Computer-aided drug screening method, system and equipment based on TRIM21 and PRMT1

Abstract

The invention discloses a computer-aided drug screening method, system and device based on TRIM21 and PRMT1, and provides the computer-aided drug screening method, system and device based on TRIM21 and PRMT1 and a computer readable storage medium for the field. The invention 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 invention belongs to the technical field of computer-assisted drug screening, and specifically, relates to a computer-assisted drug screening method, system and equipment based on TRIM21 and PRMT1. Background Art

[0002] Colorectal cancer (CRC), as one of the most common malignant tumors of the digestive tract, has an increasing incidence rate year by year and has now become the third most common malignant tumor in the world. Revealing its potential molecular mechanism is not only a need for scientific research, but also an urgent expectation for 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 certain progress has been made in the development and clinical application of new drugs such as targeted therapy and immune checkpoint inhibitors, the cure rate of colorectal cancer has not been significantly improved. Due to the insidious onset of colorectal cancer, it is usually in the late stage when local infiltration is deep, extensive lymph node metastasis in the abdominal cavity, or even distant metastasis occurs, resulting in delayed diagnosis. Therefore, in CRC research, it is crucial to identify sensitive and specific biomarkers and discover 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 inflammatory processes, cancer development, and autoimmune diseases has attracted extensive attention. During tumorigenesis and development, its function can vary depending on the specific effector factors involved in tumorigenesis 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, renal 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 are also reports that TRIM21 contributes to chemotherapeutic resistance and tumorigenesis in CRC. In summary, the exact role and potential mechanism of TRIM21 in CRC need to be further fully elucidated in order to provide new ideas and strategies for the development or screening of new drugs for the treatment of colorectal cancer.

[0004] Computer-aided Drug Design (CADD) is a method that uses computer science and bioinformatics to accelerate the drug development process. CADD is based on computer calculation and simulation technology. By learning the prior knowledge contained in massive drug data and mining the interaction between target points and candidate drugs, it can quickly select active drug molecules with drug-like properties from millions of molecules. This greatly reduces the blindness of screening candidate drug molecules and improves R&D efficiency. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a computer-assisted drug screening method, system and device based on TRIM21 and PRMT1 in the art. Computer-assisted drug screening technology is used to screen drugs for the treatment and / or prevention of colorectal cancer.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

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

[0008] Further, the method comprises:

[0009] Obtain TRIM21 protein and PRMT1 protein data;

[0010] 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 the binding site of the targeted drug;

[0011] A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

[0012] 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:

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

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

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

[0016] Furthermore, the computer-assisted drug screening method comprises:

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

[0018] Based on the spatial structure of the binding site between the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and the PRMT1 protein complex, screening small molecule compounds with similar structures in a molecular database;

[0019] The screened small molecule compounds are subjected to molecular docking with the PRMT1 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor PRMT1 protein to obtain the score of each small molecule compound, and the candidate drugs are obtained by ranking according to the score.

[0020] Furthermore, the small molecule compound includes an inorganic small molecule compound, a protein analog, a peptide analog, a polypeptide, an antibody, siRNA, shRNA, dsRNA, microRNA or an antisense nucleic acid.

[0021] A second aspect of the present invention provides a computer-assisted drug screening system based on TRIM21 and PRMT1.

[0022] Further, the system comprises:

[0023] Data acquisition unit, acquiring TRIM21 protein and PRMT1 protein data;

[0024] A site determination unit, which selects the spatial structure of the TRIM21 protein and PRMT1 protein complex, and determines the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and the PRMT1 protein complex as the binding site of the targeted drug;

[0025] The drug screening unit adopts a computer-assisted drug screening method to obtain candidate drugs targeting the binding site.

[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 truncated expression mutants of TRIM21, where n is a natural number greater than or equal to 1;

[0028] Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between TRIM21 and PRMT1;

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

[0030] Optionally, the computer-assisted drug screening method comprises:

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

[0032] Based on the spatial structure of the binding site between the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and the PRMT1 protein complex, screening small molecule compounds with similar structures in a molecular database;

[0033] The screened small molecule compounds are subjected to molecular docking with the PRMT1 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor PRMT1 protein to obtain the score of each small molecule compound, and the candidate drugs are obtained by ranking according to the score.

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

[0035] Furthermore, the device comprises:

[0036] 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 TRIM21 and PRMT1 described in the first aspect of the present invention is implemented.

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

[0038] Furthermore, the computer-readable storage medium stores a computer program, and when the computer program is executed by the processor, the computer-assisted drug screening method based on TRIM21 and PRMT1 described in the first aspect of the present invention is implemented.

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

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

[0041] (2) A drug screened by the computer-assisted drug screening method based on TRIM21 and PRMT1 according to the first aspect of the present invention.

