Polypeptide for activating Tau lactic acid and application of polypeptide in medicine

Abstract

The invention relates to a polypeptide for activating Tau lactylation and application of the polypeptide in medicine. The polypeptide comprises an amino acid sequence which has at least 80% sequence identity with SEQ ID NO: 1. The polypeptide provided by the invention can be combined with Tau in a targeted manner, competitively inhibit the combination of Tau and HDAC1 and inhibit the delactamation of Tau so as to activate the tau lactaation, has high specificity, and can safely, effectively and reversibly up-regulate the Tau lactaation level.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a polypeptide for activating Tau lactylation and application thereof in medicine. BACKGROUND

[0002] Alzheimer's Disease (AD) is a common neurodegenerative disease in the elderly, mainly manifested as cognitive decline, memory loss, behavioral disorders, and ultimately loss of independent living ability. At present, the global patient population has reached 300 million, and according to the 2023 World Alzheimer's Disease Report, it is expected to reach 139 million by 2050. In China, the number of AD patients over 65 years old exceeds 9 million, which has imposed a heavy burden on the society and the medical system. The reason is that although the pathology of AD has been studied, drugs aimed at reducing pathological features and related pathogenesis have not yet been elucidated to the extent of clinical treatment. Therefore, it is urgent to explore effective molecular targets to improve the quality of life of the elderly population and rapidly reduce the burden on families and society caused by the disease in the aging population.

[0003] Abnormal phosphorylation of Tau protein leads to microtubule disruption, impaired neuronal function, and neurofibrillary tangles. Tau abnormalities can be induced by Aβ, but can also independently trigger nerve damage. Even after Aβ clearance, Tau pathology can still persist. With the in-depth study of Tau protein, Tau has gradually been recognized as a biomarker for AD in clinical practice. p-Tau181, p-Tau217 and p-Tau231 in plasma can be used as early diagnostic markers for AD, and are highly consistent with PET and CSF detection results. At the same time, a biomarker named Alz-Tau® has been developed. This biomarker analyzes the ratio of high molecular weight Tau (HMWTau) to low molecular weight Tau (LMWTau) in platelets, which can effectively distinguish AD patients from healthy people. The levels of total Tau (t-Tau) and p-Tau in saliva are significantly increased in AD patients, which may be a non-invasive detection method. These monitoring methods targeting Tau not only contribute to the early diagnosis of AD, but also serve as important indicators of disease progression and treatment response, thereby providing support for the management of AD and the development of new therapies. However, the current clinical application of Tau mainly focuses on the early diagnosis of AD, and there is still no good exploration of drugs targeting Tau.

[0004] Lactylation modification is a newly discovered modification in recent years, but the lactylation enzyme and delactylation enzyme catalyzing Tau are not clear, since lactylation and acetylation are both modifications on lysine residues, the acetylation modification enzyme may also be the lactylation modification enzyme, and according to the modification enzyme, a related polypeptide is designed to affect the interaction between the modification enzyme and Tau, so as to affect the lactylation level of Tau, and finally affect the learning and memory of AD patients. SUMMARY

[0005] The present application aims to provide a polypeptide for activating Tau lactylation and a medical use thereof.

[0006] The technical problem of the present application is solved by the following technical solutions: In a first aspect, the present application provides a polypeptide for activating Tau lactylation, which comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is HQPGGGKVQIIN (His-Gln-Pro-Gly-Gly-Gly-Lys-Val-Gln-Ile-Ile-Asn).

[0007] In one or more embodiments of the present application, the polypeptide comprises a polypeptide sequence of an endogenous Tau protein and a polypeptide sequence of a cell-penetrating peptide TAT.

[0008] In one or more embodiments of the present application, the polypeptide sequence of the endogenous Tau protein comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1.

[0009] In one or more embodiments of the present application, the polypeptide sequence of the cell-penetrating peptide TAT comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 2 is YGRKKRRQRRR (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg).

[0010] In one or more embodiments of the present application, the polypeptide sequence of the endogenous Tau protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; and the polypeptide sequence of the cell-penetrating peptide TAT comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2.

[0011] In one or more embodiments of the present application, the polypeptide sequence of the endogenous Tau protein and the polypeptide sequence of the cell-penetrating peptide TAT are cyclized by disulfide bond. The amino acid sequence of the polypeptide sequence of Tau lactylation-activated Tau is GRKKRRQRRR-CYS 1 -HQPGGGKVQIIN-CYS 2 i.e. Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys 1 -(His-Gln-Pro-Gly-Gly-Gly-Lys-Val-Gln-Ile-Ile-Asn)-Cys 2, a disulfide bond is formed between Cys 1 and Cys 2.

