A polypeptide for inhibiting the activity of HDAC3 and its application

Abstract

The present invention discloses a polypeptide for inhibiting the activity of HDAC3 and its application, belonging to the technical field of biomedicine. The polypeptide of the present invention is TAT-nDAD, which is a combination of a polypeptide sequence derived from the endogenous NCOR2 protein and the cell-penetrating peptide TAT sequence. The polypeptide provided by the present invention can target and bind to HDAC3, competitively inhibit the binding of NCOR2 to HDAC3, inhibit the activity of HDAC3, has high specificity, can safely, effectively and reversibly reduce the level of BACE1 protein, reduce the generation of Aβ in the brain, and significantly improve the cognitive memory function of the AD model.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly relates to a polypeptide for inhibiting the activity of HDAC3 and its application. Background Art

[0002] Aging is a problem and challenge that humanity must face today, and age-related degenerative diseases are one of the most important problems among them. Alzheimer's disease (AD) is the most common and severe neurodegenerative disease mainly characterized by progressive memory impairment and cognitive dysfunction. Currently, according to incomplete statistics, there are more than 50 million AD patients globally. With the advent of the global aging era, it is estimated that by 2050, the number of AD patients will increase to 152 million. However, so far, there is still a lack of effective means for preventing and treating AD, mainly because the etiology and pathogenesis of AD have not been elucidated yet. Therefore, in order to improve the quality of life of the elderly population and rapidly reduce the burden on families and society caused by diseases in the aging population, we are urgently seeking effective molecular targets to provide a scientific basis for the treatment of AD.

[0003] In AD, although members of the HDAC family are basically related to the pathological process of the disease, histone deacetylase 3 (HDAC3) plays an important role in AD. A large number of literature reports show that the expression of HDAC3 is up-regulated in many neurodegenerative diseases including AD. The up-regulation of HDAC3 will accelerate the formation of senile plaques, neuronal death and the destruction of synaptic plasticity, while inhibiting the expression of HDAC3 can improve the learning and memory of AD mice and reduce senile plaques. At the same time, some literature confirms that HDAC3 can also cause an inflammatory response. By inhibiting HDAC3, the neuroinflammatory response can be significantly reduced, and the damage to neurons can be decreased. HDAC3 not only affects the acetylation state of histones, but may also affect the acetylation of other non-histone targets, thereby more widely regulating cell functions and gene expression. This versatility makes HDAC3 play an important role in the epigenetic regulation of AD.

[0004] Nuclear Receptor Corepressor 2 (NCOR2) is essential for the activation of histone deacetylase 3 (HDAC3) to exert its deacetylase function. The deacetylase activity of HDAC3 depends on its interaction with NCOR2. In the absence of NCOR2, the activity of HDAC3 is low. It can be seen that inhibiting the binding of NCOR2 and HDAC3 can play a role in treating Alzheimer's disease (AD) by affecting the activity of HDAC3 without affecting their respective synthesis. Peptide drugs have high selectivity and specificity, strong biological activity, low toxicity, good safety and tolerance, low production costs, and standardized synthesis methods. However, peptide drugs have certain stability problems. Peptide drugs often have poor stability in vivo and are easily degraded by enzymes, which limits their half-life and bioavailability in vivo. Moreover, since peptide drugs are easily decomposed in the gastrointestinal tract, most peptide drugs need to be administered by injection, which may lead to reduced patient compliance. At the same time, peptide drugs are often sensitive to temperature and pH conditions and need to be stored and transported under specific conditions, which increases their overall management costs. Nevertheless, peptide drugs have currently been used to treat various diseases, including diabetes, cancer, cardiovascular diseases, etc. For example, antimicrobial peptides for treating drug-resistant bacterial infections, LHRH analogs for treating certain types of prostate cancer and breast cancer, and ACE inhibitors for treating hypertension and heart failure. Peptide drugs for treating AD mainly focus on the experimental and clinical trial stages. The research and development of these peptides or peptide therapies are affected by their potential neuroprotective effects and specific biological targets (such as amyloid-beta (Aβ) and Tau proteins). Currently, HDAC3 inhibitors in clinical trials include MocetinosTAT (MGCD0103), EntinosTAT (MS-275), and DomatinosTAT (4SC-202), which target several HDACs including HDAC3 and have entered phase I / II clinical trials, mainly for Hodgkin lymphoma and non-Hodgkin lymphoma, breast cancer, non-small cell lung cancer, and skin cancer and other solid tumors. The commonly used specific inhibitor of HDAC3 is RGFP966, which is still in the in vitro or in vivo experimental stage. It can be seen that drugs specifically inhibiting the activity of HDAC3 are rare in clinical practice, and HDAC3 inhibitors specifically used to treat AD are even rarer. Therefore, polypeptides inhibiting the activity of HDAC3 have great value and potential for development as AD treatment drugs. Summary of the Invention

