A polypeptide and its application in preparing GluN2B membrane localization regulator

Abstract

The present invention discloses a polypeptide and its use in preparing a GluN2B membrane localization regulator, belonging to the field of medicine. The polypeptide disclosed herein has an amino acid sequence as shown in SEQ ID No. 1. Experimental verification has shown that the polypeptide can regulate the membrane localization of GluN2B in the lateral habenula (LHb) brain region, thereby affecting its function. By precisely regulating the membrane localization of GluN2B, the present invention is expected to improve the efficacy of antidepressant treatment and provide a new treatment option for patients with depression.

Description

Technical Field

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

[0002] Although a variety of antidepressant drugs are currently 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 hot topics in current psychiatric research.

[0003] Numerous studies have shown that overactivation of the lateral habenula (LHb) is considered a key underpinning of depressive symptoms, particularly low mood and loss of interest. Studies have also reported that clustered firing of glutamatergic neurons in the LHb is closely associated with depressive episodes. Ketamine exerts a rapid antidepressant effect by inhibiting N-methyl-D-aspartate receptors (NMDARs) in the LHb, thereby blocking clustered firing of glutamatergic neurons.

[0004] NMDARs have important physiological functions in the brain. However, excessive NMDAR blockade can cause severe neurotoxicity, posing a significant challenge to the development of direct and safe NMDAR antagonists. Ketamine, a noncompetitive NMDAR inhibitor, can cause side effects such as impaired cognitive function, dissociative symptoms, ataxia, and addiction with long-term or high-dose use. Studies have shown that ketamine has strong selectivity for the GluN2B subunit of NMDARs. By binding to GluN2B, ketamine inhibits its ion channel activity and reduces calcium influx, thereby regulating neurotransmission and synaptic plasticity. In neurobiological research, glutamate receptors, particularly the NMDA receptor subunit GluN2B, play a key role in the pathogenesis of depression. However, GluN2B has not yet been used in antidepressant settings. Summary of the Invention

[0005] The purpose of the present invention is to provide a polypeptide and its use in the preparation of a GluN2B membrane localization regulator to solve the problems existing in the above-mentioned prior art. The polypeptide can regulate the membrane localization of GluN2B in the lateral habenula (LHb) brain region. By precisely regulating the membrane localization of GluN2B, it is expected to improve the effect of antidepressant treatment and provide a new treatment option 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, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO.1.

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

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

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

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

[0012] The present invention also provides 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 shown in SEQ ID No. 1, or has at least 90% identity thereto. Experimental verification has shown that the polypeptide provided by the present invention can solve the shortcomings of the existing technology that it cannot safely and quickly treat depression. It specifically reduces the NMDAR membrane localization in the LHb region, reduces the clustered discharge of glutamatergic neurons, exerts an antidepressant effect, and cleverly avoids the defects caused by ketamine's whole-brain non-selective blockade of NMDAR, and can avoid side effects such as addiction and induction of mental symptoms. Therefore, it is shown that the polypeptide provided by the present invention can be used to prepare a regulator that reduces the membrane localization of GluN2B, so as to provide a new drug and treatment idea for antidepressant by reducing the membrane localization of GluN2B. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1This is a diagram verifying 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 test results, 1-3 correspond to Control+TAT-Control, CUMS+TAT-Control, and CUMS+Tat-GluN2B-CaMKII, respectively; B: Data statistical graph of Figure A; Control+TAT-Control indicates that mice with 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 LHb polypeptide injection 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 LHb polypeptide injection in the physiological level forced swimming experiment in Example 4 of the present invention;

[0019] Figure 4 This is a graph showing the results of LHb polypeptide injection in the horizontal tail suspension experiment of the depression model in Example 5 of the present invention;

[0020] Figure 5 This is a graph showing the results of LHb injection of polypeptides in the forced swimming test for depression model level in Example 5 of the present invention;