[0012] The polypeptide provided by the present application is a polypeptide fragment of the endogenous Tau protein, which realizes “activating Tau with Tau” and has high specificity, good safety, easy solubility in water, small molecular weight, and in order to form a cyclic structure, the peptide segment is cyclized by introducing Cys residues at both ends of the peptide chain. The disulfide bond can stabilize the cyclic structure of the peptide segment and prevent the peptide segment from being degraded by proteases. The cyclized polypeptide can interfere with the binding of HDAC1 and Tau in space by forming a stable three-dimensional structure. The cyclized peptide segment inhibits the de-lactylation function of HDAC1 by changing the local conformation or by providing a physical barrier to prevent HDAC1 from approaching and binding to the lactylation site of Tau, without significantly affecting the lactylation of p300 on Tau. With the help of the cell-penetrating peptide TAT sequence, it is easy to penetrate the blood-brain barrier, easy to hydrolyze after playing a role, the generated amino acids can nourish the nerves, synthesis, preparation is convenient, and the cost is relatively low. Compared with small molecule chemical inhibitors and monoclonal antibody drugs, the polypeptide has obvious unique advantages, and is an ideal drug for AD prevention and treatment, and has strong clinical transformation value.

[0013] The polypeptide provided by the present application can target and bind to Tau, competitively inhibit the binding of Tau and HDAC1, inhibit the de-lactylation of HDAC1 on Tau, has high specificity, can safely and effectively reversibly up-regulate the lactylation level of Tau, and significantly improve the cognitive and memory functions of AD models.

[0014] In one or more embodiments of the present application, the molecular weight of the polypeptide is 2.83 KDa, and the isoelectric point pI is 11.88. The polypeptide provided by the present application has small molecular weight and is a cyclized polypeptide, which further enhances the stability of the polypeptide, is easy to dissolve in water, has high isoelectric point pI than the pH value of blood plasma, does not ionize cations in blood, has small irritation, is easy to be degraded and removed by proteases and peptidases in the body after playing a role, is not easy to accumulate, has unique effects and economic advantages compared with small molecule inhibitors and monoclonal antibody drugs, and is a polypeptide drug that can be used for AD prevention and treatment in a true sense.

[0015] In a second aspect, the present application provides use of the polypeptide for activating Tau lactylation in the preparation of a medicament for activating Tau lactylation.

[0016] In one or more embodiments of the present application, the polypeptide activates Tau lactylation by inhibiting the binding of Tau to HDAC1.

[0017] In a third aspect, the present application provides use of the polypeptide for activating Tau lactylation in the preparation of a medicament for preventing and / or treating a neurodegenerative disease.

[0018] In one or more embodiments of the present application, the neurodegenerative disease includes Alzheimer's disease, neuroinflammation, Parkinson's disease, Huntington's disease, Lewy body dementia, amyotrophic lateral sclerosis, multiple system atrophy, spinocerebellar ataxia, and frontotemporal dementia.

[0019] In one or more embodiments of the present application, the neurodegenerative disease includes Alzheimer's disease. The polypeptide for activating Tau lactylation can effectively reduce Tau phosphorylation and improve the cognitive and memory functions of AD.

[0020] In a fourth aspect, the present application provides use of the polypeptide for activating Tau lactylation in the preparation of a medicament for preventing and / or treating a medicament for improving memory.

[0021] The present application has the following advantages: the polypeptide provided by the present application can target Tau, competitively inhibit the binding of Tau to HDAC1, inhibit the de-lactylation of Tau, and activate Tau lactylation. The polypeptide has high specificity, can safely and effectively reversibly up-regulate the level of Tau lactylation. The polypeptide provided by the present application can significantly improve the anxiety and depression of hTau mice and significantly improve the cognitive and memory functions of hTau models. The polypeptide provided by the present application can easily penetrate the blood-brain barrier, ensure its function in the brain, make its administration mode more flexible, and not be limited to intravenous injection, but can be administered by intramuscular injection. The polypeptide provided by the present application has a simple synthesis process, is easy to produce in large quantities, can be made into a lyophilized powder, has great potential in the prevention and treatment of AD, and has good promotion value and clinical transformation value. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings described below are merely exemplary. For those of ordinary skill in the art, other implementation drawings can be derived based on the provided drawings without creative work. The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read. They are not used to limit the implementation conditions of the present invention, so they have no technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed in the present invention without affecting the efficacy and purpose that can be achieved by the present invention.