[0005] To solve the above problems, the present invention provides a polypeptide inhibiting the activity of HDAC3 and its application, which can effectively reduce senile plaques generated by the metabolism of amyloid precursor protein (APP) and improve the cognitive memory function of AD.

[0006] Furthermore, the polypeptide is TAT-nDAD;

[0007] Furthermore, the polypeptide is a combination of a polypeptide sequence derived from the endogenous NCOR2 protein and a cell-penetrating peptide TAT sequence.

[0008] Furthermore, the polypeptide sequence derived from the endogenous NCOR2 protein at least includes AECVLYYYLTKKN.

[0009] Furthermore, the polypeptide sequence is TAT-AECVLYYYLTKKN, and the TAT sequence is YGRKKRRQRRR.

[0010] Furthermore, the molecular weight of the polypeptide is 3.15 KDa, and the isoelectric point pI is 10.72.

[0011] The present invention also claims the application of TAT-nDAD polypeptide as an HDAC3 inhibitor.

[0012] The present invention also claims the application of TAT-nDAD polypeptide in reducing the interaction between NCOR2 and HDAC3.

[0013] The present invention also claims the application of TAT-nDAD polypeptide in reducing APP metabolism and CTF-β.

[0014] The present invention also protects the application of a polypeptide that inhibits the activity of HDAC3 in the preparation of drugs for preventing and treating neurodegenerative diseases, anti-tumor drugs, and memory-improving drugs.

[0015] Furthermore, the neurodegenerative diseases include Alzheimer's disease, neuroinflammation, Parkinson's disease, Huntington's disease, Lewy body dementia, amyotrophic lateral sclerosis, multiple system atrophy, spinocerebellar ataxia, and frontotemporal dementia.

[0016] Furthermore, the tumors in the anti-tumor drugs include lung cancer (non-small cell lung cancer, small cell lung cancer), breast cancer, gastrointestinal tumors (gastric cancer, colorectal cancer, pancreatic cancer), liver cancer, prostate cancer, kidney cancer, ovarian cancer, brain tumors, skin cancer (melanoma, squamous cell carcinoma), leukemia (acute lymphoblastic leukemia, acute myeloid leukemia), lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma), multiple myeloma, cervical cancer, endometrial cancer, ovarian cancer, genitourinary system tumors, kidney cancer, bladder cancer, testicular cancer, bone and soft tissue tumors, osteosarcoma, soft tissue sarcoma, nasopharyngeal cancer, laryngeal cancer, oral cancer, pancreatic cancer, esophageal cancer, thyroid cancer, and other solid tumors.

[0017] The present invention also claims a polypeptide drug, comprising TAT-nDAD polypeptide and one or more pharmaceutical excipients; the pharmaceutical excipients are excipients required for preparing an injection.

[0018] Furthermore, the polypeptide drug is an injection dosage form for intravenous injection or intramuscular injection, and the injection dosage form includes powder for injection and aqueous injection.

