A computer-aided drug screening method, system and device based on TRIM56 and STING

Abstract

The present invention discloses a computer-aided drug screening method, system and device based on TRIM56 and STING. For the first time, the present invention discovers the interaction relationship that TRIM56 promotes the degradation of STING by E1A, thereby promoting adenovirus replication and transcription. Based on this, the present invention provides a computer-aided drug screening method, system, device and computer-readable storage medium based on TRIM56 and STING in the art. The present invention provides an efficient, accurate and rapid screening method for the research and development of new anti-adenovirus drugs, and has very broad application prospects in the screening of new anti-adenovirus drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of computer-aided drug screening, and specifically, relates to a computer-aided drug screening method, system and device based on TRIM56 and STING. Background Art

[0002] Human adenovirus (HAdV) is a double-stranded DNA virus belonging to the genus Mastadenovirus of the Adenoviridae family; the virus has an icosahedral structure, without an outer envelope, and the full-length genome is about 36 kb. The genomic DNA combines with viral structural proteins to form the viral core, which is covered by a capsid composed of 252 capsomeres, including 240 hexon proteins and 12 penton proteins, with a diameter of about 70-90 nm. Currently, 7 subgroups (A-G) and 67 different serotypes of adenoviruses have been discovered, among which 55 subtypes can infect humans and cause diseases. The most common infection of adenovirus in humans is the respiratory tract, leading to respiratory system diseases. In addition, some adenoviruses can also cause infections in the urinary and gastrointestinal systems.

[0003] The human adenoviruses related to respiratory diseases mainly include subgroup B (HAdV-3, 7, 11, 14, 16, 21, 55), subgroup C (HAdV-1, 2, 5, 6) and subgroup E (HAdV-4). The main types of adenoviruses prevalent in China are types 1, 3, 4, 5, 7, 11, 14, 40, 41, 55, among which types 3 and 5 adenoviruses are the main prevalent ones. Research has found that except for the hexon, fiber, and penton genes, the genomes of various types of adenoviruses are highly conserved and rarely undergo intragenic recombination. This characteristic of adenoviruses is conducive to overcoming the type-specific limitations of drugs on adenoviruses. Usually, antiviral drugs targeting the post-entry stage of adenoviruses have broad-spectrum anti-adenovirus activity within the adenovirus species.

[0004] At present, there is no specific drug for adenovirus infection clinically, and broad-spectrum antiviral therapy and immunotherapy are generally adopted. Antiviral therapy is mainly limited to non-specific medications such as ganciclovir, acyclovir, vidarabine, ribavirin, and cidofovir. Broad-spectrum antiviral drugs such as cidofovir, ribavirin, and ganciclovir have been tested for their anti-adenovirus activity in in vitro experiments and clinical settings, but the effects vary greatly and no satisfactory results have been shown. Therefore, there is an urgent need in this field to develop anti-adenovirus drugs with high specificity, safety, and bioavailability. Computer-aided drug design (CADD) is based on computer operation and simulation technologies. By learning the prior knowledge contained in a vast amount of drug data and exploring the interaction relationship between target targets and candidate drugs, it can quickly screen out drug-like active drug molecules from millions of molecules. This greatly reduces the blindness of screening candidate drug molecules and improves the R & D efficiency. Summary of the Invention

[0005] In view of this, in response to the above technical problems existing in the current development of anti-adenovirus drugs, the object of the present invention is to provide a computer-aided drug screening method, system, and device based on TRIM56 and STING. Utilize computer-aided drug screening technology to screen drugs for the treatment and / or prevention of adenovirus infection.

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

[0007] The first aspect of the present invention provides a computer-aided drug screening method based on TRIM56 and STING.

[0008] Further, the method includes:

[0009] Obtain TRIM56 protein and E1A protein data;

[0010] Select the spatial structure of the TRIM56 protein and E1A protein complex, and determine the binding site of the TRIM56 protein and E1A protein complex as the binding site for the targeted drug;

[0011] Use the method of computer-aided drug screening to obtain candidate drugs targeting the binding site.