[0023] Figure 1 As in Example 2, the effects of P300 plasmid or HDAC family inhibitors on Tau lactylation were detected by transferring them into SY5Y cells.

[0024] Figure 2 This is to detect the effect of the polypeptide on the expression of Tau lactate and the interaction between Tau and P300 as well as Tau and HDAC1 in Example 3.

[0025] Figure 3 This is the effect of TAT-H1 polypeptide in Example 4 on anxiety and depression in AD mice.

[0026] Figure 4 This is the effect of the TAT-H1 polypeptide in Example 5 on the working memory of AD mice.

[0027] Figure 5 This is the effect of TAT-H1 polypeptide on learning and memory in AD mice in the water maze experiment in Example 6.

[0028] Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0029] As used herein, the term "prevention" refers to preventing the occurrence of a disease and / or preventing the recurrence of a disease. DETAILED DESCRIPTION

[0030] While the present invention specification describes specific embodiments in detail, those skilled in the art should recognize that the following embodiments are illustrative and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that improvements and modifications to the present invention, without departing from the principles of the present invention, will be made, and that the resulting technical solutions will fall within the scope of the claims. The following examples illustrate the beneficial effects of the present invention.

[0031] Example 1

[0032] A polypeptide for activating Tau lactylation activity, comprising the following steps: (1) According to the polypeptide sequence GRKKRRQRRR-CYS 1 -HQPGGGKVQIIN-CYS 2 The first amino acid at the C-terminus is selected, 0.5 mmol of Fmoc-protected amino acid-Wang Resin is added to the solid-phase reactor. After swelling the resin in DCM for 30 minutes, it is drained and washed with DMF three times. Then, 20% piperidine (piperidine) DMF solution is added by volume ratio for 5 minutes, drained, and 20% piperidine DMF solution is added again for 10 minutes, washed with DMF once in between. After the reaction is complete, wash with DMF three times and reserve; (2) According to the amino acid sequence of the target polypeptide from the C-terminus to the N-terminus, the condensation reaction and Fmoc removal reaction of each amino acid are carried out in turn in an equal molar amount of 1.5 mmol. Condensation uses HBTU / HOBt / DIEA or other conventional condensation systems, and removal still uses 20% piperidine DMF solution. After each condensation or removal reaction, wash with DMF three times. After the condensation of the last amino acid is complete, perform another complete Fmoc removal (as described in step 1) to obtain a free N-terminus. After the reaction is complete, drain and wash with DMF three times and reserve; (3) Add 1 mmol of FITC and an appropriate amount of N-methylmorpholine (NMM) or DIEA as a catalyst to the reactor, react at room temperature for 5-10 minutes, and detect whether the reaction is complete by ninhydrin reagent. After the reaction is complete, wash with DMF and DCM alternately three times, then wash with methanol and shrinkage treatment to obtain dry polypeptide-resin; (4) Transfer the above dry polypeptide-resin to a round-bottom flask, slowly add the pre-prepared cleavage solution (volume ratio TFA: benzyl mercaptan: phenol: triisopropylsilane: water = 82.5:7.5:5:3:2) at 0°C, stir at low temperature for 0.5 hours, then continue to react at room temperature for 2 hours. After the reaction is complete, collect the cleavage solution by suction filtration, and slowly drop it into a large amount of pre-cooled anhydrous diethyl ether to precipitate the crude polypeptide. After collecting the precipitate, wash with diethyl ether three times to obtain the crude polypeptide; (5) Purify and separate the crude peptide by high-performance liquid chromatography, freeze-dry, then obtain the pure polypeptide, according to the Tau268-279 amino acid sequence to form a disulfide bond in the chain to cyclize, plus the cell-penetrating peptide TAT sequence, to synthesize the polypeptide TAT-H1 of the present application.

[0033] The polypeptide TAT-H1 can be prepared into a freeze-dried powder injection, water injection, etc. injection dosage form according to clinical needs, for administration by routes such as intramuscular injection.

