Computer-aided drug screening method, system and equipment based on SP3 and GRIK1

Abstract

The invention discloses a computer-aided drug screening method, system and equipment based on SP3 and GRIK1. According to the application, the drugs are virtually screened through a computer on the basis of the relationship that SP3 induces activation of the NLRP3 inflammasome in the GRIK1 mediated microglial cells, the screening efficiency of the inhibition drugs is improved, the screening period is shortened, and a new strategy is provided for treatment of the Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of intelligent medical treatment, and specifically relates to a computer-aided drug screening method, system and equipment based on SP3 and GRIK1. Background Art

[0002] Alzheimer's disease (AD) is a chronic neurodegenerative disease in which inflammasomes are the main players. In addition, many immune cells in the brain, especially microglia, are also involved in the pathogenesis of AD. The clinical manifestations of AD are decreased learning, memory and spatial recognition abilities, decreased ability to recover from stress stimuli, slow response, and even personality changes that progress to a state of general dementia. However, there is currently no systematic and comprehensive method for screening anti-Alzheimer's drugs.

[0003] With the development of technologies such as big data, cloud computing, and hardware acceleration, computer-aided drug design (CADD) has gradually become an important means of new drug research and development. Based on computer computing and simulation technology, by learning the prior knowledge contained in massive drug data and mining the interaction relationship between target targets and candidate drugs, active drug molecules with drug-like properties can be quickly selected from millions of molecules. This greatly reduces the blindness of screening candidate drug molecules and improves the efficiency of research and development and drug screening. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides a computer-assisted drug screening method, system and device based on SP3 and GRIK1.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a method for computer-assisted drug screening based on SP3 and GRIK1, the method comprising:

[0007] Obtain SP3 protein and GRIK1 protein data;

[0008] Determine the binding sites of the SP3 protein and the GRIK1 protein according to the SP3 protein and the GRIK1 protein data;

[0009] Candidate drugs targeting the binding sites of the two were obtained through computer-assisted screening.

[0010] Further, the process of computer-assisted screening is:

[0011] Screening of small molecule compounds based on the spatial structure of the binding sites of SP3 protein and GRIK1 protein;

[0012] The small molecule compounds obtained by screening are molecularly docked with the binding sites to obtain the scores of the docking molecules;

[0013] The candidate drugs are obtained by sorting according to the scores.

[0014] Further, the process of computer-assisted screening is:

[0015] The molecular structures of SP3 protein and GRIK1 protein are obtained and input into the pharmacophore module library for matching, and all the action sites are clustered based on the interaction patterns with SP3 and GRIK1 to obtain the pharmacophore model;

[0016] The pharmacophore model is input into a molecular compound database for high throughput screening to obtain candidate drugs.

[0017] Further, the process of computer-assisted screening is:

[0018] Obtaining the molecular structure of a small molecule inhibitor that inhibits SP3 and / or the molecular structure of a small molecule inhibitor that inhibits GRIK1;

[0019] Based on the molecular structure of the small molecule inhibitor, a small molecule library with similar structure is obtained by screening;

[0020] Performing molecular docking on the small molecule library with similar structures and the binding site to obtain a score of the docked molecule;

[0021] The candidate drugs are obtained by sorting according to the scores.

[0022] Furthermore, the candidate drugs include protein analogs, antibodies, and RNA drugs.

[0023] A second aspect of the present invention provides a system for computer-assisted drug screening based on SP3 and GRIK1, the system comprising:

[0024] Acquisition unit: acquire SP3 protein and GRIK1 protein data;

[0025] A site determination unit: determining the binding sites of the SP3 protein and the GRIK1 protein according to the SP3 protein and the GRIK1 protein data;

[0026] Screening unit: Obtain candidate drugs targeting the binding sites of the two through computer-assisted screening.

[0027] The third aspect of the present invention provides a device for computer-assisted drug screening based on SP3 and GRIK1, the device comprising:

[0028] A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the computer-assisted drug screening method based on SP3 and GRIK1 described in the first aspect of the present invention is implemented.

[0029] A fourth aspect of the present invention provides a computer-readable storage medium having a computer program thereon, the computer-readable storage medium comprising:

[0030] When the computer program is executed by a processor, the method for computer-assisted drug screening based on SP3 and GRIK1 described in the first aspect of the present invention is implemented.

[0031] The fifth aspect of the present invention provides a drug screened by the computer-assisted drug screening method based on SP3 and GRIK1 described in the first aspect of the present invention.

[0032] A sixth aspect of the present invention provides a method for regulating GRIK1 activity or GRIK1 level in a cell, the method comprising administering SP3 protein or a promoter thereof.

[0033] The activity of GRIK1 or the expression level of GRIK1 can be promoted by administering SP3 protein or its promoter.

[0034] A seventh aspect of the present invention provides a method for regulating inflammasomes in cells, the method comprising administering SP3, GRIK1 or an SP3-GRIK1 protein complex.

[0035] Furthermore, the inflammasome is the NLRP3 inflammasome.

[0036] Further, the cells are microglia.

[0037] SP3 protein or promoter, GRIK1 protein or promoter, and SP3-GRIK1 protein complex can activate inflammasomes.

[0038] The eighth aspect of the present invention provides any one of the following applications:

[0039] (1) Use of the drug, substance inhibiting SP3, substance inhibiting GRIK1 or substance inhibiting SP3-GRIK1 according to the fifth aspect of the present invention in the preparation of a drug for treating Alzheimer's disease;

[0040] (2) Use of SP3, GRIK1 or SP3-GRIK1 protein complex in regulating inflammasomes or in the preparation of drugs for regulating inflammasomes;

[0041] (3) Application of SP3 in regulating GRIK1 activity or GRIK1 level.

[0042] Furthermore, the substance that inhibits SP3, the substance that inhibits GRIK1, or the substance that inhibits SP3-GRIK1 described in (1) includes protein analogs, antibodies, and RNA drugs.

[0043] Furthermore, the drug described in (1) or (2) further includes a pharmaceutically acceptable carrier.

[0044] Furthermore, SP3 acts on the GRIK1 promoter region.

[0045] Furthermore, the inflammasome is the NLRP3 inflammasome.

[0046] Furthermore, the NLRP3 inflammasome is a NLRP3 inflammasome in microglia and / or AD.

