A computer-aided drug screening method, system and device based on TRIM21 and PRMT1

Abstract

The application discloses a computer-aided drug screening method, system and device based on TRIM21 and PRMT1, provides a computer-aided drug screening method, system, device and computer readable storage medium based on TRIM21 and PRMT1 for the field, and provides an efficient, accurate and rapid screening method for research and development of new drugs for treating colorectal cancer, and has a very wide application prospect in screening and development of new drugs for treating colorectal cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of computer-aided drug screening, and particularly relates to a computer-aided drug screening method, system and device based on TRIM21 and PRMT1. BACKGROUND

[0002] Colorectal cancer (CRC), as one of the most common malignant tumors in the digestive tract, its incidence is increasing year by year, and it has become the third most common malignant tumor in the world. Revealing its potential molecular mechanism is not only the demand of scientific research, but also the urgent expectation of clinical diagnosis and treatment. The development of colorectal cancer is a complex process involving multiple factors, multiple stages and multiple genes. In recent years, epigenetics has gradually occupied an important position in tumor research. Among them, post-translational modification of proteins, especially arginine methylation and ubiquitination, has been found to have a profound impact on the proliferation, invasion and metastasis of cancer cells. Although the research and clinical application of new drugs such as targeted therapy and immune checkpoint inhibitors have made some progress, the cure rate of colorectal cancer has not been significantly improved. Because the onset of colorectal cancer is insidious, it usually develops into an advanced stage when it has deep local infiltration, extensive lymph node metastasis in the abdominal cavity, or even distant metastasis, resulting in delayed diagnosis. Therefore, in CRC research, it is crucial to identify sensitive and specific biomarkers and find more effective therapeutic targets.

[0003] TRIM21 belongs to the TRIM family and has E3 ubiquitin ligase activity. In recent years, the function of TRIM21 in the process of inflammation, cancer development and autoimmune diseases has been widely concerned. In the process of tumor occurrence and development, its function can vary due to specific effectors involved in tumor occurrence and development and the type of tumor itself. Some studies have shown that TRIM21 plays a tumor suppressor role in specific types of cancer such as breast cancer, kidney cancer and cervical cancer. However, in other types of cancer such as glioma, nasopharyngeal carcinoma and liver cancer, TRIM21 has been found to promote tumor growth. Interestingly, TRIM21 is associated with reduced stemness and metastatic potential of colorectal cancer cells, but there have also been reports that TRIM21 contributes to CRC chemoresistance and tumorigenesis. In summary, it is necessary to further fully elucidate the exact role and potential mechanism of TRIM21 in CRC in order to provide new ideas and strategies for the development or screening of new drugs for colorectal cancer treatment.

[0004] Computer-aided Drug Design (CADD) is a method of using computer science and bioinformatics technology to accelerate the drug development process. CADD is based on computer operation and simulation technology, learns prior knowledge contained in massive drug data, and excavates the interaction relationship between target points and candidate drugs. Therefore, 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 development efficiency. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a TRIM21 and PRMT1 based computer-aided drug screening method, system and device for the field. The computer-aided drug screening technology is used to screen drugs for treating and / or preventing colorectal cancer.

[0006] The above invention purpose of the present application is realized by the following technical solutions:

[0007] The first aspect of the present application provides a TRIM21 and PRMT1 based computer-aided drug screening method.

[0008] Further, the method comprises:

[0009] Obtaining TRIM21 protein and PRMT1 protein data;

[0010] Selecting the spatial structure of the TRIM21 protein and PRMT1 protein complex, determining the binding site of the TRIM21 protein and PRMT1 protein in the TRIM21 protein and PRMT1 protein complex as the binding site of the targeted drug;

[0011] Using a computer-aided drug screening method to obtain a candidate drug targeting the binding site.

[0012] Further, the method for determining the binding site of the TRIM21 protein and PRMT1 protein in the TRIM21 protein and PRMT1 protein complex comprises:

[0013] Constructing n kinds of truncated expression mutants of TRIM21, n is a natural number greater than or equal to 1;

[0014] Based on the n kinds of truncated expression mutants, an immunoprecipitation experiment is performed to obtain the key region of the interaction between TRIM21 and PRMT1;

[0015] The spatial structure formed by the N-terminal SPRY domain of the TRIM21 and the PRMT1 is a binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex.

[0016] Further, the method for computer-aided screening of drugs comprises:

[0017] obtaining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex;

[0018] screening structurally similar small molecule compounds in a molecular database based on the spatial structure of the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex;

[0019] performing molecular docking calculation on the screened small molecule compounds and the PRMT1 protein to obtain the scores of the small molecule compounds targeting the receptor PRMT1 protein, and ranking the small molecule compounds according to the scores to obtain candidate drugs.

[0020] Further, the small molecule compounds include inorganic small molecule compounds, protein analogs, peptide analogs, polypeptides, antibodies, siRNA, shRNA, dsRNA, microRNA or antisense nucleic acids.

[0021] The second aspect of the present application provides a computer-aided drug screening system based on TRIM21 and PRMT1.

[0022] Further, the system comprises:

[0023] a data acquisition unit for acquiring TRIM21 protein and PRMT1 protein data;

[0024] a site determination unit for selecting the spatial structure of the TRIM21 protein and PRMT1 protein complex, and determining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex as a binding site of a targeted drug;

[0025] a drug screening unit for obtaining candidate drugs targeting the binding site by using the method for computer-aided screening of drugs.

[0026] Further, the method for determining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex comprises:

[0027] constructing n kinds of truncated expression mutants of TRIM21, n being a natural number greater than or equal to 1;

[0028] immunoprecipitation experiment based on the n kinds of truncated expression mutants to obtain a key region of interaction between TRIM21 and PRMT1;

[0029] The spatial structure formed by the N-terminal SPRY domain of the TRIM21 and the PRMT1 is a binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex;

[0030] Optionally, the method for computer-aided screening of drugs comprises:

[0031] obtaining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex;

[0032] screening a small molecule compound with a similar structure in a molecular database based on the spatial structure of the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex;

[0033] performing molecular docking calculation on the small molecule compound screened and the PRMT1 protein to obtain a score of the small molecule compound targeting the receptor PRMT1 protein or binding energy, and sorting the small molecule compounds according to the score to obtain a candidate drug.

[0034] A third aspect of the present application provides a computer-aided drug screening device based on TRIM21 and PRMT1.

[0035] Further, the device comprises:

[0036] a memory and a processor, the memory being used to store program instructions, and the processor being used to invoke the program instructions, when the program instructions are executed, to realize the computer-aided drug screening method based on TRIM21 and PRMT1 according to the first aspect of the present application.

[0037] A fourth aspect of the present application provides a computer-readable storage medium.

[0038] Further, the computer-readable storage medium stores a computer program, and the computer program is executed by a processor to realize the computer-aided drug screening method based on TRIM21 and PRMT1 according to the first aspect of the present application.

[0039] A fifth aspect of the present application provides any one of the following products:

[0040] (1) the TRIM21 protein and PRMT1 protein complex is the TRIM21 protein and PRMT1 protein complex described in the first aspect of the present application;

[0041] (2) a drug screened by the TRIM21 and PRMT1 based computer-aided drug screening method according to the first aspect of the present application.

