Polypeptide for inhibiting HDAC3 activity and application thereof

Abstract

The invention discloses a polypeptide for inhibiting HDAC3 activity and application thereof, and belongs to the technical field of biological medicine. The polypeptide disclosed by the invention is TAT-nDAD, and is a combination of a polypeptide sequence taken from endogenous NCOR2 protein and a cell-penetrating peptide TAT sequence. The polypeptide provided by the invention can be combined with HDAC3 in a targeted manner, competitively inhibit the combination of NCOR2 and HDAC3 and inhibit the activity of HDAC3, has high specificity, and can safely, effectively and reversibly reduce the BACE1 protein level, reduce the generation of A beta in the brain and remarkably improve the cognitive memory function of an AD model.

Description

Technical Field

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

[0002] Aging is a problem and challenge that humans must face today, and degenerative diseases related to old age are one of the most important problems. Alzheimer's disease (AD) is the most common and most serious brain degenerative disease with progressive memory impairment and cognitive dysfunction as the main clinical manifestations. At present, according to incomplete statistics, there are more than 50 million AD patients in the world. 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, to date, there is still a lack of effective means to prevent and treat AD. The main reason is that the cause and pathogenesis of AD are far from being clarified. Therefore, in order to improve the quality of life of the elderly and quickly reduce the burden of the aging population on families and society due to illness, we are eager to explore effective molecular targets to provide a scientific basis for the treatment of AD.

[0003] In AD, although HDAC family members 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 that HDAC3 expression is upregulated in many neurodegenerative diseases including AD. HDAC3 upregulation will accelerate the formation of senile plaques, neuronal death and the destruction of synaptic plasticity, while inhibiting HDAC3 expression 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 inflammatory responses. By inhibiting HDAC3, neuroinflammatory responses can be significantly reduced and damage to neurons can be reduced. HDAC3 not only affects the acetylation state of histones, but may also affect the acetylation of other non-histone targets, thereby regulating cell function and gene expression more widely. This multifaceted nature makes HDAC3 occupy an important position in the epigenetic regulation of AD.

[0004] Nuclear receptor corepressor (NCOR) 2 is necessary for the activation of HDAC3 to play the role of deacetylase. The deacetylase activity of HDAC3 depends on the 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 achieve the effect of treating 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 cost, and standardized synthesis methods. However, peptide drugs have certain stability problems. Peptide drugs are often less stable in the body and are easily degraded by enzymes, which limits their half-life and bioavailability in the body; and because 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 are currently used to treat a variety of diseases, including diabetes, cancer, cardiovascular disease, etc. For example, antimicrobial peptides are used to treat drug-resistant bacterial infections, LHRH analogs are used to treat certain types of prostate cancer and breast cancer, and ACE inhibitors are used to treat hypertension and heart failure. Peptide drugs for the treatment of AD are currently mainly concentrated in the experimental and clinical trial stages. The development of these peptides or peptide therapies is influenced by their potential neuroprotective effects and specific biological targets (such as Aβ and Tau protein). HDAC3 inhibitors currently 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's lymphoma and non-Hodgkin's lymphoma, breast cancer, non-small cell lung cancer, as well as skin cancer and other solid tumors. The currently commonly used inhibitor that specifically inhibits HDAC3 is RGFP966, which is still in the in vitro or in vivo experimental stage. It can be seen that drugs that specifically inhibit HDAC3 activity are rare in clinical practice, and HDAC3 inhibitors that are specifically used to treat AD are even rarer. Therefore, peptides that inhibit HDAC3 activity have great value and potential for development into AD therapeutic drugs. Summary of the invention

[0005] In order to solve the above problems, the present invention proposes a polypeptide that inhibits HDAC3 activity and its application, which can effectively reduce senile plaques generated by APP metabolism and improve AD cognitive memory function.

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

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

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

[0009] Furthermore, the gene sequence of the polypeptide is TAT-AECVLYYYLTKKN, and the TAT gene 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 use of TAT-nDAD polypeptide as an HDAC3 inhibitor.

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

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

[0014] The present invention also protects the use of a polypeptide that inhibits HDAC3 activity 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 drug 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 lymphocytic 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 protection for a polypeptide drug, comprising a TAT-nDAD polypeptide and one or more pharmaceutical excipients; the pharmaceutical excipients are excipients required for preparing injections.

[0018] Furthermore, the polypeptide drug is in the form of an injection for intravenous or intramuscular injection, and the injection form includes a powder injection and a water 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", is highly specific, has good safety, definite efficacy, is easily soluble in water, has a small molecular weight, and can easily penetrate the blood-brain barrier with the help of the cell-penetrating peptide TAT sequence. It is easily hydrolyzed after taking effect, and the amino acids produced can nourish nerves. It is convenient to synthesize and prepare, and has low cost. It has obvious and unique advantages over smaller molecule chemical inhibitors and monoclonal antibody drugs, and is an ideal drug for the prevention and treatment of AD, with strong clinical transformation value.