[0019] The polypeptide drug provided by the present invention is a polypeptide fragment of the endogenous NCOR2 protein, which realizes "inhibiting HDAC3 with NCOR2", has high specificity, good safety, definite curative effect, is easily soluble in water, has a small molecular weight, is easily permeable through the blood-brain barrier with the help of the cell-penetrating peptide TAT sequence, is easily hydrolyzed after exerting its effect, and the generated amino acids can nourish nerves. It is convenient for synthesis and formulation, has a low cost, and has obvious unique advantages compared with small molecule chemical inhibitors and monoclonal antibody drugs. It is an ideal drug for the prevention and treatment of AD and has strong clinical transformation value.

[0020] The polypeptide for inhibiting the activity of HDAC3 and its application according to the present invention have the following beneficial effects:

[0021] 1. The polypeptide provided by the present invention can target and bind to HDAC3, competitively inhibit the binding of NCOR2 to HDAC3, inhibit the activity of HDAC3, has high specificity, can safely, effectively and reversibly reduce the level of BACE1 protein, and reduce the generation of Aβ in the brain.

[0022] 2. The polypeptide provided by the present invention can significantly reduce the generation of CTF-β in AD and significantly improve the cognitive and memory functions of AD models.

[0023] 3. The polypeptide drug provided by the present invention is easily permeable through the blood-brain barrier, which can ensure its function in the brain, making its administration method more flexible, not limited to intravenous injection, and can be administered by intramuscular injection and other methods.

[0024] 4. The polypeptide provided by the present invention has a small molecular weight, is easily soluble in water, has an isoelectric point pI higher than the plasma pH value, does not ionize cations in the blood, has little irritation, is easily degraded and cleared by proteases and peptidases in the body after exerting its effect, is not easily accumulated, and has unique effects and economic advantages compared with small molecule inhibitors and monoclonal antibody drugs. It is a truly useful polypeptide drug for the prevention and treatment of AD.

[0025] 5. The polypeptide synthesis process provided by the present invention is simple, convenient for large-scale production, can be made into freeze-dried powder, and has great potential in the prevention and treatment of AD, with good promotion value and clinical transformation value. Since it can reduce the generation of Aβ, the disease spectrum applicable to the polypeptide may be extended to neurodegenerative diseases such as tauopathy and even the prevention and treatment of tumors in the future, with good long-term prospects. Description of the Drawings

[0026] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are merely exemplary. For those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained by extending according to the provided drawings. The structures, ratios, sizes, etc. depicted in this specification are only used to cooperate with the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they do not have technical substantial significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size, without affecting the efficacy that the present invention can produce and the purpose that can be achieved, should still fall within the scope that can be covered by the technical content disclosed in the present invention.

[0027] Figure 1 To mutate the binding site of NCOR2 and HDAC3 in Example 2 and detect the effect on the NCOR2-HDAC3 interaction in HEK cells. TAT-n in the figure is the TAT-nDAD of the present invention.

[0028] Figure 2 The site where NCOR2 binds to HDAC3 in Example 2.

[0029] Figure 3 For the effect of the TAT-nDAD polypeptide on the growth of mice in Example 3, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001. TAT-n in the figure is the TAT-nDAD of the present invention

[0030] Figure 4 For the effect of the TAT-nDAD polypeptide on the levels of APP, PS1, BACE1, and CTFβ in a murine AD cell model in Example 4, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001; TAT-n in the figure is the TAT-nDAD of the present invention.

[0031] Figure 5 For the effect of the TAT-nDAD polypeptide on the NCOR2-HDAC3 interaction in an exogenously transfected cell model in Example 5; TAT-n in the figure is the TAT-nDAD of the present invention.

[0032] Figure 6 For the effect of the TAT-nDAD polypeptide on the NCOR2-HDAC3 interaction in a murine AD cell model in Example 6; TAT-n in the figure is the TAT-nDAD of the present invention.

[0033] Figure 7To show the effect of TAT-nDAD polypeptide on anxiety and depression in AD mice in Example 7; TAT-n in the figure is TAT-nDAD of the present invention.