[0047] Advantages and beneficial effects of the present invention:

[0048] This application uses computer virtual screening of drugs based on the relationship between SP3-induced GRIK1-mediated NLRP3 inflammasome activation in microglia, which helps to improve the screening efficiency of inhibitory drugs, shorten the screening cycle, and provide a new strategy for the treatment of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a schematic diagram of the method flow of computer-assisted drug screening based on SP3 and GRIK1;

[0050] Figure 2 It is a schematic diagram of the system for computer-assisted drug screening based on SP3 and GRIK1;

[0051] Figure 3 It is a schematic diagram of the equipment for computer-assisted drug screening based on SP3 and GRIK1;

[0052] Figure 4The following is a graph showing the expression of GRIK1 in AD mice. 4A is a volcano graph of the GSE144459 dataset, 4B is a volcano graph of the GSE184942 dataset, 4C is a graph showing differentially expressed genes between brain tissues of AD mice and control mice in GSE144459, 4D is a graph showing differentially expressed genes between brain tissues of AD mice and control mice in GSE184942, and 4E is an intersection graph of AD-related genes in GSE144459, GSE184942, and Genecards datasets. 4F is a graph showing the time taken by the MWM test for normal mice and AD mice to reach the hidden platform, 4G is a graph showing the number of times mice crossed the platform by MWM, 4H is a graph showing the neuronal morphology in the hippocampus of normal group and AD group mice by HE staining, 4I is a graph showing the expression of GRIK1 mRNA in the brain tissues of normal group and AD group mice by RT-qPCR, and 4J is a graph showing the expression of GRIK1 protein in the brain tissues of normal group and AD group mice by immunohistochemical staining (n=5 / group, **p<0.01; ***p<0.001);

[0053] Figure 5 5A is a graph showing the effect of GRIK1 knockout on AD mice, wherein 5A is a graph showing the mRNA expression of GRIK1 in the brain tissues of mice with different treatments detected by RT-qPCR, 5B is a graph showing the expression of GRIK1 protein in the brain tissues of mice with different treatments detected by immunohistochemical staining, 5C is a graph showing the time taken by mice to explore new arms detected by Y-type maze, 5D is a graph showing the localization of GRIK1 and iba1 in the brain tissues detected by immunofluorescence staining, 5E is a graph showing the protein expression of CD14 and CD40 in the brain tissues of mice detected by immunoblotting, and 5F is a graph showing the expression of inflammasome components NLRP3, ASC and IL-1β in the brain tissues of mice detected by immunoblotting (n=5 / group, ***p<0.001);

[0054] Figure 6 6A is a graph showing the effect of GRIK1 silencing on NLRP3 inflammasome in BV2 cells after infection, wherein 6B is a graph showing the protein expression of NLRP3, ASC and cleaved caspase-1 in BV2 cells after infection detected by immunoblotting, 6C is a graph showing the expression of CD86 and CD206 in BV2 cells after infection detected by flow cytometry, and 6D is a graph showing the levels of IL-6 and IL-1β in BV2 cells after infection detected by ELISA (**p<0.01; ***p<0.001);

[0055] Figure 77A is a diagram of the expression of SP3 in brain tissues of AD mice and LPS-induced BV2 cells, wherein 7A is a diagram of the transcription factor binding sites of the GRIK1 promoter and AD-related genes, 7B is a diagram of the mRNA expression of SP3 in brain tissues of AD mice and normal mice detected by RT-qPCR, 7C is a diagram of the protein expression of SP3 in brain tissues of AD mice and normal mice detected by immunohistochemistry, and 7D is a diagram of the SP3 protein level in BV2 cells treated with PBS or induced by LPS detected by immunoblotting (n=5 / group, **p<0.01; ***p<0.001);

[0056] Figure 8 8A is a diagram of SP3 binding to the promoter of GRIK1 regulating the expression of GRIK1 in BV2 cells, wherein 8A is a binding matrix diagram of SP3 in the Jaspar database, 8B is a diagram of the binding sites between SP3 and the GRIK1 promoter in the Jaspar database, 8C is a diagram of the relationship between the expression of SP3 and GRIK1 in hippocampal tissue in the GEPIA database, 8D is a diagram of the expression of SP3 mRNA in BV2 cells infected with oe-SP3 or oe-NC detected by RT-qPCR, 8E is a diagram of the relative luciferase activity of GRIK1 in BV2 cells detected by dual luciferase reporter gene, 8F is a diagram of the expression of GRIK1 mRNA in BV2 cells infected with oe-SP3 or oe-NC detected by RT-qPCR, and 8G is a diagram of the enrichment of anti-SP3 or IgG on the GRIK1 promoter in BV2 cells detected by chip (**p<0.01, ***p<0.001);

[0057] Fig. 9 9A is a graph showing that upregulation of GRIK1 weakens the alleviating effect of sh-SP3 on cognitive impairment in mice, wherein 9A is a graph showing protein expression of SP3 and GRIK1 in mouse brain tissue detected by immunohistochemistry, 9B is a graph showing expression of NLRP3, ASC and Iba1 in mouse brain tissue detected by immunoblotting, 9C is a graph showing pathological morphology of neurons in mouse brain tissue detected by HE staining, and 9D is a graph showing cognitive ability of mice detected by MWM (n=5 / group, *p<0.05, ***p<0.001);

[0058] Fig.1010A is a graph showing that upregulation of GRIK1 attenuates the inhibitory effect of sh-SP3 on NLRP3 activation in vitro, wherein 10A is a graph showing the mRNA levels of SP3 and GRIK1 detected in BV2 cells by RT-qPCR, 10B is a graph showing the protein expression of NLRP3, ASC and cleaved caspase-1 detected in BV2 cells by immunoblotting, 10C is a graph showing the expression of CD86 and CD206 in BV2 cells detected by flow cytometry, and 10D is a graph showing the levels of IL-6 and IL-1β in BV2 cells detected by ELISA (**p<0.01, ***p<0.001). DETAILED DESCRIPTION

[0059] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0060] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., do not represent the order of precedence, and do not limit the "first" and "second" to be different types.

[0061] Figure 1 This is a flow chart of the method for computer-assisted drug screening based on SP3 and GRIK1, which specifically includes:

[0062] 101: Obtain SP3 protein and GRIK1 protein data;

[0063] In some embodiments, the SP3 protein and GRIK1 protein data include the molecular structure and binding site of the SP3 protein and the GRIK1 protein.

[0064] In one embodiment, the mice and treatment regimen of the present application include: Animal experiments used 8-month-old male APP / PS1 double transgenic mice and wild-type (WT) male mice of the same litter (Beijing Speif Biotechnology Co., Ltd., China). Unless otherwise specified, all mice were raised under standard laboratory conditions (22±2°C, 55±5%, 12:12 h light-dark cycle) with free access to food and water. All experiments were conducted in accordance with the Regulations on the Administration of Experimental Animals in China and approved by the Animal Ethics Committee of our hospital.

[0065] For the genetic intervention of GRIK1 and SP3 in mice, AAV vectors specifically knocking down or overexpressing GRIK1 and knocking down SP3 and their respective controls were obtained from VectorBuilder (Guangzhou, Guangdong, China). AAV vectors (1 μL) were injected into the bilateral brain using a stereotaxic instrument according to the following coordinates: anterior-posterior, −2.3 mm; mediolateral, ±1.8 mm; and dorsoventral, −2.2 mm relative to the bregma, with an injection speed of 0.1 μL / min (virus titer 5 × 10 11 GC / mL). The needle was kept in place for 5 minutes and then slowly withdrawn. The mice were placed on a heating pad until they began to recover from the surgery. Behavioral testing began 3 weeks later, and mice were euthanized 4 weeks later by intraperitoneal injection of an overdose of sodium pentobarbital (150 mg / kg) for subsequent experiments.

