Polypeptide and application thereof in preparation of GluN2B membrane positioning regulator

Abstract

The invention discloses a polypeptide and application thereof in preparation of a GluN2B membrane positioning regulator, and belongs to the field of medicines. The amino acid sequence of the polypeptide disclosed by the invention is as shown in SEQ ID No. 1. Experiments prove that the polypeptide can regulate and control membrane localization of GluN2B in a lateral habenular nucleus LHb brain region, so that the function of the polypeptide is influenced. Through precise regulation and control of GluN2B membrane positioning, the effect of anti-depression treatment is expected to be improved, and a new treatment choice is provided for depression patients.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a polypeptide and its application in the preparation of a GluN2B membrane localization regulator. Background Art

[0002] Although there are currently various antidepressant drugs available, many patients do not respond well to existing treatments, and some drugs have significant side effects. Therefore, the development of new antidepressant therapies is one of the hotspots in current psychiatric research.

[0003] Numerous studies have shown that the overactivation of the lateral habenula (LHb) is considered an important basis for depressive symptoms, especially symptoms such as low mood and loss of interest. Some research reports have pointed out that the bursting discharge of glutamatergic neurons in the LHb is closely related to depressive episodes. Ketamine exerts a rapid antidepressant effect by inhibiting the N-methyl-D-aspartate receptor (NMDAR) in the LHb region and blocking the bursting discharge of glutamatergic neurons.

[0004] NMDAR has important physiological functions in the brain. However, excessive blockade of NMDAR may cause severe neurotoxicity. Therefore, the development of direct and safe NMDAR antagonists faces great challenges. As a non-competitive NMDAR inhibitor, long-term use or high-dose application of ketamine may lead to side effects such as impaired cognitive function, dissociative symptoms, ataxia, and addiction. Research has shown that ketamine has a strong selectivity for the GluN2B subunit of NMDAR. Ketamine binds to GluN2B, inhibits its ion channel activity, reduces the influx of calcium ions, and thus regulates neurotransmission and synaptic plasticity. In neurobiological research, glutamate receptors play an important role in the pathogenesis of depression, especially the GluN2B subunit of the NMDA receptor. However, the application of GluN2B in antidepressant treatment has not been reported so far. Summary of the Invention

[0005] The object of the present invention is to provide a polypeptide and its application in the preparation of a GluN2B membrane localization regulator to solve the problems existing in the above-mentioned prior art. This polypeptide can regulate the membrane localization of GluN2B in the LHb brain region. Through the precise regulation of GluN2B membrane localization, it is expected to improve the effect of antidepressant treatment and provide new treatment options for patients with depression.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a polypeptide for regulating GluN2B membrane localization, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1.

[0008] The present invention also provides a pharmaceutical composition comprising the polypeptide.

[0009] The present invention also provides the use of the polypeptide or the pharmaceutical composition in the preparation of a GluN2B membrane localization regulator.

[0010] The present invention also provides a GluN2B membrane localization regulator, and the GluN2B membrane localization regulator uses the polypeptide as an active ingredient.

[0011] Optionally, the GluN2B membrane localization regulator further comprises a pharmaceutically acceptable excipient.

[0012] The present invention also provides the use of the GluN2B membrane localization regulator in the preparation of antidepressant drugs.

[0013] The present invention discloses the following technical effects:

[0014] The present invention provides a new polypeptide, the amino acid sequence of which is as shown in SEQ ID No.1 or has at least 90% identity therewith. Through experimental verification, the polypeptide provided by the present invention can solve the disadvantages of the prior art in terms of being unable to safely and rapidly treat depression. By specifically reducing the membrane localization of NMDAR in the LHb region and reducing the glutamatergic neuronal bursting discharge, the antidepressant effect is exerted, and the defects brought about by the non-selective blockade of NMDAR in the whole brain by ketamine are skillfully avoided, and side effects such as addiction and induction of mental symptoms can be avoided. Therefore, it shows that the polypeptide provided by the present invention can be used in the preparation of a regulator for reducing GluN2B membrane localization, so as to provide a new drug and treatment idea for treating depression by reducing GluN2B membrane localization. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings based on these drawings without creative efforts.

