N-methyl-D-aspartic acid receptor interference peptide and application thereof

Abstract

The invention discloses an N-methyl-D-aspartic acid receptor NR2B subunit interference peptide and an application of the N-methyl-D-aspartic acid receptor NR2B subunit interference peptide. Through reasonable design and synthesis, the interference peptide can be specifically combined with endogenous neurotoxic metabolite quinolinic acid (QA), and the combination between the QA and NR2B is blocked in a competitive mode, so that the NR2B-mediated excitatory neurotoxic reaction is selectively inhibited. The polypeptide can remarkably reverse neuron damage and shows an excellent nerve protection effect. In an obesity-related cognitive impairment model and an Alzheimer's disease mouse model, the interference peptide recovers neuronal synaptic injury in a hippocampus region by blocking a QA-NR2B signal channel, effectively improves cognitive hypofunction, and provides an innovative intervention strategy for prevention and treatment of neurodegenerative diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to a N-methyl-D-aspartate (NMDA) receptor interfering peptide and its application. The polypeptide analogue can specifically release endogenous neurotoxin quinolinic acid (QA) from NMDA receptor binding, inhibit excitatory neurotoxicity, and protect synapses and cognitive function. BACKGROUND

[0002] N-methyl-D-aspartate receptor (NMDA receptor) is a key ion channel complex that mediates glutamate signaling. It is highly enriched in brain regions such as cerebral cortex, hippocampus and striatum, which are closely related to high-level cognition, learning and memory, and motor control. As a core hub of excitatory neurotransmitter transmission, NMDA receptor regulates synaptic plasticity and neuronal development under physiological conditions. However, its overactivation can cause excitatory neurotoxicity, leading to neuronal damage and even death, which is directly related to the pathological mechanism of various neurodegenerative diseases.

[0003] Quinolinic acid (QA) as an endogenous neurotoxin is synthesized and released by inflammatory activated microglia, and mediates neurotoxicity by specifically activating NMDA receptor. NMDA receptor is composed of NR1, NR2(A-D) and NR3(A / B) subunits, and the NR2B subunit highly expressed in the forebrain (such as frontal cortex and hippocampus) is confirmed to be closely related to neurotoxicity. The neurotoxic effect of QA mainly depends on the abnormal activation of NR2B subunit, leading to synaptic damage and learning and memory dysfunction. The level of QA is significantly increased in patients with obesity cognitive dysfunction, Alzheimer's disease (AD) and Parkinson's disease (PD) and other neurodegenerative diseases. QA-NR2B mediated neurotoxicity plays an important role in neurodegenerative diseases. Therefore, targeting the blockade of QA-NR2B interaction is one of the important targets for the development of drugs for preventing and treating obesity cognitive dysfunction and AD neurodegenerative diseases.

[0004] Although NMDA receptor antagonists (such as memantine) have been used to treat Alzheimer's disease (AD), their modulation of NMDA receptors lacks subtype specificity and has limited therapeutic effect. Therefore, if the pathological interaction between QA and the NR2B subunit can be targeted to block and the QA-induced abnormal activation of NR2B can be specifically inhibited, the excessive influx of calcium ions and the neurotoxicity triggered thereby can be reduced, which is expected to reduce synaptic damage and improve learning and memory impairment associated with obesity and AD. However, the precise binding site of QA and the NR2B subunit is not yet known, and no drug can specifically compete with the QA binding site to block the abnormal activation of NR2B. Therefore, exploring and developing a specific decoupling interaction between QA and the NR2B subunit of the NMDA receptor provides a new path and strategy for precise intervention in the treatment of neurodegenerative diseases. SUMMARY

[0005] The purpose of this section is to outline some aspects of the embodiments of the present application and briefly introduce some preferred embodiments.

[0006] As one aspect of the present application, the present application provides an N-methyl-D-aspartate receptor interfering peptide, wherein: the amino acid sequence of the N-methyl-D-aspartate receptor interfering peptide is shown in SEQ ID NO: 1.