[0021] Figure 6 This is a graph showing the results of LHb polypeptide injection in the sugar water preference rate detection experiment for the depression model in Example 5 of the present invention. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The present invention, through whole-brain screening, has demonstrated that reducing the membrane localization of the GluN2B subunit in the LHb region can effectively and safely and rapidly exert an antidepressant effect. The present invention provides a polypeptide (shown in SEQ ID NO. 1) that overcomes the shortcomings of existing technologies in safely and rapidly treating depression. This polypeptide achieves its antidepressant effect by specifically blocking NMDAR channels in the LHb region, reducing the clustered firing of glutamatergic neurons. Unlike the side effects of ketamine's non-selective whole-brain NMDAR blockade, the polypeptide of the present invention can avoid adverse reactions such as addiction and the induction of psychiatric symptoms.

[0028] The synthesis and antidepressant function of the above polypeptides are further described below with specific examples.

[0029] Example 1 Polypeptide

[0030] This embodiment 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 was synthesized via solid-phase synthesis. Synthesis began with the first amino acid at the C-terminus and gradually lengthened the polypeptide chain to yield the target polypeptide. The synthesis process included the sequential ligation of amino acids, deprotection, washing, and cleavage, all performed on a solid support. The specific steps were:

[0033] 1. Synthesis steps

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

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

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

[0037] Following the above steps, other amino acids were added stepwise, and the condensation reaction and deprotection steps were performed in sequence until all amino acids were synthesized. DMF was used for washing after each reaction to remove impurities in the reaction.

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

[0039] After the last amino acid is synthesized, remove the FMOC protecting group from the resin using 20% ​​piperidine for 20 minutes. Wash the resin 5-6 times with DMF and drain. Cleave the crude peptide from the resin using a cleavage solution (e.g., TFA / THF solution). Filter and wash the resulting crude peptide for purification.

[0040] 2. Purification method

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

[0042] Dissolve the crude peptide obtained by cleavage in appropriate amounts of acetonitrile and water and sonicate until completely dissolved. Purify the peptide using a preparative liquid phase system. Set a gradient based on the retention time from the analytical results to isolate and purify the target peptide. Collect appropriate aliquots and analyze them to ensure the target peptide meets the required purity.

[0043] (2) Freeze-drying

[0044] The purified peptide sample was concentrated by rotary evaporation and then freeze-dried in a freeze dryer for 48 hours. The freeze-dried peptide was taken out and its final mass was weighed.

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

[0046] The preparation method of the chronic mild unpredictable stress model (CUMS) is as follows: mice are subjected to different stresses such as restraint, forced swimming, humid environment, day and night reversal, water deprivation, and fasting for 42 consecutive days. After 42 days of modeling, they show depressive behavioral manifestations such as decreased sugar water preference, prolonged tail suspension immobility time, and prolonged forced swimming immobility time.

[0047] The peptide of Example 1, namely the peptide TAT shown in SEQ ID No. 1 (50 nmol / L, 1 μL, CUMS+Tat-GluN2B-CaMKII), and the random control peptide TAT-Control (CUMS+TAT-Control, obtained by randomly scrambling the order of the peptide TAT shown in SEQ ID No. 1) were injected bilaterally in the LHb region of mice in stereotaxic manner. Samples were prepared to detect the membrane localization of GluN2B in the LHb region of mice. At the same time, mice with normal physiological levels without the CUMS model were treated with the random control peptide as blank controls (Control+TAT-Control). The results are shown in Figure 2. Figure 1 As shown, the results showed that the peptide could reduce the membrane localization of GluN2B in the LHb region.

[0048] Example 4 Antidepressant-like effects of polypeptides at physiological levels

[0049] The peptide provided in Example 1 (50 nmol / L, 1 μL, Tat-GluN2B-CaMKII) was injected into the LHb region of mice at normal physiological levels. A control group (TAT-Control) was treated with the random peptide TAT-Control using the same method. 24 hours later, the mice were subjected to a tail suspension test, a forced swim test, and a sugar water preference test.

