Method for screening AD treatment medicine by using AD transgenic zebrafish and application of CDK4 / 6-IN-2 in preparation of AD treatment medicine
AD therapeutic drugs were screened through the AD transgenic zebrafish model, and GSK-3β was targeted by small molecule compound library and computer simulation, combined with zebrafish behavioral analysis, and the problems of low screening efficiency and drug resistance of existing AD drugs were solved, and effective AD therapeutic drugs CDK4/6-IN-2 were screened out.
Patent Information
- Application Number
- CN202510554016.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-29
- Publication Date
- 2025-08-05
AI Technical Summary
The existing AD drug screening methods are not efficient in high-throughput screening, making it difficult to cope with the screening needs of large-scale drug databases, and the existing drugs have drug resistance problems during long-term use, resulting in a high failure rate of AD drug development and a lack of accurate and comprehensive screening methods.
Using the AD transgenic zebrafish model, specific compounds were screened out through the small molecule compound library, and computer simulation targeted docking GSK-3β was used. Combined with zebrafish behavioral analysis, compounds with anti-AD effects were screened out, including short-term behavioral assessment and long-term dosing assessment.
It has achieved extensive screening and accurate screening of AD therapeutic drugs, with low screening costs and screened out compounds with strong targeted binding and stable activity, significantly improving the memory and behavior of AD transgenic zebrafish, and providing new AD therapeutic targets.
Smart Images
Figure CN120420458A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medicine, and in particular to a method for screening AD therapeutic drugs using AD transgenic zebrafish, and use of CDK4 / 6-IN-2 in preparing AD therapeutic drugs. Background Art
[0002] Alzheimer's disease (AD) is the most common dementia in the elderly, accounting for approximately 60%-80% of all dementia cases. Its symptoms primarily manifest as progressively more severe cognitive impairment, including memory impairment, learning impairment, attention deficits, and spatial cognitive dysfunction. Recent advances in genomics, CRISPR gene editing technology, and induced pluripotent stem cells (iPSCs) have enabled genome-wide screening of neurons in both normal physiology and AD pathology. Currently, the mainstream view is that amyloid plaques formed by extracellular β-amyloid (Aβ) protein deposition and neurofibrillary tangles (NFTs) formed by intracellular tau protein aggregation are the primary causes of neuronal death and cognitive impairment. Individuals with abnormally folded Aβ protein in their plasma were 23 times more likely to develop AD during a 17-year follow-up compared to controls, confirming the importance of Aβ protein for early diagnosis of AD. Aβ protein can promote the accumulation of tau protein in cells, thereby accelerating the development of AD.
[0003] With the aging of the population, the incidence of AD is also increasing, and the number of patients is expected to rise to 152 million by 2050. According to literature reports, the total number of AD patients in my country has exceeded 9 million, with more than 300,000 new cases each year, accounting for a quarter of the global new cases. This has imposed a huge social and family burden, becoming a major social problem that seriously endangers the health of the Chinese people. Although drug research for AD has made some progress in the past few decades, and most drugs have shown some effectiveness in research, they remain in the research and clinical trial stages and have not been widely used for AD treatment. According to statistics, the clinical development failure rate of AD drugs reached 99.6% from 2002 to 2022, which has dealt a heavy blow to the development of drugs targeting AD. Most studies focus on short-term drug responses, ignoring the potential effects of long-term use on the model, such as drug resistance. In addition, existing screening methods are not efficient enough for high-throughput screening, making it difficult to meet the needs of screening large-scale drug libraries. Therefore, further in-depth elucidation of the molecular mechanisms of the occurrence and development of AD will open up new ideas for the research and development of new drugs, develop more accurate and comprehensive screening methods, and screen out more effective compounds for the treatment of AD, so as to improve the efficiency and success rate of Alzheimer's disease drug research and development. This will provide therapeutic targets and theoretical basis for the clinical formulation of new AD treatment plans, and has very important clinical significance for improving the prognosis of AD. Summary of the Invention
[0004] The purpose of the present invention is to address the deficiencies of the existing technology and provide a method for screening AD therapeutic drugs using AD transgenic zebrafish. The method has the advantages of a wide screening range, high screening accuracy and low screening cost, and provides a new screening idea for the development of targeted treatment for AD.