[0034] Example 2 Verification of Tau lactylation modification and de-lactylation modification enzyme

[0035] 1. Experimental group Control group: transfection of human Tau plasmid in SY5Y cells and addition of solvent; Experimental group: transfection of human Tau plasmid and P300 plasmid or HDAC family inhibitor or siRNA of HDAC1-3 in SY5Y cells 2. Test method When SY5Y cells are fused to 80%, human Tau plasmid or Tau and P300 plasmid is introduced, and protein lysate is extracted after 24 hours, and Western blot is used to detect the expression level of Tau lactylation; when SY5Y cells are fused to 80%, human Tau plasmid and control solvent or Tau plasmid and different HDAC family inhibitors are introduced, and Western blot is used to detect the expression level of Tau lactylation; when SY5Y cells are fused to 50%, siRNA of HDAC1-3 is introduced, and protein lysate is extracted after 24 hours, and Western blot is used to detect the expression level of Tau lactylation.

[0036] 3. Experimental results Figure 1 In order to detect the influence of P300 plasmid or HDAC family inhibitor introduced in SY5Y cells on Tau lactylation in Example 2. It can be known that P300 can up-regulate the expression of Tau lactylation, and the use of different HDAC family inhibitors confirms that HDAC1-3 plays an important role in the de-lactylation of Tau, and therefore the siRNA of HDAC1-3 is constructed to further determine which or which of the HDAC family plays a function in the de-lactylation of Tau, and the results show that HDAC1 plays an important role in the de-lactylation of Tau, thereby determining that HDAC1 is the de-lactylation enzyme of Tau.

[0037] Example 3 Effect of polypeptide drug TAT-H1 on Tau lactylation and Tau and its lactylation modification enzyme

[0038] In SY5Y cells, transfect Tau plasmid, add polypeptide TAT-H1 of the application at a concentration of 10 μM, treat cells for 24 h, and detect the expression level of Tau lactylation by Western blot; in SY5Y cells, transfect Tau plasmid, add TAT-H1, and perform immunoprecipitation (Co-IP) experiment. That is, 500 μg of protein lysate is added to P300 or HDAC1 antibody at 4°C overnight, the complex is boiled in 5x sample buffer at 95°C for 5 min, and the interaction between polypeptide drug TAT-H1 and Tau lactylation and P300 and HDAC1 is detected.

[0039] Figure 2 To detect the effect of the polypeptide on the expression of Tau lactylation and the interaction of Tau and P300 and Tau and HDAC1 in Example 3. TAT-H1 in the figure is TAT-Tau-H1 of the application. The results show that the polypeptide TAT-H1 of the application can up-regulate the expression of Tau lactylation, and only affect the interaction of Tau lactylation and HDAC1, but not affect the interaction of Tau and P300. Figure 3

[0040] Example 4 Verification of the effect of polypeptide drug TAT-H1 on anxiety and depression of mice

[0041] C57 mice are used as controls, and hTau (human Tau transgenic) mice are used as experimental groups. Physiological saline is used as a solvent, and the TAT-H1 administration dose is set to 5 mg / kg / d, and the same volume of physiological saline is given. Once a day, from 3 months of age, after 8 weeks of administration, the open field experiment is started. Each mouse is placed in an open field device with a size of 40 × 40 × 60 cm in turn, and freely moves for 10 minutes. After the experiment, the mouse is returned to its original cage, the open field is cleaned with 75% ethanol to remove residual odors and foreign matter, and the next mouse is tested after ensuring environmental consistency. The anxiety level of the mouse is evaluated by recording the number of times and the time of entering the central area, and the fewer the number of times and the time, the more obvious the anxiety state of the mouse.

[0042] Figure 3 To detect the effect of TAT-H1 polypeptide on anxiety and depression of AD mice in Example 4; TAT-H1 in the figure is TAT-Tau-H1 of the application. The results show that the polypeptide TAT-H1 of the application significantly improves the anxiety and depression of hTau mice in the open field.

[0043] Example 5 Verification of the effect of polypeptide drug TAT-H1 on working memory of mice

[0044] ​The test mice of Example 4 were subjected to Y-maze test one day after the open field test. The mice were placed in the Y-maze and allowed to freely explore for 6 minutes, and the activity track of the mice was automatically recorded by the ANY-maze behavior tracking system during the exploration. After each round of test, the mice were returned to the original cage, and the maze was cleaned with 75% ethanol to remove odor residues and foreign matters, so as to ensure the consistency of the test environment for the next mouse. The sequence and number of times of entering the three arms A, B and C by the mice were recorded by the system, and used to evaluate the working memory ability of the mice. The proportion of spontaneous alternation behavior, i.e. alternation index, was calculated according to the formula: Alternation = (number of spontaneous alternation times / total entering times-2) x 100%.