[0050] Tail suspension test (TST): A plastic clip was fixed to the center of the top plate of a tail suspension box (25 × 25 × 25 cm). The mouse was then suspended upside down with its head 4–5 cm above the bottom of the box. The mouse was observed for 6 minutes, and the cumulative immobility time within the last 4 minutes was recorded using an electronic stopwatch.

[0051] Forced swim test (FST): Mice were placed in cylindrical glass tanks (20 cm high and 10 cm in diameter), one per tank. The water was 10 cm deep and held at 25°C. The mice were observed for 6 minutes, and the cumulative immobility time within the last 4 minutes was recorded using a digital stopwatch. This time period was defined as the duration during which the mouse stopped struggling or remained afloat, with only minor limb movements to keep its head above water.

[0052] Depend on Figure 2 The tail suspension test shown and Figure 3As shown in the forced swimming test, the injection of the polypeptide of Example 1 into the LHb has a significant antidepressant effect at the physiological level.

[0053] Example 5 The polypeptide exerts an antidepressant effect in a depression model

[0054] Six weeks after inducing a chronic mild unpredictable stress (CUMS) model in mice (same as in Example 3), the peptide from Example 1 (50 nmol / L, 1 μL, CUMS + Tat-GluN2B-CaMKII) was injected into the LHb region of the mice. Two control groups were established: one treated with the CUMS model and a randomized control peptide, TAT-Control (CUMS + TAT-Control), and one treated with a normal physiological level of the randomized control peptide (Control + TAT-Control). Twenty-four hours later, the mice were subjected to a tail suspension test, a forced swim test, and a sugar water preference test.

[0055] The tail suspension test and forced swim test were the same as those in Example 4.

[0056] The Sugar Preference Test (SPT) for mice uses a two-bottle method, with one bottle of sugar water and one bottle of normal drinking water provided to the mice. Mice were housed individually and acclimated to drinking 1% sugar water for three days before testing. They were deprived of food and water at 10:00 PM on the night before testing. Sugar water and non-sugar water were then given at 10:00 PM on the second day. The water bottles were weighed before administration. At 8:00 AM on the third day, the sugar and non-sugar water bottles were removed and weighed. The difference between the two bottle weights was used to determine the mice's sugar and non-sugar water intake. The absolute value of sugar water intake was used to determine sugar water intake. Sugar water intake / (sugar water intake + plain water intake) = sugar water preference. After testing, the mice were returned to their original cages.

[0057] Depend on Figure 4 The tail suspension test shown, Figure 5 Forced swimming test as shown and Figure 6 As shown in the test results of the sugar water preference rate of mice, it can be seen that the injection of the polypeptide of Example 1 into the LHb has a significant antidepressant effect at the depression model level.

[0058] As demonstrated in the above examples, the antidepressant effects of the present invention are achieved by reducing the membrane localization of the GluN2B subunit in the LHb region, specifically blocking NMDAR channels in the LHb region, and reducing the burst firing of glutamatergic neurons, resulting in a rapid antidepressant effect. The drug demonstrated significant and rapid antidepressant effects in the mouse forced swim model, the mouse tail suspension model, and the sucrose preference test.

[0059] The polypeptide provided by the present invention can specifically reduce the expression of GluN2B membrane in the LHb brain region, thereby reducing the cell membrane of the LHb region to Ca 2+ permeability, reducing Ca2+ A large amount of influx reduces the cluster discharge caused by excessive excitation of LHb and exerts an antidepressant effect.

[0060] Therefore, the polypeptide provided by the present invention can reduce the membrane localization of GluN2B and reduce the cluster discharge caused by excessive excitation of LHb, without relying on the non-selective blockade of whole-brain NMDAR mechanism by ketamine, cleverly avoiding the defects caused by ketamine's whole-brain non-selective blockade of NMDAR, and can avoid side effects such as addiction and induction of psychiatric symptoms.

[0061] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a GluN2B membrane localization regulator in the preparation of an antidepressant drug, characterized in that: The GluN2B membrane localization regulator has a polypeptide as an effective active ingredient, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The GluN2B membrane localization regulator further comprises pharmaceutically acceptable excipients.

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