[0005] The technical solution for achieving the purpose of the present invention is: a method for screening AD therapeutic drugs using AD transgenic zebrafish, comprising the following steps:
[0006] 1) Selecting compounds from the small molecule compound library as preselected compounds after processing;
[0007] 2) Processing the target protein structure of GSK-3β;
[0008] 3) Performing simulated targeted docking of the preselected compounds with GSK-3β to screen out the initial screening compounds that meet the specificity requirements;
[0009] 4) Conduct toxicity tests on wild-type zebrafish using different concentrations of the initial screening compounds to determine the appropriate dosing concentration;
[0010] 5) Short-term initial screening
[0011] Add the prescreened compound at the concentration determined in step 4) to AD transgenic zebrafish. 72 hours after administration, place the zebrafish in a 48-well plate. Use a computer to establish a learning model, integrate the zebrafish behavioral data, and preliminarily evaluate and screen the prescreened compounds for therapeutic effects on behavioral abnormalities as candidate compounds.
[0012] 6) Long-term administration
[0013] The candidate compounds screened in 5) are added to the AD transgenic zebrafish group and administered continuously for 3 months. After comprehensive evaluation based on AD onset time, NFT aggregation, and behavioral indicators, the candidate compounds are observed to see whether they have an inhibitory effect on the induction of AD, and target compounds with anti-AD effects are screened.
[0014] Furthermore, in step 1), the small molecule compound library is the Specs compound library and / or the Topscience inhibitor compound entity library.
[0015] Furthermore, the processing in step 1) is to perform protonation and energy minimization processing on the compounds in the small molecule compound library using the LigPre module in the Maestro 11.9 platform, and the force field selected is OPLS3e.
[0016] Furthermore, the processing in step 2) is performed using the Maestro 11.9 platform, including removal of water and ions, protonation, addition of missing atoms, completion of missing genes, protein energy minimization, and energy minimization, and the force field selected is OPLS3e.
[0017] Further, step 3) screening method is: The Glide module in Maestro software was used to import the processed GSK-3β, and the docking site was determined according to the natural ligand U3E and used as The center of mass of the box is determined, and the processed pre-selected compounds are docked. The docking energy is scored to obtain a data set, which is screened by the SP method and sorted by score. The top M compounds determined by the SP method are screened using the XP docking template. The top M compounds are screened by MMGBSA and ADME. The top N compounds are screened by the binding energy and active site key residue evaluation methods to obtain the initial screening compounds.
[0018] Furthermore, in step 4), a toxicity test is performed according to a concentration gradient, and the IC50 value is calculated based on the mortality rate, using the following formula:
[0019] Y=Bottom+(Top-Bottom) / (1+10 (logIC50-X)*HillSlope ),
[0020] Wherein, Top is the maximum response when not inhibited, Bottom is the minimum response when completely inhibited, Hill Slope is the slope of the curve, Y is the mortality rate, and X is the drug concentration. Preferably, the determined appropriate administration concentration X is 10 uM.
[0021] Furthermore, in step 5), the corresponding concentration of the pre-screened compound is added to the AD transgenic zebrafish. 72 hours after administration, the AD transgenic zebrafish after administration and the control group zebrafish are placed in a 48-well plate, and the light and dark parameters are set using a zebrafish behavioral analyzer to detect the therapeutic effect of the pre-screened compound on the abnormal behavior of AD zebrafish. Preferably, the light and dark parameters are 10 minutes of light and 10 minutes of darkness, with a cycle of 1 hour.
[0022] Furthermore, in step 6), the primary screening compound is added to the AD transgenic zebrafish at a corresponding concentration. Three months after administration, the AD transgenic zebrafish are subjected to behavioral analysis to detect the therapeutic effect of the candidate compound on AD memory dysfunction. Preferably, the behavioral analysis is continued using a maze test.
[0023] Furthermore, in step 6), the corresponding concentration of the primary screening compound is added to the AD transgenic zebrafish. After 3 months of administration, the brain tissue of the AD transgenic zebrafish is removed and immunohistochemically stained to label the NFT protein to detect the inhibitory effect of the candidate compound on abnormal phosphorylated tau.