[0012] Further, the method for determining the binding site of the TRIM56 protein and E1A protein complex includes:

[0013] Construct n truncated expression mutants of TRIM56, where n is a natural number greater than or equal to 1;

[0014] Performing co-immunoprecipitation experiments based on the n truncated expression mutants reveals that the N-terminal region of TRIM56 is the key region interacting with E1A;

[0015] The spatial conformation formed by the interaction between the N-terminal region of TRIM56 and E1A is the binding site of the TRIM56 protein and E1A protein complex.

[0016] Furthermore, the computer-aided drug screening method includes:

[0017] Obtaining the binding site of the TRIM56 protein and E1A protein complex;

[0018] Screening for small molecule compounds with similar structures in the molecular database based on the spatial structure of the binding site of the TRIM56 protein and E1A protein complex;

[0019] Performing molecular docking of the screened small molecule compounds with the E1A protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor E1A protein, obtaining the scores of each small molecule compound, and sorting according to the scores to obtain candidate drugs.

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

[0021] Furthermore, the method further includes:

[0022] Obtaining a system expressing STING;

[0023] Treating the system expressing STING with the screened small molecule compounds to verify the effectiveness of the small molecule compounds;

[0024] Optionally, the system expressing STING includes a cell system expressing STING, a subcellular system expressing STING, a tissue system expressing STING, a solution system expressing STING, an organ system expressing STING, or an animal system expressing STING;

[0025] Optionally, screening for small molecule compounds that promote STING expression as candidate drugs.

[0026] The second aspect of the present invention provides a computer-aided drug screening system based on TRIM56 and STING.

[0027] Furthermore, the system includes:

[0028] Data acquisition unit: acquiring data of TRIM56 protein and E1A protein;

[0029] Binding site determination unit: Select the spatial structure of the TRIM56 protein and E1A protein complex, and determine the binding site of the TRIM56 protein and E1A protein complex as the binding site of the targeted drug;

[0030] Drug screening unit: Obtain candidate drugs targeting the binding site by using a computer-aided drug screening method;

[0031] Optionally, the method for determining the binding site of the TRIM56 protein and E1A protein complex includes:

[0032] Construct n truncated expression mutants of TRIM56, where n is a natural number greater than or equal to 1;

[0033] Perform co-immunoprecipitation experiments based on the n truncated expression mutants to obtain that the N-terminal region of TRIM56 is the key region interacting with E1A;

[0034] The spatial conformation formed by the interaction between the N-terminal region of TRIM56 and E1A is the binding site of the TRIM56 protein and E1A protein complex;

[0035] Optionally, the computer-aided drug screening method includes:

[0036] Obtain the binding site of the TRIM56 protein and E1A protein complex;

[0037] Screen for small molecule compounds with similar structures in the molecular database based on the spatial structure of the binding site of the TRIM56 protein and E1A protein complex;

[0038] Perform molecular docking of the screened small molecule compounds with the E1A protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor E1A protein, obtain the scores of each small molecule compound, and sort according to the scores to obtain candidate drugs;

[0039] Optionally, the system further includes a drug efficacy verification unit: Obtain a system expressing STING; Treat the system expressing STING with the screened small molecule compounds to verify the efficacy of the small molecule compounds;

[0040] Optionally, the system expressing STING includes a cell system expressing STING, a subcellular system expressing STING, a tissue system expressing STING, a solution system expressing STING, an organ system expressing STING, or an animal system expressing STING;

[0041] Optionally, screen for small molecule compounds that promote STING expression as candidate drugs.

[0042] The third aspect of the present invention provides a computer-aided drug screening device based on TRIM56 and STING.

[0043] Further, the device includes:

[0044] A memory and a processor, where 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-aided drug screening method based on TRIM56 and STING described in the first aspect of the present invention is implemented.

[0045] The fourth aspect of the present invention provides a computer-readable storage medium.

[0046] Further, a computer program is stored on the computer-readable storage medium, and when the computer program is executed by a processor, the computer-aided drug screening method based on TRIM56 and STING described in the first aspect of the present invention is implemented.

