Polypeptides that activate tau lactylation and medical uses thereof

Abstract

The application discloses a polypeptide for activating Tau lactylation and a medical application thereof. The polypeptide comprises an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 1. The polypeptide provided by the application can be targeted to bind to Tau, competitively inhibit the binding of Tau to HDAC1, inhibit the delactylation of Tau, and thus activate the lactylation of Tau. The polypeptide has high specificity, can safely and effectively reversibly increase the lactylation level of Tau, and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a polypeptide that activates Tau lactation and its application in medicine. Background Technology

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease among the elderly, mainly characterized by cognitive decline, memory loss, and behavioral disorders, ultimately leading to the loss of independent living ability. Currently, there are 30 million patients worldwide, and according to the 2023 World Alzheimer's Disease Report, this number is projected to reach 139 million by 2050. In my country, the number of AD patients aged 65 and above exceeds 9 million, placing a heavy burden on society and the healthcare system. The reason for this is that while the pathology of AD has been studied, drugs primarily aimed at alleviating pathological features and their related pathogenesis are still far from being fully elucidated to the point of clinical treatment. Therefore, exploring effective molecular targets to improve the quality of life for the elderly and rapidly reduce the burden of the disease on families and society due to the aging population is urgently needed.

[0003] Aberrant phosphorylation of Tau protein leads to microtubule damage, impaired neuronal function, and neurofibrillary tangles. Tau abnormalities can be induced by Aβ, but can also independently cause neurological damage, and Tau pathology persists even after Aβ clearance. With in-depth research on Tau protein, its use as a biomarker for Alzheimer's disease (AD) is gaining clinical acceptance. Plasma p-Tau181, p-Tau217, and p-Tau231 can serve as early diagnostic markers for AD, showing high concordance with PET and CSF results. Simultaneously, a biomarker called Alz-Tau® has been developed, which effectively distinguishes AD patients from healthy individuals by analyzing the ratio of high molecular weight Tau (HMWTau) to low molecular weight Tau (LMWTTau) in platelets. Total Tau (t-Tau) and p-Tau levels in saliva are significantly elevated in AD patients, potentially offering a non-invasive detection method. These Tau-targeted monitoring techniques not only aid in the early diagnosis of Alzheimer's disease (AD), but also serve as important indicators of disease progression and treatment response, thus supporting the management of AD and the development of new therapies. However, the current clinical application of Tau is mainly focused on the early diagnosis of AD, and drugs targeting Tau have not yet been well explored.

[0004] Lactic acidification is a newly discovered modification in recent years, but the lactylases and delactylases that catalyze Tau are still unclear. Since both lactation and acetylation are modifications on lysine residues, acetylation-modifying enzymes may also be lactation-modifying enzymes. At the same time, based on this modifying enzyme, related peptides can be designed to affect the lactation level of Tau by influencing the interaction between the modifying enzyme and Tau, in order to ultimately affect the learning and memory of AD patients. Summary of the Invention

[0005] The purpose of this invention is to provide a polypeptide that activates Tau lactation and its application in medicine.

[0006] The technical problem solved by this invention is achieved by the following technical solution: In a first aspect, the present invention provides a polypeptide that activates Tau lactation, said polypeptide comprising 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 invention, the polypeptide includes a polypeptide sequence of endogenous Tau protein and a polypeptide sequence of cell-penetrating peptide TAT.

[0008] In one or more embodiments of the present invention, 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 invention, 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 invention, the polypeptide sequence of the endogenous Tau protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; 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 invention, the polypeptide sequence of the endogenous Tau protein and the polypeptide sequence of the cell-penetrating peptide TAT are cyclized via disulfide bonds. The amino acid sequence of the polypeptide sequence that activates Tau lactation is GRKKRRQRRR-CYS. 1 -HQPGGGKVQIIN-CYS 2 That is, Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys¹-(His-Gln-Pro-Gly-Gly-Gly-Lys-Val-Gln-Ile-Ile-Asn)-Cys², where disulfide bonds are formed between Cys¹ and Cys².