[0066] In one embodiment, the cell culture and treatment method of the present application comprises: culturing the mouse BV2 microglial cell line (TW34605, TW reagent, Shanghai) in DMEM high sucrose complete medium (PM150210B, Procell, Wuhan, Hubei) containing 10% FBS, and culturing in a humidified incubator at 37°C and 5% CO2. BV2 cells were starved for 12 hours, incubated with 100 ng / mL LPS (L8880, Beijing Solebao Life Science Co., Ltd., Beijing) for 12 hours, and then stimulated with 10 μM Aβ1-42 oligomers alone for 24 hours to generate an AD model of inflammatory response in vitro.

[0067] Well-growing BV2 cells were selected for lentiviral infection. The transfected viruses were sh-GRIK1, sh-SP3, oe-SP3 and their controls (titer>10 8 TU / ml, VectorBuilder), puromycin was added for cell screening, and fresh complete culture medium was replaced after 5 days of screening for subsequent in vitro modeling.

[0068] In one embodiment, the method of the elevated plus maze in the present application includes: the elevated plus maze is used to test anxiety / emotion in mice. It consists of four arms (30×5 cm) connected by a common 5×5 cm central area. Two opposing arms are open (open arms), while the other two opposing arms are surrounded by 20 cm high walls (closed arms). The entire maze is 100 cm from the ground. The mouse is placed in the central area facing the open arm and allowed to explore the maze in a single arm for 8 minutes. Between each exploration, feces is removed from the maze and the maze floor is cleaned with 75% alcohol to remove any urine or odor clues. The time spent in the open and closed arms is recorded using an intelligent video tracking system.

[0069] In a specific embodiment, the Morris water maze (MWM) test method in the present application includes: placing mice in a circular pool filled with water (21±1°C) for experiment, placing an escape platform (4.5cm in diameter) in the pool, and the top of the platform is 1.5cm from the water surface. The MWM test includes a spatial acquisition test and an exploration test. The spatial acquisition test was performed for 5 consecutive days, with 4 tests performed every day. In each test, the mice were allowed to search for the platform for 60 seconds, and the mice were allowed to stay on the platform for 5 seconds after finding it. Mice that did not find the escape platform within 60 seconds were guided to the platform until they stayed on the platform for at least 15 seconds. The exploration test was performed 24 hours after the last training trial. During the exploration test, the platform was removed and the mice were allowed to swim freely for 60 seconds. All experiments used the MORRIS water maze video analysis system (Beijing Keyue Huacheng) to observe and record the time (in s) for each mouse to find the platform and the number of times it crossed the platform.

[0070] In a specific embodiment, the tissue collection method in the present application includes: after euthanasia of mice, brain tissue is taken and placed in 4% paraformaldehyde (441244, Merck Millipore, Darmstadt, Germany) for fixation for more than 24 hours, dehydrated with gradient alcohol, immersed in alcohol-benzene and xylene in sequence, embedded in paraffin, and the paraffin-embedded tissue is cut into 4 μm thick slices and stored at room temperature (RM).

[0071] In one embodiment, the method of RNA isolation, reverse transcription and RT-qPCR in the present application comprises: using TRIzol reagent (R0016, Shanghai Beyotime Biotechnology Co., Ltd., China) to isolate total RNA from cultured BV2 cells and brain tissue. TMThe isolated RNA was reverse transcribed into cDNA using the TaKaRa PCR Amplification Kit (R011, Dalian Takara Biotechnology Co., Ltd., Liaoning, China) and qPCR was performed on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). The primers used in qRT PCR were as follows: SP3F: 5′-GTAGCTTGCACCTGTCCCAACT-3′ (SEQ ID NO: 1), R: 5′-AATGCCAACGCAGGTGTGCTCT-3′ (SEQ ID NO: 2); GRIK1F: 5′-GTATGTGCTCCTCGCTTGCCTA-3′ (SEQ ID NO: 3), R: 5′-TTTCCACCACGTCTGAGTCAGG-3′ (SEQ ID NO: 4); GAPDH F: 5′-CATCACTGCCACCCAGAAGACTG-3′ (SEQ ID NO: 5), R: 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′ (SEQ ID NO: 6). The expression of each gene was calculated using the ΔΔCT method, with GAPDH as the internal reference gene.

[0072] In a specific embodiment, the protein blot analysis method in the present application includes: extracting the total protein of BV2 microglia and brain tissue samples cultured under different treatments with RIPA lysate (AR0105-100, Wuhan Boster Biotechnology Co., Ltd., Wuhan, Hubei), and determining the protein concentration with a BCA protein assay kit (CW0014S, Beijing Xiwei Biotechnology Co., Ltd., China). A total of 20 μg of protein was loaded onto a 12% SDS-PAGE gel and then transferred to a PVDF membrane (ab133411, Abcam). The PVDF membrane was sealed with 5% skim milk powder and detected with a primary antibody at 4°C overnight. After washing, the membrane was detected with a specific secondary antibody at RM for 1 hour. The immunoreactive bands were visualized by BeyoECL Plus (P0018S, Beyotime). Primary primary antibodies: GRIK1 (1:1000, NBP1-76849, Novus Biological Inc., Littleton, CO, USA), SP3 (1:1000, PA5-40700, Thermo Fisher Scientific), CD14 (1:1000, ab221678, Abcam), CD40 (1:1000, ab188181, Abcam), NLRP3 (1:1000, ab263899, Abcam), ASC (1:1000, PA5-50915, Novus Biological), IL-1β (1:1000, NB600-633, Novus Biological), Caspase-1p20 (Cleaved Asp296) (1:1400, PA5-99390, Thermo Fisher Scientific), Iba1 (1:1200, ab178846, Abcam), GAPDH (1:1200, NB100-56875, Novus Biological). The secondary antibody used was horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1000, HAF008, Novus Biological).

[0073] In one embodiment, the method of chromatin immunoprecipitation (ChIP) in the present application includes: ChIP detection is performed using a Pierce Magnetic ChIP kit (26157, Thermo Scientific). BV2 cells are cross-linked on ice containing 2% formaldehyde for 30 minutes, lysed on ice for 10 minutes, and then the cross-linked complex is extracted and lysed. The complex is then incubated with anti-SP3 (1:120, sc-28305, Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) at 4°C overnight. The antigen-antibody complex is then combined with the protein A / G magnetic beads in the kit at RM for 1 hour. The antigen / antibody complex is eluted by washing the microbeads twice with IP lysis / wash buffer and once with purified water. The separated and purified DNA is then amplified by qPCR.

[0074] In a specific embodiment, the method of dual luciferase reporter gene detection in the present application includes: inserting the GRIK1 promoter fragment into the pGL3-Basic (Promega, Madison, Wisconsin, USA) vector, constructing a GRIK1 promoter firefly luciferase reporter plasmid, co-transfecting the reporter plasmid with the Renilla luciferase reporter plasmid into BV2 cells that overexpress or do not overexpress SP3, and detecting after 48 hours using a dual luciferase reporter gene detection kit (RG027, Beyotime).

