Cl), Tyr(3,5-F), Tyr(3,5-Br), Tyr(3,5-I), Phe(P 1 X2 includes one of the following: Lys, Tyr, Arg, Phe(4-NH2), Trp(6-Cl), Lys(Met)2, Leu(4,5-dehydro); X3 includes one of the following: Pro, Leu; X4 includes one of the following: Gln, Asn, Arg. Y1 includes one of the following: Tyr, Tyr(3-Cl), Tyr(3-F), Tyr(3-I), Tyr(3-Br), Tyr(3,5-Cl), Tyr(3,5-F), Tyr(3,5-Br), Tyr(3,5-I), Phe, Phe(P 1 Y2 includes one of the following: Pro, His; Y3 includes one of the following: Lys, Tyr; Y4 includes one of the following: Lys, Asn, Phe(P 1 ), Glu, HoSer, Ala, Ala(P 2 Y5 includes one of the following: D-Asn, L-Asn; the Phe(P 1 The phenyl ring in phenylalanine is substituted; the Ala(P) 2 This includes the substitution of the methyl group of alanine.

[0005] The Phe(P) 1 The following are included: the benzene ring in phenylalanine is replaced by a heterocyclic ring; the benzene ring in phenylalanine is replaced by a substituted hydrocarbon group; the benzene ring in phenylalanine is replaced by a substituted benzene ring; the benzene ring in phenylalanine is replaced by a biphenyl ring; or the benzene ring in phenylalanine is replaced by a substituted naphthalene ring.

[0006] The Ala(P) 2 The following are included: the methyl group of alanine is replaced by a hydrogen atom, the methyl group of alanine is replaced by a substituted hydrocarbon group, the methyl group of alanine is replaced by a substituted heterocyclic group, and the methyl group of alanine is replaced by a benzene ring.

[0007] The number of substituents in the substituted hydrocarbon group is 1-5.

[0008] Optionally, the number of substituents in the substituted hydrocarbon group is 1 to 3.

[0009] The substituted hydrocarbon group includes one of the following substituents: halogen, nitro, cyano, oxo, hydroxyl, or halogenated C1-C6 alkoxy.

[0010] Optionally, the peptide agonist includes one of PEP1-14: PEP1: Asn-Trp-Thr-Leu-Asn-Ala-Met-Leu-Tyr(3-Cl)-Leu-Lys-Gly-Ala-Gln-NH2; PEP2: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Leu-Gly-Pro-NH2; PEP3: Asn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Leu-Gly-Pro-NH2; PEP4: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Leu-Gly-His-NH2; PEP5: Asn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Leu-Gly-His-NH2; PEP6: dAsn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Leu-Gly-His-NH2; PEP7: dAsn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Leu-Gly-Pro-NH2; PEP8: Asn-Trp-Thr-Pro-Gln-Ala-Met-Leu-Tyr(3-Cl)-Leu-Lys-Gly-Ala-Gln-NH2; PEP9: Asn-Trp-Thr-Pro-Asn-Ala-Met-Leu-Tyr(3-Cl)-Leu-Lys-Gly-Ala-Gln-NH2; PEP10: Asn-Trp-Thr-Leu-Gln-Ala-Met-Leu-Tyr(3-Cl)-Leu-Lys-Gly-Ala-Gln-NH2; PEP11: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Lys-Gly-Pro-NH2; PEP12: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Tyr-Gly-Pro-NH2; PEP13: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Leu-Gly-Pro(4-OH)-NH2; PEP14: Asn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Tyr-Gly-His-NH2.

[0011] The PEP1-14 sequences are numbered SEQ ID NO: 1-14 respectively.

[0012] The purity of the polypeptide agonist is ≥98%.

[0013] The second aspect of the present invention provides a method for preparing a GALR2 selective peptide agonist, comprising the following steps: taking a peptide corresponding to Formula I or Formula II, obtaining a resin for crude peptide by solid-phase synthesis using a peptide synthesizer, cutting and processing the resin for crude peptide to obtain crude peptide, and purifying the crude peptide by reversed-phase high-performance liquid chromatography.

[0014] A third aspect of the present invention provides an application of the aforementioned GALR2 selective peptide agonist in the preparation of drugs for ischemic brain injury.