[0012] The polypeptide drug provided by this invention is a polypeptide fragment of endogenous Tau protein, achieving "Tau lactation activation via Tau". It is highly specific, has good safety profile, is readily soluble in water, and has a small molecular weight. To form a cyclic structure, Cys residues are introduced at both ends of the peptide chain, cyclizing the peptide segment. Disulfide bonds stabilize the cyclic structure of the peptide segment, preventing degradation by proteases. The cyclized polypeptide, by forming a stable three-dimensional structure, can spatially interfere with the binding of HDAC1 to Tau. The cyclized peptide inhibits delactation by preventing HDAC1 from approaching and binding to the lactation site of Tau through local conformational changes or by providing a physical barrier, without significantly affecting the lactation of Tau by p300. With the help of the cell-penetrating peptide TAT sequence, it easily crosses the blood-brain barrier. After exerting its effect, it is easily hydrolyzed, and the resulting amino acids can nourish nerves. It is convenient to synthesize and formulate, has low cost, and exhibits significant unique advantages over smaller molecule chemical inhibitors and monoclonal antibodies, making it an ideal drug for the prevention and treatment of Alzheimer's disease (AD) and possessing strong clinical translational value.

[0013] The peptide provided by this invention can target and bind to Tau, competitively inhibit the binding of Tau to HDAC1, and inhibit the delactation of Tau by HDAC1. It has high specificity, can safely, effectively and reversibly upregulate Tau lactation levels, and significantly improve cognitive and memory functions in AD models.

[0014] In one or more embodiments of the present invention, the polypeptide has a molecular weight of 2.83 kDa and an isoelectric point (pI) of 11.88. The polypeptide provided by the present invention has a small molecular weight and is a cyclized polypeptide, further enhancing its stability. It is readily soluble in water, has an isoelectric point (pI) higher than the plasma pH, does not ionize into cations in the blood, has low irritation, and is easily degraded and cleared by proteases and peptidases in the body after exerting its effect, making it less prone to accumulation. It possesses unique effects and economic advantages compared to smaller molecule inhibitors and monoclonal antibodies, making it a truly viable polypeptide drug for the prevention and treatment of Alzheimer's disease (AD).

[0015] In a second aspect, the present invention provides the use of the above-mentioned peptide that activates Tau lactation in the preparation of a drug that activates Tau lactation.

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

[0017] In a third aspect, the present invention provides the use of the above-mentioned peptide that activates Tau lactation in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases.

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

[0019] In one or more embodiments of the present invention, the neurodegenerative disease includes Alzheimer's disease. The aforementioned peptides that activate Tau lactation can effectively reduce Tau phosphorylation and improve cognitive and memory function in AD.

[0020] In a fourth aspect, the present invention provides the use of the above-mentioned peptide that activates Tau lactation in the preparation of a drug for the prevention and / or treatment of memory improvement.

[0021] The present invention offers the following beneficial effects: The peptides provided by this invention can target and bind to Tau, competitively inhibiting the binding of Tau to HDAC1, inhibiting Tau delactation and thus activating Tau lactation. This demonstrates high specificity and can safely, effectively, and reversibly upregulate Tau lactation levels. The peptides provided by this invention can significantly improve anxiety and depression in hTau mice and significantly improve cognitive and memory functions in the hTau model. The peptide drugs provided by this invention easily cross the blood-brain barrier, ensuring their effectiveness in the brain and allowing for more flexible administration methods, not limited to intravenous injection, but also via intramuscular injection. The peptides provided by this invention have a simple synthesis process, are easy to mass-produce, can be made into lyophilized powder, and have great potential in the prevention and treatment of AD, possessing significant promotional and clinical translational value. Attached Figure Description

[0022] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are merely exemplary. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort. The structures, proportions, sizes, etc., depicted in this specification are only used to complement the content disclosed in the specification for those skilled in the art to understand and read, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size, without affecting the effects and objectives achieved by the present invention, should still fall within the scope of the technical content disclosed in the present invention.

