A small peptide and its use in the preparation of a medicament for treating alzheimer's disease

Abstract

The application discloses a small peptide with 30 amino acids, and the sequence of the small peptide is shown as SEQ ID NO:1. The small peptide specifically acts on P25 protein, inhibits the combination of the P25 protein and CDK5 kinase, reduces the Tau phosphorylation catalyzed by the CDK5 kinase, reduces the neurofibrillary tangle caused by the excessive phosphorylation of the Tau protein, and improves the clinical symptoms of AD patients, and has a good clinical application prospect in the treatment of AD.

Description

Technical Field

[0001] This invention relates to a small peptide for the treatment of Alzheimer's disease, belonging to the field of biomedicine. Background Technology

[0002] Alzheimer's disease (AD) is a common, age-related, slowly progressive neurodegenerative disease. Two characteristic pathological features of AD are extracellular deposition of amyloid Aβ and intracellular neurofibrillary tangles caused by excessive phosphorylation of Tau protein. According to reports, the incidence of AD is increasing year by year and is exacerbated by the aging population, thus imposing a significant social burden.

[0003] Neurofibrillary tangles are a characteristic pathological feature of Alzheimer's disease (AD). While extensive neuronal death was observed in the brains of AD patients as early as 1906, neurofibrillary tangles were not isolated from the brains of AD patients until 1974. In 1985, it was first discovered that the cause of these erroneous tangles was hyperphosphorylation of Tau protein on neurons. In the normal brain, the microtubule system is the skeletal structure of neurons, and Tau protein can bind to tubulin, promoting the formation of the microtubule system. Therefore, Tau protein plays a crucial role in maintaining the normal morphology and function of neurons. Tau protein has multiple phosphorylation sites, and under normal circumstances, Tau protein maintains its normal physiological function through phosphorylation. However, in the brains of AD patients, Tau protein exhibits hyperphosphorylation. Hyperphosphorylated Tau protein is unable to bind to tubulin to perform its normal function, ultimately leading to extensive neuronal death and the formation of neurofibrillary tangles.

[0004] Tau protein contains multiple phosphorylated kinases. Among them, cyclin-dependent kinase 5 (CDK5) is considered to play a key role in the course of Alzheimer's disease (AD). Normally, CDK5 needs to bind to its chaperone protein P35 to exert its kinase function; this complex has a short half-life. However, in the pathological state of AD, calpain is activated and hydrolyzes P35 into a smaller protein, P25. At this point, CDK5 pathologically binds to P25, extending the half-life of this complex by more than tenfold. This leads to excessive phosphorylation of Tau protein by CDK5, ultimately resulting in neurofibrillary tangles. Therefore, inhibiting the binding of CDK5 and P25, or increasing the degradation rate of the CDK5-P25 complex, would help alleviate the excessive phosphorylation of Tau protein, thereby contributing to the treatment of AD.

[0005] In recent years, the construction of specific functional peptides based on protein degradation targeting chimeras (PROTAC) technology for the treatment of various neurological diseases has become a research hotspot. PROTACs typically consist of three parts: an E3 ligase-binding domain, a target protein-binding domain, and a linker between the two. The mechanism of action of PROTAC peptides is to bind to the target protein and transport it to the proteasome for degradation, thereby directionally reducing the content of the target protein. Compared to traditional small molecule drugs, PROTACs have advantages such as easy penetration of the blood-brain barrier and easy binding to target proteins.

[0006] CN 114736264 A discloses a visualized PROTAC degradation compound for Tau protein; CN 111518215 A discloses a chimera that specifically degrades Tau protein and its encoding gene. In existing literature, PROTAC peptides are designed to directly bind to Tau protein and degrade it via the proteasome pathway. However, due to the lack of a specific target phosphorylated Tau protein, this may lead to the degradation of normal Tau protein, causing side effects. Summary of the Invention

[0007] The purpose of this invention is to provide a novel small peptide designed based on PROTAC technology, which can specifically bind to and induce the degradation of P25 protein, thereby reducing the level of CDK5-catalyzed Tau phosphorylation, reducing phosphorylated Tau protein in the brain, and improving the symptoms of AD patients.