[0015] The GALR2 selective peptide agonist provided by this invention can serve as a candidate new drug for treating ischemic brain injury. The sequence of this selective peptide agonist is derived from the endogenous GALR2 ligand. This invention explored and optimized each amino acid in the peptide sequence, introducing non-standard amino acids to improve the activity and metabolic stability of the peptide ligand, while also considering the selectivity of the peptide molecule for different GALR1, GALR2, and GALR3 subtypes, ultimately obtaining the optimal GALR2 selective peptide agonist of this invention. The GALR2 selective peptide agonist molecule of this invention has high affinity for GALR2, strong agonistic activity, and can significantly improve cell viability after oxidative stress injury in HT22 cells, reduce neurological deficits after cerebral ischemia-reperfusion injury in mice, and decrease cerebral infarction volume, thus serving as a potential candidate drug for treating ischemic brain injury.

[0016] The ischemic brain injury pharmaceutical composition contains the aforementioned polypeptide agonist and a pharmaceutically acceptable carrier.

[0017] The pharmaceutical composition can be formulated into various conventional dosage forms known in the art, including but not limited to tablets (including various coated tablets, sustained-release or controlled-release tablets), lozenges, capsules (including soft capsules and hard capsules), granules, dispersible powders, aqueous or oily suspensions, emulsions, elixirs or syrups, etc., suitable for oral administration; creams, ointments, gels, aqueous or oily solutions or suspensions, etc., suitable for topical application; powders or liquid aerosols suitable for inhalation; sterile aqueous or oily intravenous, subcutaneous or intramuscular injections, suppositories suitable for parenteral administration.

[0018] Those skilled in the art will recognize that the appropriate amounts of the GALR2 selective peptide agonist molecule for ischemic brain injury and the pharmaceutically acceptable carrier, which are the active ingredients in the pharmaceutical compositions of the present invention, can be determined according to conventional methods in the art.

[0019] Beneficial effects 1. The polypeptide agonist provided by this invention has both high activity and high selectivity.

[0020] 2. The peptide agonist provided by this invention is derived from the endogenous GALR2 ligand and can target and activate GALR2. While introducing non-standard amino acids to improve the activity and metabolic stability of the peptide ligand, it also takes into account the selectivity of the peptide molecule for different isoforms of GALR1, GALR2 and GALR3.

[0021] 3. The GALR2 selective peptide agonist molecule provided by this invention has high affinity for GALR2 and strong agonistic activity.

[0022] 4. The polypeptide agonist provided by the present invention can be used to prepare a drug composition for treating ischemic brain injury, and the composition can be prepared in various dosage forms for the treatment of neuronal injury.

[0023] 5. The GALR2 selective peptide agonist provided by the present invention can significantly improve the cell viability of HT22 cells after oxidative stress injury, the neurological deficits after cerebral ischemia-reperfusion injury in mice, and reduce the volume of cerebral infarction, and can be used as a potential candidate drug for anti-ischemic brain injury. Attached Figure Description

[0024] Figure 1 Experimental results of PEP1-6 activation of GALR2 coupled with Gαq.

[0025] Figure 2 Experimental results on PEP1-6 activation of GALR2 to recruit β-arrestin2.

[0026] Figure 3 For PEP6 to GALR1 ( Figure 3 a) GALR2 ( Figure 3 b), GALR3 ( Figure 3 c) Results of selective testing experiments.

[0027] Figure 4 Experimental results on the improvement of cell viability of HT22 cells after oxidative stress injury by PEP6.

[0028] Figure 5 The results of experiments on PEP6 improving neurological deficits in model mice.

[0029] Figure 6 The results of the experiment on reducing cerebral infarction volume in PEP6 model mice are shown, where a) is a stained brain slice photograph; b) is a bar chart of cerebral infarction volume. Detailed Implementation

[0030] Example 1 A selective GALR2 peptide agonist, PEP1: Asn-Trp-Thr-Leu-Asn-Ala-Met-Leu-Tyr(3-Cl)-Leu-Lys-Gly-Ala-Gln-NH2; A method for preparing a GALR2 selective peptide agonist comprises the following steps: 1.1 Solid-phase synthesis of PEP1 Using N,N-dimethylformamide (DMF) as solvent, the concentrations of various α-amino groups protected by Fmoc were 0.25 mol / L, the concentration of piperidine solution was 20%, the concentration of diisopropylcarbodiimide (DIC) solution was 0.5 mol / L, and the concentration of 1-hydroxyphenyl-4,5-diphenylimidazole (Oxyma) solution was 1 mol / L.