[0042] A sixth aspect of the present invention provides any of the following applications:

[0043] (1) An application of a protein complex, wherein the complex is the TRIM21 protein and PRMT1 protein complex described in the fifth aspect of the present invention, and the application includes: application of the complex in screening anti-colorectal cancer drugs;

[0044] (2) A use of a drug, wherein the drug is the drug described in the fifth aspect of the present invention, and the use includes: use of the drug in the preparation of an anti-colorectal cancer drug preparation;

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

[0046] (4) Use of TRIM21 or TRIM21 promoter in the preparation of anti-colorectal cancer drugs;

[0047] (5) Application of PRMT1 inhibitors in the preparation of anti-colorectal cancer drugs.

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

[0049] The present invention clarifies for the first time the molecular mechanism by which TRIM21 inhibits the development of colorectal cancer by ubiquitinating PRMT1. Based on this, the present invention provides a computer-assisted drug screening method, system, equipment and computer-readable storage medium based on TRIM21 and PRMT1 in the art. The present invention provides an efficient, accurate and rapid screening method for the research and development of new drugs for the treatment of colorectal cancer, and has very broad application prospects in the screening and development of new drugs for the treatment of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0051] Figure 1: A schematic flow chart of a computer-assisted drug screening method based on TRIM21 and PRMT1 provided in an embodiment of the present invention;

[0052] Figure 2 : A schematic diagram of a computer-assisted drug screening system based on TRIM21 and PRMT1 provided in an embodiment of the present invention;

[0053] Figure 3 : A schematic diagram of a computer-aided drug screening device based on TRIM21 and PRMT1 provided in an embodiment of the present invention;

[0054] Figure 4 :Decreased expression of TRIM21 is associated with poor survival in CRC patients, including: A: protein expression levels of TRIM21 in normal colon tissues and colorectal cancer tissues, data from the CPTAC database; B: Representative immunohistochemical images of TRIM21 protein expression in 427 cases of colorectal cancer tissues and paired paracancerous tissues; C, D: Comparative analysis of staining intensity and immunohistochemical scores of TRIM12 in colorectal cancer tissues and paired paracancerous tissues, T: tumor tissue; N: paired paracancerous non-cancerous tissue; E, F: Kaplan-Meier survival curves of overall survival and disease-specific survival of colorectal cancer patients were drawn according to the stratification of TRIM21 protein expression levels in colorectal cancer tissues. Data are 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 detection of the effect of knocking down TRIM21 on the proliferation of HCT-116 and LoVo cells; C, D: CCK-8 detection of the effect of overexpressing TRIM21 on the proliferation of HCT-116 and LoVo cells; E, F: Western blot detection of the effect of knocking down or overexpressing TRIM21 on major cyclins, data are expressed as mean±SD (*P<0.05, ***P<0.001);

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

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

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

[0059] Fig. 9 : PRMT1 is required for TRIM21-mediated CRC progression, wherein, Figure A: Western blot detection of the overexpression efficiency of TRIM21 and PRMT1 in HCT-116 cells, and quantification of the relative intensity of EZH2 protein by Image J software; Figures B and C: Cloning assay and CCK-8 assay were used to evaluate the effects of TRIM21 overexpression, PRMT1 overexpression, or both overexpression on HCT-116 cell proliferation; Figures D and E: Representative images and statistical results of migration and invasion assays, used to evaluate the migration and invasion abilities of HCT-116 cells overexpressing TRIM21, PRMT1, or both overexpression, data are expressed as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001);

[0060] Fig.10 : TRIM21 affects CRC progression in a PRMT11-dependent manner, wherein, AC: tumor tissue images, tumor volume statistical curves, and tumor weights of HCT116 tumor-bearing mice (n=6); D: macroscopic inspection of lung metastatic nodules after sacrificing nude mice (n=6), red arrows indicate metastatic nodules; E: Bouin staining and HE staining of lung metastases after tail vein injection in BALB / C nude mice; 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 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.

[0062] In some of the processes described in the specification and claims of the present invention and the above-mentioned drawings, multiple operations appearing 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 document or may be executed in parallel. 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 may include more or fewer operations, and these operations may be executed in sequence or in parallel.