[0016] Figure 1This is a diagram showing the effect of the polypeptide on the membrane localization of GluN2B in the LHb region in Example 3 of the present invention; A: Western blotting detection results, 1-3 correspond to Control+TAT-Control, CUMS+ TAT-Control, and CUMS+Tat-GluN2B-CaMKII respectively; B: Statistical chart of the data in Figure A; Control+TAT-Control indicates that mice at normal physiological levels were treated with the random control peptide TAT-Control, CUMS+ TAT-Control indicates that CUMS model mice were treated with the random control peptide, and CUMS+Tat-GluN2B-CaMKII indicates that CUMS model mice were treated with the polypeptide of the present invention;

[0017] Figure 2 This is a diagram showing the results of injecting the polypeptide into the LHb in the physiological-level tail suspension experiment in Example 4 of the present invention;

[0018] Figure 3 This is a diagram showing the results of injecting the polypeptide into the LHb in the physiological-level forced swimming experiment in Example 4 of the present invention;

[0019] Figure 4 This is a diagram showing the results of injecting the polypeptide into the LHb in the tail suspension experiment at the depression model level in Example 5 of the present invention;

[0020] Figure 5 This is a diagram showing the results of injecting the polypeptide into the LHb in the forced swimming experiment at the depression model level in Example 5 of the present invention;

[0021] Figure 6 This is a diagram showing the results of injecting the polypeptide into the LHb in the sucrose preference rate detection experiment at the depression model level in Example 5 of the present invention. Detailed implementation manners

[0022] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0025] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0026] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0027] Through whole-brain screening, the results of this invention show that reducing the membrane localization of the GluN2B subunit in the LHb region can effectively play a safe and rapid antidepressant effect. This invention provides a polypeptide (as shown in SEQ ID NO.1) that can overcome the disadvantages of the prior art in being unable to treat depression safely and rapidly. This polypeptide specifically blocks the NMDAR channels in the LHb region, reduces the bursting discharge of glutamatergic neurons, and thus achieves antidepressant efficacy. Different from the side effects of ketamine's non-selective blockade of NMDAR in the whole brain, the polypeptide of this invention can avoid adverse reactions such as addiction and induction of mental symptoms.

[0028] The specific synthesis and antidepressant function of the above polypeptide are further illustrated by specific examples below.

[0029] Example 1 Polypeptide

[0030] This example provides a polypeptide, whose amino acid sequence (SEQ ID NO.1) is: YGRKKRRQRRRKAQKKNRNKLRRQHSYDTFVDL.

[0031] Example 2 Synthesis of Polypeptide

[0032] The polypeptide sequence shown in SEQ ID NO.1 is synthesized by solid-phase synthesis. The synthesis starts from the first amino acid at the C-terminus and gradually lengthens the polypeptide chain until the target polypeptide is obtained. The synthesis process includes steps such as step-by-step connection of amino acids, deprotection, washing, and cleavage, and all reactions are carried out on a solid-phase support. The specific steps are as follows:

[0033] 1. Synthesis steps

[0034] (1)Resin pretreatment and first amino acid linkage

[0035] Take FMOC-Lys(Boc)-Wang resin (resin for polypeptide synthesis), swell the resin with dichloromethane (DCM) for 30 minutes, and drain. Remove the FMOC protecting group on the resin with 20% piperidine solution, and react for 20 minutes. Wash the resin with N,N-dimethylformamide (DMF) 6 times, and drain. Add Fmoc-Ala-OH (protected amino acid), react with TBTU, HOBT, and morpholine, use DMF as the solvent, and react for 40 minutes. After the reaction, wash the resin with DMF 6 times, and drain. Use 20% piperidine to remove the FMOC protecting group, and react for 20 minutes. Wash the resin with DMF 6 times, and drain to prepare for the next reaction.