[0023] Figure 1 In Example 2, the effect on Tau lactation was detected by transfecting SY5Y cells with P300 plasmid or an inhibitor of the HDAC family.

[0024] Figure 2 The purpose of Example 3 was to detect the effect of the peptide on Tau lactation expression and its interaction with Tau and P300, as well as with Tau and HDAC1.

[0025] Figure 3 The effect of TAT-H1 peptide on anxiety and depression in AD mice in Example 4.

[0026] Figure 4 The effect of TAT-H1 peptide on working memory in AD mice in Example 5.

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

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

[0029] As described in this invention, the term "prevention" refers to preventing the occurrence of disease and / or preventing the recurrence of disease. Detailed Implementation

[0030] This specification provides a detailed description of specific embodiments. Those skilled in the art should recognize that the following embodiments are exemplary and should not be construed as limiting the invention. For those skilled in the art, various improvements and modifications can be made to the invention without departing from its principles; such improvements and modifications also fall within the scope of protection of the claims. The beneficial effects of the invention are illustrated below through specific examples.

[0031] Example 1

[0032] A polypeptide that activates Tau lactation activity, comprising the following steps: (1) Based on the polypeptide sequence GRKKRRQRRR-CYS 1 -HQPGGGKVQIIN-CYS 2 The first amino acid at the C-terminus was selected, and 0.5 mmol of the Fmoc-protected amino acid, Wang Resin, was added to a solid-phase reactor. The resin was swollen with DCM for 30 minutes, then dried under vacuum and washed three times with DMF. Next, a 20% (v / v) piperidine DMF solution was added and reacted for 5 minutes, dried under vacuum, and then another 20% piperidine DMF solution was added and reacted for 10 minutes, washing once with DMF in between. After the reaction was complete, the mixture was washed three times with DMF and set aside. (2) Following the amino acid sequence of the target peptide from C-terminus to N-terminus, perform condensation and Fmoc removal reactions sequentially at equimolar amounts of 1.5 mmol for each amino acid. Condensation is performed using HBTU / HOBt / DIEA or other conventional condensation systems, while removal is performed using 20% ​​piperidine DMF solution. After each condensation or removal reaction, wash three times with DMF. After the last amino acid condensation is complete, perform a complete Fmoc removal reaction (as described in step 1) to obtain the free N-terminus. After the reaction is complete, dry the mixture and wash three times with DMF for later use. (3) Add 1 mmol FITC and an appropriate amount of N-methylmorpholine (NMM) or DIEA as catalyst to the reactor and react at room temperature for 5–10 minutes. Check the completeness of the reaction using ninhydrin reagent. After the reaction is complete, wash three times alternately with DMF and DCM, then wash with methanol and shrink to obtain dry polypeptide-resin; (4) Transfer the dried polypeptide-resin to a round-bottom flask, and slowly add the pre-prepared lysis buffer (volume ratio TFA: benzyl sulfide: phenol: triisopropylsilane: water = 82.5:7.5:5:3:2) at 0°C. After stirring at low temperature for 0.5 hours, restore to room temperature and continue the reaction for 2 hours. After the reaction is complete, collect the lysis buffer by filtration and slowly add it dropwise to a large amount of pre-cooled anhydrous ice-cold ether to precipitate crude polypeptide. After collecting the precipitate, wash it three times with ice-cold ether to obtain crude polypeptide; (5) The crude peptide was purified and separated by high performance liquid chromatography, lyophilized, and then the pure peptide was obtained. The peptide was cyclized according to the intrachain disulfide bond formed by the Tau268-279 amino acid sequence, and the cell membrane peptide TAT sequence was added to synthesize the peptide TAT-H1 of the present invention.

[0033] The polypeptide TAT-H1 can be prepared into lyophilized powder injections, aqueous injections, and other injection formulations according to clinical needs, for administration via intramuscular injection and other routes.