[0063] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

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

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

[0066] In one embodiment, the TRIM21 protein is an E3 ubiquitin ligase that plays a vital role in immune response by targeting intracellular pathogens for degradation. TRIM21 protein is involved in the recognition and clearance of viral particles. The TRIM21 (tripartite motif containing 21 [Homo sapiens (human)]) GeneID in NCBI is 6737, and its 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 protein arginine methyltransferase 1, which is a major member of the protein arginine methyltransferase family. PRMT1 has a variety of histidine and non-histidine substrates, and it regulates protein-protein and protein-nucleic acid interactions by methylating these substrates, exerting epigenetic transcription regulation, RNA splicing, RNA output and signal transduction functions. The Gene ID of the PRMT1 (protein arginine methyltransferase 1 [Homo sapiens (human)]) in NCBI is 3276, and its corresponding detailed information (including sequence information, etc.) can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0068] In one embodiment, the present invention demonstrates that TRIM21 expression is reduced 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 Proteomics Cancer 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 a constructed colorectal cancer tissue chip: 485 pairs of colorectal cancer tissue specimens were collected from the Affiliated Hospital of Wannan Medical College from 2005 to 2008. The patient's clinical pathological information was obtained from the hospital's medical records. On a tissue chip containing 427 CRC tissue samples with complete clinical information and their paired adjacent cancer tissues, the expression level of TRIM21 was detected and scored using immunohistochemical staining. (3) Analysis of the correlation between TRIM21 expression and clinicopathological characteristics and prognosis of patients with colorectal cancer: Fisher's exact test was used to analyze the correlation between TRIM21 expression in colorectal cancer (CRC) and clinicopathological characteristics, including tumor volume, tumor differentiation, lymph node metastasis, distant metastasis, and TNM stage. Overall survival (OS) time and disease-specific survival (DSS) time from the start of surgery to death due to CRC were calculated. Kaplan-Meier survival analysis and log-rank test were used to explore the correlation between TRIM21 expression and overall survival (OS) and disease-specific survival (DSS) of CRC patients.

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

[0070] In addition, the inventors used Fisher's exact test to explore the correlation between TRIM21 expression and clinical pathological characteristics in colorectal cancer (CRC) (Table 1) to further understand the clinical significance of TRIM21 in CRC. The results showed that low TRIM21 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 association was found between TRIM21 expression and age or gender.

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

[0072]

[0073]

[0074] *P value is derived from χ 2 test.

[0075] At the same time, the inventors also used Kaplan-Meier survival analysis and log-rank test to explore the correlation between TRIM21 expression and overall survival (OS) and disease-specific survival (DSS) of CRC patients. Compared with patients with high TRIM21 expression, patients with low TRIM21 expression had worse OS and DSS ( Figure 4 E, F).

[0076] In one embodiment, the present invention demonstrates that TRIM21 inhibits the proliferation of colorectal cancer cells. The specific experimental method is as follows: (1) Whether silencing endogenous TRIM21 promotes the proliferation of colorectal cancer cells (HCT116, LoVo): small interfering RNA fragments (siTRIM21-1, siTRIM21-2) of TRIM21 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 hours, cells were counted. The cells were planted in three replicates in a 96-well plate, with 3000 cells inoculated in each well. The cell growth rate was evaluated using the CCK8 kit at 0, 1, 2, 3, and 4 days after inoculation. (2) Whether the introduction of exogenous TRIM21 promotes the inhibition of colorectal cancer cell (HCT116, LoVo) proliferation: Negative control plasmid (NC) and TRIM21 overexpression plasmid (OE-TRIM21) were transfected into HCT116 and LoVo cells, respectively. After 48 hours, the cells were counted. The cells were seeded into 96-well plates in triplicate, with 3000 cells inoculated per well. The cell growth rate was evaluated using the CCK8 kit at 0, 1, 2, 3, and 4 days after inoculation. (3) Immunoblotting to detect the regulation of proliferation-related proteins by high and low expression of TRIM21: TRIM21 small interfering fragment and TRIM21 overexpression plasmid were transfected into HCT116 and LoVo cells, respectively. After 48 hours of transfection, the proteins were collected. Immunoblotting verified the transfection efficiency and detected 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 conducted 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), while overexpression of TRIM21 weakened the cell proliferation ability ( Figure 5 C, D).

[0078] TRIM21 can ubiquitinate the cyclin-dependent kinase inhibitor p21, which can inhibit the G1 / S cell cycle process. Therefore, the inventors speculated that TRIM21 may inhibit tumor proliferation by affecting the cell cycle process. Western blot analysis was then performed to study the expression levels of p21 and two major cell cycle proteins, cyclin D1 and cyclin E2. Cyclin D1, as an important positive regulator of the cell cycle, shortens the G1 phase, prompts cells to enter the S phase early, accelerates the cell cycle process, and leads to uncontrolled proliferation. Cyclin E2 is the main rate-limiting factor for G1 / S conversion. It is overexpressed in a variety of tumors and promotes the occurrence and development of cancer. The above analysis results show that TRIM21 deficiency leads to increased levels of cyclin E2 and cyclin D1, while decreased levels of p21 ( Figure 5 E). In contrast, overexpression of TRIM21 showed the opposite results ( Figure 5 F).