[0024] A second object of the present invention is to provide a use of CDK4 / 6-IN-2 in the preparation of a drug for treating Alzheimer's disease, wherein CDK4 / 6-IN-2 is obtained by screening using any of the above-mentioned screening methods.
[0025] The above technical solution has the following beneficial effects:
[0026] 1. GSK-3β (glycogen synthase kinase-3β) is a serine / threonine kinase that was originally identified as a key regulator of glycogen metabolism, specifically as an inhibitor of glycogen synthesis. GSK-3β regulates the activity, stability, and subcellular localization of target proteins by phosphorylating specific serine and threonine residues. This fine-tuning of post-transcriptional modification affects cellular processes, from gene expression to cell proliferation and apoptosis. GSK-3β plays a key role in a variety of diseases. Its dysregulation is closely associated with the occurrence and progression of various cancers, developmental diseases, neurodevelopmental disorders, and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Inhibition or regulation of GSK-3β has potential therapeutic effects in the treatment of a variety of diseases such as cancer and neurodegenerative diseases.
[0027] 2. The present invention uses compounds (>200,000) in the small molecule compound library as the screening target group, first uses computer molecular docking technology to simulate the targeted docking of these compounds with GSK-3β, and through scoring and sorting, screens out small molecule compounds with higher specificity to make preliminary screening compounds. Subsequently, the concentration of the preliminary screening compounds is measured using wild-type zebrafish as candidate compounds to obtain a suitable dosing concentration. Subsequently, AD transgenic zebrafish are used to quickly and efficiently perform behavioral analysis on the preliminary screening compounds in a 48-well plate to screen out candidate anti-AD compounds. The entire screening process is simple, reliable, and extremely low-cost. The target compounds obtained by screening have a strong targeted binding effect with GSK-3β, and have strong binding affinity and stable activity. This targeted binding inhibits the formation of GSK-3β and thus realizes potential therapeutic effects in the treatment of AD.
[0028] 3. CDK4 / 6-IN-2, obtained by screening using the screening method of the present invention, is a GSK-3β inhibitor that can inhibit the phosphorylation of GSK-3β substrates, primarily by inhibiting the phosphorylation of tau, thereby playing a therapeutic role in AD. Although CDK4 / 6-IN-2 also has a certain inhibitory effect on the phosphorylation of other GSK-3β substrates (such as β-catenin, Cyclin-D1, and eIF2β), compared with the preclinical compounds Tideglusib and Alsterpaullone, CDK4 / 6-IN-2 has outstanding advantages: ① The inhibition rate of CDK4 / 6-IN-2 on phosphorylated tau (S396) is close to 100%, significantly higher than Tideglusib and Alsterpaullone; ② The inhibition rate of CDK4 / 6-IN-2 on other GSK-3β substrates is significantly lower than Tideglusib and Alsterpaullone, which can reduce the side effects of the drug.
[0029] Animal experiments conducted by the applicant have shown that the memory and anxiety-like behaviors of AD transgenic zebrafish treated with the CDK4 / 6-IN-2 of the present invention are alleviated to a certain extent, and the motor ability is improved, and the antipsychotic-like activity of AD can be significantly inhibited.
[0030] The following is a further description with reference to the accompanying drawings and specific implementations. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1Molecular docking model and analysis of GSK-3β and target protein ((A) The overall three-dimensional structure of the GSK-3β complex, with the protein backbone shown as a tubular model (bright cyan) and the target protein shown as a stick model (red); (B) A local view of the active site and ligand binding, with key residues interacting with the ligand shown as stick models (green); (C) A two-dimensional protein-ligand interaction diagram of the ligand-GSK-3β complex, with protein residues represented by circles and colored according to their properties: green (hydrophobic residues) and purple (polar residues); (D) The re-docking result of the ligand U3E (red) with GSK-3β, showing the binding conformation of the ligand in the active site;
[0032] Figure 2 、 Figure 3 The binding mode of GSK-3β with inhibitors and candidate compounds;
[0033] Figure 4 Flowchart for the establishment of a transgenic zebrafish model of AD;
[0034] Figure 5 Screening of candidate compounds for AD transgenic zebrafish;
[0035] Figure 6 This is a western blot analysis of the inhibition of CDK4 / 6-IN-2 on GSK-3β substrate;
[0036] Figure 7 CDK4 / 6-IN-2 treatment significantly improved the tau pathological phenotype and memory function of AD zebrafish. DETAILED DESCRIPTION
[0037] In the present invention, the materials used are:
[0038] 1.CDK4 / 6-IN-2 was purchased from MedChemexpress Biotechnology Company.