[0008] To achieve the above objectives, the applicant designed the sequence of the small peptide as YGRKKRRQRRR using computer simulation. LARAFGIPVRCYSAE RRRG consists of three sequences: the first is the binding domain peptide sequence of CDK5 and P25, the second is the transmembrane peptide sequence, and the third is the ubiquitination-inducing degradation peptide sequence. The three sequences are linked together through organic synthesis.

[0009] Molecular docking results showed that the small peptide could specifically bind to the P25 protein, and the binding ability of the small peptide to the P25 protein was further verified in vitro using the HEK-293 cell line stably transfected with Tau.

[0010] Next, the Morris water maze test was conducted to investigate the effect of the small peptide on the AD model animal P301S mice. The results showed that the small peptide could significantly improve the cognitive and learning abilities of the P301S mouse model. Immunological detection of Tau protein phosphorylation in the brain of the treated mice revealed that the small peptide could significantly reduce the phosphorylation level of Tau protein at multiple sites in the brain of P301S mice.

[0011] Finally, to confirm that the small peptide can cross the blood-brain barrier from peripheral blood vessels to reach the central nervous system, we attached a fluorescent label to the peptide and injected it into test mice via the tail vein. In vivo imaging was then performed on the animals at different time points. The results showed a significant fluorescent signal in the mouse brain, demonstrating the peptide's ability to penetrate the blood-brain barrier. Simultaneously, imaging of the liver, stomach, lungs, and brain of the injected mice revealed that the fluorescent signal gradually disappeared in these tissues over time, indicating that the small peptide can be excreted through metabolic pathways within the body.

[0012] In summary, the small peptide provided by this invention specifically acts on the P25 protein, inhibiting the binding of the P25 protein to CDK5 kinase, reducing Tau phosphorylation catalyzed by CDK5 kinase, reducing neurofibrillary tangles caused by excessive phosphorylation of Tau protein, and improving the clinical symptoms of AD patients. Moreover, it can penetrate the blood-brain barrier and be excreted from the body through metabolic pathways, showing great promise for clinical application.

[0013] For more detailed technical solutions, please refer to the specific embodiments. Attached Figure Description

[0014] Figure 1 It shows the structure of the TPP peptide and its molecular docking with the P25 protein.

[0015] Figure 2 This is the HPLC chromatogram of TPP peptide.

[0016] Figure 3 This is the mass spectrum of the TPP peptide.

[0017] Figure 4 This is an immunoblotting experiment and its analysis results after TPP peptide was reacted with HEK-293 cell line stably transfected with Tau. ★★ P<0.01.

[0018] Figure 5 These are the results of the Morris water maze test for four groups of mice. In the figure, A is the time to reach the platform area; B is the number of times the mouse crossed the platform; and C is the duration of time spent in the platform quadrant. ★★ P<0.01, ★★★ P<0.005.

[0019] Figure 6 These are typical trajectory diagrams of the movement of mice in each group on the MWM platform.

[0020] Figure 7 This is the result of Western blot analysis of phosphorylation levels of Tau protein sites in the brains of mice in the P301S+Veh and P301S+TPP groups. ★ P<0.05, ★★ P<0.01.

[0021] Figure 8 This image shows the immunofluorescence results of phosphorylation levels of the Tau protein Ser404 site in the CA3, DG, and cortical regions of the brains of mice in the P301S+Veh and P301S+TPP groups. In the figure, A is the immunofluorescence staining photograph; B is the immunofluorescence analysis result. ★ P<0.05.

[0022] Figure 9 This is an in vivo imaging image of a mouse after the tail vein is injected with a fluorescently labeled TPP peptide. Detailed Implementation

[0023] The present invention will be described in detail below through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and not for limiting the scope of protection. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the scope of protection of the present invention. Experimental methods not described in detail in the embodiments can be conventional practices in the art or implemented according to reference books such as "Pharmacological Experimental Methodology".

[0024] Key material sources and their descriptions:

[0025] TPP peptide: synthesized by Wuhan Institute of Biomedical Research, Jianghan University.

[0026] HEK-293 cells: human embryonic kidney cells. The cells used in this experiment expressed the stably transferred Tau gene and were provided by the Wuhan Institute of Biomedical Research, Jianghan University.

[0027] P301S mice: mutant Tau protein transgenic mice, which have been widely used in research on Tau-related diseases (including Alzheimer's disease), provided by Wuhan Institute of Biomedical Research, Jianghan University.