[0079] In one embodiment, the present invention demonstrates that TRIM21 inhibits the migration and invasion of colorectal cancer cells. The specific experimental methods are as follows: (1) Whether silencing endogenous TRIM21 promotes the migration and invasion of colorectal cancer cells (HCT116, LoVo): In vitro cell migration and invasion experiments were performed using a Transwell chamber with a polyethylene terephthalate membrane (24-well insert, 12.0 μm; Corning). Small interfering RNA fragments of TRIM21 (siTRIM21-1, siTRIM21-2) (designed and synthesized using conventional methods) were transfected into HCT116 and LoVo cells, respectively, and nonspecific siRNA (siCtrl) was used as a negative control. After 48 hours, cell counting was performed. For the migration experiment, 2×10 4 For invasion assays, 2×10 4Cells were seeded into the upper chamber coated with Matrigel. Complete culture medium was added to the lower chamber to stimulate migration or invasion. After incubation for 24-48 hours, the cells were stained with 0.1% crystal violet. Five fields of view were counted for each filter. (2) Whether the introduction of exogenous TRIM21 inhibits the migration and invasion of rectal cancer cells (HCT116, LoVo): Negative control plasmid (NC) and TRIM21 overexpression plasmid (OE-TRIM21) were transfected into HCT116 and LoVo cells, respectively. After 48 hours, the cells were counted. Cell migration and invasion experiments were performed in the same manner as above. (3) Immunoblotting was used to detect the regulation of epithelial-mesenchymal transition-related proteins by high and low expression of TRIM21: TRIM21 small interfering fragment and TRIM21 overexpression plasmid were transfected into HCT116 and LoVo cells, respectively. After 48 hours of transfection, the protein was collected. Immunoblotting was used to verify the transfection efficiency and detect the expressions 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 E-cadherin expression is a major hallmark of EMT. Transwell assay results showed that TRIM21 knockdown promoted the migration of CRC cells ( Figure 6 A) and invasive ability ( Figure 6 B), while TRIM21 overexpression inhibits migration and invasion ability ( Figure 6 C, D). Correspondingly, TRIM21 deficiency leads to increased levels of mesenchymal markers N-cadherin and Snail, while 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 invention demonstrates that TRIM21 binds to PRMT1 and reduces its protein stability. The specific experimental method is as follows: (1) Immunoprecipitation to detect whether endogenous TRIM21 and PRMT1 bind: HCT116 and LoVo cells were co-transfected with HA-TRIM21 and Flag-PRMT1 overexpression plasmids, respectively. After 48 hours of transfection, the cells were lysed, and immunoprecipitation was performed with anti-Flag antibody, and immunoblotting was performed with anti-HA antibody to detect whether PRMT1 binds to TRIM21; immunoprecipitation was performed with anti-HA antibody, and immunoblotting was performed with anti-Flag antibody to detect whether TRIM21 binds to PRMT1. (2) Immunoblotting to detect the effects of high and low expression of TRIM21 on PRMT1 protein: HCT116 and LoVo cells were transfected with small interfering RNA fragments of TRIM21 (siTRIM21-1 and siTRIM21-2), and nonspecific siRNA (siCtrl) was used as a negative control. After 48 hours, immunoblotting was used to verify the transfection efficiency and detect the expression of PRMT1; HCT116 and LoVo cells were transfected with TRIM21 overexpression plasmid (OE-TRIM21) 0, 2, 4 μg and HA empty plasmid (4, 2, 0 μg), respectively, and immunoblotting was used to verify the transfection efficiency and detect the expression of PRMT1. (3) qRT-PCR to detect the effects of high and low expression of TRIM21 on PRMT1 mRNA levels: HCT116 and LoVo cells were transfected with TRIM21 small interfering fragments and TRIM21 overexpression plasmids, respectively, and mRNA was extracted 48 hours after transfection. qRT-PCR was used to verify the transfection efficiency and detect the expression of PRMT1 gene. (4) Protein synthesis inhibitor CHX tracking experiment was used to detect the effect of TRIM21 on the half-life of PRMT1 protein: Negative control plasmid (NC) and TRIM21 overexpression plasmid (OE-TRIM21) were transfected into HCT116 and LoVo cells, respectively. After 48 hours, the cells were treated with CHX (100 μg / mL). After 0, 4, 6, and 8 hours of continuous culture, the protein was collected, and the PRMT1 protein level was detected by immunoblotting, and the time curve was drawn.

[0082] To study the tumor suppressor effect of TRIM21 in colorectal cancer, the inventors transfected control vector plasmids and TRIM21 overexpression plasmids into HCT-116 cells. Protein samples were then subjected to mass spectrometry analysis to explore potential downstream targets of TRIM21. Among the 760 candidate targets, this study focused on arginine methyltransferase PRMT1. The inventors' research group previously discovered that PRMT1 mediates asymmetric methylation modification of the R342 site of EZH2, thereby increasing the stability of the EZH2 protein and promoting the metastasis of breast cancer. 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, we 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, 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. When HCT-116 cells were treated with the protein synthesis inhibitor cycloheximide (CHX), the degradation rate of PRMT1 protein in the TRIM21 overexpression group was higher than that in the control group, resulting in a shortened half-life ( Figure 7 G). These findings suggest that TRIM21 reduces PRMT1 stability in CRC cells.