[0007] As a preferred aspect of the N-methyl-D-aspartate receptor interfering peptide of the present application: further comprising a cell-penetrating peptide sequence.

[0008] As a preferred aspect of the N-methyl-D-aspartate receptor interfering peptide of the present application: the amino acid sequence of the N-methyl-D-aspartate receptor interfering peptide is shown in SEQ ID NO: 2.

[0009] The present application also provides the use of the N-methyl-D-aspartate receptor interfering peptide in the preparation of a medicament for treating obesity-induced cognitive impairment and Alzheimer's disease.

[0010] The N-methyl-D-aspartate receptor interfering peptide can reverse obesity-induced hippocampal neuronal damage.

[0011] The N-methyl-D-aspartate receptor interfering peptide can alleviate obesity-induced cognitive decline and improve cognitive impairment caused by Alzheimer's disease.

[0012] The beneficial effects of the present application: the present application uses molecular simulation docking computational biology technology to systematically analyze the interaction mechanism of QA and NR2B subunit. The present application researches and finds that QA mainly dynamically binds to the R1-R2 connecting region of the NR2B extracellular amino-terminal domain (ATD), forms a specific binding site with key amino acids Gly-123 and Ser-126, and realizes the activation of the NR2B receptor. Based on this molecular interaction mechanism, the present application designs and synthesizes the interfering peptide TAT-NR2B123-126. The polypeptide can specifically bind to QA to competitively block the interaction between QA and NR2B, and then selectively inhibit the NR2B-mediated excitatory neurotoxicity response and reverse the neuron damage, and exhibits excellent neuroprotective effect. In the models of obesity and Alzheimer's disease related cognitive impairment, the TAT-NR2B123-126 polypeptide effectively improves the cognitive impairment of obesity and Alzheimer's disease mice by blocking the QA-NR2B signaling pathway, and provides an innovative intervention strategy for the prevention and treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows:

[0014] Figure 1 The figure is the result of NR1-NR2B protein structure analysis, QA and NR2B molecular simulation docking, and polypeptide NR2B123-126 sequence design.

[0015] Figure 2 The figure is the result of TAT-NR2B123-126 improving the cognitive impairment of mice induced by high-fat diet.

[0016] Figure 3 The figure is the result of TAT-NR2B123-126 improving the hippocampal neuron damage of mice induced by high-fat diet.

[0017] Figure 4 The figure is the result of TAT-NR2B123-126 improving the cognitive impairment of Alzheimer's disease mice.

[0018] Figure 5 The figure is the result of TAT-NR2B123-126 reducing the increase of intracellular calcium ion concentration induced by QA. DETAILED DESCRIPTION

[0019] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below.

[0020] The present application is based on high-resolution analysis of NR1-NR2B protein structure (PDB: 4TLL, PDB database) by cryo-EM technology, combined with molecular docking simulation and other computational biology methods, to deeply study the interaction mechanism of quinolinic acid (QA) and NR2B protein. The study found that QA mainly moves in the R1-R2 linking region of the extracellular amino-terminal domain (ATD) of NR2B, and forms a specific binding site with key amino acids Gly-123, Ser-126 on NR2B. This finding clearly identifies the key role region of QA-NR2B interaction, providing an important molecular target and theoretical basis for subsequent product development.

[0021] According to the above research results, a small molecule polypeptide TAT-NR2B123-126 is designed and synthesized. The amino acid sequence of the polypeptide is Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Ile-Leu-Gly-Ile-His-Gly-Gly-Ser-Ser-Met-Ile-Met-Ala-Asp. The design closely surrounds the binding site of QA and NR2B, aiming to compete with QA for binding to NR2B, block the QA-NR2B mediated neurotoxicity signaling pathway, and thus play a potential neuroprotective role.