[0047] The fifth aspect of the present invention provides any one of the following products:

[0048] (1) A complex of TRIM56 protein and E1A protein, and the complex of TRIM56 protein and E1A protein is the complex of TRIM56 protein and E1A protein described in the first aspect of the present invention;

[0049] (2) A drug screened according to the computer-aided drug screening method based on TRIM56 and STING described in the first aspect of the present invention.

[0050] The sixth aspect of the present invention provides any one of the following applications:

[0051] (1) An application of a protein complex, where the complex is the complex of TRIM56 protein and E1A protein described in the fifth aspect of the present invention, and the application includes: the application of the complex in regulating adenovirus replication and / or transcription;

[0052] (2) An application of a drug, where the drug is the drug described in the fifth aspect of the present invention, and the application includes: the application of the drug in anti-adenovirus.

[0053] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0054] The present invention first discovers the interaction relationship that TRIM56 promotes the degradation of STING by E1A, thereby promoting adenovirus replication and transcription. Based on this, the present invention provides a computer-aided drug screening method, system, device, and computer-readable storage medium based on TRIM56 and STING in the art. The present invention provides an efficient, accurate, and rapid screening method for the research and development of new anti-adenovirus drugs, and has a very broad application prospect in the screening of new anti-adenovirus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0056] Figure 1 : Schematic flow chart of a computer-aided drug screening method based on TRIM56 and STING provided by an embodiment of the present invention;

[0057] Figure 2 : Schematic diagram of a computer-aided drug screening system based on TRIM56 and STING provided by an embodiment of the present invention;

[0058] Figure 3 : Schematic diagram of a computer-aided drug screening device based on TRIM56 and STING provided by an embodiment of the present invention;

[0059] Figure 4:Results graph corresponding to the promotion of adenovirus replication by overexpression of TRIM56 provided by the embodiments of the present invention. (A) Mass spectrometry analysis results of 786-O cells overexpressing E1A and control cells. (B) An A549 cell line stably overexpressing TRIM56 was constructed, and the virus replicated in A549 cells overexpressing TRIM56. A549 cells overexpressing TRIM56 or control A549 cells were infected with HAdV-C5 at an MOI of 5 or 20, and the supernatant was collected at specific time points. The virus titer was measured by the TCID50 assay. ***, P < 0.001; ****, P < 0.0001. (C) The cell viability of A549 cells overexpressing TRIM56 was detected by the CCK-8 assay, and the data were expressed as the mean ± standard deviation (SD) of three transfections. (D and E) The virus-infected cells were observed using a fluorescence microscope, scale bar, 200 μm. Western blot analysis of the virus-infected cells was performed using an anti-GFP antibody. (F) The expression of TRIM56 in A549 cells was inhibited using siRNA. After A549 cells were transfected with siRNA targeting TRIM56 or control siRNA for 48 h, the cell lysates were collected and subjected to Western blot analysis. (G) The effect of siRNA-mediated knockdown of TRIM56 on the viability of A549 cells was detected by the CCK-8 assay. (H) The virus replication in A549 cells treated with siRNA targeting TRIM56. A549 cells with TRIM56 knockdown were infected with HAdV-C5 at an MOI of 5 and 20, and the virus titers were detected at 24 h and 48 h after infection, respectively. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. At least three independent experiments were performed;

[0060] Figure 5: Results graphs corresponding to the promotion of adenovirus gene transcription by overexpression of TRIM56 provided in the embodiments of the present invention. (A, B, and C) A549 cells with overexpressed TRIM56 or control A549 cells were infected with HAdV-C5 at an MOI of 20. At 24 h and 48 h after infection, the RNA levels of E1A, DBP, and Hexon genomic transcripts were detected by RT-qPCR. The results are expressed as the mean ± SD of three independent experiments normalized according to the GAPDH results. **, P < 0.01; ****, P < 0.0001. (D) RT-qPCR confirmed the stable overexpression of TRIM56. ****, P < 0.0001. (E, F, and G) A549 cells were transfected with siRNA targeting TRIM56 or control siRNA for 36 h, and then infected with HAdV-C5 at an MOI of 20. At the indicated time points after infection, the RNA levels of E1A, DBP, and Hexon genomic transcripts were detected by RT-qPCR. The results are expressed as the mean ± SD of three independent experiments normalized according to the GAPDH results. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (H) RT-qPCR confirmed that siRNA knocked down TRIM56 in A549 cells. ****, P < 0.0001. At least three independent experiments were performed;