[0039] 2. The applicant used Gal4 / UAS to establish an AD transgenic zebrafish model, specifically Tau-P301S transgenic zebrafish:
[0040] The zebrafish neuron-specific promoter huc was used to mediate the expression of the human TAU P301S mutant protein (the protein mutated at the 301st amino acid position). Transgenic zebrafish stably expressing the AD-related protein TAU P301S were generated. Two expression vectors, Driver and Responder, were constructed using the medaka Tol2 transposable element, and the Gal4 / UAS expression system was integrated into the two vectors ( Figure 4). Among them, the Driver vector contains the zebrafish neuronal promoter huc, which controls the expression of the Gal4-VP16 fusion protein, thereby effectively activating and amplifying the protein expression on the response element UAS. In order to achieve transgenic expression in two directions, we inserted two minimal promoters Eb1 on both sides of the UAS sequence, expressing the human TAUP301L protein in one direction and the fluorescent reporter gene eGFP in the other direction. This expression pattern allows eGFP to track the expression of TAU P301S. Subsequently, the Driver, Responder and transposase mRNA were simultaneously injected into the one-cell stage of zebrafish ( Figure 4 ).
[0041] Example 1: Using computer molecular docking to target and dock the compounds in the small molecule compound library (>200,000) with GSK-3β to screen for small molecule compounds with high specificity
[0042] The small molecule compound library was primarily sourced from the Specs compound library (210,068 compounds) and the Topscience inhibitor library (7,618 compounds) (https: / / www.specs.net / , https: / / www.targetmol.cn / ). All compounds were protonated and energy minimized using the LigPre module in the Maestro 11.9 platform, using the OPLS3e force field.
[0043] The GSK-3β target protein structure (PDBID: 8DJD) was processed on the Maestro 11.9 platform, including removal of water and ions, protonation, addition of missing atoms, completion of missing groups, protein energy minimization, and energy optimization. The force field was selected as OPLS3e.
[0044] Virtual screening processing and optimization are performed by The Glide module in Maestro software was used for protein processing. Protein Preparation Wizard module was used for pre-processing, optimization and minimization of the receptor (constrained minimization using OPLS3e force field). All compounds were prepared according to the default settings of LigPre module. When screening in Glide module, the prepared receptor was imported to specify the appropriate position in the receptor grid generation. The docking site was determined based on the natural ligand U3E of the protein and used as The center of mass of the box is (x = 4.4, y = -0.16, z = 33.88). The ligands were first redocked to confirm the feasibility of the chosen docking method. The dataset was then screened using SP docking. The SP docking template is suitable for screening large quantities of compounds. The XP docking template was then used to screen for high-scoring preligands identified by the SP method. The XP method is designed to provide a better correlation between good poses and scores. Finally, MMGBSA and ADME screening were performed.
[0045] The crystal structure of GSK-3β protein is highly accurate, with no missing key residues and a clear active site. The GSK-3β active site is mainly composed of VAL-135, LYS-85, PHE-67, VAL-70, ALA-83, LEU-188, CYS-199, ASP-133, etc. The reference compound selected for this study is the natural ligand U3E of GSK-3β protein, and its binding mode is as follows: Figure 1-3 As shown. U3E can form strong hydrogen bond interactions with key residues VAL-135 and LYS-85, and the nitrogen heterocyclic ring and benzene ring of U3E can also form conjugated interactions with amino acids PHE-67, VAL-70, ALA-83, LEU-188, CYS-199, and ASP-133. In particular, the benzene ring of the compound forms a pi-pi conjugated interaction with PHE-67. These interactions play an important role in stabilizing the ligand ( Figure 1 1B and 1C in the ), further indicating that these amino acids will play an important role in the screening process. In order to determine the appropriate docking scheme for screening potential active compounds, the known original ligand was docked to the GSK-3β binding site, and the binding pose had good overlap with the ligand in the previous complex ( Figure 1 1D in ), which shows that the screening method is effective and reasonable.