[0042] The sixth aspect of the present application provides any of the following aspects:

[0043] (1) an application of a protein complex, the complex being the TRIM21 protein and PRMT1 protein complex described in the fifth aspect of the present application, the application comprising: the application of the complex in screening anti-colorectal cancer drugs;

[0044] (2) an application of a drug, the drug being the drug described in the fifth aspect of the present application, the application comprising: the application of the drug in preparing anti-colorectal cancer drug preparations;

[0045] (3) an application of TRIM21 or a TRIM21 promoter in regulating PRMT1;

[0046] (4) an application of TRIM21 or a TRIM21 promoter in preparing anti-colorectal cancer drugs;

[0047] (5) an application of a PRMT1 inhibitor in preparing anti-colorectal cancer drugs.

[0048] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0049] The present application first clarifies the molecular mechanism that TRIM21 inhibits the development of colorectal cancer by ubiquitinating PRMT1, and based on this, the present application provides a TRIM21 and PRMT1 based computer-aided drug screening method, system, device and computer readable storage medium for the field, and the present application provides a high-efficiency, accurate and rapid screening method for the research and development of new drugs for the treatment of colorectal cancer, and has a very broad application prospect in the screening and development of new drugs for the treatment of colorectal cancer. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0051] Figure 1A flow chart of a computer-aided drug screening method based on TRIM21 and PRMT1 is provided in the embodiments of the present application.

[0052] Figure 2 A schematic diagram of a computer-aided drug screening system based on TRIM21 and PRMT1 is provided in the embodiments of the present application.

[0053] Figure 3 A schematic diagram of a computer-aided drug screening device based on TRIM21 and PRMT1 is provided in the embodiments of the present application.

[0054] Figure 4 TRIM21 expression is reduced and is related to poor survival of CRC patients, wherein, A: protein expression level of TRIM21 in normal colon tissue and colorectal cancer tissue, data from CPTAC database; B: representative immunohistochemical images of TRIM21 protein expression in 427 cases of colorectal cancer tissue and paired adjacent cancer tissue; C, D: comparison and analysis of staining intensity and immunohistochemical score of TRIM12 in colorectal cancer tissue and paired adjacent cancer tissue, T: tumor tissue; N: paired adjacent non-cancerous tissue; E, F: according to the TRIM21 protein expression level in colorectal cancer tissue, the Kaplan-Meier survival curve of the overall survival and disease-specific survival of colorectal cancer patients is drawn. Data is expressed as mean±SD (**P<0.01, ***P<0.001);

[0055] Figure 5 TRIM21 inhibits the proliferation of colorectal cancer cells, wherein, A, B: CCK-8 detects the effect of TRIM21 knockdown on the proliferation of HCT-116 and LoVo cells; C, D: CCK-8 detects the effect of overexpression of TRIM21 on the proliferation of HCT-116 and LoVo cells; E, F: Western blot detects the effect of TRIM21 knockdown or overexpression on the main cycle proteins, data is expressed as mean±SD (*P<0.05, ***P<0.001);

[0056] Figure 6 TRIM21 inhibits the migration and invasion ability of colorectal cancer cells, wherein, A, B: Transwell matrix gel migration and invasion experiment detects the migration and invasion ability of CRC cells after TRIM21 knockdown; C, D: Transwell matrix gel migration and invasion experiment detects the migration and invasion ability of CRC cells after overexpression of TRIM21; E, F: Western blot detects EMT markers in CRC cells after TRIM21 knockdown or TRIM21 overexpression. Data is expressed as mean±SD (**P<0.01);

[0057] Figure 7 TRIM21 reduces the protein stability of PRMT1 in CRC cells, wherein, A, B panels: the interaction between TRIM21 and PRMT1 was detected by Co-IP experiment; C, D panels: Western blot experiment analysis detected the expression of PRMT1 protein in CRC cells after knocking down TRIM21 or overexpressing TRIM21; E, F panels: qPCR analysis detected the expression of PRMT1 mRNA in CRC cells after knocking down TRIM21 or overexpressing TRIM21; G panel: the effect of TRIM21 on the protein half-life of PRMT1 in HCT-116 cells overexpressing TRIM21 and treated with CHX. Data are expressed as mean ± SD (**P < 0.01);

[0058] Figure 8 TRIM21 mediates the ubiquitination degradation of K48-linked PRMT1 in CRC cells, wherein, A panel: TRIM21 mediates the ubiquitination degradation of PRMT1 in HCT-116 and LoVo cells, Input: whole cell lysate, positive control; IP: immunoprecipitation, with anti-Flag antibody; B panel: Western blot analysis showed the effect of proteasome inhibitor MG132 (10 μM, 6 h) treatment on the expression amount of PRMT1 protein in HCT-116 and LoVo cells, the relative intensity of PRMT1 protein was quantified by software Image J; C panel: schematic diagram of TRIM21 truncation mutants; D panel: HCT-116 cells were transfected with wild-type TRIM21 or the mutants to detect the potential domain interacting with PRMT1; E panel: 293T cells were co-transfected with K48-only and K63-only ubiquitin plasmids alone or with TRIM21-HA plasmid for 48 h, then the cells were harvested and subjected to myc IP;

[0059] Figure 9 PRMT1 is essential for TRIM21-mediated CRC progression, wherein, A panel: Western blot detected the overexpression efficiency of TRIM21 and PRMT1 in HCT-116 cells, the relative intensity of EZH2 protein was quantified by software Image J; B, C panels: clonogenic assay and CCK-8 assay were used to evaluate the effect of TRIM21 overexpression, PRMT1 overexpression or both on the proliferation of HCT-116 cells; D, E panels: representative images and statistical results of migration experiment and invasion experiment were used to evaluate the migration and invasion ability of HCT-116 cells overexpressing TRIM21, PRMT1 or both, data are expressed as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001);

[0060] Figure 10 Figure 3: TRIM21 affects CRC progression in a PRMT1 -dependent manner, wherein A-C: tumor tissue images, tumor volume statistical curve and tumor weight (n = 6) of HCT116 tumor-bearing mice; D: macroscopic examination of lung metastatic nodules after the nude mice were sacrificed (n = 6), red arrows indicate metastatic nodules; E: Bouin staining and HE staining of lung metastatic tumors after BALB / C nude mice were injected through the tail vein; F: statistical analysis of the number of lung nodules, data are expressed as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001). DETAILED DESCRIPTION

[0061] In order to enable persons skilled in the art to better understand the technical solutions of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present application.

[0062] In some of the processes described in the specification and the attached drawings, a plurality of operations are included in a specific order, but it should be clearly understood that these operations can be performed in the order in which they appear in this text or in parallel, and the serial numbers of the operations such as S101, S102, etc. are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes can include more or fewer operations, and the operations can be performed in sequence or in parallel.