[0061] Figure 6 : Results graphs corresponding to the interaction between TRIM56 and adenovirus E1A provided in the embodiments of the present invention. (A) TRIM56-V5 and E1A in HAdV-C5 were co-transfected into HEK293T cells. Cell lysates were immunoprecipitated (IP) using anti-E1A antibody (left panel), anti-V5 antibody (right panel), or control IgG, and blotted as indicated. (B) During the infection of A549 cells with HAdV-C5 at an MOI of 20, cell lysates were collected at specific time points and immunoprecipitated using anti-E1A antibody (left panel), anti-TRIM56 antibody (right panel), or control IgG, and blotted as indicated. (C) E1A and TRIM56 were co-transfected into HeLa cells for 36 h, and their co-localization was determined by confocal microscopy. The cells were fixed with 4% paraformaldehyde, incubated with anti-E1A and anti-TRIM56 antibodies, and finally analyzed by confocal microscopy. (D) Schematic diagram of truncated mutants of TRIM56 with V5 tags (Del R, Del BB, Del CC, Del N363) constructed. (E) E1A and V5-tagged TRIM56 mutants were co-transfected into HEK293T cells. Cell lysates were immunoprecipitated using anti-E1A antibody, and detected by Western blotting. At least three independent experiments were performed;

[0062] Figure 7 : Results graph showing that TRIM56 provided in the embodiments of the present invention promotes the stability of E1A protein. (A) HEK293T cells were co-transfected with E1A and TRIM56-V5 (gradually increasing TRIM56-V5), and cell lysates were immunoblotted with anti-E1A antibody and anti-V5 antibody. (B) Gray value calculation of three independent experiments in (A). ****, P<0.0001. (C) A549 cells overexpressing TRIM56 and control cells were infected with HAdV-C5, and cells were collected at different infection time points, and the expression of E1A viral protein was detected by IB analysis. (D) A549 cells were infected with HAdV-C5 at an MOI of 5, and whole cell lysates were collected at 0, 12, 24, 36, 48, and 72 h after infection, and immunoblotting was performed with mouse anti-E1A antibody and rabbit anti-TRIM56 antibody, with at least three independent experiments;

[0063] Figure 8 : Results graph showing that TRIM56 provided in the embodiments of the present invention promotes the degradation of STING by E1A. (A) E1A of HAdV-C5 and STING plasmid with Flag tag were co-transfected into HEK293T cells, and cell lysates were immunoprecipitated with anti-E1A antibody and control IgG, and blotted as indicated. (B) TRIM56-V5 and STING-Flag were co-transfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody or control IgG, and blotted as indicated. (C) HEK293T cells were co-transfected with STING-Flag and gradually increasing TRIM56-V5. Cell lysates were immunoblotted with the indicated antibodies. (D) HEK293T cells were co-transfected with STING-Flag and E1A (gradually increasing E1A), and cell lysates were immunoblotted with the indicated antibodies. (E) Gray value calculation of three independent experiments in (D). ****, P<0.0001. (F) HEK293T cells were co-transfected with STING-Flag, E1A, and TRIM56-V5, cell lysates were collected, and IB analysis was performed with the indicated antibodies. (G) Gray value calculation of three independent experiments in (F). ****, P<0.0001. At least three independent experiments were performed. Detailed implementation manners

[0064] In order to enable those skilled in the art to better understand the solution 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.

[0065] In some of the processes described in the specification, claims, and above-mentioned drawings of the present invention, a plurality of operations appear in a specific order. However, it should be clearly understood that these operations may not be executed in the order in which they appear herein 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.