[0075] In a specific embodiment, the method of hematoxylin-eosin (HE) staining in the present application includes: dewaxing the slices with xylene (2 times, 20 minutes each time) and gradient ethanol (100%, 2 times, 5 minutes; 75%, 1 time, 5 minutes), washing with water and then staining with a HE staining kit (C0105S, Beyotime) according to the instructions, the method is: staining with hematoxylin staining solution for 5 to 8 minutes, soaking in tap water for about 10 minutes to wash away excess staining solution, rinsing with distilled water (several seconds), staining with eosin staining solution for 30 to 60 seconds, washing twice with 70% ethanol, and observing under an optical microscope.

[0076] In a specific embodiment, the method of immunohistochemical staining in the present application includes: the fixed brain slices are dewaxed and rehydrated with xylene and gradient ethanol, and then placed in citric acid antigen retrieval buffer (P0081, Beyotime) for antigen retrieval, then soaked in PBS, oscillated on a decolorization shaker 3 times, 5 minutes each time, incubated in RM in the dark with 3% hydrogen peroxide for 25 minutes, permeabilized with proteinase K in PBS (10401ES60, Shanghai Yasen Biotechnology Co., Ltd.), sealed with 5% goat serum, and incubated overnight at 4°C with GRIK1 (1:50, PA5-20447, Thermo Fisher Scientific) and SP3 (1:200, PA5-40700, Thermo Fisher Scientific) antibodies. The sections were incubated with HRP-labeled secondary antibody goat anti-rabbit IgG (1:1000, HAF008, Novus Biological) at room temperature for 50 min, developed with diaminobenzidine (D8001-1G, Merck Millipore, Darmstadt, Germany), counterstained with hematoxylin (C0107, Beyotime), mounted with neutral resin, and finally observed under an optical microscope.

[0077] In one embodiment, the method of immunofluorescence labeling in the present application includes: the procedure of immunofluorescence staining is similar to immunohistochemistry. The primary antibody and secondary antibody are GRIK1 (1:50, AGC-008-200UL, Thermo Fisher Scientific), Iba1 (1:100, ab283319, Abcam) and goat anti-rabbit IgG H&L (Alexa Fluor 488) (1:200, ab150077, Abcam) or goat anti-mouse IgG H&L (Alexa Fluor 647) (1:500, ab150115, Abcam). DAPI (OMDCM007-1ml, Oumarsi, Shanghai, China) is used to counterstain the cell nucleus.

[0078] In one embodiment, the flow cytometry method in the present application includes: detecting the expression of CD86 and CD206 in BV2 cells by flow cytometry. The cells were inoculated in a 24-well plate and starved for 24 hours. Subsequently, the cell suspension (100 μL) was incubated with APC-anti-CD86 (ab218757, Abcam) or PE-CD206 (12-2061-82, Thermo Fisher Scientific Inc) at 4°C in the dark for 20 minutes and then loaded into the flow cytometer.

[0079] In one embodiment, the method of enzyme-linked immunosorbent assay (ELISA) in the present application includes: centrifuging the cell and tissue samples at 1000×g for 10 minutes at 4°C, collecting the supernatant of the lysate, and adding a sterile protease mixture inhibitor to prevent protein degradation. The levels of IL-6 and IL-1β in microglia and the content of IL-1β in mouse brain tissue were detected using a mouse IL-6 ELISA kit (ab100713, Abcam) and a mouse IL-1β ELISA kit (ab197742, Abcam). Finally, the OD value was measured at 450nm using a microplate reader.

[0080] In a specific embodiment, the method of statistical analysis in the present application includes: the data values ​​obtained are expressed as mean ± SEM. All statistical analyses were performed using GraphPad Prism 8.0.2 (GraphPad, San Diego, California, USA). Unpaired t-tests were used to compare a variable under two conditions. For experiments comparing a variable under more than two conditions, one-way analysis of variance and Tukey's multiple comparison test were used. Two-way analysis of variance and Tukey's multiple comparison test were used to compare two variables under multiple conditions. Probability values ​​of p < 0.05 were considered statistically significant.

[0081] In a specific embodiment, one result in the present application is that GRIK1 expression in the brain tissue of APP / PS1 mice is significantly increased, specifically: firstly, the brain tissue of 3xTg-AD mice and control mice in the GSE144459 dataset ( Figure 4 A) and hippocampal tissues of AD patients and healthy controls in the GSE184942 dataset ( Figure 4 The differentially expressed genes in the two datasets were screened out using the Benjamini&Hochberg method for p value correction, with p adj < 0.05 as the threshold. Figure 4 C) and 29 differentially expressed genes ( Figure 4 D). Then, AD-related genes were downloaded from GeneCards (https: / / www.genecards.org / ) using the keyword "Alzheimer's disease". The 5000 genes with the highest correlation scores were intersected with the differentially expressed genes in the two datasets, and a total of 5 genes were found ( Figure 4 E), POSTN, GRIK1, EEF1G, RPS2, and SCN1A, respectively.

[0082] To determine whether the model was successfully constructed, the MWM test was first performed. Compared with PBS-injected (normal) mice, APP / PS1 mice took longer to reach the hidden platform ( Figure 4 F), fewer platforms are crossed ( Figure 4 G), which indicates cognitive dysfunction. In addition, HE staining showed that the neurons in the hippocampus of AD mice were significantly atrophied ( Figure 4 H). Using RT-qPCR ( Figure 4 I) and immunohistochemistry ( Figure 4 J) Examination of GRIK1 expression in mouse brain tissues. Upregulation of GRIK1 was confirmed in the brain tissues of APP / PS1 mice.

[0083] In one embodiment, a result in the present application is that knocking down GRIK1 can improve cognitive dysfunction in APP / PS1 mice. Specifically, to study the functional role of GRIK1 in AD, lentiviral vectors carrying sh-GRIK1 or sh-NC were injected into mice. As expected, sh-GRIK1 treatment had an effect on the transcriptional level ( Figure 5 A) and translation level ( Figure 5 B) The above results showed that the expression of GRIK1 in the brain tissue of APP / PS1 mice was downregulated.

[0084] The elevated plus maze test showed that the time that APP / PS1 mice spent exploring the open arms was shorter than that of normal mice. In contrast, mice with GRIK1 knockdown spent more time exploring the open arms ( Figure 5 C). Double labeling with anti-GRIK1 and anti-Iba1 (a microglial marker) in brain tissues of mice receiving different treatments. As expected, silencing GRIK1 reduced Aβ-induced microglial activation ( Figure 5 D). The microglial activation marker proteins CD14 and CD40 in the brain tissue of AD mice were significantly increased, while knocking down GRIK1 could reduce the expression of CD14 and CD40 proteins ( Figure 5 E). In the brain tissue of AD mice, the expression of inflammasome components (including NLRP3, ASC, and IL-1β) was upregulated, while knockdown of GRIK1 downregulated the expression of NLRP3, ASC, and IL-1β ( Figure 5 F).