[0022] Studies on obese cognitive impairment and Alzheimer's disease model mice showed that TAT-NR2B123-126 can effectively alleviate cognitive decline and reduce neuronal damage. It is suitable for the adjuvant therapy of neurodegenerative diseases (such as metabolic disease-related cognitive impairment, Alzheimer's disease, Parkinson's disease, etc.) caused by QA-NR2B mediated neurotoxicity, and provides new treatment strategies and options for patients.

[0023] Example 1: The design method of the polypeptide sequence is as follows:

[0024] 1. Based on the NR1-NR2B protein complex structure (PDB number: 4TLL, from PDB database) obtained by cryo-EM analysis, it is found by molecular docking simulation analysis that the small molecule quinolinic acid (QA) is mainly combined with the R1-R2 connecting region of the extracellular amino-terminal domain (ATD) of NR2B ( Figure 1 A). Further analysis shows that QA forms a stable binding site with key amino acid residues Gly-123 and Ser-126 of NR2B ( Figure 1 B). Figure 1 The three-dimensional structure analysis of NR1-NR2B protein and the molecular docking results of QA are shown.

[0025] 2. Based on the above binding site, the corresponding NR2B small molecule polypeptide is designed. The specific method is: from the Gly-123 residue to the N terminal, 6 amino acids are traced back, and the Pro at the 117th position is taken as the starting residue; 5 amino acids are extended to the C terminal, and the Asp at the 131st position is taken as the terminal residue. The final 15 amino acid sequence (SEQ ID NO: 1) is: Pro-Ile-Leu-Gly-Ile-His-Gly-Gly-Ser-Ser-Met-Ile-Met-Ala-Asp (PILGIHGGSSMIMAD), that is, the fragment covering the 117th to 131st of NR2B, as shown in FIG. C. Figure 1

[0026] 3. In order to endow the polypeptide with transmembrane delivery ability, it is connected with TAT transmembrane peptide to form a fusion polypeptide TAT-NR2B123-126. The TAT sequence is derived from the 47th to 57th of HIV-1 virus Tat protein, which has good cell membrane penetration ability, and the amino acid sequence is: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (YGRKKRRQRRR). Therefore, the complete amino acid sequence (SEQ ID NO: 2) of the final constructed interference peptide TAT-NR2B123-126 is: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Ile-Leu-Gly-Ile-His-Gly-Gly-Ser-Ser-Met-Ile-Met-Ala-Asp

[0027] That is, (YGRKKRRQRRR-PILGIHGGSSMIMAD).

[0028] Example 2:

[0029] TAT-NR2B 123-126 polypeptide is applied to the treatment of obesity-induced cognitive impairment mice:

[0030] A total of 60 eight-week-old wild type mice (C57BL / 6J) were given high-fat diet (HFD) (45) or normal diet (15) for 12 weeks. The 45 mice of the high-fat diet group were divided into three subgroups after being fed with high-fat diet for 8 weeks: control ddH2O, blank peptide TAT, and TAT-NR2B123-126 interference peptide (2 nmol / g), each group of 15, tail vein injection of corresponding solvent for 4 weeks; then the cognitive behavior of the mice was detected, and the morphology and dendritic spine of the hippocampal neurons of the mice were analyzed.

[0031] ​1. TAT-NR2B123-126 improves the cognitive impairment induced by high-fat obesity in mice. Behavioral experiments include: time memory experiment, new place recognition experiment, new object recognition experiment, Y maze spontaneous alternation experiment, Y maze spatial location recognition experiment.