[0084] S102: 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 the binding site of the targeted drug;

[0085] In one embodiment, the method for 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, where n is a natural number greater than or equal to 1;

[0087] Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between TRIM21 and PRMT1;

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

[0089] In one embodiment, the present invention demonstrates 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: HA-TRIM21 and Flag-PRMT1 overexpression plasmids were co-transfected into HCT116 and LoVo cells, respectively. After 48 hours of transfection, the cells were lysed, and immunoprecipitation was performed with anti-Flag antibody, and immunoblotting was performed with anti-Ubiquitin antibody to detect whether the ubiquitination level of PRMT1 was enhanced. (2) The proteasome inhibitor MG132 was added to verify whether TRIM21 regulates PRMT1 through the proteasome pathway: negative control plasmid (NC) and TRIM21 overexpression plasmid (OE-TRIM21) were transfected into HCT116 and LoVo cells, respectively. After 48 hours, MG132 (10 μM) was used to treat for 6 hours, the cells were lysed, and the expression of PRMT1 was detected by immunoblotting. (3) Detection of the region and mode of TRIM21-mediated ubiquitination of PRMT1: Study the region where TRIM21 interacts with PRMT1: HA-TRIM21 truncated mutants (△RIGN, △B-BOX, △SPRY, △PRY) and complete WT plasmids were used to transfect HCT116 and LoVo cells, respectively. Anti-HA was used for immunoprecipitation and PRMT1 antibody was used for immunoblotting to detect the region where TRIM21 interacts with PRMT1. Study the mode of TRIM21-mediated ubiquitination of PRMT1: HCT116 and LoVo cells were co-transfected with HA-TRIM21 and Flag-ubiquitin-K48 / K63, respectively. Anti-Flag was used for immunoprecipitation and PRMT1 antibody was used for immunoblotting to detect the mode of TRIM21-mediated ubiquitination of PRMT1.

[0090] TRIM21 is an E3 ubiquitin ligase that participates in a variety of 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 the Flag-PRMT1 plasmid into HCT-116 and LoVo cells, and then transfected the control plasmid and TRIM21 overexpression plasmid. The results of Co-IP analysis showed that compared with the control group, the ubiquitination level of PRMT1 in the TRIM21 overexpression group was significantly increased, indicating that TRIM21 can mediate the ubiquitination modification of PRMT1 in CRC cells ( Figure 8A) After the addition of proteasome inhibitor MG132, the downregulation of PRMT1 protein level by TRIM21 was reversed ( Figure 8 B), indicating that TRIM21 regulates the stability of PRMT1 protein through the ubiquitination-proteasome pathway.

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

[0092] There are many types of ubiquitination modifications, which can be classified according to the different positions of the lysine residues of the ubiquitin molecules. Among them, polyubiquitination at the K48 and K63 sites 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 assays confirmed that TRIM21 mainly induces K48-linked ubiquitination of PRMT1 ( Figure 8 E).

[0093] Together, these results suggest that TRIM21 has a pervasive regulatory effect on PRMT1 ubiquitination in CRC cells. The interaction between the SPRY domain of TRIM21 and PRMT1 promotes the delivery of K48-ubiquitin to PRMT1, leading to proteasome recognition and degradation of PRMT1.

[0094] In one embodiment, the present invention demonstrates that PRMT1 is required for TRIM21-mediated CRC progression in vitro. The specific experimental methods are as follows: (1) Four groups of HCT116 stable cell lines were constructed: empty, overexpressing TRIM21, overexpressing PRMT1, and co-expressing TRIM21 and PRMT1. Lentiviral packaging and transfection technology was used to obtain 4 different HCT-116 stable cell lines: 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. Construct the corresponding viral vector plasmids and package the corresponding viruses respectively; infect HCT116 cells; the construction is completed after two weeks of puromycin selection; qRT-PCR detects TRIM21 and PRMT1 mRNA expression, and Western Blot detects TRIM21 and PRMT1 protein expression. (2) Detect the proliferation ability of four groups of HCT116 stable cell lines: The proliferation ability of four groups of HCT116 stable cell lines was detected by CCK-8 kit and clone formation experiment, and the specific steps are as described above. (3) Detect the migration and invasion ability of four groups of HCT116 stable cell lines Transwell experiment was performed on the four groups of cells to evaluate their migration and invasion ability, and the specific steps are as described above.

[0095] Previous studies by the inventors' team have shown that PRMT1 plays a promoting role in the proliferation and metastasis of breast cancer and colorectal cancer. In this study, TRIM21 was found to have 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. The transfection efficiency of TRIM21 and PRMT1 in four groups of HCT-116 cells was confirmed by Western blotting experiments, proving that the corresponding cell lines were successfully constructed ( Fig. 9 A).