[0063] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0064] Figure 1 Figure 1 is a flowchart of a computer-aided drug screening method based on TRIM21 and PRMT1 provided by an embodiment of the present application. Specifically, the method comprises the following steps:

[0065] S101: obtaining TRIM21 protein and PRMT1 protein data;

[0066] In one embodiment, the TRIM21 protein is an E3 ubiquitin ligase that plays a crucial role in immune responses by targeting intracellular pathogens for degradation. The TRIM21 protein is involved in the recognition and clearance of viral particles. The TRIM21 (tripartite motif containing 21 [Homo sapiens (human)]) has a Gene ID of 6737 in NCBI, and the corresponding detailed information (including sequence information, etc.) can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0067] In one embodiment, the PRMT1 (Protein arginine methlytransferase 1, PRMT1) refers to a protein arginine methyltransferase 1, which is a major member of the protein arginine methyltransferase family. PRMT1 has multiple histidine and non-histidine substrates, and it regulates protein-protein and protein-nucleic acid interactions by methylating these substrates, and plays a role in epigenetic transcriptional regulation, RNA splicing, RNA export and signaling functions. The PRMT1 (protein arginine methyltransferase 1 [Homo sapiens (human)]) has a Gene ID of 3276 in NCBI, and the corresponding detailed information (including sequence information, etc.) can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0068] In one embodiment, the present application demonstrates that TRIM21 expression is decreased in colorectal cancer and is associated with poor prognosis. The specific experimental methods are as follows: (1) The protein expression level of TRIM21 in colon cancer patients was analyzed using the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database: The proteomic data of 97 colon cancer tissue samples and 100 control samples were found in the CPTAC database, and statistical analysis was performed using GraphPad Prism 7 software. (2) The expression of TRIM21 was detected using the constructed colorectal cancer tissue chip: 485 pairs of colorectal cancer tissue samples from the Affiliated Hospital of Wannan Medical College from 2005 to 2008 were collected. The clinical and pathological information of the patients was obtained from the medical records. The expression level of TRIM21 was detected and scored using immunohistochemical staining on the tissue chip containing 427 CRC tissue samples with complete clinical information and their paired adjacent cancer tissues. (3) Analysis of the correlation between TRIM21 expression and the clinical and pathological characteristics and prognosis of colorectal cancer patients: Fisher's exact test was used to analyze the correlation between the expression of TRIM21 in colorectal cancer (CRC) and the clinical and pathological characteristics, including tumor volume, tumor differentiation, lymph node metastasis, distant metastasis, and TNM stage. The overall survival (OS) time was calculated according to the date of surgery to the date of death, and the disease-specific survival (DSS) time was calculated from the start of surgery to the death caused by CRC. Kaplan-Meier survival analysis and log-rank test were used to explore the correlation between TRIM21 expression and the overall survival (OS) and disease-specific survival (DSS) of CRC patients.

[0069] The inventors analyzed the protein expression level of TRIM21 in colon cancer patients using the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. The results showed that the expression of TRIM21 in colon cancer tissue was significantly decreased compared to normal tissue Figure 4 A) To confirm these findings, we performed IHC staining of TRIM21 protein on a tissue chip containing 427 colorectal cancer (CRC) tissue samples and their paired adjacent cancer tissues. The expression of TRIM21 was decreased in colorectal cancer compared to normal tissue Figure 4 B-D). This is consistent with the results of database analysis.

[0070] In addition, the inventors explored the correlation between the expression of TRIM21 and clinicopathological features in colorectal cancer (CRC) using Fisher's exact test (Table 1) to further understand the clinical significance of TRIM21 in CRC. The results showed that TRIM21 low expression was significantly positively correlated with large tumor volume (P < 0.001), poor tumor differentiation (P < 0.001), lymph node metastasis (P < 0.001), distant metastasis (P < 0.001), and high TNM stage (P < 0.001), and no correlation between TRIM21 expression and age or gender was found.

[0071] Table 1 Relationship between TRIM21 expression and clinicopathological features in patients with colorectal cancer

[0072]

[0073]

[0074] P values were derived from χ2tests. 2

[0075] Meanwhile, the inventors also explored the correlation between the expression of TRIM21 and the overall survival (OS) and disease-specific survival (DSS) of CRC patients using Kaplan-Meier survival analysis and log-rank test. Patients with low expression of TRIM21 had worse OS and DSS than patients with high expression of TRIM21 (Figures E and F). Figure 4

[0076] ​​In one embodiment, the present application demonstrates that TRIM21 inhibits the proliferation of colorectal cancer cells. The specific experimental methods are as follows: (1) Whether silencing endogenous TRIM21 promotes the proliferation of colorectal cancer cells (HCT116, LoVo): Transfect TRIM21 small interfering RNA fragments (siTRIM21-1, siTRIM21-2) into HCT116 and LoVo cells respectively (obtained by conventional method of design and synthesis), use non-specific siRNA (siCtrl) as negative control, 48h later, cell counting. Cells were planted in 96-well plates in three replicate wells, 3000 cells were inoculated in each well. On 0, 1, 2, 3, 4 days after inoculation, the cell growth rate was evaluated by CCK8 kit. (2) Whether the introduction of exogenous TRIM21 inhibits the proliferation of colorectal cancer cells (HCT116, LoVo): Transfect negative control plasmid (NC) and TRIM21 overexpression plasmid (OE-TRIM21) into HCT116 and LoVo cells respectively, 48h later, cell counting. Cells were planted in 96-well plates in three replicate wells, 3000 cells were inoculated in each well. On 0, 1, 2, 3, 4 days after inoculation, the cell growth rate was evaluated by CCK8 kit. (3) Western blotting detects the regulation of high and low expression of TRIM21 on proliferation-related proteins: Transfect TRIM21 small interfering fragments and TRIM21 overexpression plasmid into HCT116 and LoVo cells respectively, 48h after transfection, collect protein. Western blotting verifies the transfection efficiency and detects the expression of proliferation-related cell cycle proteins p21, cyclin D1 and cyclin E2.

[0077] To further study the function of TRIM21 in colorectal cancer, the inventors performed CCK-8 cell proliferation experiments after knockdown and overexpression in colon cancer cells HCT-116 and LoVo cells. The results showed that when TRIM21 was knocked down, the cell proliferation ability was enhanced Figure 5 A, B), and when TRIM21 was overexpressed, the cell proliferation ability was weakened Figure 5 C, D).

[0078] TRIM21 can ubiquitinate cyclin-dependent kinase inhibitor p21, which can inhibit G1 / S cell cycle progression. Therefore, the inventors speculated that TRIM21 might inhibit tumor proliferation by affecting cell cycle progression. Next, Western blot analysis was performed to study the expression levels of p21 and two major cyclins, cyclin D1 and cyclin E2. Cyclin D1 is an important positive regulator of the cell cycle, which shortens the G1 phase, promotes cells to enter the S phase in advance, accelerates the cell cycle progression, and leads to uncontrolled proliferation. Cyclin E2 is a major rate-limiting factor for G1 / S transition, which is overexpressed in various tumors and promotes the occurrence and development of cancer. The above analysis results show that TRIM21 deletion leads to increased levels of cyclin E2 and cyclin D1, and decreased levels of p21 Figure 5 E). On the contrary, overexpression of TRIM21 showed the opposite results Figure 5 F).