[0028] C57 mice: These are the background mice for P301S mice, providing a genetic background for the mutated gene. They were provided by the Wuhan Institute of Biomedical Research, Jianghan University.

[0029] All other materials not specified are conventional materials in this field.

[0030] Example 1: Synthesis and Characterization of Peptides

[0031] By searching the UniProt database, the applicant found that the CDK5-P25 binding domain peptide is LARAFGIPVRCYSAE. This peptide, along with the transmembrane peptide YGRKKRRQRRR and the ubiquitination-inducing degradation peptide RRRG, was linked together via organic synthesis to obtain the TPP peptide (sequence YGRKKRRQRRRLARAFGIPVRCYSAERRRG, e.g., ...). Figure 1 As shown in Figure A). Computer simulations of protein-protein docking results show that this peptide can specifically bind to the P25 protein (e.g., ...). Figure 1 (as shown in B).

[0032] The transmembrane peptide acts to induce the peptide to cross the phospholipid bilayer of the cell membrane, thereby entering the cell interior; the PTM peptide acts to induce ubiquitination modification of the peptide, and then induce P25 degradation through the proteasome pathway.

[0033] The designed peptide was artificially synthesized, and its structure was characterized by HPLC-MS. The HPLC results of the TPP peptide are as follows: Figure 2 As shown, the mass spectrometry data are as follows: Figure 3 As shown.

[0034] Example 2: Evaluation of peptide efficacy

[0035] 1. In vitro efficacy evaluation of TPP peptides

[0036] The binding affinity of TPP peptide to P25 was evaluated in vitro using a stable Tau-transfected HEK-293 cell line. TPP peptide at concentrations of 0, 50, and 150 μM was added to wells of HEK-293 cells cultured with stable Tau transfection. After 24 hours, cells were collected and proteins were extracted. Western blot results were presented. Figure 4 A) indicates that, compared to the control group, the P25 protein concentration was significantly reduced in both the 50 μM and 150 μM administration groups. Figure 4 B), while the concentration of P35 protein is unaffected. Figure 4 C). This indicates that the TPP peptide can directionally reduce the content of P25 protein in cells, but does not affect the content of P35 protein.

[0037] 2. Morris water maze experiment

[0038] In this experiment, male 6-month-old AD model mice (P301S mice) were randomly divided into two groups of 15 mice each. One group (P301S+TPP group) received a tail vein injection of 20 mg / kg body weight of TPP every 6 days; the other group (P301S+Veh group) received the same amount of saline via tail vein injection every 6 days. Simultaneously, 30 male 6-month-old C57 mice were also randomly divided into two groups of 15 mice each. One group (C57+TPP group) received a tail vein injection of 20 mg / kg body weight of TPP every 6 days; the other group (C57+Veh group) received the same amount of saline via tail vein injection every 6 days. The drug administration continued for one month, after which the four groups of mice were tested.

[0039] (1) Morris water maze test

[0040] The Morris water maze is a classic animal experimental platform for testing spatial cognition and learning abilities in mice. In this experiment, mice in four groups—P301S+TPP, P301S+Veh, C57+TPP, and C57+Veh—were administered medication, and their cognitive abilities were tested using the Morris water maze (MWM). The MWM consists of a circular water tank and a platform. During five days of training, each mouse was placed into the maze from four different quadrants for 60 seconds of free exploration, and the escape latency upon reaching the platform was recorded. On the day of the exploration test, the platform was removed, and each mouse was placed into the maze from a diagonal area of ​​the platform for 60 seconds of free exploration. The time the mice spent in the platform area and the number of times they crossed the platform area were recorded. All behavioral test data were recorded using SuperMaze software.

[0041] During the first 5 days of training, there were no significant differences in escape latency and swimming speed among the four groups of mice. In the detection test on day 6, the time taken for mice in the P301S+TPP group to find the platform area was significantly shorter than that in the P301S+Veh group. Figure 5 A). Meanwhile, the number of times mice in the P301S+TPP group crossed the platform and the duration of time in the platform quadrant were both higher than those in the P301S+Veh group ( Figure 5 B and Figure 5 C). Typical trajectories of mice in each group moving on the MWM platform are shown below. Figure 6 As shown in the Morris water maze results, the invented TPP peptide significantly improved spatial cognition and learning abilities in P301S model mice.