[0034] Figure 8 To show the effect of TAT-nDAD polypeptide on working memory in AD mice in Example 8, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001; TAT-n in the figure is TAT-nDAD of the present invention.

[0035] Figure 9 To show the effect of TAT-nDAD polypeptide on learning and memory in AD mice in the water maze experiment of Example 9, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001; TAT-n in the figure is TAT-nDAD of the present invention.

[0036] Figure 10 To show the effect of TAT-nDAD polypeptide on spatial memory in AD mice in the Barnes maze experiment of Example 9, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001; TAT-n in the figure is TAT-nDAD of the present invention.

[0037] Figure 11 To show the effect of TAT-nDAD polypeptide on the levels of APP, PS1, BACE1 and CTFβ in the hippocampus of AD mice in Example 11, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001; TAT-n in the figure is TAT-nDAD of the present invention. Detailed implementation mode

[0038] The present invention will be further described in detail below with reference to specific embodiments.

[0039] Example 1

[0040] A polypeptide for inhibiting the activity of HDAC3, comprising the following steps:

[0041] (1). According to the first amino acid at the C-terminus of the polypeptide sequence, select 0.5 mmol of the corresponding Fmoc-protected amino acid-Wang Resin, add it to a solid-phase reactor, add DCM to swell the resin for 30 min, then drain, wash it three times with DMF, add a 20% v / v solution of piperidine in DMF, react for 5 min, add the 20% v / v solution of piperidine in DMF again and react for 10 min, wash it once with DMF in the middle, drain after the reaction, and wash it 3 times with DMF;

[0042] (2) Sequentially perform condensation reactions and Fmoc deprotection reactions alternately with a reaction amount of 1.5 mmol according to the sequence of the polypeptide from the C-terminus to the N-terminus until all the remaining amino acids of the polypeptide are condensed onto the resin. After the last amino acid is condensed, add a 20% (by volume) solution of piperidine in DMF, react for 5 min, add the 20% (by volume) solution of piperidine in DMF again and react for 10 min, wash once with DMF in the middle, dry by suction after the reaction ends, and wash 3 times with DMF;

[0043] (3) Add 1 mmol of FITC and N-methylmorpholine to the reactor, react for 5 - 10 min, and use ninhydrin to detect whether the reaction is complete. After the reaction is complete, wash alternately 3 times with DMF and DMC, and then wash and contract with methanol to obtain dry polypeptide-resin.

[0044] (4) Put the polypeptide-resin obtained in step (3) into a round-bottom flask, slowly add the prepared cleavage solution (the volume ratio of the reagents in the cleavage solution is TFA:benzyl methyl sulfide:phenol:triisopropylsilane:water = 82.5:7.5:5:3:2) at 0 °C, stir slowly, react for 0.5 h at low temperature, react for 2 h at room temperature, filter to obtain the cleavage solution, slowly add the cleavage solution to anhydrous ice ether and stir, filter to separate the crude polypeptide, and wash 3 times with ice ether to obtain the crude peptide;

[0045] (5) Purify and separate the crude peptide by high-performance liquid chromatography, freeze-dry, and then obtain the pure polypeptide. According to the NCOR2464 - 476 amino acid sequence, add the cell-penetrating peptide TAT sequence to synthesize the polypeptide TAT-nDAD of the present invention.

[0046] The polypeptide TAT-nDAD obtained in the present invention can be prepared into muscle injection dosage forms such as powder injections and aqueous injections according to existing standards.

[0047] Example 2

[0048] Verification of the site where NCOR2 binds to HDAC3 is mutated

[0049] Control group: Transfect HA-NCOR2 WT plasmid and MYC-HDAC3 plasmid into HEK293 cells;

[0050] Experimental group: Transfect HA-NCOR2 Y470A mutant plasmid and MYC-HDAC3 plasmid into HEK293 cells

[0051] Experimental method: When HEK293 cells were fused to 80%, the HA-NCOR2 WT plasmid and the MYC-HDAC3 plasmid were transfected, or the HA-NCOR2 Y470A mutant plasmid and the MYC-HDAC3 plasmid were transfected. After 24 hours, protein lysates were extracted for co-immunoprecipitation (Co-IP) experiments. That is, 500 μg of protein lysate was added with MYC or HA tags and incubated overnight at 4°C. The complex was boiled in 5x sample buffer at 95°C for 5 minutes. Western blot was used to detect the interaction between HDAC3 and NCOR2.