[0096] The results of CCK-8 and colony formation experiments showed that PRMT1 overexpression promoted cell proliferation. In contrast, TRIM21 overexpression inhibited cell proliferation, and in cells overexpressing TRIM21, this inhibition could be reversed by overexpressing PRMT1 ( Fig. 9B, C). Subsequently, the four groups of cells were subjected to Transwell assay to evaluate their migration and invasion abilities. The results showed that TRIM21 overexpression inhibited the migration and invasion abilities of cells, while PRMT1 overexpression promoted them. It is worth noting that in cells overexpressing TRIM21, PRMT1 overexpression restored the migration ability of cells ( Fig. 9 D, E). These findings suggest that the mechanism by which TRIM21 inhibits CRC cell proliferation and metastasis in vitro is dependent on PRMT1.

[0097] In one embodiment, the present invention demonstrates that TRIM21 affects the development of CRC in vivo in a PRMT1-dependent manner. The specific experimental methods are as follows: (1) Nude mouse subcutaneous tumor model to study the effects of TRIM21 and PRMT1 on colorectal cancer tumorigenesis: Four groups of HCT-116 stable cell lines (NC, OE-TRIM21, OE-PRMT1 and OE-TRIM21+OE-PRMT1) were cultured at 5×10 6 Each cell was inoculated into the ventral region of the mouse, and the tumor volume was calculated weekly according to the formula V = a × (b × b) / 2, where "a" represents the maximum diameter and "b" represents the minimum diameter. At the sixth week of inoculation, the tumor was removed and weighed, and the subcutaneous tumor tissue was stained with IHC to analyze the expression of TRIM21, PRMT1 and the proliferation marker Ki-67. (2) Tail vein lung metastasis model to study the effect of TRIM21 and PRMT1 on colorectal cancer metastasis: Four groups of HCT-116 stable cell lines (NC, OE-TRIM21, OE-PRMT1 and OE-TRIM21+OE-PRMT1) were inoculated at 5×10 6 The cells were implanted into mice by tail vein injection. After 10 weeks, the nude mice were euthanized, and their lung tissues were removed and Bouin staining was performed to observe the number of metastatic foci in the lungs.

[0098] To investigate the potential inhibitory effects of TRIM21 on CRC tumorigenesis and metastasis in vivo, two animal models were constructed in this study: a nude mouse subcutaneous tumor model and a tail vein pulmonary 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 functions in a PRMT1-dependent manner, this study restored PRMT1 expression in the OE-TRIM21 group, thereby reversing the tumor inhibitory effect of TRIM21 ( Fig.10 A and B). Subcutaneous tumor tissues were then subjected to IHC staining to analyze the expression of TRIM21, PRMT1, and Ki-67 ( Fig.10C). The results showed that both TRIM21 and PRMT1 were stably overexpressed in the corresponding groups. The results of Ki67 staining (an important indicator of cell proliferation) 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 increased significantly compared with the OE-TRIM21 group.

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

[0100] In the present invention, the inventors explored the role of TRIM21 in mediating the ubiquitination and degradation of PRMT1, a process that led to a decrease in PRMT1 protein expression and subsequently inhibited 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 therapeutic approach for the treatment of colorectal cancer and opening up new avenues for clinical intervention. This study first discovered that TRIM21, as a tumor suppressor gene in colorectal cancer, acts as an E3 ubiquitin ligase to promote 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 invention provides for the first time in the art a computer-assisted drug screening method, system, device and computer-readable storage medium based on TRIM21 and PRMT1, which has broad application prospects in the technical field of screening and development of new drugs for the treatment of colorectal cancer.

[0101] S103: using a computer-assisted drug screening method to obtain a candidate drug targeting the binding site;

[0102] In one embodiment, the computer-assisted 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] Based on the spatial structure of the binding site between the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and the PRMT1 protein complex, screening small molecule compounds with similar structures in a molecular database;

[0105] The screened small molecule compounds are subjected to molecular docking with the PRMT1 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor PRMT1 protein to obtain the score of each small molecule compound, and the candidate drugs are obtained by ranking according to the score.

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

[0107] It should be noted that the present invention has no particular restrictions on the specific types of the small molecule compounds, and any small molecule compounds that may be used in the computer-assisted drug screening method provided by the present invention and may produce corresponding effects fall within the protection scope of the present invention.

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

[0109] Obtain a system expressing PRMT1;

[0110] Treating the PRMT1-expressing system with the screened small molecule compound to verify the effectiveness of the small molecule compound;

[0111] Optionally, the system expressing PRMT1 includes a cell system expressing PRMT1, a subcellular system expressing PRMT1, a tissue system expressing PRMT1, a solution system expressing PRMT1, an organ system expressing PRMT1 or an animal system expressing PRMT1;

[0112] Optionally, small molecule compounds that inhibit PRMT1 expression are screened as candidate drugs.