[0034] Example 2 Verification of Tau lactation and delactation enzymes

[0035] 1. Experimental Groups Control group: SY5Y cells transfected with human Tau plasmid and with added solvent; Experimental group: SY5Y cells were transfected with human Tau plasmid and P300 plasmid or HDAC family inhibitors, or HDAC1-3 siRNA. 2. Test Methods When SY5Y cells reached 80% confluence, they were transfected with human Tau plasmid or Tau and P300 plasmid. After 24 hours, protein lysates were extracted, and Tau lactation expression levels were detected by Western blot. When SY5Y cells reached 80% confluence, they were transfected with human Tau plasmid and control solvent or Tau plasmid and inhibitors of different HDAC families. Tau lactation expression levels were detected by Western blot. When SY5Y cells reached 50% confluence, they were transfected with siRNA of HDAC1-3 respectively. After 24 hours, protein lysates were extracted, and Tau lactation expression levels were detected by Western blot.

[0036] 3. Experimental Results Figure 1 In Example 2, the effects of transfecting SY5Y cells with the P300 plasmid or HDAC family inhibitors on Tau lactation were examined. It was found that P300 can upregulate Tau lactation expression. Furthermore, the use of different HDAC family inhibitors confirmed that HDAC1-3 play a crucial role in Tau delactation. Therefore, siRNAs for each of HDAC1-3 were constructed to further determine which HDAC family(s) function in Tau delactation. The results showed that HDAC1 plays a crucial role in Tau delactation, thus identifying HDAC1 as the delactylase of Tau.

[0037] Example 3 The effects of the peptide drug TAT-H1 on Tau lactation and on Tau and its lactation-modifying enzymes

[0038] In SY5Y cells, Tau plasmid was transfected, and the peptide TAT-H1 of this invention was added at a concentration of 10 μM. Cells were treated for 24 h, and Western blot was used to detect the expression level of Tau lactation. In SY5Y cells, Tau plasmid was transfected, and TAT-H1 was added for co-immunoprecipitation (Co-IP) experiments. Specifically, 500 μg of protein lysis buffer was added to P300 or HDAC1 antibody and incubated overnight at 4°C. The complex was then boiled in 5x sample buffer at 95°C for 5 min. The interaction between the peptide drug TAT-H1 and Tau lactation was detected, along with that between P300 and HDAC1.

[0039] Figure 2 This example illustrates the effect of the peptide on Tau lactation expression and its interaction with Tau and P300, as well as with HDAC1, as determined in Example 3. In the figure, TAT-H1 represents the TAT-Tau-H1 strain of this invention. Results are as follows... Figure 3 As shown, the present invention’s peptide TAT-H1 can upregulate the expression of Tau lactation and only affects the interaction between Tau lactation and HDAC1, without affecting the interaction between Tau and P300.

[0040] Example 4 Validation of the effects of the peptide drug TAT-H1 on anxiety and depression in mice

[0041] C57 mice were used as controls, and hTau (human Tau transgenic) mice were used as the experimental group. TAT-H1 was administered at a dose of 5 mg / kg / day, with the same volume of saline solution given once daily. Starting at 3 months of age, after 8 weeks of administration, the open field test was conducted. Each mouse was individually placed in a 40 × 40 × 60 cm open field device and allowed free movement for 10 minutes. After the experiment, the mice were returned to their original cages, and the open field was cleaned with 75% ethanol to remove residual odors and foreign objects, ensuring environmental consistency, before testing the next mouse. Anxiety levels were assessed by recording the number of times the mouse entered the central area and the duration of its stay; fewer entries and shorter stays indicated greater anxiety.

[0042] Figure 3 The figure shows the effect of the TAT-H1 peptide on anxiety and depression in AD mice in Example 4; TAT-H1 in the figure is the TAT-Tau-H1 of this invention. The results show that the TAT-H1 peptide of this invention significantly improved anxiety and depression in hTau mice in an open field.