[0085] In one embodiment, a result in the present application is that knocking down GRIK1 can inhibit the activation of NLRP3 inflammasome in BV2 cells, specifically: BV2 cells were infected with GRIK1 shRNA lentivirus and then treated with LPS. First, compared with PBS treatment, the expression of GRIK1 protein in microglia after LPS induction was significantly increased. Pre-infection with sh-GRIK1 can significantly inhibit the expression of GRIK1 protein ( Figure 6 A). After LPS induction, the expression of NLRP3, ASC and Cleaved caspase-1 proteins in BV2 cells was increased, while these proteins were decreased after GRIK1 knockdown ( Figure 6 B). In addition, the expression of M1 (CD86) and M2 markers (CD206) was detected by flow cytometry. The results showed that compared with the PBS group, the expression of CD86 increased and the expression of CD206 decreased after LPS induction; compared with the sh-NC group, the expression of CD86 decreased and the expression of CD206 increased after sh-GRIK1 treatment ( Figure 6 C). Finally, ELISA was used to detect the changes in pro-inflammatory factors IL-6 and IL-1β. After LPS treatment of BV2 cells, the levels of IL-6 and IL-1β increased, while knockdown of GRIK1 significantly reduced them ( Figure 6 D).

[0086] In a specific embodiment, one result in the present application is that SP3 expression is significantly increased in the brain tissue of AD-like mice. Specifically, in order to clarify the mechanism of GRIK1 overexpression in AD, transcription factors with binding sites to the GRIK1 promoter were downloaded from GeneCards, and then these transcription factors were intersected with the top 5000 AD-related genes predicted by GeneCards. The results showed that there were 4 intersections ( Figure 7 A): SIN3A, NRF1, SP3 and EZH2.

[0087] SP3 mRNA expression in brain tissue of AD mice ( Figure 7 B) and the number of positive cells ( Figure 7 C) were significantly increased, and upregulation of SP3 protein was also detected in LPS-induced BV2 cells ( Figure 7 D).

[0088] In a specific embodiment, a result in the present application is that SP3 promotes the transcriptional activation of GRIK1 by binding to the GRIK1 promoter, specifically: the binding matrix of SP3 was downloaded from the Jaspar database (https: / / jaspar.genereg.net / ) Figure 8 A), multiple binding sites were found between SP3 and the GRIK1 promoter ( Figure 8 B), GEPIA database (http: / / gepia.cancer-pku.cn / index.html) showed that the expression of SP3 and GRIK1 in the hippocampus was significantly positively correlated ( Figure 8 C).

[0089] BV2 cells were infected with a lentivirus overexpressing SP3, and then LPS was used to induce inflammasome activation. RT-qPCR results showed that the mRNA expression of SP3 in BV2 cells infected with oe-SP3 was significantly higher than that in the oe-NC group ( Figure 8 D). After overexpression of SP3, the relative luciferase activity of the GRIK1 promoter increased significantly ( Figure 8 E). Subsequently, the mRNA expression of GRIK1 in BV2 cells of the oe-SP3 group was increased compared with that of the oe-NC group ( Figure 8 F). In addition, ChIP experiments showed that anti-SP3 was significantly enriched in the GRIK1 promoter in BV2 cells ( Figure 8 G).

[0090] In a specific embodiment, one result in the present application is that overexpression of GRIK1 reverses the alleviating effect of sh-SP3 on AD-like mice, specifically: In order to further study the role of SP3 in AD, APP / PS1 mice were treated with sh-NC, sh-SP3, sh-SP3+oe-NC and sh-SP3+oe-GRIK1, respectively. Immunohistochemical staining showed that the levels of SP3 and GRIK1 proteins in brain tissue decreased after knocking down SP3, while there was no significant change in the level of SP3 protein after overexpression of GRIK1, and the level of GRIK1 protein in brain tissue increased significantly ( Fig. 9 A). The MWM test was used to study the cognitive ability of mice. After knocking down SP3, the time it took for mice to reach the hidden platform and the number of times they crossed the platform were both better, while on this basis, the results of mice that overexpressed GRIK1 were worse ( Fig. 9 B). Western blot experiments showed that knockdown of SP3 downregulated the expression of NLRP3, ASC, and Iba1 in brain tissue. In contrast, overexpression of GRIK1 upregulated the expression of NLRP3, ASC, and Iba1 ( Fig. 9 C). HE staining was used to observe the pathological morphology of neurons. Knockdown of SP3 improved the atrophy of brain neurons, while overexpression of GRIK1 significantly atrophied the neurons in the brain ( Fig. 9 D).

[0091] In one embodiment, one result in the present application is that GRIK1 overexpression induces NLRP3 inflammasome activation in BV2 cells, specifically: BV2 cells were infected with SP3shRNA lentivirus, followed by LPS induction, and the same 4 groups as in the in vivo study were set up. First, RT-qPCR assay showed that after knocking down SP3, the mRNA expression of SP3 and GRIK1 in BV2 cells was significantly reduced. On this basis, overexpression of GRIK1 increased the mRNA expression of GRIK1, while the mRNA level of SP3 did not change significantly ( Fig.10A). After knockdown of SP3, the expression of NLRP3, ASC, and Cleaved caspase-1 in BV2 cells was significantly reduced, while overexpression of GRIK1 enhanced these expressions ( Fig.10 B). In addition, the expression of M1 and M2 markers was detected by flow cytometry. Knockdown of SP3 resulted in a significant downregulation of CD86 expression and a significant upregulation of CD206 expression in BV2 cells. Overexpression of GRIK1 then polarized BV2 cells to the M1 phenotype ( Fig.10 C). Finally, ELISA was used to detect the changes in pro-inflammatory factors IL-6 and IL-1β. After knocking down SP3, the expression of IL-6 and IL-1β in microglia was significantly reduced, while overexpression of GRIK1 increased the expression of IL-6 and IL-1β in microglia ( Fig.10 D).

[0092] 102: Determine the binding sites of the SP3 protein and the GRIK1 protein based on the SP3 protein and the GRIK1 protein data

[0093] In some embodiments, virtual screening is an important method in targeted drug design, and its process is as follows:

[0094] First, based on the known complex structure of the drug and the target protein, the binding mode of the drug and the target protein is studied to determine the key amino acid residues. This step helps to understand how the drug interacts with the target protein and provides a basis for subsequent virtual screening.

[0095] Next, a large number of small molecule compounds are virtually screened using computer-aided drug design methods. In this process, the binding ability of small molecule compounds to target proteins will be predicted and evaluated. Generally, small molecules with low binding energy and good shape complementarity will be selected as potential drug candidates.

[0096] Finally, the screened candidate drugs are further experimentally verified to confirm their interaction with the target protein and their biological activity.

[0097] In some embodiments, the binding site is a specific part of a molecule that can form a stable interaction with a ligand. The binding site of a protein is usually formed by a certain number of separated amino acid residues on a polypeptide chain that are gathered together in space through the folding of the polypeptide chain to form a specific spatial arrangement.

[0098] Once the binding site is determined, the computer-aided drug design method can be used to dock the compounds in the small molecule compound library with the target protein. During the docking process, the computer simulates the interaction between the compound and the binding site and evaluates their binding ability. Based on the docking results, small molecule compounds with strong binding ability to the target protein can be screened as potential drug candidates. The determination of virtual screening binding sites is crucial to the success of drug design. Therefore, when determining the binding site, it is necessary to consider a variety of factors, such as the structural characteristics of the target protein, the binding mode of known ligands, etc. At the same time, in the virtual screening process, the binding site also needs to be reasonably processed and optimized to improve the accuracy and efficiency of the screening.