[0079] In one embodiment, the present application proves that TRIM21 inhibits the migration and invasion of colorectal cancer cells. The specific experimental method is as follows: (1) Whether silencing endogenous TRIM21 promotes the migration and invasion of colorectal cancer cells (HCT116, LoVo): Transwell chambers with polyethylene terephthalate films (24-well insert, 12.0 μm; Corning) were used for in vitro cell migration and invasion experiments. TRIM21 small interfering RNA fragments (siTRIM21-1, siTRIM21-2) were transfected into HCT116 and LoVo cells (designed and synthesized by conventional methods), and non-specific siRNA (siCtrl) was used as a negative control. After 48 h, cell counting was performed. For the migration experiment, 2 x 10 4 cells were added to the upper chamber. For the invasion experiment, 2 x 10 4Cells were seeded into the upper chamber coated with Matrigel. Complete medium was added to the lower chamber to stimulate migration or invasion. After 24-48h incubation, cells were stained with 0.1% crystal violet. Five fields were counted for each filter. (2) Whether the introduction of exogenous TRIM21 inhibits the migration and invasion of colorectal cancer cells (HCT116, LoVo): HCT116 and LoVo cells were transfected with negative control plasmid (NC) and TRIM21 overexpression plasmid (OE-TRIM21), respectively. After 48h, cell counting was performed. Cell migration and invasion experiments were performed as described above. (3) Western blotting to detect the regulation of epithelial-mesenchymal transition-related proteins by high and low expression of TRIM21: HCT116 and LoVo cells were transfected with TRIM21 small interfering fragments and TRIM21 overexpression plasmid, respectively. After 48h of transfection, proteins were collected. Western blotting was used to verify the transfection efficiency and detect the expression of epithelial-mesenchymal transition-related proteins N-cadherin, Snail and E-cadherin.

[0080] Epithelial-mesenchymal transition (EMT) is considered an important process of tumor cell metastasis, and loss of expression of cadherin (E-cadherin) is a major marker of EMT. Transwell experiment analysis results show that TRIM21 knockdown promotes the migration Figure 6 A) and invasion ability Figure 6 B) of CRC cells, while TRIM21 overexpression inhibits the migration and invasion ability Figure 6 C, D). Correspondingly, TRIM21 deletion leads to increased levels of mesenchymal markers N-cadherin and Snail, and decreased levels of epithelial marker E-cadherin Figure 6 E). The opposite is true when TRIM21 is overexpressed Figure 6 F).

[0081] In one embodiment, the present application proves that TRIM21 binds to PRMT1 and reduces its protein stability. The specific experimental methods are as follows: (1) Immunoprecipitation detects whether endogenous TRIM21 and PRMT1 are combined: co-transfect HA-TRIM21 and Flag-PRMT1 overexpression plasmids in HCT116 and LoVo cells, respectively, 48 h after transfection, lyse the cells, use anti-Flag antibody for immunoprecipitation, use anti-HA antibody for immunoblotting, detect whether PRMT1 is combined with TRIM21; use anti-HA antibody for immunoprecipitation, use anti-Flag antibody for immunoblotting, detect whether TRIM21 is combined with PRMT1. (2) Western blotting detects the effect of high and low expression of TRIM21 on PRMT1 protein: transfect siTRIM21-1 and siTRIM21-2 in HCT116 and LoVo cells, respectively, use non-specific siRNA (siCtrl) as a negative control, 48 h later, Western blotting verifies the transfection efficiency and detects PRMT1 expression; transfect OE-TRIM21 in HCT116 and LoVo cells, respectively, 0, 2, and 4 μg of HA empty plasmid (4, 2, and 0 μg), Western blotting verifies the transfection efficiency and detects PRMT1 expression. (3) qRT-PCR detects the effect of high and low expression of TRIM21 on PRMT1 mRNA level: transfect TRIM21 small interfering fragments and TRIM21 overexpression plasmids in HCT116 and LoVo cells, respectively, 48 h after transfection, extract mRNA. qRT-PCR verifies the transfection efficiency and detects PRMT1 gene expression. (4) Protein synthesis inhibitor CHX tracking experiment detects the effect of TRIM21 on PRMT1 protein half-life: transfect negative control plasmid (NC) and TRIM21 overexpression plasmid (OE-TRIM21) in HCT116 and LoVo cells, respectively, 48 h later, CHX (100 μg / mL) is used to treat cells, continue to culture for 0, 4, 6, and 8 h, collect proteins, and use Western blotting to detect PRMT1 protein level, and draw a time curve.

[0082] To investigate the tumor suppressive role of TRIM21 in colorectal cancer, the inventors transfected control vector plasmid and TRIM21 overexpression plasmid into HCT-116 cells. Protein samples were then subjected to mass spectrometry analysis to explore potential downstream targets of TRIM21. Among 760 candidate targets, the inventors focused on arginine methyltransferase PRMT1. The inventors' group previously found that PRMT1 mediates asymmetric methylation modification at R342 site of EZH2, thereby increasing the stability of EZH2 protein and promoting breast cancer metastasis. In addition, PRMT1 can promote the proliferation of breast cancer cells by inhibiting the transcription and expression of p16 and p21.

[0083] To investigate the potential interaction between TRIM21 and PRMT1, the inventors first performed Co-IP experiments in HCT-116 and LoVo cells to confirm the mutual binding of endogenous TRIM21 and PRMT1 Figure 7 A, B). Subsequently, Western blot experiments were performed, and it was found that TRIM21 negatively regulated the protein level of PRMT1 in HCT-116 and LoVo cells Figure 7 C, D). However, the knockdown or overexpression of TRIM21 did not significantly affect the mRNA level of PRMT1 Figure 7 E, F), indicating that TRIM21 mainly regulates the post-translational modification of PRMT1. Treatment of HCT-116 cells with the protein synthesis inhibitor cycloheximide (CHX) resulted in a higher degradation rate of PRMT1 protein in the TRIM21 overexpression group compared to the control group, leading to a shorter half-life Figure 7 G). These findings suggest that TRIM21 reduces the stability of PRMT1 in CRC cells.

[0084] S102: selecting a spatial structure of the TRIM21 protein and PRMT1 protein complex, and determining a binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex as a binding site of a targeted drug;

[0085] In one embodiment, the method of determining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex comprises:

[0086] constructing n truncated expression mutants of TRIM21, n being a natural number greater than or equal to 1;

[0087] performing immunoprecipitation experiments based on the n truncated expression mutants to obtain a key region of interaction between TRIM21 and PRMT1;

[0088] The spatial structure formed by the N-terminal SPRY domain of the TRIM21 interacting with the PRMT1 is a binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex.

[0089] In one embodiment, the present application proves that TRIM21 mediates the ubiquitination degradation of PRMT1 in CRC cells. The specific experimental methods are as follows: (1) Whether the introduction of exogenous TRIM21 promotes the ubiquitination degradation of PRMT1: HCT116 and LoVo cells are respectively co-transfected with HA-TRIM21 and Flag-PRMT1 overexpression plasmids, and 48 h after transfection, the cells are lysed, anti-Flag antibody is used for immunoprecipitation, and anti-Ubiquitin antibody is used for immunoblotting to detect whether the ubiquitination level of PRMT1 is enhanced. (2) Adding proteasome inhibitor MG132 to verify whether TRIM21 regulates PRMT1 through the proteasome pathway: HCT116 and LoVo cells are respectively transfected with negative control plasmid (NC) and TRIM21 overexpression plasmid (OE-TRIM21), and 48 h later, treated with MG132 (10 μM) for 6 h, the cells are lysed, and immunoblotting is used to detect the expression of PRMT1. (3) Detecting the region and mode of TRIM21-mediated PRMT1 ubiquitination: studying the region of TRIM21 interacting with PRMT1: using HA-TRIM21 truncated mutants (△RIGN, △B-BOX, △SPRY, △PRY) and complete WT plasmid, HCT116 and LoVo cells are respectively transfected, anti-HA is used for immunoprecipitation, and PRMT1 antibody is used for immunoblotting to detect the region of TRIM21 interacting with PRMT1. Study the mode of TRIM21-mediated PRMT1 ubiquitination: HCT116 and LoVo cells are respectively co-transfected with HA-TRIM21, Flag-ubiquitin-K48 / K63, anti-Flag is used for immunoprecipitation, and PRMT1 antibody is used for immunoblotting to detect the mode of TRIM21-mediated PRMT1 ubiquitination.