[0032] Figure 2 TAT-NR2B123-126 improves the cognitive impairment induced by high-fat obesity in mice. (A) Time memory experiment (B) New place recognition experiment (C) New object recognition experiment (D) Y maze spontaneous alternation experiment (E) Y maze spatial location recognition experiment. n = 15. *P < 0.05, ＊＊ P < 0.01, ＊＊＊ P < 0.001, compared with the blank control. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the HFD group. As Figure 2 shown, the experimental results show that: TAT-NR2B123-126 intervention can reverse the decrease of time memory index, new position discrimination index, and new object discrimination index caused by high-fat obesity (P < 0.05, Figure 2 A, B, C), increase the decrease of Y maze spontaneous alternation rate of high-fat obese mice (P < 0.05, Figure 2 D), and up-regulate the decrease of the proportion of exploring new and different walls caused by high-fat obesity in mice (P < 0.05, Figure 2 E). These results prove that the TAT-NR2B123-126 interfering peptide designed based on the QA-NR2B binding site effectively reverses the cognitive impairment induced by high-fat obesity.

[0033] 2. TAT-NR2B123-126 improves the hippocampal neuron damage induced by high-fat obesity in mice. TAT-NR2B123-126 reverses the decrease of neurite length (P < 0.01), neurite number (P < 0.01), maximum intersection radius of neurons (P < 0.01), and total intersection number of neurons (P < 0.01) in mice induced by high-fat obesity, and significantly improves the decrease of dendritic spine density of hippocampal neurons (P < 0.001).

[0034] Figure 3 TAT-NR2B123-126 improves the hippocampal neuron damage induced by high-fat obesity in mice. (A) Golgi silver staining to observe the neurons in the hippocampal region of mice, and Ima geJ software to analyze the morphology of neurons. (B) Total neurite length (C) Neurite number (D) Average neurite length (E) Maximum intersection radius of neurons (F) Total intersection number of neurons (G) Sholl analysis reflecting the complexity of hippocampal neurons (H) Microscope photograph of hippocampal neuron dendritic spine (I) Statistical graph of neuron dendritic spine density. *P < 0.05, ＊＊P<0.01, ＊＊＊ P<0.001 compared with blank control. #P<0.05, ##P<0.01, ###P<0.001 compared with HFD group.

[0035] Example 3: TAT-NR2B 123-126 polypeptide is applied to the treatment of Alzheimer's disease model (5xFAD) mice:

[0036] Take 4-month-old normal wild-type mice (C57BL / 6J, n=12) as a control group, and 4-month-old 5xFAD mice are 36 in total, which are randomly divided into 3 groups (n=12 each): ddH2O group, blank TAT peptide group, and TAT-NR2B 123-126 interfering peptide group (2 nmol / g). Each group of mice is injected with the corresponding reagent through the tail vein, and the intervention is continued for 4 weeks. After the intervention, the cognitive behavior of the mice is detected. The behavior experiment includes: time sequence memory experiment, new position recognition experiment, new object recognition experiment, Y maze spontaneous alternation experiment, and Y maze spatial location recognition experiment.

[0037] Figure 4 TAT-NR2B 123-126 improves the cognitive function decline of 5xFAD mice. (A) Time sequence memory experiment (B) New position recognition experiment (C) New object recognition experiment (D) Y maze spontaneous alternation experiment (E) Y maze spatial location recognition experiment. n=12. *P<0.05, ＊＊ P<0.01, ＊＊＊ P<0.001 compared with blank control. #P<0.05, ##P<0.01 compared with 5xFAD group. As Figure 4 shown, the experimental results show that: TAT-NR2B 123-126 intervention can reverse the decline of time sequence memory index, new position discrimination index, and new object discrimination index of 5xFAD mice (P<0.05, Figure 4 A, B, C), significantly increase the decrease of Y maze spontaneous alternation rate of 5xFAD mice (P<0.05, Figure 4 D), and reduce the decrease of the proportion of exploring new and different walls of 5xFAD mice (P<0.05, Figure 4 E). These results prove that the TAT-NR2B 123-126 interfering peptide designed based on the QA-NR2B binding site can effectively reverse the cognitive function damage of 5xFAD mice by specifically blocking the interaction of QA and NR2B.