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

[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, have high specificity, and 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 memory function of the AD model.

[0023] 3. The polypeptide drug provided by the present invention can easily pass through the blood-brain barrier, which can ensure that it works 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 into cations in the blood, has low irritation, is easily degraded and removed by proteases and peptidases in the body after exerting its effect, and is not easy to accumulate. It has unique effects and economic advantages over smaller molecule inhibitors and monoclonal antibody drugs, and is a polypeptide drug that can be used for the prevention and treatment of AD in a true sense.

[0025] 5. The polypeptide synthesis process provided by the present invention is simple, easy to produce in large quantities, and can be made into freeze-dried powder. It has great potential in the prevention and treatment of AD and has good promotion value and clinical transformation value. Since it can reduce the generation of Aβ, the spectrum of diseases applicable to the polypeptide may be expanded to neurodegenerative diseases such as tau disease and even tumor prevention and treatment, and has a good long-term prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the implementation mode of the present invention or the technical solution in the prior art, the drawings required for the implementation mode or the prior art description are briefly introduced below. Obviously, the drawings described below are only exemplary. For ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work. The structure, proportion, size, etc. illustrated in this specification are only used to match the content disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions that the present invention can be implemented, so it has no technical substantive significance. Any structural modification, change in proportional relationship or adjustment of size, without affecting the effect that the present invention can produce and the purpose that can be achieved, should still fall within the scope that the technical content disclosed by the present invention can cover.

[0027] Figure 1 The binding sites of NCOR2 and HDAC3 mutated in Example 2 were tested in HEK cells for the effect on NCOR2-HDAC3 interaction. TAT-n in the figure is TAT-nDAD of the present invention.

[0028] Figure 2 This is the site where NCOR2 binds to HDAC3 in Example 2.

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

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

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

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

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

[0034] Figure 8 The effect of the TAT-nDAD polypeptide on the working memory of 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] Fig. 9 The effect of TAT-nDAD polypeptide on learning and memory of 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] Fig.10 This is the effect of TAT-nDAD polypeptide on spatial memory of AD mice in the Barnes maze test in Example 9. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001; TAT-n in the figure is TAT-nDAD of the present invention.

[0037] Fig.11 The effect of the TAT-nDAD polypeptide in Example 11 on the levels of APP, PS1, BACE1 and CTFβ in the hippocampus of AD mice, * 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 DESCRIPTION

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

[0039] Example 1

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

[0041] (1) According to the first amino acid at the C-terminus of the polypeptide sequence, 0.5 mmol of the corresponding Fmoc-protected amino acid-Wang Resin was selected and added to the solid phase reactor. After adding DCM to swell the resin for 30 minutes, the resin was drained and washed three times with DMF. A 20% volume ratio of hexahydropyridine DMF solution was added and reacted for 5 minutes. A 20% volume ratio of hexahydropyridine DMF solution was added again and reacted for 10 minutes. The resin was washed once with DMF in the middle. After the reaction was completed, the resin was drained and washed three times with DMF.

[0042] (2) According to the sequence of the peptide from C-terminus to N-terminus, the condensation reaction and Fmoc removal reaction were carried out alternately in a reaction amount of 1.5 mmol, and all the remaining amino acids of the peptide were condensed on the resin. After the last amino acid was condensed, a 20% by volume solution of hexahydropyridine in DMF was added and reacted for 5 minutes. A 20% by volume solution of hexahydropyridine in DMF was added again and reacted for 10 minutes. The mixture was washed once with DMF in the middle. After the reaction was completed, the mixture was drained and washed 3 times with DMF.

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

[0044] (4) The polypeptide-resin obtained in step (3) is placed in a round-bottom flask, and the prepared lysis solution (the volume ratio of the reagent formula of the lysis solution is TFA: thioanisole: phenol: triisopropylsilane: water = 82.5:7.5:5:3:2) is slowly added at 0°C and stirred slowly, and the mixture is reacted at low temperature for 0.5 hour and at room temperature for 2 hours, and the lysis solution is filtered to obtain the lysis solution, and the lysis solution is slowly added to anhydrous ice ether and stirred, and the crude polypeptide is separated by filtration, and washed with ice ether three times to obtain the crude peptide;

[0045] (5) The crude peptide was purified and separated by high performance liquid chromatography, and freeze-dried to obtain a pure peptide. The peptide TAT-nDAD of the present invention was synthesized based on the amino acid sequence of NCOR2464-476 and the cell-penetrating peptide TAT sequence.