[0090] TRIM21 is an E3 ubiquitin ligase involved in various important biological functions by promoting ubiquitination modification. In order to study whether TRIM21 mediates the ubiquitination degradation of PRMT1 through the ubiquitination-proteasome pathway, the inventors introduced Flag-PRMT1 plasmid into HCT-116 and LoVo cells, and then transfected control plasmid and TRIM21 overexpression plasmid. The results of Co-IP analysis showed that the ubiquitination level of PRMT1 in the TRIM21 overexpression group was significantly higher than that in the control group, indicating that TRIM21 can mediate the ubiquitination modification of PRMT1 in CRC cells Figure 8A) The downregulation of PRMT1 protein levels by TRIM21 was reversed upon addition of the proteasome inhibitor MG132 (B), indicating that TRIM21 regulates the stability of PRMT1 protein through the ubiquitination-proteasome pathway. Figure 8 B), indicating that TRIM21 regulates the stability of PRMT1 protein through the ubiquitination-proteasome pathway.

[0091] The structure of TRIM21 contains a C-terminal ring finger domain, zinc finger domain (B-box) and coiled coil (CC) region, and an N-terminal SPRY domain and PRY domain. To determine the domain in TRIM21 responsible for PRMT1 interaction and degradation, the inventors used single amino acid mutants of TRIM21 that cannot bind zinc ions (C). The results of the study showed that the truncated mutants lacking the SPRY domain exhibited impaired ability to interact with PRMT1 (D), indicating that the N-terminal SPRY of TRIM21 is a major requirement for TRIM21 interaction with PRMT1. Figure 8 C) The structure of TRIM21 contains a C-terminal ring finger domain, zinc finger domain (B-box) and coiled coil (CC) region, and an N-terminal SPRY domain and PRY domain. To determine the domain in TRIM21 responsible for PRMT1 interaction and degradation, the inventors used single amino acid mutants of TRIM21 that cannot bind zinc ions (C). The results of the study showed that the truncated mutants lacking the SPRY domain exhibited impaired ability to interact with PRMT1 (D), indicating that the N-terminal SPRY of TRIM21 is a major requirement for TRIM21 interaction with PRMT1. Figure 8 D) The structure of TRIM21 contains a C-terminal ring finger domain, zinc finger domain (B-box) and coiled coil (CC) region, and an N-terminal SPRY domain and PRY domain. To determine the domain in TRIM21 responsible for PRMT1 interaction and degradation, the inventors used single amino acid mutants of TRIM21 that cannot bind zinc ions (C). The results of the study showed that the truncated mutants lacking the SPRY domain exhibited impaired ability to interact with PRMT1 (D), indicating that the N-terminal SPRY of TRIM21 is a major requirement for TRIM21 interaction with PRMT1.

[0092] Ubiquitination modification has multiple types, which can be classified according to the different lysine residue positions of the linked ubiquitin molecules. Among them, K48 and K63 site polyubiquitination is the most common. Previous studies have shown that K48-linked polyubiquitination mainly regulates protein degradation and stability, while K63-linked polyubiquitination mainly regulates intracellular signal transduction. Co-IP assay confirmed that TRIM21 mainly induced K48-linked ubiquitination of PRMT1 (E). Figure 8 E) Ubiquitination modification has multiple types, which can be classified according to the different lysine residue positions of the linked ubiquitin molecules. Among them, K48 and K63 site polyubiquitination is the most common. Previous studies have shown that K48-linked polyubiquitination mainly regulates protein degradation and stability, while K63-linked polyubiquitination mainly regulates intracellular signal transduction. Co-IP assay confirmed that TRIM21 mainly induced K48-linked ubiquitination of PRMT1 (E).

[0093] In summary, these results indicate that TRIM21 has a general regulatory role on PRMT1 ubiquitination in CRC cells. The interaction between the SPRY domain of TRIM21 and PRMT1 promotes the transfer of K48-ubiquitin to PRMT1, leading to proteasome recognition and degradation of PRMT1.

[0094] In one embodiment, the present application proves that PRMT1 is necessary for TRIM21-mediated CRC progression in vitro. The specific experimental methods are as follows: (1) Four groups of HCT116 stable cell lines are constructed: empty load, overexpression of TRIM21, overexpression of PRMT1, and co-expression of TRIM21 and PRMT1. Slow virus packaging and transfection technology is used to obtain four different HCT-116 stable cell strains: control group (NC): vector+vector; TRIM21 overexpression group (OE-TRIM21): vector+OE-TRIM21; PRMT1 overexpression group (OE-PRMT1): vector+OE-PRMT1; TRIM21 and PRMT1 co-overexpression group (TRIM21+PRMT1): OE-TRIM21+OE-PRMT1. The corresponding viral vector plasmid is constructed, and the corresponding virus is packaged; HCT116 cells are infected; after two weeks of puromycin screening, construction is completed; qRT-PCR detects TRIM21 and PRMT1 mRNA expression, and Western Blot detects TRIM21 and PRMT1 protein expression. (2) The proliferation ability of the four groups of HCT116 stable cell lines is detected: the proliferation ability of the four groups of HCT116 stable cell lines is detected by CCK-8 kit and colony formation experiment, and the specific steps are as described above. (3) The migration and invasion ability of the four groups of HCT116 stable cell lines is detected: Transwell experiment is performed on the four groups of cells to evaluate their migration and invasion ability, and the specific steps are as described above.

[0095] The inventors' team's previous research showed that PRMT1 plays a promoting role in the proliferation and metastasis of breast cancer and colorectal cancer. In this study, it was found that TRIM21 has the ability to inhibit the proliferation and metastasis of CRC. At the same time, TRIM21 can ubiquitinate and degrade PRMT1. Based on these findings, the inventors hypothesized that TRIM21 may play a tumor suppressor role in CRC by negatively regulating PRMT1. Western blotting experiments confirmed the transfection efficiency of TRIM21 and PRMT1 in the four groups of HCT-116 cells, proving that the corresponding cell lines were successfully constructed Figure 9 A).

[0096] The results of CCK-8 and colony formation experiments showed that PRMT1 overexpression promoted cell proliferation. On the contrary, TRIM21 overexpression inhibited cell proliferation, and this inhibition could be reversed in TRIM21 overexpressing cells by overexpressing PRMT1 Figure 9B, C). Subsequently, Transwell assay was performed to evaluate the migration and invasion ability of the four groups of cells. The results showed that overexpression of TRIM21 inhibited the migration and invasion ability of the cells, while overexpression of PRMT1 promoted the migration and invasion of the cells. Notably, in the TRIM21 overexpressing cells, the migration ability of the cells was restored after overexpression of PRMT1 Figure 9 D, E). These findings suggest that the mechanism by which TRIM21 inhibits CRC cell proliferation and metastasis in vitro depends on PRMT1.