[0031] Weigh 1g of Fmoc (fluorenebenzyloxycarbonyl)-protected Rink resin preloaded with Gln and place it in the reactor. Weigh the corresponding amino acid raw materials in the polypeptide chain and prepare a 0.2mol / L solution with DMF. Place the prepared 20% piperidine solution, activator DIC, and activator base Oxyma solution into their respective reagent bottles on the polypeptide solid-phase synthesizer. Following the instrument software's preset program, after the resin swells for 5 minutes, deprotection with 20% piperidine is performed, followed by sequential condensation, deprotection, and condensation of the amino acid sequence in PEP1. After synthesis is complete, wash the resin with DMF for 2-3 minutes, filter, discard the filtrate, and obtain resin containing crude polypeptide.

[0032] 1.2 Cutting of Excess Groups and Resin The dried resin was placed in a 25 mL round-bottom flask, and 15 mL of resin cutting solution (95% trifluoroacetic acid + 2.5% triisopropylsilane + 2.5% water, by weight) was slowly added dropwise under an ice-water bath. The mixture was shaken and reacted at room temperature for 2-3 hours. After the reaction was complete, the reaction solution and resin were transferred to a vacuum filtration device to remove the resin. The collected filtrate was placed in a round-bottom flask, allowed to settle in ice-cold ether, and then heated at 4°C and 7000× g Centrifuge at 15 min, discard the supernatant, repeat the operation 3 times, and finally dry in a vacuum drying oven at 50℃ for 48 h to obtain approximately 0.03 g of crude peptide.

[0033] 1.3 Purification and Identification of PEP1 The peptide was purified using reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 preparative column was used as the stationary phase, and the mobile phase consisted of an aqueous phase and an organic phase. The aqueous phase was deionized water (containing 0.1% trifluoroacetic acid by mass); the organic phase was an aqueous solution containing 80% acetonitrile (containing 0.1% trifluoroacetic acid by mass). The flow rate was 20.00 mL / min; the elution gradient was: aqueous phase (90%→10%), organic phase (10%→90%). The eluent at the highest peak in the HPLC chromatogram was collected, and acetonitrile was removed from the eluent using a rotary evaporator at 50 °C. The remaining aqueous solution was rapidly frozen and then refrigerated to remove moisture, yielding a white, fluffy powder. HPLC-MS (High-Performance Liquid Chromatography-Mass Spectrometry) verified that the solid purity was greater than 98%, ESI: 829.1 [M+2H]. 2+ .

[0034] Example 2: PEP2 was prepared using the same method as in Example 1: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Leu-Gly-Pro-NH2, with a solid purity greater than 98% and ESI: 748.6 [M+2H]. 2+ .

[0035] Example 3: PEP3 was prepared using the same method as in Example 1: Asn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Leu-Gly-Pro-NH2, with a solid purity greater than 98% and ESI: 778.5 [M+2H]. 2+ .

[0036] Example 4: PEP4 was prepared using the same method as in Example 1: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Leu-Gly-His-NH2, with a solid purity greater than 98% and ESI: 768.1 [M+2H]. 2+ .

[0037] Example 5: PEP5 was prepared using the same method as in Example 1: Asn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Leu-Gly-His-NH2, with a solid purity greater than 98% and ESI: 798.7 [M+2H]. 2+ .

[0038] Example 6: PEP6 was prepared using the same method as in Example 1: dAsn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Leu-Gly-His-NH2, with a solid purity greater than 98% and ESI: 798.2 [M+2H]. 2+ .

[0039] Performance testing methods and data 1. Evaluation of the in vitro activity of PEP1-6 in conjugating Gαq to GALR2 and recruiting β-arrestin2 using the BRET-based method.

[0040] (1) Plasmid construction: The wild-type GALR2 plasmid was purchased from the SnapGene gene library. This plasmid contains the codon-optimized full-length GALR2 gene. Primers were designed and the target fragment was amplified by PCR. The full-length GALR2 gene was inserted between the BamHI and EcoRI restriction sites of the pcDNA3.1 vector. The HiBiT tag, Igl signal peptide tag and Kozak start sequence were connected to the N-terminus of the GALR2 gene by a flexible linker. For the plasmid used in the β-arrestin2 recruitment experiment, the RLuc8 luciferase gene plasmid was connected to the full-length GALR2 gene by a flexible linker. After the plasmid was constructed, it was used after first-generation sequencing.