[0042] (2) TPP peptides reduced Tau phosphorylation at animal levels.

[0043] To further verify that the TPP peptide can reduce the phosphorylation level of Tau protein in the brain of P301S mice, the phosphorylation level of Tau protein in the brains of P301S+TPP and P301S+Veh mice was detected by immunoblotting and immunofluorescence.

[0044] The immunoblotting method was as follows: Mice were anesthetized and euthanized with isoflurane gas. Brain tissue was removed from the mice on ice, and hippocampal tissue was isolated. Total protein from the hippocampus was extracted using RIPA (strong) lysis buffer. The immunoblotting method was as follows: Sample proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then immediately transferred to a nitrocellulose (NC) membrane, blocked with 5% skim milk at room temperature for 1 hour, and then incubated overnight with the corresponding primary antibody. The next day, the NC membrane was washed three times with TBST, incubated for 1 hour with the corresponding HRP-labeled secondary antibody at room temperature, washed three times with TBST, and finally, the NC membrane was photographed using chemiluminescent detection solution to detect the expression of the target proteins.

[0045] The immunofluorescence method was as follows: Mice were anesthetized and euthanized with isoflurane gas. The mouse brains were removed on ice and fixed in 4% paraformaldehyde. The fixed brains were sectioned into 30μm sections using a vibratory microtome (Leica VT1000S, Wetzlar, Germany). The sections were washed three times with phosphate buffer solution and then blocked with 0.1% Triton X-100 containing 5% bovine serum albumin. The brain sections were then incubated with the corresponding primary antibody overnight. The next day, the brain sections were washed three times with TBST and incubated with the corresponding fluorescently labeled secondary antibody at 37°C for 1 hour. The sections were washed three times with TBST, stained with Hoechst for 10 minutes, and washed three times with PBS. Finally, the sections were sealed with an anti-fluorescence quencher and the brain sections were photographed and analyzed using a laser scanning confocal microscope (Leica SP8, Germany).

[0046] Experimental results are as follows Figure 7 As shown in Figure A, immunoblotting revealed that, compared to the P301S+Veh group, the P301S+TPP group showed a significant decrease in phosphorylation of Tau protein at both Ser199 and Ser214 sites in the brain. Figure 7 B). Immunofluorescence staining was used to detect phosphorylation of Tau protein Ser404 in the CA3, DG, and cortical regions of the brains of both groups of mice. The results showed that, compared with the P301S+Veh group, phosphorylation of Tau protein Ser404 in the brains of the P301S+TPP group was significantly decreased in the CA3 and cortical regions. Figure 8 A and Figure 8B). Based on the above results, the invented TPP peptide can significantly reduce the phosphorylation level of Tau protein at multiple sites in the brain of P301S mice.

[0047] 3. Animal live imaging

[0048] To confirm that the TPP peptide can cross the blood-brain barrier from peripheral blood vessels to reach the central nervous system, the TPP peptide was tagged with the red fluorescent marker cy3. Mice were injected with TPP-cy3 via the tail vein (10 mg / kg body weight), and in vivo images were taken at 2, 4, 6, 8, 12, 24, 48, 96, 120, and 144 hours using an animal in vivo imaging device. Results are as follows: Figure 9 As shown in Figure A, after TPP was injected via the tail vein, a distinct red signal appeared in the mouse brain, demonstrating that the invented TPP peptide has the ability to cross the blood-brain barrier. Simultaneously, mice injected with TPP-cy3 were euthanized at 6, 36, and 144 hours, and their livers, stomachs, lungs, and brains were photographed. The results are as follows. Figure 9 As shown in Figure B, red signals were detected in mouse liver, stomach, lung, and brain samples at 6 and 36 hours post-injection, and signals were also detected in the cortex and hippocampus. However, no significant red signals were detected in mouse liver, stomach, lung, and brain tissue at 144 hours post-injection, indicating that TPP can be normally excreted through metabolism.

Claims

1. A small peptide having the amino acid sequence shown in SEQ ID NO:

1.

2. The use of the small peptide according to claim 1 in the preparation of a drug for treating Alzheimer's disease.

3. A medicine for treating Alzheimer's disease, the medicine comprising the small peptide of claim 1.

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