[0046] The docked data were used to screen out the top 223 compounds based on energy scores. Finally, 40 compounds were obtained through MMGBSA and ADME, combining the energy scores and the evaluation of key residues in the active sites.
[0047] Table 1 Virtual screening of compounds in the small molecule library that bind to the GSK-3β target
[0048]
[0049]
[0050]
[0051] Example 2 Animal Experiment
[0052] Wild zebrafish were used to test the concentrations of the 40 compounds screened (0, 0.5, 1, 5, 10, 50 μM) to evaluate the appropriate dosing concentrations, and the final dosing concentrations were determined by calculating the median lethal dose (IC50) (Table 3).
[0053] Table 3 Appropriate compound concentrations determined for wild-type zebrafish
[0054]
[0055]
[0056]
[0057] Example 3 Preliminary screening of candidate drugs for effective treatment of AD using AD transgenic zebrafish
[0058] AD transgenic zebrafish embryos were exposed to 40 primary screening compounds at concentrations as shown in Table 3 for the final dosing concentrations. AD transgenic zebrafish embryos were placed in 10 cm culture dishes, with 100 eggs per dish, and dosing was performed for 72 hours. Subsequently, the juveniles were transferred to 48-well plates, with one juvenile per well, 8 compounds per plate, and 6 replicates per compound (e.g. Figure 5 The 48-well plate was placed in a zebrafish behavioral analyzer and set to the parameters (10 minutes light / 10 minutes dark, for a total of 60 minutes), and the movement trajectory of the AD transgenic zebrafish was measured. The results clearly show that CDK4 / 6-IN-2, AT057, AN988, and AZD5597 have a significant therapeutic effect on the movement behavior of AD zebrafish.
[0059] Example 4 Screening of anti-AD compounds using AD transgenic zebrafish
[0060] The candidate compounds CDK4 / 6-IN-2, AT057, AN988, and AZD5597 were used to treat AD transgenic zebrafish for 3 months to test the inhibitory effects of the four compounds on GSK-3β substrates. Figure 6 As shown in the results, although AT057 and AN988 compounds can effectively inhibit the phosphorylation of tau (S396), they also have a strong inhibitory effect on other substrates of GSK-3β substrates. Therefore, AD transgenic zebrafish treated with CDK4 / 6-IN-2 and AZD5597 were finally selected to evaluate the anti-AD effect. After a comprehensive evaluation of indicators such as the onset time of AD transgenic zebrafish, NFT aggregation, and behavior (T-maze), it was observed whether the candidate drugs had an inhibitory effect on the induction of AD in zebrafish. The final results showed that compared with AD transgenic zebrafish, the tau pathological phenotype and memory ability of AD transgenic zebrafish were significantly improved after CDK4 / 6-IN-2 treatment (such as Figure 7), and the therapeutic effect of AZD5597 was significantly weaker than that of CDK4 / 6-IN-2, indicating that CDK4 / 6-IN-2 may become a potential small molecule drug for the treatment of Alzheimer's disease, providing a therapeutic target and theoretical basis for the clinical formulation of new AD treatment plans.
[0061] As can be seen from the above examples, the present invention employs a drug screening method utilizing a transgenic zebrafish model for Alzheimer's disease. This method uses computer simulation to perform molecular docking of candidate compounds with GSK-3β, and then conducts rapid initial drug screening in the AD transgenic zebrafish model. Once candidate compounds are obtained, pharmacodynamic evaluation is performed on the AD transgenic zebrafish using long-term administration to identify the optimal therapeutic drug. This method successfully identified CDK4 / 6-IN-2 as an effective drug for treating Alzheimer's disease.