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

[0047] Example 2

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

[0049] Control group: HEK293 cells were transfected with HA-NCOR2 WT plasmid and MYC-HDAC3 plasmid;

[0050] Experimental group: HEK293 cells were transfected with HA-NCOR2 Y470A mutant plasmid and MYC-HDAC3 plasmid

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

[0052] The results are as follows Figure 1 and Figure 2 As shown, HDAC3 requires NCOR2 binding for activation, so HA-NCOR2WT and HA-NCOR2 Y470A mutant plasmids were constructed to reduce the interaction between NCOR2 and HDAC3 and affect BACE1, thereby determining the effect of inhibiting the binding of the complex on AD.

[0053] Example 3

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

[0055] WT + control peptide: WT mice were injected with control peptide once a day starting from two months of age until the end of the experiment. Body weight was recorded once a week from the injection of peptide to the start of behavioral experiments.

[0056] WT + experimental peptide: WT mice were injected with experimental peptides once a day starting from two months of age until the end of the experiment. Body weight was recorded once a week from the injection of peptides to the start of behavioral experiments.

[0057] AD+control peptide: The control peptide was injected into AD mice from two months of age, once a day by intraperitoneal injection until the end of the experiment. The body weight was recorded once a week from the injection of peptide to the start of behavioral experiments.

[0058] AD+experimental peptide: AD mice were injected with experimental peptides starting from two months old, intraperitoneally once a day until the end of the experiment, and body weight was recorded once a week from the time of peptide injection to the start of behavioral experiments.

[0059] The initial body weight of the mice was weighed, and during the administration of the peptide drug TAT-nDAD, the body weight of mice of different ages was weighed and monitored. The results were as follows: Figure 3 As shown. Figure 3 It can be seen that WT mice are heavier than AD mice, but the peptide drug TAT-nDAD does not affect the growth of mice.

[0060] Example 4

[0061] The peptide drug TAT-nDAD affects the levels of APP metabolism-related enzymes in the mouse AD cell model. Control group: N2A (N2A APP ) cells, and given the control peptide TAT-scramble;

[0062] Experimental group: N2A with stable APP (N2A APP ) cells, and given the experimental peptide 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 was performed to detect the levels of APP, PS1, BACE1 and CTF-β. The results were as follows Figure 4 As shown, the polypeptide TAT-nDAD of the present invention can significantly reduce the levels of BACE1 and CTFβ in the mouse 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 peptide drug TAT-nDAD on the interaction between NCOR2 and HDAC3 of exogenous transfected plasmid

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

[0067] The results are as follows Figure 5 As 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 peptide drug TAT-nDAD on the interaction between NCOR2 and HDAC3 in mouse AD cell model

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

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

[0072] Example 7

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

[0074] C57 mice were used as the control group, and AD (APP23 / PS45 double transgenic) mice were used as the experimental group. Using normal saline as the solvent, the TAT-nDAD dosage was set at 5 mg / kg / d, and the same volume of normal saline was given once a day. Starting from 2 months of age, the open field test was started 12 weeks after administration. Each mouse was placed in an open box of 40×40×60 cm in turn, and allowed to explore freely for 10 minutes. After the exploration, the mouse was put back in the cage, and the odor in the open field was eliminated with 75% ethanol, foreign objects were removed, and the next mouse was placed in. The number of times and time the mouse reached the middle was recorded. The fewer times and time it reached the middle, the more anxious it was. The results are as follows Figure 7 As 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 peptide drug TAT-nDAD on working memory in mice

[0077] The experimental mice of Example 7 started the Y-maze experiment one day after the open field experiment. In the Y-maze maze, mice were placed and allowed to explore freely for 10 minutes, and the ANY-maze tracking system was used to record the results. After the exploration, the mice were returned to the cage, 75% ethanol was used to eliminate the odor, foreign matter was removed, and the next mouse was placed in. The ANY-maze tracking system was used to record the number of times the mice entered the three arms ABC. After the experiment, the working memory index of the mice was counted, and the cycle index Alteration = (spontaneous alternation number / total number of entries-2) × 100% was calculated.

[0078] The results are as follows Figure 8 As 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 peptide drug TAT-nDAD on spatial learning and memory in mice