[0041] (2) HEK293T cells were injected at a concentration of 1.2 × 10⁻⁶. 6The cells were seeded at a density of 10 cells per 6 cm cell culture dish, and 5 ml of DMEM medium was added. The cells were then incubated at 37°C for 24 h.

[0042] (3) Replace with fresh complete culture medium 2 hours before transfection. Prepare transfection solution by adding 250 µL of Opti-MEM containing the following components to a 6 cm culture dish. The components of the transfection system for G protein dissociation detection are shown in Table 1, and the components of the transfection system for β-arrestin2 recruitment detection are shown in Table 2.

[0043] Table 1

[0044] Table 2

[0045] (4) 24 h after transfection, discard the original culture medium, digest the cells with trypsin, add 500 μL of digestion solution to each dish, incubate at 37°C for 2 min, transfer the cells to centrifuge tubes, and centrifuge at 200 g for 5 min. Discard the supernatant, resuspend in DMEM medium, mix well by pipetting, and count the cells. Spread the cell suspension at 3 × 10⁻⁶ cells / mL. 4 100 µL of cells were seeded into 96-well pure white cell culture plates. After mixing, the plates were incubated at 37°C.

[0046] (5) After culturing for 24 hours, the culture medium in the 96-well plate was aspirated, and 80 µL of BRET assay buffer was added to each well for equilibration. Then, 20 µL of 5× analyte (diluted with assay buffer) was added, and the plate was incubated for 3-5 minutes. Finally, 20 µL of coelenterate 400a working solution (working concentration 5 µM) was added, and the fluorescence value was measured using the chemiluminescence module of the microplate reader. The microplate reader was set to switch filters for each well to read fluorescence emission values ​​at two wavelengths. The 410 nm wavelength filter was used to detect the fluorescence emission value of Rluc8, and the 515 nm wavelength filter was used to detect the fluorescence emission value of GFP2. The fluorescence value at 515 nm was divided by the fluorescence value at 410 nm to calculate the BRET ratio. The background BRET ratio of the model group was subtracted to obtain the net BRET value (NetBRET). The netBRET value was used to fit the dose-response curve. The experimental results are as follows: Figure 1 , 2 As shown.

[0047] 2. The selectivity of PEP6 for GALR1, GALR2, and GALR3 was detected using a BRET-based method (the procedure was the same as in test 1). The experimental results are as follows: Figure 3 As shown.

[0048] 3. The effect of PEP6 on the cell viability of HT22 cells after oxidative stress injury was detected using the CCK8 assay. The experimental results are as follows: Figure 4 As shown.

[0049] (1) Select HT22 cells with good growth status and confluence of about 90%, collect the cells after trypsin digestion, centrifuge and resuspend them to make a single-cell suspension, count the cells and then divide them into 1.5×10⁻⁶ cells. 4 / well / 100 μL was seeded into clear 96-well cell culture plates and cultured in a CO2 incubator for 24 h as usual.

[0050] (2) Discard the original culture medium in each well, rinse twice with PBS, add fresh DMEM complete culture medium containing 100 μM H2O2 to the model group cells, add DMEM complete culture medium to the normal group cells, and incubate in a CO2 incubator for 2 hours.

[0051] (3) After 1.5 hours of modeling, the original culture medium was discarded. Normal group and model group were added to normal DMEM complete culture medium, and drug intervention group was added to DMEM complete culture medium containing compounds of different concentration gradients. They were cultured in a CO2 incubator for 24 hours.

[0052] (4) Remove the original culture medium and add 100 μL CCK8 working solution (DMEM complete culture medium: CCK8 reagent = 9:1) to each well, and incubate in a CO2 incubator in the dark for 30-60 min; (5) Absorbance value detection: Select the absorption light reading module in the multi-functional microplate reader, set the parameters as follows: temperature 37℃, wavelength 450nm, double-rail horizontal vibration for 10s before reading the plate, and measure the absorbance value of each well.

[0053] (6) Calculate cell viability = [(As-Ab) / (Ac-Ab)] × 100% As: Absorbance of experimental wells (including cells, culture medium, CCK-8 solution, and drug treatment groups); Ac: Absorbance of control wells (including cells, culture medium, and CCK-8 solution, but excluding drug-treated groups); Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drugs).

[0054] 4. Evaluate the effect of PEP6 on neurological deficits in the model group mice. The experimental results are as follows: Figure 5 As shown.