[0113] In one embodiment, the cell system expressing PRMT1, the subcellular system expressing PRMT1, the tissue system expressing PRMT1, the solution system expressing PRMT1, the organ system expressing PRMT1 or the animal system expressing PRMT1 can be obtained through conventional purchasing channels known to those skilled in the art, or can be constructed and obtained through conventional construction methods known to those skilled in the art.

[0114] In one embodiment, the animals include but are not limited to mice, rats, guinea pigs, rabbits, pigs, chickens, pigeons, monkeys and dogs.

[0115] In one embodiment, 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).

[0116] In the process of molecular docking, affinity depends on the interaction between the molecule and the receptor, including hydrogen bonding, van der Waals forces, electrostatic interactions, etc. These interactions jointly 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 interaction 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.

[0117] In one embodiment, the modes of drug screening using computer-assisted drug screening technology include one or more of the following: protein-small molecule docking, protein-protein docking, and protein-nucleic acid docking.

[0118] In one embodiment, the 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 interaction 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 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.

[0120] In one embodiment, the 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 interaction between proteins and possible biological functions.

[0121] In protein-protein docking, RosettaDock is a commonly used 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 energy. 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.

[0122] In one embodiment, the 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.

[0123] 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.

[0124] In one embodiment, drug virtual screening is an important method in the field of targeted drug design. The process is as follows: 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. 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 good shape complementarity will be selected as potential candidate drugs. Finally, the screened candidate drugs are further experimentally verified to confirm their interaction with the target protein 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 the small molecule compound. In order to perform receptor-based virtual screening, it is first necessary to determine the binding site of the target protein. 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 between the compound and 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 out as potential candidate drugs. The determination of the binding site for drug virtual screening 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, it is also necessary to reasonably process and optimize the binding site to improve the accuracy and efficiency of the screening.

[0126] In one embodiment, computer-aided drug screening is a technology for drug screening using computer-aided drug design methods, which 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.

[0127] In one embodiment, 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 its 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. The molecular docking method mainly focuses on spatial matching and energy matching. Spatial matching refers to the geometric complementarity between drug molecules and receptor proteins, while energy matching refers to the minimization of interactions between drug molecules and receptor proteins. For the calculation of geometric matching, grid point calculation, fragment growth and other methods are usually used, while energy calculation uses simulated annealing, genetic algorithms and other methods. 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 conformation involved in the docking does not change during the calculation process, only the spatial position and posture of the molecule are changed. Semi-flexible docking allows some conformations to change during the calculation. Flexible docking allows more conformational changes.

[0128] In one embodiment, the molecular libraries used in drug virtual screening are mainly the following: ZINC: contains more than 250 million commercially available 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 often used for drug virtual screening.

[0129] Figure 2 Schematic diagram of a computer-assisted drug screening system based on TRIM21 and PRMT1 provided by an embodiment of the present invention. Specifically, the system includes:

[0130] Data acquisition unit, acquiring TRIM21 protein and PRMT1 protein data;

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

[0132] The drug screening unit adopts a computer-assisted drug screening method to obtain candidate drugs targeting the binding site.

[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, where n is a natural number greater than or equal to 1;

[0135] Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between TRIM21 and PRMT1;

[0136] The spatial structure formed by the interaction between the N-terminal SPRY domain of 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-assisted 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] Based on the spatial structure of the binding site between the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and the PRMT1 protein complex, screening small molecule compounds with similar structures in a molecular database;

[0140] The screened small molecule compounds are subjected to molecular docking with the PRMT1 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor PRMT1 protein to obtain the score of each small molecule compound, and the candidate drugs are obtained by ranking according to the score.

[0141] Figure 3 Schematic diagram of a computer-assisted drug screening device based on TRIM21 and PRMT1 provided by an embodiment of the present invention. Specifically, the device includes:

[0142] 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 TRIM21 and PRMT1 as described above of the present invention is implemented.

[0143] An embodiment of the present invention further provides a computer-readable storage medium having a computer program stored thereon. When the computer program is executed by a processor, the computer-assisted drug screening method based on TRIM21 and PRMT1 as described above in the present invention is implemented.

[0144] The present invention also provides any of the following products:

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

[0146] (2) A drug screened by the computer-assisted drug screening method based on TRIM21 and PRMT1 as described above according to the present invention.

[0147] The embodiments of the present invention also provide any 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 includes: application of the complex in screening anti-colorectal cancer drugs;

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

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

[0151] (4) Use of TRIM21 or TRIM21 promoter in the preparation of anti-colorectal cancer drugs;

[0152] (5) Application of PRMT1 inhibitors in the preparation of anti-colorectal cancer drugs.