[0097] In one embodiment, the present application demonstrates that TRIM21 affects the development of CRC in vivo in a PRMT1-dependent manner. The specific experimental methods are as follows: (1) Subcutaneous tumor model in nude mice to study the effect of TRIM21 and PRMT1 on the tumorigenesis of colorectal cancer: four groups of HCT-116 stable cell lines (NC, OE-TRIM21, OE-PRMT1 and OE-TRIM21+OE-PRMT1) were inoculated with 5x10 6 cells into the ventral region of the mice, and the tumor volume was calculated every week according to the formula V = a x (b x b) / 2, where "a" represents the maximum diameter and "b" represents the minimum diameter. At the sixth week of inoculation, the tumor was removed, weighed, and IHC staining was performed on the subcutaneous tumor tissue to analyze the expression of TRIM21, PRMT1 and the proliferation index Ki-67. (2) Tail vein lung metastasis model to study the effect of TRIM21 and PRMT1 on the metastasis of colorectal cancer: four groups of HCT-116 stable cell lines (NC, OE-TRIM21, OE-PRMT1 and OE-TRIM21+OE-PRMT1) were injected into the mice through the tail vein with 5x10 6 cells. After 10 weeks, the nude mice were euthanized, dissected and the lung tissue was removed for Bouin staining to observe the number of metastatic foci in the lung.

[0098] To study the potential inhibitory effect of TRIM21 on CRC tumor occurrence and metastasis in vivo, two animal models were constructed: subcutaneous tumor model in nude mice and tail vein lung metastasis model. In the subcutaneous tumor model, it was observed that the tumor weight and volume in the OE-TREIM21 group were significantly lower than those in the control group. However, in the OE-PRMT1 group, the tumor weight and volume were larger. To determine whether TRIM21 acts in a PRMT1-dependent manner, the expression of PRMT1 was restored in the OE-TRIM21 group, thereby reversing the tumor inhibitory effect of TRIM21 Figure 10 A and B). Subsequently, IHC staining was performed on the subcutaneous tumor tissue to analyze the expression of TRIM21, PRMT1 and Ki-67 Figure 10C). The results showed that both TRIM21 and PRMT1 were stably overexpressed in the corresponding groups. Ki67 staining (an important indicator of cell proliferation) results showed that the number of Ki67 positive cells in the OE-TRIM21 group was significantly lower than that in the control group. In contrast, the number of Ki67 positive cells in the OE-PRMT1 group was higher. Interestingly, the number of Ki67 positive cells in the OE-TRIM21+OE-PRMT1 group was significantly increased compared with the OE-TRIM21 group.

[0099] For the lung metastasis model, the staining results showed that the number of metastatic foci in the OE-TRIM21 group was significantly reduced compared with the control group, while the number of metastatic foci in the OE-PRMT1 group was increased. In addition, the number of metastatic foci in the OE-TRIM21+OE-PRMT1 group was significantly higher than that in the OE-TRIM21 group Figure 10 D and E). The above results indicate that TRIM21 can inhibit the tumorigenicity and metastatic ability of CRC cells in vivo in a PRMT1-dependent manner.

[0100] In the present application, the inventors explored the role of TRIM21 in mediating the ubiquitination and degradation of PRMT1, which leads to a decrease in PRMT1 protein expression and subsequently inhibits the growth and metastasis of CRC cells. These results provide insights into the potential mechanisms of PRMT1 and potential solutions to the challenges associated with anti-PRMT1 therapy. In addition, this study highlights the possibility of developing targeted drugs that specifically activate TRIM21, providing a promising treatment for colorectal cancer and opening up new avenues for clinical intervention. For the first time, it was found that TRIM21, as a tumor suppressor gene in colorectal cancer, acts as an E3 ubiquitin ligase, promoting the ubiquitination and degradation of the oncogene PRMT1. This mechanism plays a key role in inhibiting the proliferation and metastasis of colorectal cancer cells in vitro and in vivo. This study explores the clinical significance of the TRIM21-PRMT1 axis in colorectal cancer and provides new insights into the treatment of this disease. Based on this, the present application provides, for the first time in the art, a computer-aided drug screening method, system, device and computer-readable storage medium based on TRIM21 and PRMT1, which has a broad application prospect in the technical field of screening and developing new drugs for the treatment of colorectal cancer.

[0101] S103: obtaining a candidate drug targeting the binding site by using the computer-aided drug screening method;

[0102] In one embodiment, the computer-aided drug screening method comprises:

[0103] obtaining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex;

[0104] screening structural analog small molecule compounds in a molecular database based on the spatial structure of the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex;

[0105] performing molecular docking calculation on the small molecule compounds screened and the PRMT1 protein to obtain the score of each small molecule compound targeting the receptor PRMT1 protein in terms of affinity or binding energy, and ranking the small molecule compounds according to the score to obtain a candidate drug.

[0106] In one embodiment, the small molecule compound includes an inorganic small molecule compound, a protein analog, a peptide analog, a polypeptide, an antibody, an siRNA, an shRNA, a dsRNA, a microRNA, or an antisense nucleic acid.

[0107] It should be noted that the specific type of the small molecule compound is not particularly limited in the present application, and any small molecule compound that can be used in the computer-aided drug screening method provided by the present application and can produce a corresponding effect falls within the protection scope of the present application.

[0108] In one embodiment, the method further comprises:

[0109] obtaining a PRMT1 expression system;

[0110] treating the PRMT1 expression system with the small molecule compound screened to verify the effectiveness of the small molecule compound;

[0111] Optionally, the PRMT1 expression system includes a PRMT1 expression cell system, a PRMT1 expression subcellular system, a PRMT1 expression tissue system, a PRMT1 expression solution system, a PRMT1 expression organ system, or a PRMT1 expression animal system.

[0112] Optionally, the small molecule compound with the function of inhibiting PRMT1 expression is used as a candidate drug.

[0113] In one embodiment, the PRMT1 expression cell system, the PRMT1 expression subcellular system, the PRMT1 expression tissue system, the PRMT1 expression solution system, the PRMT1 expression organ system, or the PRMT1 expression animal system can be obtained through a conventional purchase route known to those skilled in the art, or constructed through a conventional construction method known to those skilled in the art.

[0114] In one embodiment, the animal includes, but is not limited to, a mouse, a rat, a guinea pig, a rabbit, a pig, a chicken, a pigeon, a monkey, a dog, and the like.

[0115] In one embodiment, in molecular docking, affinity refers to the tightness of the binding of a molecule to a receptor. High affinity means that the binding is more stable, while low affinity indicates that the binding is less stable. Affinity is usually calculated, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd).

[0116] In molecular docking, affinity depends on the interactions between the molecule and the receptor, including hydrogen bonds, van der Waals forces, electrostatic interactions, etc. These interactions together determine the binding mode between the molecule and the receptor, thereby affecting the affinity. In order to evaluate the affinity, some scoring systems or scoring methods are usually used to quantify the interactions between the molecule and the receptor. These scoring methods are based on different algorithms and physical models, which can reflect the binding energy, interaction type and affinity between the molecule and the receptor, etc.

[0117] In one embodiment, the mode of screening drugs using computer-aided drug screening technology includes one or more of the following: protein-small molecule docking, protein-protein docking, protein-nucleic acid docking.

[0118] In one embodiment, the protein-small molecule docking refers to a computational simulation process that uses certain algorithms and programs to dock the structures of proteins and small molecules (such as drug molecules) together. This process can be used to study the interactions between proteins and small molecules, as well as possible biological functions.