[0055] (1) Animal pre-treatment: Healthy male ICR mice weighing 26-30g were selected as experimental subjects and randomly divided into sham-operated group, model group, and model group + PEP6 (0.1, 1, 10 mg / kg) dosage group, with 6-10 mice in each group. After 3 days of acclimatization, the mice in the drug test group were treated with different concentrations of PEP6 (0.1, 1, 10 mg / kg, tail vein injection), while the mice in the sham-operated group and model group were given the same volume of physiological saline. The drugs were administered once a day for 7 consecutive days.

[0056] (2) Mouse model preparation: The mouse model was established 2 hours after the end of drug administration on day 7. Mice were anesthetized with 2.5% isoflurane and then maintained with 0.5% isoflurane. The mice were fixed on the operating table in a supine position. The neck was shaved and disinfected with povidone-iodine. The skin was cut along the midline of the neck and the tissues were separated to separate the common carotid artery, external carotid artery and internal carotid artery. The proximal end of the common carotid artery and the distal end of the internal carotid artery were clamped with arterial clamps, and a suture was placed below the external carotid artery for later use. A "V" shaped incision was made on the external carotid artery, and the suture plug was held with ophthalmic forceps and slowly pushed into the external carotid artery and common carotid artery. Then the internal carotid artery clamp was released, and the suture plug was slowly inserted into the internal carotid artery. When the suture plug entered the middle cerebral artery through the internal carotid artery and could not continue to advance, the insertion was stopped. Finally, the suture plug was fixed with the suture below the external carotid artery and the arterial clamp of the common carotid artery was removed. Blood clots were cleaned with medical cotton swabs, the skin was sutured, and the area was disinfected with povidone-iodine. The model mice were placed on a 37°C constant temperature blanket to wait for awakening. 1.5 hours later, the outer end of the suture plug was grasped with tweezers and pulled out. The mice in the sham-operated group did not have the suture plug inserted, but the rest of the procedure was the same.

[0057] (3) The Bederson score was used to score the neurological deficit of the model group mice. The scoring criteria were as follows: 0 points: The mice were normal with no neurological deficit. The mice extended their forelimbs symmetrically forward without any abnormalities. 1 point: Mild neurological deficit. When the mice were lifted, the contralateral forelimb could not be fully extended. The forelimbs were adducted and flexed, and the extension range was significantly smaller than that of the normal side, but there was no twisting of the body to one side. 2 points: Moderate neurological deficit. When the mice were lifted, not only could the contralateral forelimb not be fully extended, but the body also twisted to the opposite side. The neurological deficit was more obvious. 3 points: Severe neurological deficit. The mice fell to the side of injury when walking. 4 points: The mice lost their ability to move independently and were in a coma. They had a weak or no response to external stimuli.

[0058] 5. Evaluate the effect of PEP6 on the infarct volume of the model group mice. The experimental results are as follows: Figure 6 As shown.

[0059] (1) The mice were pre-treated and modeled as in Example 10 (1) (2).

[0060] (2) TTC staining method for detecting ischemic cerebral infarction volume in mice: 24 h after modeling, mice were anesthetized with isoflurane and decapitated to remove the brain. After removing the cerebellum and brainstem, the brain was neatly placed in a brain groove and frozen at -80℃ for 10-15 min. The brain slices were then cut into 2 mm thick slices and completely immersed in TTC staining solution. The slices were stained at 37℃ in the dark for 20-30 min. The brain slices were rotated to ensure uniform staining on both sides by observing the staining depth. After staining, the TTC staining solution was recovered, and the brain slices were fixed with 4% paraformaldehyde for 24 h. The stained brain slices were then photographed, and the cerebral infarction volume was statistically analyzed using ImageJ software. Cerebral infarction volume (%) = [(contralateral brain tissue volume - ipsilateral brain tissue volume + ipsilateral brain tissue infarction volume) / contralateral brain tissue volume] × 100%.

[0061] like Figure 1 As shown, PEP1-6 can activate GALR2-coupled Gαq signaling in a dose-dependent manner. As the peptide concentration (the horizontal axis represents the logarithmic concentration of the drug, covering a concentration range from nanomolar to micromolar) increases, the NetBRET value shows a decreasing trend. Moreover, each peptide can reach obvious signal saturation within the effective concentration range, confirming that PEP1-6 all have in vitro activity in activating the GALR2-coupled Gαq pathway, laying the foundation for subsequent screening of advantageous peptides.

