Discovery of Alzheimer's disease target spot and drug research, development and application based on target spot

Abstract

The invention discloses an Alzheimer's disease target spot, the target spot is Aurora A kinase, and the expression of the Aurora A kinase and the expression of p-Aurora-A (Thr288) kinase in the active form of the Aurora A kinase in the brain of an AD patient are increased. Compared with the prior art, the application has the advantages that the provided Aurora A kinase can be used as a new target spot for treating AD, targeted treatment of AD is expected to be performed through an antibody and a vaccine related to the Aurora A kinase or a selective inhibitor adopting the target spot, and a new major breakthrough is made in AD treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an Alzheimer's disease target point discovery and drug research and development application based on the target point. BACKGROUND

[0002] Alzheimer's disease (AD) is a central nervous system degenerative disease, and its main clinical manifestations are progressive memory impairment, cognitive dysfunction, personality changes, mental symptoms, and loss of daily living ability, etc., which is the most common form of dementia in the elderly.

[0003] The incidence of AD in people over 65 years old is about 5%, and the incidence in people over 80 years old is as high as 20%, and the disease is gradually becoming younger. At present, AD has become the fourth killer after cardiovascular disease, malignant tumor, stroke, etc., which seriously endangers human health.

[0004] The pathological features of AD patients mainly include extracellular amyloid β protein (Aβ) aggregation, intracellular neurofibrillary tangles, selective neuron loss, and chronic neuroinflammatory response. At present, there is still no safe and effective therapeutic drug in the world. Therefore, finding new targets for AD and safe and reliable AD prevention and effective treatment methods are the urgent needs at present.

[0005] Aurora kinase belongs to the serine / threonine kinase family, which has three highly homologous members: Aurora A, Aurora B and Aurora C. Among them, Aurora A kinase is a centrosome-associated kinase that exists in eukaryotic cells and plays a crucial role in important mitotic processes such as centrosome replication, bipolar spindle formation, chromosome rearrangement and chromosome checkpoint monitoring. It has been found that Aurora-A kinase is highly expressed in various tumors, and some Aurora-A kinase selective inhibitors have been used in clinical tumor treatment. SUMMARY

[0006] The technical problem to be solved by the present application is to provide an Alzheimer's disease target point discovery and drug research and development application based on the target point based on Aurora A kinase research, which becomes a new potential target for AD treatment.

[0007] To solve the above technical problems, the technical scheme provided by the present application is as follows: an Alzheimer's disease target point, the target point is Aurora A kinase.

[0008] In another aspect, the present application discloses the use of Alzheimer's disease target, the Aurora A kinase is abnormally increased in the brain of Alzheimer's disease patients.

[0009] Preferably, the Aurora A kinase is associated with the expression of p-Aurora-A (Thr288) kinase in its active form.

[0010] In another aspect, the present application discloses an Alzheimer's disease treatment drug, which comprises a selective inhibitor of Aurora A kinase, an antibody associated with Aurora A kinase, and a vaccine or a viral vector carrying a fragment of Aurora A kinase gene. The drug can inhibit the expression level of Aurora A kinase and its active form p-Aurora-A (Thr288) kinase.

[0011] In another aspect, the present application discloses a method for preparing an Alzheimer's disease treatment drug, comprising the following steps: S1: screening a selective inhibitor of Aurora A kinase; S2: mixing the screened inhibitor with a pharmaceutically acceptable carrier to prepare a drug preparation; S3: preparing an antibody associated with Aurora A kinase, and a vaccine or a viral vector carrying a fragment of Aurora A kinase gene.

[0012] In another aspect, the present application discloses the use of an Alzheimer's disease treatment drug in the prevention or treatment of Alzheimer's disease.

[0013] Compared with the prior art, the present application has the advantages that: the present application first uses the tumor-related cell cycle protein Aurora A kinase in the field of AD research, which is expected to become a new potential target for AD treatment. The high-selectivity and specific inhibitor, the antibody associated with Aurora A kinase, and the vaccine can become an AD treatment drug, and provide a new direction for AD treatment. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The expression of Aurora A kinase and p-Aurora A kinase (Thr288) in the brains of mice of different months (1A-1C) (W: Wildtype mice; A: AD mice; n=8) and the hippocampus of late AD patients (1D-1G) (N: Normal control; A: AD patient; n=15) is shown.

[0015] Figure 2 The expression of p-Aurora-A kinase (Thr288) in the brains of four genotypes of mice is shown.

[0016] Figure 3 This study demonstrated that half-knockout of A kinase in Aurora reduced Aβ deposition and oligomer formation in the brains of 9-month-old APP / PS1 mice.

[0017] Figure 4 This study demonstrated that partial knockout of Aurora A kinase improved learning and memory and enhanced synaptic plasticity in APP / PS1 mice. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to the accompanying drawings.

[0019] In this invention, it was found that Aurora A kinase and its active form were abnormally elevated in the brains of AD transgenic mice of different ages and patients with advanced AD. (1) It was found that the expression of Aurora A kinase and p-Aurora-A(Thr288) kinase in the brains of AD transgenic mice of different ages was significantly higher than that in age-matched WT mice; in the brains of elderly patients with advanced AD, the expression of Aurora A kinase and p-Aurora-A(Thr288) kinase was significantly higher than that in age-matched healthy individuals, and the difference was highly significant. Figure 1 ).