[0099] In some embodiments, the molecular libraries used in virtual screening mainly include the following:

[0100] ZINC: contains more than 250 million commercially available compounds for small molecule virtual screening.

[0101] PubChem: contains bioactive substances for small molecule virtual screening.

[0102] DrugBank: Contains drugs and small molecules for drug design and discovery.

[0103] ChEMBL: Contains small molecules and is used for drug discovery and chemical genomics research.

[0104] ChemDB: Contains a large number of known small molecules for chemical genomics research and drug discovery.

[0105] HMDB: Contains a large number of known small molecules for use in chemical genomics research and drug discovery.

[0106] BindingDB: Contains a large number of known small molecules for chemical genomics research and drug discovery.

[0107] SMPDB: Contains a large number of known small molecules for chemical genomics research and drug discovery.

[0108] In addition, there are some commercial databases such as ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas-M and Interbioscreen, which are also often used for virtual screening.

[0109] 103: Obtaining candidate drugs targeting the binding sites of the two through computer-assisted screening

[0110] In some embodiments, the process of computer-assisted screening is:

[0111] Screening of small molecule compounds based on the spatial structure of the binding sites of SP3 protein and GRIK1 protein;

[0112] The small molecule compounds obtained by screening are molecularly docked with the binding sites to obtain the scores of the docking molecules;

[0113] The candidate drugs are obtained by sorting according to the scores.

[0114] In other embodiments, the computer-assisted screening process is:

[0115] The molecular structures of SP3 protein and GRIK1 protein are obtained and input into the pharmacophore module library for matching, and all the action sites are clustered based on the interaction patterns with SP3 and GRIK1 to obtain a pharmacophore model;

[0116] The pharmacophore model is input into a molecular compound database for high throughput screening to obtain candidate drugs.

[0117] In other embodiments, the computer-assisted screening process is:

[0118] Obtaining the molecular structure of a small molecule inhibitor that inhibits SP3 and / or the molecular structure of a small molecule inhibitor that inhibits GRIK1;

[0119] Based on the molecular structure of the small molecule inhibitor, a small molecule library with similar structure is obtained by screening;

[0120] Performing molecular docking on the small molecule library with similar structures and the binding site to obtain a score of the docked molecule;

[0121] The candidate drugs are obtained by sorting according to the scores.

[0122] In some embodiments, the candidate drugs include protein analogs, antibodies, and RNA drugs.

[0123] In some embodiments, computer-aided drug screening is a technology that uses computer-aided drug design methods to screen drugs. It can help researchers quickly screen candidate drugs with strong binding ability to target proteins and potential efficacy from a large number of small molecule compounds.

[0124] In some embodiments, molecular docking is a method for drug design based on the characteristics of receptors and the interaction between receptors and drug molecules. It mainly studies the interaction between molecules (such as ligands and receptors) and predicts their binding mode and affinity. This method is widely used in the early stages of drug development and can help researchers quickly screen out compounds with potential efficacy.

[0125] Molecular docking methods mainly focus on spatial matching and energy matching. Spatial matching refers to the geometric complementarity between the drug molecule and the receptor protein, while energy matching refers to the minimization of the interaction between the drug molecule and the receptor protein. For the calculation of geometric matching, grid point calculation, fragment growth and other methods are usually used, while energy calculation uses simulated annealing, genetic algorithm and other methods.

[0126] According to the degree and method of simplification, molecular docking methods can be divided into rigid docking, semi-flexible docking and flexible docking. In the rigid docking method, the molecular conformations involved in the docking do not change during the calculation process, only the spatial position and posture of the molecules are changed. Semi-flexible docking allows some conformations to change during the calculation. Flexible docking allows more conformational changes.

[0127] In some embodiments, in molecular docking, affinity refers to how tightly a molecule binds to a receptor. A high affinity means that the binding is more stable, while a low affinity means that the binding is less stable. Affinity is usually calculated, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd).

[0128] In the process of molecular docking, affinity depends on the interaction between molecules and receptors, including hydrogen bonds, van der Waals forces, electrostatic interactions, etc. These interactions jointly determine the binding mode between molecules and receptors, thus affecting affinity.

[0129] In order to evaluate affinity, some scoring systems or scoring methods are usually used to quantify the interaction between molecules and receptors. These scoring methods are based on different algorithms and physical models, which can reflect the binding energy, interaction type and affinity between molecules and receptors.

[0130] In some embodiments, the process and method of designing protein analogs, antibodies and other drugs based on proteins can be summarized as follows:

[0131] Determine the target protein: First, you need to determine the target protein for drug design, that is, the target. The target can be a known disease-related protein, viral antigen or other biological molecule.

[0132] Protein structure analysis: Perform structural analysis on the target protein to understand its three-dimensional conformation, surface configuration, subdomain structure and other characteristics. This can be obtained through X-ray crystal diffraction, nuclear magnetic resonance and other technologies.

[0133] Determine drug-protein interactions: Study the interaction mechanism between the drug and the target protein, including binding sites, binding modes, and binding kinetics. This can be done through computer simulations, laboratory experiments, and other methods.

[0134] Drug design: Based on the interaction mechanism between the drug and the target protein, design drug molecules that can specifically bind to the target protein. This includes selecting the appropriate drug type, designing the chemical structure of the molecule, and optimizing the drug efficacy and pharmacokinetic properties of the molecule.

[0135] Synthesize and validate drugs: Prepare drug molecules through chemical synthesis and other methods, and validate their biological activity, safety and pharmacokinetic properties. This includes different stages such as cell experiments, animal experiments and clinical trials.

[0136] In some embodiments, protein analogs refer to substances that have similar structures and functions to certain proteins in the body. They can mimic the functions of proteins and thus play an important role in disease treatment.

[0137] Antibodies are immunoglobulins produced by plasma cells differentiated from B lymphocytes under antigen stimulation, which can specifically bind to the corresponding antigen. The structure of antibodies is mainly divided into two parts: constant region and variable region. In a given species, the constant regions of different antibody molecules have the same or almost the same amino acid sequence. The variable region is located at the ends of the two arms of the "Y".

[0138] RNA drugs are a class of drugs designed based on the properties of RNA and its mechanism of action in cells. RNA can act as messenger RNA (mRNA) to guide protein synthesis in cells, or as microRNA (miRNA) to regulate gene expression. Therefore, rationally designed RNA drugs can be used to regulate gene expression in cells to achieve the purpose of treating diseases. For example, many studies are exploring the use of mRNA vaccines to prevent and treat various infectious diseases.

[0139] In some embodiments, the general steps for performing an inhibitory activity experiment on a small molecule compound are as follows:

[0140] Select the appropriate target protein: According to the research purpose and disease target, select the appropriate target protein as the experimental object. Make sure that the target protein has potential interaction with the small molecule compound being studied.