[0062] like Figure 2 As shown, PEP1-6 all dose-dependently promoted the recruitment of β-arrestin2 by GALR2: as peptide concentration (the horizontal axis represents the logarithmic concentration of the drug) increased, the NetBRET value increased significantly and tended to saturate, and the recruitment activity of each peptide was related to... Figure 1 The consistent trend of Gαq-coupled activity indicates that PEP1-6 can not only activate the G protein-dependent signaling pathway of GALR2, but also trigger the non-G protein-dependent signaling pathway mediated by β-arrestin2, which is consistent with the typical activation characteristics of G protein-coupled receptors (GPCRs), further verifying the integrity of its GALR2 agonistic activity.

[0063] like Figure 3 As shown, PEP6 exhibits significant dose-dependent activation only in GALR2: with increasing PEP6 concentration, the NetBRET value in the GALR2 group decreases significantly and reaches signal saturation; while for the GALR1 and GALR3 groups, even under high concentrations of PEP6, the NetBRET value remains close to background levels, with no significant activation effect. This result directly confirms that PEP6 has high GALR2 subtype selectivity, avoiding off-target effects caused by acting on GALR1 and GALR3, which is a key characteristic for its potential as a targeted therapeutic drug candidate.

[0064] like Figure 4As shown, the cell viability in the model group was significantly lower than that in the normal group (demonstrating successful modeling of oxidative stress injury); while the cell viability in the PEP6 intervention group showed a significant dose-dependent increase with increasing peptide concentration: the viability was slightly improved at low concentrations (0.0032 μM, 0.016 μM), and significantly increased at medium and high concentrations (0.08 μM, 0.4 μM, 10 μM). Moreover, the cell viability in the 10 μM PEP6 group was close to that of the normal group, confirming that PEP6 can effectively improve oxidative stress injury in HT22 cells, providing cellular-level experimental evidence for its neuroprotective effect.

[0065] like Figure 5 As shown, the Bederson score in the model group was significantly higher than that in the sham-operated group (reflecting neurological function damage caused by ischemia in mice); while the scores of each PEP6 dose group decreased in a dose-dependent manner with increasing dose: the score in the 0.1 mg / kg group decreased slightly, and the scores in the 1 mg / kg and 10 mg / kg groups decreased significantly, with the 10 mg / kg group showing the most significant effect. This indicates that PEP6 can effectively improve neurological function deficits after cerebral ischemia-reperfusion in animals, verifying its neuroprotective activity in vivo.

[0066] like Figure 6 As shown, the proportion of cerebral infarction volume in the model group mice was significantly higher than that in the sham-operated group (indicating successful establishment of the cerebral ischemia-reperfusion injury model); while the proportion of cerebral infarction volume in mice in each PEP6 dose group showed a significant dose-dependent decrease with increasing dose: the cerebral infarction volume in the 0.1 mg / kg group was slightly reduced, while the cerebral infarction volume in the 1 mg / kg and 10 mg / kg groups was significantly reduced, with the 10 mg / kg group showing the smallest proportion of cerebral infarction volume. This directly confirms that PEP6 can effectively reduce the cerebral infarction range after cerebral ischemia-reperfusion injury in mice in vivo, demonstrating its effectiveness in treating ischemic brain injury.

Claims

1. A selective GALR2 peptide agonist, characterized in that, The polypeptide agonist includes one of the following: a polypeptide with an amino acid sequence of Formula I, a polypeptide salt with an amino acid sequence of Formula I, a polypeptide with an amino acid sequence of Formula II, or a polypeptide salt with an amino acid sequence of Formula II. Asn-Trp-Thr-X3-X4-Ala-Met-Leu-X1-Leu-X2-Gly-Ala-Gln-NH2, formula I; Y5-Trp-Thr-Leu-Asn-Y4-Ala-Leu-Y1-Leu-Y3-Gly-Y2-NH2, formula II; X1 includes one of the following: Tyr, Tyr(3-Cl), Tyr(3-F), Tyr(3-I), Tyr(3-Br), Tyr(3,5-Cl), Tyr(3,5-F), Tyr(3,5-Br), Tyr(3,5-I), Phe(P 1 X2 includes one of the following: Lys, Tyr, Arg, Phe(4-NH2), Trp(6-Cl), Lys(Met)2, Leu(4,5-dehydro); X3 includes one of the following: Pro, Leu; X4 includes one of the following: Gln, Asn, Arg. Y1 includes one of the following: Tyr, Tyr(3-Cl), Tyr(3-F), Tyr(3-I), Tyr(3-Br), Tyr(3,5-Cl), Tyr(3,5-F), Tyr(3,5-Br), Tyr(3,5-I), Phe, Phe(P 1 Y2 includes one of the following: Pro, His; Y3 includes one of the following: Lys, Tyr; Y4 includes one of the following: Lys, Asn, Phe(P 1 ), Glu, HoSer, Ala, Ala(P 2 Y5 includes one of the following: D-Asn, L-Asn; the Phe(P 1 The phenyl ring in phenylalanine is substituted; the Ala(P) 2 This includes the substitution of the methyl group of alanine.