[0066] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0067] Figure 1 It is a schematic flowchart of a computer-aided drug screening method based on TRIM56 and STING provided by an embodiment of the present invention. Specifically, the method includes the following steps:

[0068] S101: Obtain TRIM56 protein and E1A protein data;

[0069] In one embodiment, the TRIM56 is one of the TRIM family members, and includes a RING domain, a B-box domain, and a coiled-coil domain. As a member of the TRIM family that mostly functions as an E3 ubiquitin ligase, the conserved RING domain of TRIM56 confers its E3 ubiquitin ligase activity. The Gene ID of the TRIM56 (tripartite motif containing 56 [Homo sapiens (human)]) in NCBI is 81844. The detailed information (including sequence information, etc.) of the above gene can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / . Currently, there is no relevant research or report on the application of TRIM56 in screening anti-adenovirus drugs.

[0070] In one embodiment, the present invention proves that overexpression of TRIM56 promotes adenovirus replication. First, the differentially expressed genes between the E1A overexpression group and the control group were analyzed by mass spectrometry. The results showed that the expression level of TRIM56 in cells overexpressing E1A was significantly increased ( Figure 4 A). Combining the results of mass spectrometry analysis, we speculate that TRIM56 may regulate HAdV infection.

[0071] To investigate the role of TRIM56 in the viral life cycle, A549 cells with control and overexpressed TRIM56 were infected with HAdV-C5 at an MOI of 5 or 20. Overexpression of TRIM56 significantly promoted viral replication ( Figure 4 B). Especially when the MOI was 5, the viral titer in cells with overexpressed TRIM56 increased by approximately 10-fold at 48 h compared to that in control cells. The results of the CCK-8 assay showed that overexpression of TRIM56 had no obvious effect on cell viability ( Figure 4 C). In addition, A549 cells with overexpressed TRIM56 were infected with HAdV-C5-GFP virus. The results showed that after overexpression of TRIM56, the number of virus-infected cells increased and the fluorescence intensity was significantly enhanced ( Figure 4 D). The Western blot results showed that when TRIM56 was overexpressed, the levels of E1A viral protein and GFP increased significantly ( Figure 4 E).

[0072] Next, the effect of TRIM56 deficiency on cells was determined by knocking down TRIM56 in A549 cells mediated by small interfering RNA (siRNA) ( Figure 4 F), and the sequences of the siRNA targeting TRIM56 and the control siRNA are shown in Table 1 below. The results of the CCK-8 assay showed that knocking down TRIM56 did not affect cell viability ( Figure 4 G). Cells with knocked-down TRIM56 were infected with HAdV-C5 at an MOI of 5 and 20, and the viral titers were detected at 24 h and 48 h after infection, respectively. The results showed that when TRIM56 was knocked down, at an MOI of 5, the replication titer of the virus decreased by 10-fold at 48 h ( Figure 4 H). These results together indicate that TRIM56 positively regulates the replication of HAdV-C5 and promotes the production of progeny virus particles.

[0073] Table 1 Sequence information of siRNA targeting TRIM56 and control siRNA

[0074]

[0075] In one embodiment, the present invention demonstrates that overexpression of TRIM56 promotes adenovirus gene transcription. TCID50 assay revealed that TRIM56 could increase the virus titer during HAdV-C5 infection. Therefore, we examined whether TRIM56 could promote the transcription and genomic replication of HAdV-C5. A549 cell lines with overexpression of TRIM56 and control were infected with HAdV-C5. Genomic transcripts of E1A, Hexon, and DBP of HAdV-C5 were detected by qPCR. Compared with the control cells, the levels of E1A, Hexon, and DBP in the TRIM56-overexpressing cells were significantly increased at 24 or 48 h post-infection ( Figure 5 A, B, C). In addition, the results of real-time quantitative PCR showed that the expression level of TRIM56 in the cell line overexpressing TRIM56 was significantly higher than that in the control cell line ( Figure 5 D).

[0076] In addition, we used siRNA targeting TRIM56 to silence TRIM56 or treated A549 cells with control siRNA. The sequences of the siRNA targeting TRIM56 and the control siRNA are shown in Table 1, and then the cells were infected with HAdV-C5 virus. The results showed that the transcriptional levels of E1A, Hexon, and DBP in the HAdV-C5-infected cells treated with TRIM56-specific siRNA were significantly decreased compared with those in the A549 cells treated with control siRNA ( Figure 5 E, F, G). The results of Real-time PCR showed that the expression of TRIM56 in the A549 cells treated with siRNA targeting TRIM56 was decreased compared with that in the A549 cells treated with control siRNA ( Figure 5 H). These results together indicate that overexpression of TRIM56 promotes the transcription of the adenovirus genome, while knockdown of TRIM56 does not.