[0020] (2) AD transgenic mice (APPswe / PS1ΔE9, B6C3) were compared with Aurora-A + / - (C) 57 The mice were hybridized to obtain four genotypes: APP / PS1-Aurora-A + / - APP / PS1, WT and Aurora-A + / - Mice. Western blot analysis of brain tissue from four genotype mice at 4 months of age revealed APP / PS1-Aurora-A... + / - The expression of p-Aurora-A (Thr288) in the mouse brain was significantly lower than that in APP / PS1 mice, and a successful model mouse was obtained for experimental research. Figure 2 ).

[0021] (3) APP / PS1-Aurora-A + / - Deposition of Aβ plaques in the mouse brain was significantly reduced, and APP / PS1-Aurora-A + / - The production of Aβ oligomers in the mouse brain was significantly reduced. Figure 3 ).

[0022] (4) APP / PS1-Aurora-A + / -The learning and memory ability of the mice was significantly improved compared with the same age APP / PS1 mice Figure 4 ).

[0023] Example 1 Expression of Aurora A kinase and p-Aurora-A (Thr288) kinase

[0024] The expression of Aurora A kinase and p-Aurora-A (Thr288) kinase in the brain of AD transgenic mice of different ages and in the brain of old AD patients was detected by western blot method.

[0025] Example 2 APP / PS1-Aurora-A + / - Establishment of model mice AD transgenic mice (APPswe / PS1ΔE9, B6C3) were crossed with Aurora-A + / - (C57) to obtain four genotypes of mice, namely APP / PS1-Aurora-A + / - , APP / PS1, WT and Aurora-A + / - mice. The genotype of the mice was determined by PCR after tail clipping and DNA extraction, and the genotyped mice were labeled by ear punching and then housed separately.

[0026] Example 3 Verification of the effect of Aurora-A kinase knockout in half in the brain of AD transgenic mice

[0027] 3.1 Aβ deposition experiment: 9-month-old mice were anesthetized with isoflurane, and the brain tissue was fixed by perfusion with 5% paraformaldehyde to make paraffin sections (6 µm), which were then subjected to DAB staining with 6E10 antibody, and then laser confocal imaging was performed to observe the deposition of Aβ in the cerebral cortex and hippocampus of the mice.

[0028] 3.2 Dotblot method was used to detect Aβ oligomer formation experiment: the brain tissue of the four genotypes of mice was homogenized in ice PBS, and then centrifuged at 13,000 xg for 0.5 h, and the supernatant was taken for protein quantification with BCA kit. 2 μg of protein was taken by microinjection and dropped onto a nitrocellulose membrane, and protein immunoblotting was performed with Aβ oligomer specific antibody A11.

[0029] 3.3 Morris water maze: Morris water maze is a commonly used tool for animal behavior research, which can effectively be used to study the spatial learning and memory ability of animals. The position of the water maze and its surrounding environment remain unchanged throughout the experiment. One day before the experiment, the mice were placed in the water tank without the platform for 2 minutes to adapt to the maze environment and swimming. The experiment lasted for 9 days, and the first 5 days were directional navigation experiments. The mice were placed in the water tank from the entry point, and the time required for the mice to climb from the entry point to the platform within 120 seconds was recorded, which was the latency period. Each mouse was trained 4 times a day, i.e., 1 time from each of the 4 different quadrant entry points. During training, if the mouse found the platform within 120 seconds, it was allowed to stand on the platform for 10 seconds; if it did not, it was guided onto the platform with a stick and allowed to stand for 20 seconds, and the latency period was recorded as 120 seconds. The mice rested for 2 days on days 6-7. On days 8-9, the spatial exploration experiment was conducted, the platform was removed, and the mice were placed in the water tank from the second quadrant entry point. The swimming path of the mouse within 120 seconds was recorded, and the percentage of the swimming path length in the platform quadrant to the total path length was analyzed. Data collection and processing were completed by the Morris water maze image automatic monitoring and processing system.

[0030] The above describes the present application and its embodiments, which are not limited, and the embodiments shown in the drawings are only one of the embodiments of the present application, and the actual structure is not limited thereto. In summary, if a person skilled in the art is inspired by it, without departing from the purpose of the present application, without creative design, similar structure and embodiments of the technical solution can be designed, which should belong to the protection scope of the present application.

Claims

1. A target for Alzheimer's disease, characterized in that: The target is Aurora A kinase.

2. An application of the Alzheimer's disease target as described in claim 1, characterized in that: The Aurora A kinase is abnormally elevated in the brains of Alzheimer's patients.

3. The application as described in claim 2, characterized in that: The expression of Aurora A kinase and its active form, p-Aurora-A(Thr288) kinase, is increased.

4. An Alzheimer's disease treatment drug, based on the Aurora A kinase of claim 1, characterized in that: The drug comprises a selective inhibitor of Aurora A kinase, an antibody or vaccine associated with Aurora A kinase, or an Aurora A kinase gene fragment carried by various viral vectors, and the drug is capable of inhibiting the expression level of Aurora A kinase and its active form, p-Aurora-A(Thr288) kinase.

5. A method for preparing the Alzheimer's disease treatment drug as described in claim 4, characterized in that: Includes the following steps: S1: Screening for selective inhibitors of Aurora A kinase; S2: The screened inhibitors are mixed with a pharmaceutically acceptable carrier to prepare a drug formulation; S3: Prepare antibodies and vaccines related to Aurora A kinase or Aurora A kinase gene fragments carried by various viral vectors.

6. The use of the Alzheimer's disease treatment drug as described in claim 4 in the prevention or treatment of Alzheimer's disease.

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