[0081] The Morris water maze experiment was used to verify that the spatial learning and memory ability of mice was tested in a circular pool with a radius of 1.5 meters and a height of 60 cm. The pool was divided into four quadrants, marked as NE, NW, SW, SE, or quadrants 1, 2, 3, and 4. Markers of different shapes were affixed to the pool wall of each quadrant to help mice locate in space. In order to create a relatively independent experimental environment, blue curtains were hung around the pool, and markers of corresponding shapes were affixed to different positions of the curtains. In the experiment, a camera installed above the pool was used to track the movement trajectory of the mice. The first day was the adaptation stage. The mice were gently placed in the pool, starting from the first quadrant, facing the pool wall, and freely explored for 120 seconds to adapt to the pool and the surrounding environment. No platform was placed underwater at this time. After the exploration, the mice were taken out and dried, and then put back into the cage. Days 2-6 are the learning phase, during which an underwater hidden platform is placed in the center of the third quadrant, with the top of the platform 1 cm below the water surface. During the experiment, mice are placed into the pool from different quadrants facing the pool wall. The mice need to find the platform within 120 seconds and stay for 2 seconds to be considered a successful escape, and their escape latency (i.e., the time required to find the platform) is recorded. If the mouse fails to find the platform within the specified time, it is guided to the platform and stays on the platform for 20 seconds, and the latency is then recorded as 120 seconds. Learning is performed four times a day, and the average of the four escape latencies is calculated as the escape latency for the day. Day 7 is the testing phase, during which the platform is removed and the mice are placed into the water from the first quadrant facing the pool wall. Their behavioral trajectories within 120 seconds are recorded, and the number of times they cross the platform area is counted. The results are as follows: Fig. 9 As shown, the polypeptide TAT-nDAD of the present invention significantly improved the spatial learning and memory of AD mice in the water maze.

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

[0083] Day 1 is the adaptation stage. The mouse is placed in the escape hole for 3 minutes. After the experiment, the mouse is taken out and put back into the cage. 75% alcohol is used to remove the smell and remove foreign objects, and then the experiment with the next mouse is carried out. Days 2-6 are the training stage. The mouse is first placed in the center of the maze and covered with a black plastic box for 10 seconds. Then its exploration behavior in the maze is recorded. If the mouse finds the target hole and enters the escape hole within 3 minutes, its escape latency is recorded. If the mouse fails to find the target hole within the specified time, it is guided into the escape hole and allowed to stay for 1 minute. Training is carried out twice a day. The disc is randomly rotated each time, but the position of the escape hole remains unchanged. After the experiment, 75% alcohol is also used to remove the smell. Day 7 is the test stage. The escape room is removed and the mouse freely explores the maze for 3 minutes. The accuracy rate of finding the target hole (i.e. the number of times the head explores the escape hole / the total number of times)*100% is recorded. The results are as follows Fig.10 As shown, 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 effect of peptide drug TAT-nDAD on the levels of APP, PS1, BACE1 and CTF-β in the hippocampus of AD mice

[0086] WT + control peptide: The control peptide was injected into WT mice starting from two months of age, intraperitoneally once a day until the end of the experiment.

[0087] WT+experimental peptide: WT mice were injected with experimental peptides starting from two months of age, intraperitoneally once a day until the end of the experiment.

[0088] AD+control peptide: The control peptide was injected into AD mice starting from two months old, intraperitoneally once a day until the end of the experiment.

[0089] AD+experimental peptide: Experimental peptides were injected into AD mice starting from two months old, intraperitoneally once a day until the end of the experiment.

[0090] The mouse hippocampus tissues after the above experiment were lysed, the total hippocampal protein was extracted, and the Western blot experiment was performed. Fig.11 As shown, the experimental peptide TAT-nDAD had no significant 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 technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A polypeptide for inhibiting HDAC3 activity, characterized in that: It is TAT-nDAD and includes at least the gene segment -AECVLYYYLTKKN.

2. The polypeptide for inhibiting HDAC3 activity according to claim 1, characterized in that: The polypeptide 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 an endogenous NCOR2 protein at least includes AECVLYYYLTKKN.

3. The polypeptide for inhibiting HDAC3 activity according to claim 2, characterized in that: The polypeptide gene sequence is TAT-AECVLYYYLTKKN, and the TAT gene sequence is YGRKKRRQRRR.

4. Use of the polypeptide according to any one of claims 1 to 3 in inhibiting HDAC3.

5. Use of the polypeptide according to any one of claims 1 to 3 in reducing the interaction between NCOR2 and HDAC3.

6. Use of the polypeptide according to any one of claims 1 to 3 in reducing APP metabolism and CTF-β.

7. Use of the polypeptide according to any one of claims 1 to 3 in the preparation of drugs for preventing and treating neurodegenerative diseases, anti-tumor drugs, and memory-improving drugs.

8. The use according to claim 7, characterized in that: The neurodegenerative diseases include Alzheimer's disease, neuroinflammation, Parkinson's disease, Huntington's disease, dementia with Lewy bodies, amyotrophic lateral sclerosis, multiple system atrophy, spinocerebellar ataxia, and frontotemporal dementia.

9. The use according to claim 7, characterized in that: The tumors described in the anti-tumor drug include non-small cell lung cancer, small cell lung cancer, breast cancer, gastrointestinal tumors, liver cancer, prostate cancer, kidney cancer, ovarian cancer, brain tumors, skin cancer, leukemia, 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, and thyroid cancer.

10. A polypeptide drug, characterized in that: It comprises the polypeptide described in claims 1 to 3 and / or 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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