[0043] Example 5 Validation of the effect of peptide drug TAT-H1 on working memory in mice

[0044] One day after the open field experiment, the mice in Example 4 began the Y-maze experiment. The mice were placed in a Y-shaped maze and allowed to explore freely for 6 minutes, during which their activity was automatically recorded by the ANY-maze behavior tracking system. After each round of the experiment, the mice were returned to their original cages, and the maze was cleaned with 75% ethanol to remove odor residue and foreign objects, ensuring consistency of the testing environment for the next mouse. The system recorded the order and number of times the mice entered arms A, B, and C to assess their working memory capacity. The proportion of spontaneous alternation behaviors, i.e., the alternation score, was calculated using the formula: Alternation = (Number of spontaneous alternations / Total number of entries - 2) × 100%.

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

[0046] Example 6 Validation of the effect of the peptide drug TAT-H1 on spatial learning and memory in mice

[0047] The Morris water maze test was used to assess the spatial learning and memory abilities of mice. The experiment was conducted in a circular pool with a radius of 1.5 meters and a height of 60 centimeters, divided into four quadrants labeled NE, NW, SW, and SE, or numbered 1 to 4. Visual markers of varying shapes were placed above the pool wall corresponding to each quadrant to assist the mice in spatial localization. To create a stable spatial cue environment, blue curtains were hung around the pool, with corresponding graphic markers attached at different locations. A camera was installed above the pool to record the mice's movements in real time. Adaptation phase: Mice were gently placed into the pool from quadrant 1, facing the pool wall, and allowed to explore freely for 120 seconds without an underwater platform, allowing them to become familiar with the pool environment and surrounding spatial cues. After exploration, the mice were removed, gently dried with a towel, and returned to their cages. Learning and training phase: An underwater hiding platform was placed in the center of quadrant 3, with the platform surface approximately 1 cm above the water surface. Mice were placed into the pool from different quadrants each day, always facing the pool wall. Each experiment lasted 120 seconds. If a mouse found the platform within the allotted time and stayed on it for more than 2 seconds, it was considered a successful escape, and its escape latency (i.e., the time taken to find the platform) was recorded. If the mouse failed to find the platform within the time limit, it was guided to the platform and forced to stay there for 20 seconds; the escape latency was recorded as 120 seconds. Training was conducted four times a day. Spatial memory test phase: The underwater platform was removed, and the mouse was placed in the water from the first quadrant facing the pool wall, swimming freely for 120 seconds. Its spatial memory retention ability was assessed by tracking its movement trajectory and counting the number of times it crossed the original platform location area.

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

[0049] This invention specification provides a detailed description of specific embodiments. Those skilled in the art should recognize that the above embodiments are exemplary and should not be construed as limiting the invention. For those skilled in the art, various improvements and modifications can be made to the invention without departing from its principles, and the resulting technical solutions also fall within the scope of protection of the claims of this invention.

Claims

1. The use of a polypeptide that activates Tau lactation in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease, characterized in that, The polypeptide comprises a polypeptide sequence of endogenous Tau protein and a polypeptide sequence of cell-penetrating peptide TAT cyclized by disulfide bonds. The amino acid sequence of the peptide that activates Tau lactation is GRKKRRQRRR-CYS 1 -HQPGGGKVQIIN-CYS 2 That is, Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys¹-(His-Gln-Pro-Gly-Gly-Gly-Lys-Val-Gln-Ile-Ile-Asn)-Cys², where disulfide bonds are formed between Cys¹ and Cys².

2. The application of a polypeptide that activates Tau lactation in the preparation of drugs for improving memory, characterized in that, The polypeptide comprises a polypeptide sequence of endogenous Tau protein and a polypeptide sequence of cell-penetrating peptide TAT cyclized by disulfide bonds. The amino acid sequence of the peptide that activates Tau lactation is GRKKRRQRRR-CYS 1 -HQPGGGKVQIIN-CYS 2 That is, Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys¹-(His-Gln-Pro-Gly-Gly-Gly-Lys-Val-Gln-Ile-Ile-Asn)-Cys², where disulfide bonds are formed between Cys¹ and Cys².

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