[0052] The results are shown in Figure 1 and Figure 2 As shown, HDAC3 needs to bind to NCOR2 to be activated. Therefore, HA-NCOR2WT and HA-NCOR2 Y470A mutant plasmids were constructed to reduce the interaction between NCOR2 and HDAC3, which had an impact on BACE1, so as to determine the effect of inhibiting the binding of the complex on AD.

[0053] Example 3

[0054] Verification of the effect of polypeptide on animal growth

[0055] WT + control peptide: Starting from two months of age, WT mice were injected with the control polypeptide once a day intraperitoneally until the end of the experiment. The body weight was recorded once a week from the time of polypeptide injection until before the behavioral experiment.

[0056] WT + experimental peptide: Starting from two months of age, WT mice were injected with the experimental polypeptide once a day intraperitoneally until the end of the experiment. The body weight was recorded once a week from the time of polypeptide injection until before the behavioral experiment.

[0057] AD + control peptide: Starting from two months of age, AD mice were injected with the control polypeptide once a day intraperitoneally until the end of the experiment. The body weight was recorded once a week from the time of polypeptide injection until before the behavioral experiment.

[0058] AD + experimental peptide: Starting from two months of age, AD mice were injected with the experimental polypeptide once a day intraperitoneally until the end of the experiment. The body weight was recorded once a week from the time of polypeptide injection until before the behavioral experiment.

[0059] The initial body weight of the mice was measured, and during the administration of the polypeptide drug TAT-nDAD, the body weight of mice at different ages was measured and monitored. The results are shown in Figure 3 As shown. It can be seen from Figure 3 that WT mice are heavier than AD mice, but the polypeptide drug TAT-nDAD does not affect the growth of mice.

[0060] Example 4

[0061] Verification of the effect of the polypeptide drug TAT-nDAD on the levels of APP metabolism-related enzymes in a murine AD cell model. Control group: N2A cells stably transfected with APP (N2A APP ) were treated with the control polypeptide TAT-scramble;

[0062] Experimental group: N2A cells stably transfected with APP (N2A APP ) were treated with the experimental polypeptide TAT-nDAD;

[0063] The drug concentration was 10 μM, and the cells were treated for 24 h. Then, the total cell protein was extracted, and Western blot experiments were performed to detect the levels of APP, PS1, BACE1, and CTF-β. The results are as Figure 4 shown. The polypeptide TAT-nDAD of the present invention can significantly reduce the levels of BACE1 and CTFβ in the murine AD cell model, while the polypeptide TAT-scramble in the control group does not affect the expression of BACE1.

[0064] Example 5

[0065] Verification of the effect of the polypeptide drug TAT-nDAD on the interaction between NCOR2 and HDAC3 of an exogenous transfected plasmid

[0066] In HEK cells, HA-NCOR2 WT and MYC-HDAC3 plasmids were transfected. The polypeptide TAT-nDAD of the present invention was added at a drug concentration of 10 μM, and the cells were treated for 24 h. Then, the cell protein was extracted using IP lysis buffer, and co-immunoprecipitation (Co-IP) experiments were performed to detect the interaction between NCOR2 and HDAC3.

[0067] The results are as Figure 5 shown. The polypeptide TAT-nDAD of the present invention can reduce the exogenous interaction between NCOR2 and HDAC3.

[0068] Example 6

[0069] Verification of the effect of the polypeptide drug TAT-nDAD on the interaction between NCOR2 and HDAC3 in a murine AD cell model

[0070] Using N2A APP cells as the experimental group, the polypeptide TAT-nDAD of the present invention and the control peptide TAT-NC were both added at a concentration of 10 μM to the culture medium, and the cells were treated for 24 h. Then, the cell protein was extracted using IP lysis buffer, and co-immunoprecipitation (Co-IP) experiments were performed to detect the interaction between NCOR2 and HDAC3.