2. The GALR2 selective peptide agonist according to claim 1, characterized in that, The Phe(P) 1 The following are included: the benzene ring in phenylalanine is replaced by a heterocyclic ring; the benzene ring in phenylalanine is replaced by a substituted hydrocarbon group; the benzene ring in phenylalanine is replaced by a substituted benzene ring; the benzene ring in phenylalanine is replaced by a biphenyl ring; or the benzene ring in phenylalanine is replaced by a substituted naphthalene ring.

3. The GALR2 selective peptide agonist according to claim 2, characterized in that, The Ala(P) 2 The following are included: the methyl group of alanine is replaced by a hydrogen atom, the methyl group of alanine is replaced by a substituted hydrocarbon group, the methyl group of alanine is replaced by a substituted heterocyclic group, and the methyl group of alanine is replaced by a benzene ring.

4. The GALR2 selective peptide agonist according to claim 2 or 3, characterized in that, The number of substituents in the substituted hydrocarbon group is 1-5.

5. The GALR2 selective peptide agonist according to claim 4, characterized in that, The number of substituents in the substituted hydrocarbon group is 1-3.

6. The GALR2 selective peptide agonist according to claim 5, characterized in that, The substituted hydrocarbon group includes one of the following substituents: halogen, nitro, cyano, oxo, hydroxyl, or halogenated C1-C6 alkoxy.

7. The GALR2 selective peptide agonist according to claim 1 or 6, characterized in that, The peptide agonist includes one of PEP1-14: PEP1: Asn-Trp-Thr-Leu-Asn-Ala-Met-Leu-Tyr(3-Cl)-Leu-Lys-Gly-Ala-Gln-NH2; PEP2: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Leu-Gly-Pro-NH2; PEP3: Asn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Leu-Gly-Pro-NH2; PEP4: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Leu-Gly-His-NH2; PEP5: Asn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Leu-Gly-His-NH2; PEP6: dAsn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Leu-Gly-His-NH2; PEP7: dAsn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Leu-Gly-Pro-NH2; PEP8: Asn-Trp-Thr-Pro-Gln-Ala-Met-Leu-Tyr(3-Cl)-Leu-Lys-Gly-Ala-Gln-NH2; PEP9: Asn-Trp-Thr-Pro-Asn-Ala-Met-Leu-Tyr(3-Cl)-Leu-Lys-Gly-Ala-Gln-NH2; PEP10: Asn-Trp-Thr-Leu-Gln-Ala-Met-Leu-Tyr(3-Cl)-Leu-Lys-Gly-Ala-Gln-NH2; PEP11: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Lys-Gly-Pro-NH2; PEP12: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Tyr-Gly-Pro-NH2; PEP13: Asn-Trp-Thr-Leu-Asn-Ser-Ala-Leu-Tyr(3-Cl)-Leu-Leu-Gly-Pro(4-OH)-NH2; PEP14: Asn-Trp-Thr-Leu-Asn-Ser-Met-Leu-Tyr(3-Cl)-Leu-Tyr-Gly-His-NH2.

8. The GALR2 selective peptide agonist according to claim 7, characterized in that, The purity of the polypeptide agonist is ≥98%.

9. A method for preparing a GALR2 selective peptide agonist according to any one of claims 1-8, characterized in that, Includes the following steps: Take the polypeptide corresponding to Formula I or Formula II, and use a polypeptide synthesizer to obtain the resin of crude polypeptide by solid phase synthesis. After cutting and processing the resin of crude polypeptide, crude peptide is obtained, and the crude peptide is purified by reversed-phase high-performance liquid chromatography.

10. The application of a GALR2 selective peptide agonist according to any one of claims 1-8, characterized in that, Applications in the preparation of drug compositions for ischemic brain injury.

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