[0141] Preparation of small molecule compounds: Synthesize or purchase the required small molecule compounds and ensure their purity and structural accuracy. If necessary, the small molecule compounds can be modified or transformed to optimize their inhibitory activity.

[0142] Enzyme activity assay: Design appropriate enzyme activity assays to evaluate the inhibitory activity of small molecule compounds on target proteins. This can be achieved through techniques such as fluorescence resonance energy transfer (FRET), radioisotope labeling, and enzyme-linked immunosorbent assay (ELISA). Ensure that the assay is reliable, sensitive, and can accurately reflect the interaction between small molecule compounds and target proteins.

[0143] Experimental operation: Mix small molecule compounds of different concentrations with the target protein, incubate for a period of time under appropriate conditions, and then measure the enzyme activity. According to the experimental design, a control group and an experimental group can be set up to compare the inhibitory activity of different small molecule compounds.

[0144] Data analysis: The IC50 value of the small molecule compound (i.e., the concentration of the compound required to inhibit 50% of the enzyme activity) is determined by statistical analysis of the experimental data. The smaller the IC50 value, the stronger the inhibitory activity of the small molecule compound. In addition, a dose-effect curve can be drawn to intuitively display the inhibitory activity of the small molecule compound.

[0145] Interpretation and discussion of results: Based on the experimental results, analyze the inhibitory activity of small molecule compounds on target proteins and discuss them in combination with their structures and properties. Explore possible binding modes, mechanisms of action, and comparisons with known inhibitors. In addition, the effects of small molecule compounds on cells or organisms can be further studied to evaluate their potential drug candidates.

[0146] In some embodiments, co-immunoprecipitation (Co-IP) is a classic method for studying protein interactions based on the specific interaction between antibodies and antigens. It is an effective method to determine the physiological interaction between two proteins in intact cells. The principle is to use target protein-specific antibodies to indirectly capture proteins bound to specific target proteins to identify relevant protein-protein interactions in vivo, which is a classic method for studying protein interactions. Specific antibodies capture target proteins in samples to form antibody-target protein complexes, and then use bead supports that can bind to antibodies (such as Nanoab-Agarose) to fix and precipitate the complexes. At the same time, proteins that interact with the target protein will also be precipitated. Finally, non-specific proteins bound to the target protein are washed away, and then analyzed by SDS-PAGE and Western blot.

[0147] In some embodiments, co-transfection refers to the simultaneous transfection of two independent nucleic acid molecules, such as plasmid DNA and siRNA, and is a common procedure for stable transfection. The physical process of co-transfection is to integrate the two nucleic acid molecules into the same integration sequence and express them in the same transfected cell. It involves selecting the right co-transfection reagent and using advanced lipid nanoparticle technology to achieve excellent transfection performance and reproducible results. The purpose of co-transfection is to make cells express two different genetic markers at the same time, such as resistance to a certain toxin.

[0148] In some embodiments, the computer-assisted drug screening technology for drug screening includes one or more of the following: protein-small molecule docking, protein-protein docking, protein-nucleic acid docking.

[0149] In some embodiments, protein-small molecule docking refers to a computational simulation process of docking the structures of proteins and small molecules (such as drug molecules) together through certain algorithms and programs. This process can be used to study the interactions between proteins and small molecules, as well as possible biological functions.

[0150] In protein-small molecule docking, software such as DOCK is usually used for computational simulation. DOCK is a highly automated drug design software that can achieve docking between small molecule ligands and biomacromolecule receptors. It uses a fragment-based scoring method to achieve fast and accurate docking. The basic algorithm of DOCK software includes two stages. The first stage is the low-precision stage, which mainly searches for a rough match between small molecule ligands and biomacromolecule receptors. The second stage is the high-precision stage, in which all side chain conformations are considered to calculate more accurate interaction energy. In the low-precision stage, DOCK software will randomly translate and rotate small molecule ligands, perform a certain number of rigid body moves, and then calculate the interaction energy. After outputting the lowest conformation, it enters the high-precision stage. In the high-precision stage, the program will perform more optimization and adjustments to achieve more accurate docking.

[0151] In some embodiments, protein-protein docking refers to a computational simulation process of docking the structures of two proteins together through certain algorithms and programs. This process can be used to study the interactions between proteins and possible biological functions.

[0152] RosettaDock is a commonly used protein-protein docking software that uses a fragment-based scoring method to achieve fast and accurate protein docking. The software can achieve precise adjustment of side chain conformations during docking and consider a variety of complex interactions, such as hydrogen bonds, ionic bonds, hydrophobic interactions, etc. The basic algorithm of RosettaDock includes two stages. The first stage is the low-precision stage, which mainly searches for the degree of adaptation of the backbone shapes between the two proteins. The second stage is the high-precision stage, in which all side chain conformations are considered to calculate more accurate interaction energies. In the low-precision stage, the program randomly translates and rotates a component of a protein molecule, performs a certain number of rigid body moves, and then calculates the interaction energy. After outputting the lowest conformation, it enters the high-precision stage. In the high-precision stage, the program performs 50 MCMPCycles: rearranges the conformation and minimizes the interaction energy, which is used as the initial starting conformation.

[0153] In some embodiments, protein-nucleic acid docking refers to a computational simulation process of docking the structures of proteins and nucleic acids (such as DNA or RNA) together through certain algorithms and programs. This process can be used to study the interactions between proteins and nucleic acids, as well as possible biological functions.

[0154] In protein-nucleic acid docking, software such as NAflex is usually used for computational simulation. NAflex is a software developed specifically for nucleic acid structure prediction and design, which can achieve accurate modeling and docking of DNA or RNA molecules. NAflex software uses a fragment-based scoring method to achieve fast and accurate docking. It takes into account a variety of complex interactions, such as hydrogen bonds, ionic bonds, hydrophobic interactions, etc., and can achieve precise adjustment of side chain conformations during docking. The basic algorithm of NAflex software includes two stages. The first stage is the low-precision stage, which mainly searches for rough matches between proteins and nucleic acids. The second stage is the high-precision stage, in which all side chain conformations are considered and more accurate interaction energies are calculated. In the low-precision stage, NAflex software randomly translates and rotates nucleic acid molecules, performs a certain number of rigid body moves, and then calculates the interaction energy. After outputting the lowest conformation, it enters the high-precision stage. In the high-precision stage, the program will perform more optimization and adjustments to achieve more accurate docking.

[0155] Figure 2 Schematic diagram of a system for computer-assisted drug screening based on SP3 and GRIK1 provided by the present invention, specifically comprising:

[0156] 201: Acquisition unit: Acquisition of SP3 protein and GRIK1 protein data

[0157] 202: Site determination unit: determining the binding sites of the SP3 protein and the GRIK1 protein according to the SP3 protein and the GRIK1 protein data

[0158] 203: Screening Unit: Obtain candidate drugs targeting the binding sites of the two through computer-assisted screening

[0159] Figure 3 Schematic diagram of a device for computer-assisted drug screening based on SP3 and GRIK1 provided by the present invention, specifically comprising:

[0160] A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the above-mentioned computer-assisted drug screening method based on SP3 and GRIK1 is implemented.