[0153] The validation results of this validation example show that assigning intrinsic weights to indications can moderately improve the performance of the method relative to the default setting.

[0154] Those skilled in the art can clearly understand that, for the convenience and brevity 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.

[0155] In the several embodiments provided in the present 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. 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 mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0156] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0157] 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.

[0158] A person of ordinary skill in the art can understand that all or part of the steps in the various methods of the above embodiments can be completed by instructing related hardware through a program, and the program can 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.

[0159] A person skilled in the art can understand that all or part of the steps in the above-mentioned embodiment method can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium. The above-mentioned storage medium can be a read-only memory, a disk or an optical disk, etc.

[0160] The above is a detailed introduction to the computer-assisted drug screening method, system and equipment based on TRIM21 and PRMT1 provided by the present invention. For those skilled in the art, according to the ideas of the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A computer-assisted drug screening method based on TRIM21 and PRMT1, characterized in that: The method comprises: Obtain TRIM21 protein and PRMT1 protein data; 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 the binding site of the targeted drug; A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

2. The computer-assisted drug screening method based on TRIM21 and PRMT1 according to claim 1, characterized in that: The method for determining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex comprises: constructing n truncated expression mutants of TRIM21, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between TRIM21 and PRMT1; The spatial structure formed by the interaction between the N-terminal SPRY domain of TRIM21 and the PRMT1 is the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex.

3. The computer-assisted drug screening method based on TRIM21 and PRMT1 according to claim 1, characterized in that: The computer-assisted drug screening method comprises: Obtaining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex; Based on the spatial structure of the binding site between the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and the PRMT1 protein complex, screening small molecule compounds with similar structures in a molecular database; The screened small molecule compounds are subjected to molecular docking with the PRMT1 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor PRMT1 protein to obtain the score of each small molecule compound, and the candidate drugs are obtained by ranking according to the score.

4. The computer-assisted drug screening method based on TRIM21 and PRMT1 according to claim 3, characterized in that: The small molecule compound includes an inorganic small molecule compound, a protein analog, a peptide analog, a polypeptide, an antibody, a siRNA, a shRNA, a dsRNA, a microRNA or an antisense nucleic acid.

5. A computer-assisted drug screening system based on TRIM21 and PRMT1, characterized in that: The system comprises: a data acquisition unit, which acquires TRIM21 protein and PRMT1 protein data; A site determination unit, which selects the spatial structure of the TRIM21 protein and PRMT1 protein complex, and determines the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and the PRMT1 protein complex as the binding site of the targeted drug; The drug screening unit adopts a computer-assisted drug screening method to obtain candidate drugs targeting the binding site.

6. The computer-aided drug screening system based on TRIM21 and PRMT1 according to claim 5, characterized in that: The method for determining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex comprises: constructing n truncated expression mutants of TRIM21, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between TRIM21 and PRMT1; The spatial structure formed by the interaction between the N-terminal SPRY domain of TRIM21 and the PRMT1 is the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and PRMT1 protein complex; Optionally, the computer-assisted drug screening method comprises: Obtaining the binding site of the TRIM21 protein and the PRMT1 protein in the TRIM21 protein and the PRMT1 protein complex; screening small molecule compounds with similar structures 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 the PRMT1 protein complex; The screened small molecule compounds are subjected to molecular docking with the PRMT1 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor PRMT1 protein to obtain the score of each small molecule compound, and the candidate drugs are obtained by ranking according to the score.

7. A computer-aided drug screening device based on TRIM21 and PRMT1, 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 TRIM21 and PRMT1 described in any one of claims 1 to 4 is implemented.

8. A computer-readable storage medium, characterized in that: The computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the computer-assisted drug screening method based on TRIM21 and PRMT1 described in any one of claims 1 to 4 is implemented.

9. Any of the following products: (1) A complex of TRIM21 protein and PRMT1 protein, characterized in that: The TRIM21 protein and PRMT1 protein complex is the TRIM21 protein and PRMT1 protein complex described in claim 1; (2) A drug screened by the computer-assisted drug screening method based on TRIM21 and PRMT1 according to any one of claims 1 to 4.

10. Any of the following applications: (1) An application of a protein complex, characterized in that: The complex is the TRIM21 protein and PRMT1 protein complex described in claim 9, and the application includes: application of the complex in screening anti-colorectal cancer drugs; (2) A use of a drug, characterized in that the drug is the drug described in claim 9, and the use comprises: Use of the drug in preparing an anti-colorectal cancer drug preparation; (3) The application of TRIM21 or TRIM21 promoter in regulating PRMT1; (4) Use of TRIM21 or TRIM21 promoter in the preparation of anti-colorectal cancer drugs; (5) Application of PRMT1 inhibitors in the preparation of anti-colorectal cancer drugs.

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