[0036] (2)Repeat the above steps until the synthesis of all amino acids is completed

[0037] Add other amino acids step by step according to the above steps, and carry out condensation reaction and deprotection steps in turn until the synthesis of all amino acids is completed. After each reaction, wash with DMF to remove impurities in the reaction.

[0038] (3)Final removal of FMOC and cleavage of peptide chain

[0039] After the synthesis of the last amino acid is completed, remove the FMOC protecting group on the resin with 20% piperidine, and react for 20 minutes. Wash the resin with DMF 5-6 times, and drain. Use a cleavage solution (such as TFA / THF solution) to cleave the crude peptide from the resin. Filter and wash the obtained crude peptide to prepare for purification.

[0040] 2. Purification method

[0041] (1)Dissolution and purification of crude peptide

[0042] Dissolve the obtained crude peptide with an appropriate amount of acetonitrile and water, and ultrasonically treat until completely dissolved. Use a liquid-phase preparation system for purification, set the gradient according to the retention time in the analysis result, and separate and purify the target polypeptide. Collect appropriate vial samples and analyze to ensure that the purity of the target polypeptide meets the requirements.

[0043] (2)Lyophilization treatment

[0044] After the purified polypeptide sample is concentrated by rotary evaporation, perform lyophilization treatment with a lyophilizer for 48 hours. Take out the lyophilized polypeptide and weigh its final mass.

[0045] Example 3 Effect of polypeptide on GluN2B membrane localization in LHb region

[0046] The preparation method of the chronic unpredictable mild stress model (CUMS) is as follows: Mice are continuously subjected to different stresses such as restraint fixation, forced swimming, humid environment, circadian rhythm reversal, water deprivation, and food deprivation for 42 days. After 42 days of modeling, depressive behavior manifestations such as decreased sucrose preference rate, prolonged immobility time in the tail suspension test, and prolonged immobility time in the forced swimming test are shown.

[0047] Bilaterally stereotaxically inject the polypeptide of Example 1 into the LHb region of mice, that is, the polypeptide TAT shown in SEQ ID No.1 (50 nmol / L, 1 μL, CUMS+Tat-GluN2B-CaMKII). Randomly control the polypeptide TAT-Control (CUMS+TAT-Control, which is obtained by randomly scrambling the order of the polypeptide TAT shown in SEQ ID No.1). Take the LHb region of mice and prepare samples to detect the membrane localization of GluN2B. At the same time, use normal physiological level mice that have not constructed the CUMS model and treat them with the randomly controlled polypeptide as a blank control (Control+TAT-Control). The results are as Figure 1 shown, and the results show that the polypeptide can reduce the membrane localization of GluN2B in the LHb region.

[0048] Example 4 Antidepressant-like effect of the polypeptide at physiological level

[0049] Inject the polypeptide provided in Example 1 (50 nmol / L, 1 μL, Tat-GluN2B-CaMKII) into the LHb region of mice at normal physiological level, and set up 1 control example, that is, use the random polypeptide TAT-Control to be treated in the same way (TAT-Control). After 24 hours, conduct the tail suspension test, forced swimming test, and detection of the sucrose preference rate of mice.

[0050] Tail suspension test (TST): Fix a plastic clip at the center of the top plate of the tail suspension box (25×25×25 cm). During the test, clamp the tip of the mouse's tail about 1 cm with the clip and hang the mouse upside down so that the head is 4-5 cm away from the bottom of the box. Observe for 6 minutes, and use an electronic stopwatch to record the cumulative immobility time within the last 4 minutes.

[0051] Forced swimming test (FST): Put the mouse into a cylindrical glass cylinder with a height of 20 cm and a diameter of 10 cm. One mouse is placed in each cylinder, and the water depth in the cylinder is 10 cm, and the water temperature is 25°C. Observe for 6 minutes, and use an electronic stopwatch to record the cumulative immobility time within the last 4 minutes, that is, the duration when the mouse stops struggling in the water or floats, and only has small limb movements to keep the head floating on the water surface.