Claims
1. A method for screening AD therapeutic drugs using AD transgenic zebrafish, characterized in that: The following steps are involved: 1) Selecting compounds from the small molecule compound library as preselected compounds after processing; 2) Processing the target protein structure of GSK-3β; 3) Performing simulated targeted docking of the preselected compounds with GSK-3β to screen out the initial screening compounds that meet the specificity requirements; 4) Conduct toxicity tests on wild-type zebrafish using different concentrations of the initial screening compounds to determine the appropriate dosing concentration; 5) Short-term initial screening The pre-screened compound at the concentration determined in step 4) is added to the AD transgenic zebrafish group. 72 hours after administration, the zebrafish are placed in a 48-well plate. A learning model is established using a computer, and the zebrafish behavioral data are integrated to preliminarily evaluate and screen the pre-screened compounds for therapeutic effects on behavioral abnormalities as candidate compounds. 6) Long-term administration The candidate compounds screened in 5) are added to the AD transgenic zebrafish group and administered continuously for 3 months. After comprehensive evaluation based on AD onset time, NFT aggregation, and behavioral indicators, the candidate compounds are observed to see whether they have an inhibitory effect on the induction of AD, and target compounds with anti-AD effects are screened.
2. The method for screening AD therapeutic drugs using AD transgenic zebrafish according to claim 1, characterized in that: Step 1) The small molecule compound library is the Specs compound library and / or the Topscience inhibitor compound entity library.
3. The method for screening AD therapeutic drugs using AD transgenic zebrafish according to claim 1, characterized in that: The processing in step 1) is to perform protonation and energy minimization processing on the compounds in the small molecule compound library using the LigPre module in the Maestro 11.9 platform, and the force field selected is OPLS3e.
4. The method for screening AD therapeutic drugs using AD transgenic zebrafish according to claim 1, characterized in that: The processing in step 2) is performed using the Maestro 11.9 platform, including removal of water and ions, protonation, addition of missing atoms, completion of missing genes, protein energy minimization, and energy minimization, with the force field selected as OPLS3e.
5. The method for screening AD therapeutic drugs using AD transgenic zebrafish according to claim 1, characterized in that: Step 3) screening method is: The Glide module in Maestro software was used to import the processed GSK-3β, and the docking site was determined according to the natural ligand U3E and used as The center of mass of the box is determined, and the processed pre-selected compounds are docked. The docking energy is scored to obtain a data set, which is screened by the SP method and sorted by score. The top M compounds determined by the SP method are screened using the XP docking template. The top M compounds are screened by MMGBSA and ADME. The top N compounds are screened by the binding energy and active site key residue evaluation methods to obtain the initial screening compounds.
6. The method for screening AD therapeutic drugs using AD transgenic zebrafish according to claim 1, characterized in that: Step 4) Toxicity test was performed according to the concentration gradient, and the IC50 value was calculated based on the mortality rate. The formula is as follows: Y=Bottom+(Top-Bottom) / (1+10 (logIC50-X)*HillSlope ), Wherein, Top is the maximum response when not inhibited, Bottom is the minimum response when completely inhibited, Hill Slope is the slope of the curve, Y is the mortality rate, and X is the drug concentration. Preferably, the determined appropriate administration concentration X is 10 uM.
7. The method for screening AD therapeutic drugs using AD transgenic zebrafish according to claim 1, characterized in that: Step 5) Add the corresponding concentration of the primary screening compound to the AD transgenic zebrafish. 72 hours after administration, place the AD transgenic zebrafish and the control group zebrafish in a 48-well plate, set the light and dark parameters using a zebrafish behavioral analyzer, and detect the therapeutic effect of the primary screening compound on the abnormal behavior of AD zebrafish. Preferably, the light and dark parameters are 10 minutes of light and 10 minutes of darkness, with a cycle of 1 hour.
8. The method for screening AD therapeutic drugs using AD transgenic zebrafish according to claim 1, characterized in that: Step 6) Add the primary screening compound at a corresponding concentration to the AD transgenic zebrafish. After 3 months of administration, perform behavioral analysis on the AD transgenic zebrafish to detect the therapeutic effect of the candidate compound on AD memory dysfunction. Preferably, the behavioral analysis is continued using a maze test.
9. The method for screening AD therapeutic drugs using AD transgenic zebrafish according to claim 1, characterized in that: Step 6) Add the corresponding concentration of the primary screening compound to the AD transgenic zebrafish. After 3 months of administration, the brain tissue of the AD transgenic zebrafish is removed and immunohistochemically stained to label the NFT protein to detect the inhibitory effect of the candidate compound on abnormal phosphorylated tau.
10. Use of CDK4 / 6-IN-2 in the preparation of a drug for treating Alzheimer's disease, wherein CDK4 / 6-IN-2 is obtained by screening using any screening method according to claims 1-9.