[0045] Figure 4 For the effect of TAT-H1 polypeptide on the working memory of AD mice in Example 5, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001; and TAT-H1 in the figure is TAT-Tau-H1 of the application. The results show that the polypeptide TAT-H1 of the application can improve the working memory of hTau mice.

[0046] Example 6 Verification of the effect of polypeptide drug TAT-H1 on the spatial learning and memory of mice

[0047] The Morris water maze experiment is used to evaluate the spatial learning and memory ability of mice. The experiment is carried out in a circular pool with a radius of 1.5 meters and a height of 60 centimeters, which is divided into four quadrants, marked as NE, NW, SW and SE, or numbered as 1 to 4 quadrants. Each quadrant corresponds to the pool wall above which a visual mark with different shapes is set to assist the mouse in spatial positioning. To create a stable spatial cue environment, a blue cloth curtain is hung around the pool, and corresponding graphic marks are added at different positions. A camera is installed above the pool to record the movement trajectory of the mouse in real time. Adaptation stage: the mouse is gently placed in the pool from the first quadrant, facing the pool wall, and freely explores for 120 seconds without an underwater platform, aiming to familiarize it with the pool environment and surrounding spatial cues. After the exploration, the mouse is taken out, dried with a towel and put back into the feeding cage. Learning and training stage: an underwater hidden platform is set in the center of the third quadrant, with the platform surface about 1 cm from the water surface. The mouse is placed in different quadrants in turn, all facing the pool wall. Each experiment lasts for 120 seconds, and if the mouse finds the platform and stays on it for more than 2 seconds within the specified time, it is considered a successful escape, and the escape latency (i.e. the time taken to find the platform) is recorded. If it fails to find the platform within the specified time, it is guided to the platform and forced to stay for 20 seconds, and the escape latency is recorded as 120 seconds. A total of 4 training sessions are conducted each day. Spatial memory test stage: the underwater platform is removed, and the mouse is placed in the water from the first quadrant facing the pool wall, and freely swims for 120 seconds. By tracking its movement trajectory, the number of times it crosses the original platform location area is counted to evaluate its spatial memory retention ability.

[0048] Figure 5 For the effect of TAT-H1 polypeptide on the learning and memory of AD mice in the water maze experiment of Example 6, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001; TAT-H1 in the figure is the TAT-Tau-H1 of the present application. The results show that the polypeptide TAT-H1 of the present application significantly improves the spatial learning and memory of hTau mice in the water maze.

[0049] The present application describes specific embodiments in detail, and those skilled in the art should recognize that the above embodiments are exemplary and cannot be understood as limiting the present application. For those skilled in the art, without departing from the principles of the present application, through several improvements and modifications of the present application, the technical solutions obtained by the improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A polypeptide that activates Tau lactylation, characterized in that The polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that The polypeptide includes the polypeptide sequence of endogenous Tau protein and the polypeptide sequence of cell-penetrating peptide TAT.

3. The polypeptide according to claim 2, characterized in that The polypeptide sequence of the endogenous Tau protein includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

1.

4. The polypeptide according to claim 2, characterized in that The polypeptide sequence of the cell-penetrating peptide TAT includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

2.

5. The polypeptide according to claim 2, characterized in that The polypeptide sequence of the endogenous Tau protein includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; the polypeptide sequence of the cell-penetrating peptide TAT includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

2.

6. The polypeptide according to any one of claims 2 to 5, characterized in that The polypeptide sequence of the cell-penetrating peptide TAT is cyclized via a disulfide bond.

7. Use of the polypeptide according to any one of claims 1 to 6 in the preparation of a drug for activating Tau lactylation.

8. The use according to claim 7, wherein the polypeptide activates Tau lactylation by inhibiting the binding of Tau to HDAC1.

9. Use of the polypeptide according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating a neurodegenerative disease, preferably, the neurodegenerative disease is Alzheimer's disease, neuroinflammation, Parkinson's disease, Huntington's disease, Lewy body dementia, amyotrophic lateral sclerosis, multiple system atrophy, spinocerebellar ataxia or frontotemporal dementia.

10. Use of the polypeptide according to any one of claims 1 to 6 in the preparation of a drug for preventing and / or treating memory improvement.

Images