[0119] 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 biological macromolecular receptors. It uses a scoring method based on fragments, which can 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 rough matching between small molecule ligands and biological macromolecular receptors. The second stage is the high-precision stage, which considers all side chain conformations and calculates more accurate interaction energies. In the low-precision stage, DOCK software randomly translates and rotates small molecule ligands and performs a certain number of rigid body movements, 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 more optimization and adjustment to achieve more accurate docking.

[0120] In one embodiment, the protein-protein docking refers to a computational simulation process that uses certain algorithms and programs to dock the structures of two proteins together. This process can be used to study the interactions between proteins, as well as possible biological functions.

[0121] In protein-protein docking, RosettaDock is a commonly used docking software that employs a fragment-based scoring method to achieve fast and accurate protein docking. The software can accurately adjust the side chain conformation during the docking process and consider various complex interactions such as hydrogen bonds, ionic bonds, and hydrophobic interactions. The basic algorithm of RosettaDock includes two stages. The first stage is the low-precision stage, which mainly searches for the degree of shape adaptation between the two proteins. The second stage is the high-precision stage, which considers all side chain conformations and calculates more accurate interaction energies. In the low-precision stage, the program randomly translates and rotates a certain component of a protein molecule and performs a certain number of rigid body movements, 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, taking it as the initial starting conformation.

[0122] In one embodiment, the protein-nucleic acid docking refers to a computational simulation process that docks 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 and possible biological functions.

[0123] In protein-nucleic acid docking, software such as NAflex is commonly used for computational simulation. NAflex is a software specifically developed for nucleic acid structure prediction and design, which can accurately model and dock DNA or RNA molecules. NAflex software employs a fragment-based scoring method to achieve fast and accurate docking. It considers various complex interactions such as hydrogen bonds, ionic bonds, and hydrophobic interactions, and can accurately adjust the side chain conformation during the docking process. The basic algorithm of NAflex software includes two stages. The first stage is the low-precision stage, which mainly searches for the rough match between proteins and nucleic acids. The second stage is the high-precision stage, which considers all side chain conformations and calculates more accurate interaction energies. In the low-precision stage, NAflex software randomly translates and rotates the nucleic acid molecule and performs a certain number of rigid body movements, then calculates the interaction energy. After outputting the lowest conformation, it enters the high-precision stage. In the high-precision stage, the program performs more optimization and adjustment to achieve more accurate docking.

[0124] In one embodiment, drug virtual screening is an important method in the field of targeted drug design. The process is roughly as follows: first, based on the known complex structure of drugs and target proteins, the binding mode of drugs and target proteins is studied, and the key amino acid residues are determined. This step helps to understand how drugs interact with target proteins and provides a basis for subsequent virtual screening. Next, using computer-aided drug design methods, a large number of small molecule compounds are screened. In this process, the binding ability of small molecule compounds and target proteins is predicted and evaluated. Generally, small molecules with good shape complementarity are selected as potential candidate drugs. Finally, the selected candidate drugs are further experimentally verified to confirm their interaction with target proteins and biological activity.

[0125] In one embodiment, in drug virtual screening, the binding site refers to the area on the target protein that binds to small molecule compounds. In order to carry out receptor-based virtual screening, the binding site of the target protein needs to be determined first. This is usually achieved by analyzing and studying the structure of the target protein. Once the binding site is determined, the compounds in the small molecule compound library can be docked with the target protein using computer-aided drug design methods. During the docking process, the computer simulates the interaction of the compounds with the binding site and evaluates their binding ability. According to the docking results, small molecule compounds with strong binding ability to the target protein can be screened as potential candidate drugs. The determination of the binding site in drug virtual screening is crucial to the success of drug design. Therefore, when determining the binding site, various factors need to be considered, such as the structural characteristics of the target protein, the binding mode of known ligands, etc. At the same time, during the virtual screening process, the binding site also needs to be reasonably processed and optimized to improve the accuracy and efficiency of the screening.

[0126] In one embodiment, computer-aided drug screening is a technology that uses computer-aided drug design methods to screen drugs. It can help researchers quickly screen out candidate drugs with strong binding ability to target proteins and potential drug effects from a large number of small molecule compounds.

[0127] In one embodiment, molecular docking is a method of drug design by the characteristics of the receptor and the interaction between the receptor and the drug molecule. It is a theoretical simulation method that 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 compounds with potential efficacy. The molecular docking method mainly focuses 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 geometric matching calculations, methods such as grid calculation and fragment growth are commonly used, while energy calculations use methods such as simulated annealing and genetic algorithms. According to the degree and method of simplification, molecular docking methods can be divided into rigid docking, semi-flexible docking, and flexible docking. In rigid docking methods, the conformation of the molecules involved in docking does not change during the calculation, only the spatial position and attitude of the molecules are changed. Semi-flexible docking allows some conformation changes during calculation. Flexible docking allows more conformation changes.

[0128] In one embodiment, the molecular library used in drug virtual screening mainly includes the following: ZINC: contains more than 250 million purchasable compounds for small molecule virtual screening; PubChem: contains bioactive substances for small molecule virtual screening; DrugBank: contains drugs and small molecules for drug design and discovery; ChEMBL: contains small molecules for drug discovery and chemical genomics research; ChemDB: contains a large number of known small molecules for chemical genomics research and drug discovery; HMDB: contains a large number of known small molecules for chemical genomics research and drug discovery; BindingDB: contains a large number of known small molecules for chemical genomics research and drug discovery; SMPDB: contains a large number of known small molecules for chemical genomics research and drug discovery. In addition, there are some commercial databases such as ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas-M, and Interbioscreen, which are also commonly used for drug virtual screening.

[0129] Figure 2 is a computer-aided drug screening system based on TRIM21 and PRMT1 provided by an embodiment of the present application, specifically, the system comprises:

[0130] a data acquisition unit for acquiring TRIM21 protein and PRMT1 protein data;

[0131] A site determination unit selects a spatial structure of the TRIM21 protein and PRMT1 protein complex, and determines a binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex as a binding site of a targeted drug.

[0132] A drug screening unit obtains a candidate drug targeting the binding site by using a computer-aided drug screening method.

[0133] The method for determining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex comprises:

[0134] Constructing n truncated expression mutants of TRIM21, n being a natural number greater than or equal to 1;

[0135] Obtaining a key region of interaction between TRIM21 and PRMT1 based on the n truncated expression mutants by performing an immunoprecipitation experiment;

[0136] The spatial structure formed by the N-terminal SPRY domain of the TRIM21 and the PRMT1 is the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex;

[0137] Optionally, the computer-aided drug screening method comprises:

[0138] Obtaining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex;

[0139] Screening structurally similar small molecule compounds in a molecular database based on the spatial structure of the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex;

[0140] Performing molecular docking calculation on the screened small molecule compounds and the PRMT1 protein to obtain scores of the small molecule compounds targeting the receptor PRMT1 protein, and obtaining a candidate drug according to the scores.

[0141] Figure 3 is a computer-aided drug screening device based on TRIM21 and PRMT1 provided by an embodiment of the application, specifically, the device comprises:

[0142] A memory and a processor, the memory is used to store program instructions; the processor is used to call program instructions, when the program instructions are executed, the computer-aided drug screening method based on TRIM21 and PRMT1 of the application is realized.

[0143] The embodiment of the present application also provides a computer readable storage medium, wherein the computer readable storage medium stores a computer program, and the computer program is executed by a processor to implement the TRIM21 and PRMT1 based computer-aided drug screening method as described above.