[0071] The results are as Figure 6As shown, the polypeptide TAT-nDAD of the present invention can significantly reduce the interaction between NCOR2 and HDAC3 in a murine AD cell model.

[0072] Example 7

[0073] Verification of the effect of the polypeptide drug TAT-nDAD on anxiety and depression in mice

[0074] Using C57 mice as the control and AD (APP23 / PS45 double transgenic) mice as the experimental group. Using physiological saline as the solvent, the administration dose of TAT-nDAD was set at 5 mg / kg / d, and the same volume of physiological saline was given, once a day. Starting from 2 months of age, after 12 weeks of administration, the open field test was carried out. Each mouse was sequentially placed in an open box of 40×40×60 cm and allowed to freely explore for 10 min. After the exploration, the mouse was returned to the cage, and the odor in the open field was eliminated with 75% ethanol, foreign objects were removed, and then the next mouse was placed in. The number of times and the time of the mouse reaching the middle were recorded. The fewer the number of times and the time of reaching the middle, the more anxious it represented. The results are as Figure 7 shown, the polypeptide TAT-nDAD of the present invention can significantly improve the anxiety and depression of AD mice.

[0075] Example 8

[0076] Verification of the effect of the polypeptide drug TAT-nDAD on working memory in mice

[0077] One day after the open field test of the test mice in Example 7, the Y-maze test was started. In the Y-maze, the mouse was placed and allowed to freely explore for 10 min, and the ANY-maze tracking system was used for recording. After the exploration, the mouse was returned to the cage, the odor was eliminated with 75% ethanol, foreign objects were removed, and then the next mouse was placed in. And the ANY-maze tracking system was used to record the number of times of entering the three arms of ABC. After the experiment, the working memory index of the mouse was statistically analyzed, and the cycle index Alteration = (number of spontaneous alternations / total number of entries - 2) × 100% was calculated.

[0078] The results are as Figure 8 shown, the polypeptide TAT-nDAD of the present invention has no effect on the working memory of AD mice.

[0079] Example 9

[0080] Verification of the effect of the polypeptide drug TAT-nDAD on spatial learning and memory in mice

[0081] Verified by the Morris water maze experiment, in the Morris water maze experiment, the experiment was carried out in a circular pool with a radius of 1.5 meters and a height of 60 centimeters, aiming to detect the spatial learning and memory ability of mice. The pool was divided into four quadrants, labeled NE, NW, SW, SE, or quadrants 1, 2, 3, 4 respectively. Different-shaped markers were pasted above the pool wall of each quadrant to help mice with spatial positioning. To create a relatively independent experimental environment, blue curtains were hung around the pool, and corresponding-shaped markers were pasted at different positions on the curtains. During the experiment, a camera installed above the pool was used to track the movement trajectory of mice. The first day was the adaptation stage. Mice were gently placed into the pool, starting from the first quadrant, facing the pool wall, and allowed to freely explore for 120 s to adapt to the pool and the surrounding environment. At this time, no platform was placed underwater. After the exploration, the mice were taken out, dried, and then put back into the cage. The second to sixth days were the learning stage. An underwater hidden platform was placed in the center of the third quadrant, and the top of the platform was 1 cm below the water surface. During the experiment, mice were placed into the pool from different quadrants facing the pool wall. The mice needed to find the platform within 120 s and stay on it for 2 s to be considered a successful escape, and their escape latency (i.e., the time required to find the platform) was recorded. If the mice failed to find the platform within the specified time, they were guided to the platform and stayed on it for 20 s, and then the latency was recorded as 120 s. Four learning sessions were carried out every day, and the average value of the four escape latencies was calculated as the escape latency for that day. The seventh day was the testing stage. The platform was removed, and the mice were placed into the water from the first quadrant facing the pool wall. Their behavioral trajectories within 120 s were recorded, and the number of times they crossed the platform area was counted. The results are as Figure 9 shown that the polypeptide TAT-nDAD of the present invention in the water maze significantly improved the spatial learning and memory of AD mice.