[0038] Example 4: TAT-NR2B 123-126 specifically inhibits QA-induced intracellular calcium ion elevation:

[0039] In HT22 cells, TAT (10 μM) and TAT-NR2B123-126 (10 μM) were added respectively for 2 hours of pretreatment, and then control group (Control), quinolinic acid (QA, 50 μM) was given respectively for 24 hours of stimulation, and the change of intracellular calcium ion concentration was detected.

[0040] Figure 5 It is shown that TAT-NR2B123-126 can significantly inhibit the increase of intracellular calcium ion concentration induced by QA, indicating that it can intervene in the activation of QA-NR2B mediated calcium signal pathway. (A) The intracellular calcium ion image of HT22 cells treated with TAT, TAT123-126 and QA was observed by fluorescence microscope; (B) is the quantitative result of corresponding calcium ion fluorescence intensity (n=6). *P<0.05, compared with blank control. #P<0.05, compared with QA.

[0041] The statistical results show that, compared with the control group (Control), the intracellular calcium ion concentration of the QA treatment group is significantly increased (P<0.05, Figure 5 A, B); and the treatment of TAT-NR2B123-126 can significantly inhibit the increase of calcium ion caused by QA (P<0.05, Figure 5 A, B), which indicates that the polypeptide can specifically inhibit the abnormal calcium signal related to excitotoxicity by specifically blocking the interaction of QA-NR2B.

[0042] In summary, based on the deep analysis of molecular docking technology, it is found that the specific binding of quinolinic acid (QA) and NR2B subunit presents a highly precise molecular interaction mode. QA mainly moves dynamically in the R1-R2 connecting region of the NR2B extracellular amino terminal domain (ATD), and forms a stable connection with Gly-123 and Ser-126, two key amino acid residues, to realize the selective activation of NR2B receptor. This unique binding site gives the possibility of targeted intervention, that is, by designing a polypeptide molecule that competes with QA for binding, the QA-NR2B signal pathway can be specifically blocked.

[0043] The application designs and synthesizes a novel transmembrane interfering peptide TAT-NR2B123-126, which is derived from a key binding sequence of NMDA receptor NR2B subunit mediating neurotoxicity and can simulate the spatial conformation when it binds with quinolinic acid (QA). The polypeptide competes with QA and NR2B by binding with QA, thereby playing an antagonistic effect. Its specificity is reflected in effectively preventing the pathological binding between QA and NR2B by using a steric hindrance mechanism, thereby relieving the QA-induced overactivation of NR2B and its downstream excitotoxicity response. In animal experiments, the TAT-NR2B123-126 interfering peptide significantly improves the spatial learning and memory ability of a mouse model of cognitive impairment induced by a high-fat diet and a mouse model of Alzheimer's disease, and effectively relieves neuronal damage. The strategy has high targeting and selectivity, can specifically block the neurotoxicity mediated by the QA-NR2B pathway, and provides a new intervention strategy and technical path for the precise treatment of obesity-related cognitive impairment and neurodegenerative diseases.

[0044] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. An N-methyl-D-aspartate receptor interfering peptide, characterized in that: The amino acid sequence of the N-methyl-D-aspartate receptor interfering peptide is shown as SEQ ID NO:

1.

2. The N-methyl-D-aspartate receptor interfering peptide of claim 1, wherein: It also comprises a cell-penetrating peptide sequence.

3. The N-methyl-D-aspartate receptor interfering peptide of claim 2, wherein: The amino acid sequence of the N-methyl-D-aspartate receptor interfering peptide is shown as SEQ ID NO:

2.

4. The N-methyl-D-aspartate receptor interfering peptide according to claim 1 for use in the preparation of a medicament for treating obesity-induced cognitive impairment and Alzheimer's disease.

5. Use according to claim 4, characterized in that: The N-methyl-D-aspartate receptor interfering peptide can reverse obesity-induced hippocampal neuron damage.

6. Use according to claim 4, characterized in that: The N-methyl-D-aspartate receptor interfering peptide can alleviate obesity-induced cognitive decline and improve cognitive impairment caused by Alzheimer's disease.

Images