[0144] The embodiment of the present application also provides any one of the following products:

[0145] (1) a TRIM21 protein and PRMT1 protein complex, wherein the TRIM21 protein and PRMT1 protein complex is the TRIM21 protein and PRMT1 protein complex as described above;

[0146] (2) a drug screened by the TRIM21 and PRMT1 based computer-aided drug screening method as described above.

[0147] The embodiment of the present application also provides any one of the following applications:

[0148] (1) an application of a protein complex, wherein the complex is the TRIM21 protein and PRMT1 protein complex as described above, and the application comprises: application of the complex in screening an anti-colorectal cancer drug;

[0149] (2) an application of a drug, wherein the drug is the drug as described above, and the application comprises: application of the drug in preparation of an anti-colorectal cancer drug preparation;

[0150] (3) application of TRIM21 or a TRIM21 promoter in regulating PRMT1;

[0151] (4) application of TRIM21 or a TRIM21 promoter in preparation of an anti-colorectal cancer drug;

[0152] (5) application of a PRMT1 inhibitor in preparation of an anti-colorectal cancer drug.

[0153] The verification result of the verification embodiment shows that assigning inherent weights to indications can moderately improve the performance of the method compared with the default setting.

[0154] Those skilled in the art can clearly understand the specific working process of the system, device and unit described above for the convenience and brevity of description, and the corresponding process in the foregoing method embodiments can be referred to, and will not be described herein.

[0155] In several embodiments provided in the present application, it should be understood that the disclosed system, device and method can be implemented in other manners. For example, the described device embodiment is merely illustrative. For example, the division of the units is only a logical function division. There can be another division manner for the actual implementation, for example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. In addition, the displayed or discussed mutual couplings or direct couplings or communication connections between different units, can be indirect couplings or communication connections through some interfaces, devices or units, and can be electrical, mechanical or in other forms.

[0156] The units described as separated components can or can not be physically separated, and the components displayed as units can or can not be physical units, i.e., can be located in one place, or can be distributed on a plurality of network units. Some or all of the units can be selected according to actual needs to achieve the purposes of the embodiments.

[0157] In addition, each functional unit in the various embodiments of the present application can be integrated in one processing unit, or each unit can exist physically, or two or more units can be integrated in one unit. The integrated unit can be implemented in the form of hardware, or in the form of software functional units.

[0158] Those skilled in the art can understand that all or part of the steps in the above-mentioned embodiments can be completed by a program instructing relevant hardware, and the program can be stored in a computer readable storage medium, which can include read only memory (ROM), random access memory (RAM), magnetic disk or optical disk, etc.

[0159] Those skilled in the art can understand that all or part of the steps in the above-mentioned embodiments can be completed by a program instructing relevant hardware, and the program can be stored in a computer readable storage medium, which can include read only memory (ROM), random access memory (RAM), magnetic disk or optical disk, etc.

[0160] The above provides a computer-aided drug screening method, system and device based on TRIM21 and PRMT1. For those skilled in the art, according to the idea of the embodiments of the present application, there will be changes in specific implementation and application range. In view of the above, the content of the specification should not be understood as a limitation of the present application.

Claims

1. A computer-aided screening method based on TRIM21 and PRMT1 for the treatment and / or prevention of colorectal cancer, characterized in that, The method includes: Obtain data on TRIM21 and PRMT1 proteins; The spatial structure of the TRIM21 protein and PRMT1 protein complex was selected, and the binding sites of TRIM21 protein and PRMT1 protein in the TRIM21 protein and PRMT1 protein complex were determined as the binding sites of the targeted drug. Candidate drugs targeting the binding site were obtained using a computer-aided drug screening method. The method for determining the binding sites of TRIM21 and PRMT1 proteins in the TRIM21 and PRMT1 protein complex includes: Construct n truncated expression mutants of TRIM21, where n is a natural number greater than or equal to 1; Immunoprecipitation experiments were performed on the n truncated expression mutants to obtain the key regions of TRIM21-PRMT1 interaction. The spatial structure formed by the interaction between the N-terminal SPRY domain of TRIM21 and PRMT1 is the binding site of TRIM21 and PRMT1 proteins in the TRIM21 and PRMT1 protein complex. The computer-aided drug screening method includes: Obtain the binding sites of TRIM21 and PRMT1 proteins in the TRIM21 and PRMT1 protein complex; Based on the spatial structure of the binding sites of TRIM21 and PRMT1 proteins in the TRIM21 and PRMT1 protein complex, small molecule compounds with similar structures were screened from molecular databases. The small molecule compounds selected through screening are molecularly docked with the PRMT1 protein to calculate the affinity or binding energy of the small molecule compounds to the PRMT1 protein receptor, and a score is obtained for each small molecule compound. Candidate drugs are obtained by sorting the small molecule compounds according to the scores.

2. The computer-aided screening method for drugs used to treat and / or prevent colorectal cancer based on TRIM21 and PRMT1 according to claim 1, characterized in that, The small molecule compounds include inorganic small molecule compounds, protein analogs, peptide analogs, polypeptides, antibodies, siRNA, shRNA, dsRNA, microRNA, or antisense nucleic acids.

3. A system for computer-aided screening of drugs for the treatment and / or prevention of colorectal cancer based on TRIM21 and PRMT1, characterized in that, The system includes: The data acquisition unit acquires data on TRIM21 and PRMT1 proteins. The site determination unit selects the spatial structure of the TRIM21 protein and PRMT1 protein complex and determines the binding sites of TRIM21 protein and PRMT1 protein in the TRIM21 protein and PRMT1 protein complex as the binding sites of the targeted drug. The drug screening unit uses a computer-aided drug screening method to obtain candidate drugs that target the binding site; The method for determining the binding sites of TRIM21 and PRMT1 proteins in the TRIM21 and PRMT1 protein complex includes: Construct n truncated expression mutants of TRIM21, where n is a natural number greater than or equal to 1; Immunoprecipitation experiments were performed on the n truncated expression mutants to obtain the key regions of TRIM21-PRMT1 interaction. The spatial structure formed by the interaction between the N-terminal SPRY domain of TRIM21 and PRMT1 is the binding site of TRIM21 and PRMT1 proteins in the TRIM21 and PRMT1 protein complex. The computer-aided drug screening method includes: Obtain the binding sites of TRIM21 and PRMT1 proteins in the TRIM21 and PRMT1 protein complex; Based on the spatial structure of the binding sites of TRIM21 and PRMT1 proteins in the TRIM21 and PRMT1 protein complex, small molecule compounds with similar structures were screened from molecular databases. The small molecule compounds selected through screening are molecularly docked with the PRMT1 protein to calculate the affinity or binding energy of the small molecule compounds to the PRMT1 protein receptor, and a score is obtained for each small molecule compound. Candidate drugs are obtained by sorting the small molecule compounds according to the scores.

4. A device for computer-aided screening of drugs for the treatment and / or prevention of colorectal cancer based on TRIM21 and PRMT1, characterized in that, The device includes: The system includes a memory and a processor, wherein the memory is used to store program instructions; and the processor is used to invoke the program instructions, which, when executed, implement the computer-aided drug screening method based on TRIM21 and PRMT1 as described in claim 1 or 2.

5. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores a computer program that, when executed by a processor, implements the computer-aided screening method for the treatment and / or prevention of colorectal cancer based on TRIM21 and PRMT1 as described in claim 1 or 2.

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