[0077] In one embodiment, the E1A (adenovirus early region 1) refers to the adenovirus E1A gene. E1A plays a crucial role in the life cycle of adenovirus (AdV), including adenovirus nuclear transport, transcription, and viral genome replication. The E1A protein is essential for the efficient transcription of early type 5 adenovirus mRNA and can stimulate its transcription. E1A initiates adenovirus transcription by binding to and altering the functions of a large number of cellular target proteins. E1A is the earliest transcribed gene in the adenovirus genome, and its encoded product binds to the host cell pRb protein, resulting in the separation of the E2F transcription factor from the pRb protein, thereby activating the transcription of other early transcription genes (E1B, E2, E3, E4) mediated by E2F and initiating the replication of viral DNA.

[0078] In one embodiment, the adenovirus, especially Human adenovirus (HAdV), belongs to the genus Mastadenovirus of the family Adenoviridae. Adenovirus infections are relatively common clinically, and the organ systems where infections often occur are the cornea and the respiratory tract. Adenovirus infections are self-limiting, but in some special populations such as immunocompromised individuals and children, the infections can lead to more serious consequences. Since the first isolation of adenovirus, a total of seven species, A - G, and more than 100 serotypes of adenovirus have been identified. Among them, there are 52 types of human adenoviruses, named AdV1 - AdV52 respectively, and they are divided into six subgroups: A, B, C, D, E, and F. With the continuous development of molecular biology techniques and the continuous exploration of adenoviruses, the structure of adenoviruses and their genomic replication mechanisms have been studied relatively clearly. Human adenovirus infections are widespread worldwide and can induce various inflammatory diseases in the human body.

[0079] S102: Select the spatial structure of the TRIM56 protein and E1A protein complex, and determine the binding site of the TRIM56 protein and E1A protein complex as the binding site for the targeted drug;

[0080] In one embodiment, the method for determining the binding site of the TRIM56 protein and E1A protein complex includes:

[0081] Construct n truncated expression mutants of TRIM56, where n is a natural number greater than or equal to 1;

[0082] Based on the n truncated expression mutants, perform co-immunoprecipitation experiments to obtain that the N-terminal region of TRIM56 is the key region interacting with E1A;

[0083] The spatial conformation formed by the interaction between the N-terminal region of TRIM56 and E1A is the binding site of the TRIM56 protein and E1A protein complex.

[0084] In one embodiment, when it has been proven that TRIM56 can significantly promote the replication of HAdV-C5, it was further verified whether this is related to the correlation between TRIM56 and E1A. We co-transfected TRIM56 with a V5 tag and E1A of HAdV-C5 into HEK293T cells to detect their interaction. The results showed that in HEK293T cells, there was a specific interaction between TRIM56 and the full-length E1A protein ( Figure 6 A). In addition, during the infection of HAdV-C5, there was a specific interaction between the viral E1A and endogenous TRIM56 ( Figure 6 B).

[0085] We co-transfected E1A and TRIM56 into HeLa cells and then observed by confocal microscopy that the two proteins were mainly co-localized in the cytoplasm ( Figure 6 C). These results indicate that there is a correlation between TRIM56 and E1A of HAdV-C5. Next, we constructed truncated mutants of TRIM56 with V5 tags (Del R, Del BB, Del CC, Del N363)( Figure 6 D) to locate the interaction region between TRIM56 and E1A. Co-immunoprecipitation experiments found that Del N363 had little effect on the interaction between TRIM56 and E1A. On the contrary, the interaction occurred only when other domains co-existed( Figure 6 E). The interaction between E1A and TRIM56 may require the RING, BBox, and coiled-coil domains to form a certain spatial conformation. In contrast, the interaction alone does not occur.

[0086] In summary, the interaction between TRIM56 and E1A is achieved by forming a spatial conformation in the N-terminal region of TRIM56.