[0161] The present invention provides use of the above-mentioned drug, a substance inhibiting SP3, a substance inhibiting GRIK1 or a substance inhibiting SP3-GRIK1 in preparing a drug for treating Alzheimer's disease.

[0162] The medicine also includes a pharmaceutically acceptable carrier.

[0163] In some embodiments, a pharmaceutically acceptable carrier is used to refer to a material that is compatible with a recipient, preferably a mammal, more preferably a human, and is suitable for delivering an active agent to a target site without terminating the activity of the agent. Toxicity or side effects (if any) associated with the pharmaceutically acceptable carrier are preferably commensurate with a reasonable risk / benefit ratio for the intended use of the active agent.

[0164] Pharmaceutically acceptable carriers include, but are not limited to, diluents, adhesives, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants. These carriers are used to help the stability of the formulation or to help improve the activity or its biological effectiveness or to produce an acceptable taste or smell in the case of oral administration. The preparations that can be used in this medicine can be in the form of the original compound itself or optionally in the form of a pharmaceutically acceptable salt thereof. The pharmaceutical composition thus prepared can be administered in any appropriate manner known to those skilled in the art as needed.

[0165] Among them, the diluent includes but is not limited to lactose, sodium chloride, glucose, urea, starch, and water.

[0166] Binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

[0167] Surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, and cetyl alcohol.

[0168] Humectants include, but are not limited to, glycerin.

[0169] Adsorption carriers include, but are not limited to, bentonite, silica gel, kaolin, and bentonite.

[0170] Lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium dodecyl sulfate.

[0171] Fillers include, but are not limited to, mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugars, coupling sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, and sodium bicarbonate.

[0172] Disintegrants include, but are not limited to, cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, and soybean polysaccharides.

[0173] The verification results of this verification embodiment show that assigning inherent weights to indications can improve the performance of the method relative to the default setting. Those skilled in the art can clearly understand that for the convenience and simplicity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments, and will not be repeated here. In the several embodiments provided in this application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic, for example, the division of the units is only a logical function division, and there may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the coupling or direct coupling or communication connection between each other shown or discussed can be through some interfaces, indirect coupling or communication connection of devices or units, which can be electrical, mechanical or other forms. The unit described as a separate component may or may not be physically separated, and the component displayed as a unit may or may not be a physical unit, that is, it may be located in one place, or it may be distributed on multiple network units. Some or all of the units can be selected according to actual needs to achieve the purpose of the scheme of this embodiment. In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units. A person of ordinary skill in the art may understand that all or part of the steps in the various methods of the above-mentioned embodiments may be completed by instructing the relevant hardware through a program, and the program may be stored in a computer-readable storage medium, and the storage medium may include: a read-only memory (ROM), a random access memory (RAM), a disk or an optical disk, etc.

[0174] A person skilled in the art can understand that all or part of the steps in the above-mentioned embodiment method can be implemented by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium. The above-mentioned medium storage can be a read-only memory, a disk or an optical disk, etc.

[0175] The above is a detailed introduction to a computer device provided by the present invention. For a person skilled in the art, according to the concept of the embodiments of the present invention, there may be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for computer-assisted drug screening based on SP3 and GRIK1, characterized in that: The method comprises: Obtain SP3 protein and GRIK1 protein data; Determine the binding sites of the SP3 protein and the GRIK1 protein according to the SP3 protein and the GRIK1 protein data; Candidate drugs targeting the binding sites of the two were obtained through computer-assisted screening.

2. The method for computer-assisted drug screening based on SP3 and GRIK1 according to claim 1, characterized in that: The process of computer-assisted screening is: Screening of small molecule compounds based on the spatial structure of the binding sites of SP3 protein and GRIK1 protein; The small molecule compounds obtained by screening are molecularly docked with the binding sites to obtain the scores of the docking molecules; Sorting according to the scores to obtain candidate drugs; Preferably, the process of computer-assisted screening is: The molecular structures of SP3 protein and GRIK1 protein are obtained and input into the pharmacophore module library for matching, and all the action sites are clustered based on the interaction patterns with SP3 and GRIK1 to obtain a pharmacophore model; Inputting the pharmacophore model into a molecular compound database for high-throughput screening to obtain candidate drugs; Preferably, the process of computer-assisted screening is: Obtaining the molecular structure of a small molecule inhibitor that inhibits SP3 and / or the molecular structure of a small molecule inhibitor that inhibits GRIK1; Based on the molecular structure of the small molecule inhibitor, a small molecule library with similar structure is obtained by screening; Performing molecular docking on the small molecule library with similar structures and the binding site to obtain a score of the docked molecule; The candidate drugs are obtained by sorting according to the scores.

3. The method for computer-assisted drug screening based on SP3 and GRIK1 according to claim 2, characterized in that: The candidate drugs include protein analogs, antibodies, and RNA drugs.

4. A computer-assisted drug screening system based on SP3 and GRIK1, characterized in that: The system comprises: Acquisition unit: acquire SP3 protein and GRIK1 protein data; A site determination unit: determining the binding sites of the SP3 protein and the GRIK1 protein according to the SP3 protein and the GRIK1 protein data; Screening unit: Obtain candidate drugs targeting the binding sites of the two through computer-assisted screening.

5. A device for computer-assisted drug screening based on SP3 and GRIK1, characterized in that: The device comprises: A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the computer-assisted drug screening method based on SP3 and GRIK1 according to any one of claims 1 to 3 is implemented.

6. A computer-readable storage medium having a computer program thereon, characterized in that: The computer readable storage medium comprises: When the computer program is executed by a processor, the method for computer-assisted drug screening based on SP3 and GRIK1 according to any one of claims 1 to 3 is implemented.

7. A drug screened by the computer-assisted drug screening method based on SP3 and GRIK1 according to any one of claims 1 to 3.

8. A method for regulating GRIK1 activity or GRIK1 level in a cell, characterized in that: The method comprises administering the SP3 protein or a promoter thereof.

9. A method for regulating inflammasomes in cells, characterized in that: The method comprises administering SP3, GRIK1, or an SP3-GRIK1 protein complex; Preferably, the inflammasome is the NLRP3 inflammasome; Preferably, the cells are microglial cells.

10. Any of the following applications: (1) Use of the drug, substance inhibiting SP3, substance inhibiting GRIK1 or substance inhibiting SP3-GRIK1 according to claim 7 in the preparation of a drug for treating Alzheimer's disease; (2) Use of SP3, GRIK1 or SP3-GRIK1 protein complex in regulating inflammasomes or in the preparation of drugs for regulating inflammasomes; (3) Application of SP3 in regulating GRIK1 activity or GRIK1 levels; Preferably, the substance inhibiting SP3, the substance inhibiting GRIK1 or the substance inhibiting SP3-GRIK1 in (1) comprises a protein analog, an antibody or an RNA drug; Preferably, the drug described in (1) or (2) further comprises a pharmaceutically acceptable carrier; Preferably, SP3 acts on the GRIK1 promoter region; Preferably, the inflammasome is the NLRP3 inflammasome; Preferably, the NLRP3 inflammasome is a NLRP3 inflammasome in microglia and / or AD.

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