[0052] From Figure 2 the tail suspension test shown and Figure 3As can be seen from the forced swimming experiment shown, injecting the polypeptide of Example 1 into the LHb has an obvious antidepressant effect at the physiological level.

[0053] Example 5 The polypeptide exerts an antidepressant effect at the level of the depression model

[0054] After 6 weeks of establishing the chronic mild unpredictable stress model (CUMS, the same as Example 3) in mice, inject the polypeptide of Example 1 (50 nmol / L, 1 μL, CUMS+Tat-GluN2B-CaMKII) into the LHb region of the mice, and set up 2 control groups, namely, the CUMS model is treated with the random control peptide TAT-Control (CUMS+TAT-Control), and the normal physiological level mice are treated with the random control peptide (Control+TAT-Control). After 24 hours, perform the tail suspension test, forced swimming test, and detection of the sucrose preference rate of the mice.

[0055] The tail suspension test and forced swimming test are the same as in Example 4.

[0056] Detection of the sucrose preference rate of mice (SPT): The double-bottle method is used, that is, one bottle of sucrose solution and one bottle of normal drinking water are supplied to the mice for drinking at the same time. Before the test, the mice are housed individually in cages. The mice are adapted to drink 1% sucrose solution for 3 days, and are deprived of water and food at 10:00 pm the day before the test. At 10:00 pm the next day, sucrose solution and non-sucrose solution are given. The weights of the water bottles are weighed before giving them to the mice. At 8:00 am on the third day, the sucrose solution and non-sucrose solution are collected and the weights of the water bottles are weighed. The sucrose and non-sucrose intakes of the mice are obtained by the difference in the weights of the water bottles twice. The absolute value of the sucrose solution intake is the sucrose intake. Sucrose intake / (sucrose intake + plain water intake) = sucrose preference rate. After the test, the mice are put back into the original cages for breeding.

[0057] From Figure 4 the tail suspension test shown, Figure 5 the forced swimming test shown, and Figure 6 the results of the detection time of the sucrose preference rate of the mice shown, it can be seen that injecting the polypeptide of Example 1 into the LHb has an obvious antidepressant effect at the level of the depression model.

[0058] As can be seen from the above examples, the antidepressant effect of the present invention is achieved by reducing the membrane localization of the GluN2B subunit in the LHb region, specifically blocking the NMDAR channel in the LHb region, reducing the bursting discharge of glutamatergic neurons, and rapidly exerting an antidepressant effect. In the mouse forced swimming model, mouse tail suspension model, and sucrose preference rate test, a significant and rapid antidepressant effect is shown.

[0059] The polypeptide provided by the present invention can specifically reduce the membrane expression of GluN2B in the LHb brain region, thereby reducing the permeability of the cell membrane in the LHb region to Ca 2+ and reducing Ca2+ A large amount of inward current reduces the bursting discharges generated by the excessive excitation of the LHb, exerting an antidepressant effect.

[0060] Therefore, the polypeptide provided by the present invention can reduce GluN2B membrane localization and the bursting discharges generated by the excessive excitation of the LHb, without relying on the non-selective blockade of the whole-brain NMDAR mechanism of ketamine, skillfully avoiding the defects brought about by the non-selective blockade of NMDAR in the whole brain by ketamine, and can avoid side effects such as addiction and induction of mental symptoms.

[0061] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A polypeptide for regulating GluN2B membrane localization, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A pharmaceutical composition, characterized in that, Comprising the polypeptide according to claim 1.

3. Use of the polypeptide according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a GluN2B membrane localization regulator.

4. A GluN2B membrane localization regulator, characterized in that, The GluN2B membrane localization regulator uses the polypeptide according to claim 1 as an active ingredient.

5. The GluN2B membrane localization regulator according to claim 4, wherein The GluN2B membrane localization regulator further comprises a pharmaceutically acceptable excipient.

6. Use of the GluN2B membrane localization regulator according to claim 4 or 5 in the preparation of an antidepressant drug.

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