[0087] In one embodiment, considering the correlation between TRIM56 and E1A, we first detected the effect of TRIM56 on the protein stability of HAdV-C5 E1A by a dose-dependent experiment. The results showed that the protein stability of E1A increased with the increase in the expression level of TRIM56( Figure 7 A). In addition, the gray value showed that the expression level of E1A increased to the maximum value, and at this time, the expression level of TRIM56 was also the highest( Figure 7 B).

[0088] In addition, A549 cells overexpressing TRIM56 and control cells were infected with HAdV-C5, and cells were collected at different infection time points to detect the expression of E1A viral protein. Compared with the control group, overexpression of TRIM56 promoted the replication of HAdV-C5 and produced more E1A viral proteins, especially at 48 h after infection( Figure 7 C).

[0089] In addition, A549 cells infected with HAdV-C5 were collected at 0, 12, 24, 36, 48, and 72 h after infection, respectively, to detect the expression levels of E1A from HAdV-C5 and endogenous TRIM56. The results showed that with the increase in the infection time of HAdV-C5, the expression levels of E1A viral protein and endogenous TRIM56 increased sequentially and were consistent( Figure 7 D).

[0090] These results indicate that during HAdV-C5 infection, TRIM56 promotes the stability of the E1A viral protein and enhances the replication of HAdV-C5.

[0091] In one embodiment, when a DNA virus infects a cell and transmits a signal to induce the production of downstream interferon, STING is activated. Previous studies have confirmed that the oncogenes E7 and E1A of the DNA tumor viruses human papillomavirus 18 and adenovirus can inhibit the cGAS-STING pathway. We will explore whether STING is involved in the regulation of E1A by TRIM56. First, the E1A of HAdV-C5 was co-transfected with the STING plasmid with a Flag tag into HEK293T cells for co-IP experiments. The results confirmed a specific interaction between the E1A of HAdV-C5 and STING ( Figure 8 A). Similarly, we performed immunoprecipitation experiments to determine the interaction between TRIM56 and STING. The results showed an interaction between TRIM56 and STING ( Figure 8 B).

[0092] Considering the relationship among E1A, TRIM56, and STING, we first investigated whether TRIM56 regulates E1A by affecting the protein expression of STING. The dose-dependent experiment showed that the protein stability of STING was not affected by the increase in TRIM56 ( Figure 8 C). However, the dose-dependent experiment found that with the increase in the level of the E1A viral protein, the protein stability of STING decreased ( Figure 8 D). At the same time, the gray value also confirmed that when E1A expression was the highest, STING expression was the lowest ( Figure 8 E). These results indicate that the E1A of HAdV-C5 can significantly degrade STING, while TRIM56 does not affect the stability of STING.

[0093] In addition, Western blot analysis suggested that E1A degrades STING, while TRIM56 can significantly increase the expression of E1A, indirectly enhancing the degradation of STING by the E1A protein and playing an antagonistic role against STING ( Figure 8 F and G). In summary, TRIM56 promotes the expression of HAdV-C5 E1A and promotes the degradation of STING by E1A, thereby enhancing the replication of HAdV-C5.

[0094] The above results demonstrated the interaction relationship that TRIM56 promotes adenovirus replication and transcription by promoting the degradation of STING by E1A. Based on this, the present invention developed for the first time a computer-aided drug screening method based on TRIM56 and STING.

[0095] S103: Obtain candidate drugs targeting the binding site by using a computer-aided drug screening method;

[0096] In one embodiment, the computer-aided drug screening method includes:

[0097] Obtain the binding site of the TRIM56 protein and the E1A protein complex;

[0098] Based on the spatial structure of the binding site of the TRIM56 protein and the E1A protein complex, screen for small molecule compounds with similar structures in a molecular database;

[0099] Perform molecular docking of the screened small molecule compounds with the E1A protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor E1A protein, obtain the scores of each small molecule compound, and sort according to the scores to obtain candidate drugs.

[0100] In one embodiment, the small molecule compounds include siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog, or inorganic small molecule compound. It should be noted that the present invention does not particularly limit the specific type of the small molecule compound, and any small molecule compound that may be used in the computer-aided drug screening method provided by the present invention and may produce corresponding effects falls within the protection scope of the present invention.