[0082] The Barnes maze experiment was carried out in the Barnes maze to detect the spatial learning and memory retrieval functions of mice.

[0083] On the first day, it was the adaptation stage. The mice were placed in the escape hole for 3 minutes. After the experiment ended, the mice were taken out and put back into the cage. 75% alcohol was used to remove the smell and foreign objects, and then the experiment on the next mouse was carried out. From the 2nd to the 6th day was the training stage. First, the mice were placed in the center of the maze and covered with a black plastic box for 10 seconds, and then their exploratory behaviors in the maze were recorded. If the mice found the target hole and entered the escape hole within 3 minutes, their escape latency was recorded. If the mice failed to find the target hole within the specified time, they were guided into the escape hole and allowed to stay for 1 minute. The training was carried out 2 times a day, and the disc was randomly rotated each time, but the position of the escape hole remained unchanged. After the experiment, 75% alcohol was also used to remove the smell. On the 7th day was the testing stage. The escape room was removed, and the mice freely explored in the maze for 3 minutes. The correct rate of finding the target hole was recorded (i.e., the number of times of exploring the escape hole with the head / the total number of times) * 100%. The results were as Figure 10 shown that the polypeptide TAT-nDAD of the present invention in Barnes significantly improved the spatial learning and memory of AD mice.

[0084] Example 10

[0085] Verification of the effects of the polypeptide drug TAT-nDAD on the levels of APP, PS1, BACE1 and CTF-β in the hippocampus of AD mice

[0086] WT + control peptide: From the second month of WT mice, the control polypeptide was injected intraperitoneally once a day until the end of the experiment.

[0087] WT + experimental peptide: From the second month of WT mice, the experimental polypeptide was injected intraperitoneally once a day until the end of the experiment.

[0088] AD + control peptide: From the second month of AD mice, the control polypeptide was injected intraperitoneally once a day until the end of the experiment.

[0089] AD + experimental peptide: From the second month of AD mice, the experimental polypeptide was injected intraperitoneally once a day until the end of the experiment.

[0090] The hippocampal tissues of the mice at the end of the above experiments were lysed, the total hippocampal proteins were extracted, and Western blot experiments were carried out. The results were as Figure 11 shown that the experimental peptide TAT-nDAD had no obvious effect on the levels of APP and PS1 in the hippocampus of AD mice, but reduced the expression of BACE1 and CTF-β.

[0091] The above has introduced in detail the technical solutions provided by the embodiments of the present invention. Specific examples are used herein to elaborate on the principles and implementation manners of the embodiments of the present invention. The description of the above embodiments is only applicable to helping understand the principles of the embodiments of the present invention. At the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A polypeptide for inhibiting HDAC3 activity, characterized in that: The polypeptide is TAT-nDAD, and is a combination of a polypeptide sequence taken from an endogenous NCOR2 protein and a cell-penetrating peptide TAT sequence; the polypeptide sequence taken from the endogenous NCOR2 protein at least includes AECVLYYYLTKKN; and at least includes a fragment sequence -AECVLYYYLTKKN; The TAT-nDAD sequence is TAT-AECVLYYYLTKKN, and the TAT sequence is YGRKKRRQRRR.

2. Use of the polypeptide according to claim 1 in preparing a drug for preventing and treating Alzheimer's disease.

3. The use according to claim 2, characterized in that: The application of the drug includes inhibiting HDAC3, reducing the interaction between NCOR2 and HDAC3, and reducing APP metabolism and CTF-β.

4. A polypeptide drug, characterized in that: It comprises the polypeptide described in claim 1 and one or more pharmaceutical excipients; the pharmaceutical excipients are excipients required for preparing injections; the polypeptide drug is an injection form for intravenous injection or intramuscular injection, and the injection form includes powder injection and water injection.

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