[0101] In one embodiment, in molecular docking, affinity refers to the tightness of the binding between a molecule and a receptor. High affinity means more stable binding, while low affinity means less stable binding. Affinity is usually calculated, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd).

[0102] During the molecular docking process, the affinity depends on the interaction 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.

[0103] To evaluate the affinity, some scoring systems or scoring methods are usually adopted to quantify the interaction between the molecule and the receptor. These scoring methods are based on different algorithms and physical models and can reflect the binding energy, interaction type, and affinity between the molecule and the receptor.

[0104] In one embodiment, the modes of screening drugs by using computer-aided drug screening technology include one or more of the following: protein-small molecule docking, protein-protein docking, protein-nucleic acid docking.

[0105] In one embodiment, the protein-small molecule docking refers to a computational simulation process that docks the structures of a protein and a small molecule (such as a drug molecule) together through certain algorithms and programs. This process can be used to study the interactions between proteins and small molecules, as well as possible biological functions.

[0106] 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 the docking between small molecule ligands and biomacromolecular receptors. It adopts a fragment-based scoring method and can achieve fast and accurate docking. The basic algorithm of the DOCK software includes two stages. The first stage is the low-precision stage, which mainly searches for a rough match between the small molecule ligand and the biomacromolecular receptor. 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, the DOCK software randomly translates and rotates the small molecule ligand 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 will perform more optimizations and adjustments to achieve more accurate docking.

[0107] In one embodiment, the protein-protein docking refers to a computational simulation process that docks the structures of two proteins together through certain algorithms and programs. This process can be used to study the interactions between proteins, as well as possible biological functions.

[0108] In protein-protein docking, RosettaDock is a commonly used docking software. It adopts a fragment-based scoring method and can achieve fast and accurate protein docking. This software can accurately adjust the side chain conformations during the docking process and considers various 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 fit between the backbone shapes of the two proteins. 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, the program randomly translates and rotates a certain component of a protein molecule 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 will perform 50 MCMP Cycles: rearrange the conformation and minimize the interaction energy, and use this as the initial starting conformation.

[0109] 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 their possible biological functions.

[0110] 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, capable of accurately modeling and docking DNA or RNA molecules. The NAflex software adopts a fragment-based scoring method, enabling fast and accurate docking. It takes into account various complex interactions, such as hydrogen bonds, ionic bonds, hydrophobic interactions, etc., and can precisely adjust the side-chain conformations during the docking process. The basic algorithm of the NAflex software includes two stages. The first stage is the low-precision stage, mainly searching for a rough match between the protein and the nucleic acid. 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, the NAflex software randomly translates and rotates the nucleic acid molecule 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 will perform more optimizations and adjustments to achieve more precise docking.

[0111] In one embodiment, virtual drug screening is an important method in the field of targeted drug design. Its process is roughly as follows:

[0112] First, based on the known complex structure of a drug and its target protein, study the binding mode between the drug and the target protein 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.

[0113] Next, use computer-aided drug design methods to perform virtual screening on a large number of small molecule compounds. During this process, the binding ability of the small molecule compounds to the target protein is predicted and evaluated. Generally, small molecules with low binding energy and good shape complementarity are selected as potential candidate drugs.

[0114] Finally, conduct further experimental verification on the selected candidate drugs to confirm their interactions with the target protein and biological activities.

[0115] In one embodiment, in virtual drug screening, the binding site refers to the region on the target protein that binds to small molecule compounds. 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.

[0116] 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 will simulate the interaction between the compound and the binding site and evaluate their binding ability. Based on 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 virtual drug screening is crucial for 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.

[0117] In one embodiment, computer-aided drug screening is a technology that uses computer-aided drug design methods for drug screening. It can help researchers quickly screen out candidate drugs with strong binding ability to the target protein and potential drug efficacy from a large number of small molecule compounds.

[0118] In one embodiment, molecular docking is a method of drug design based on the characteristics of the receptor and the interaction mode between the receptor and the drug molecule. It mainly studies the interaction between molecules (such as ligands and receptors) and predicts their binding mode and affinity through a theoretical simulation method. This method is widely used in the early stage of drug research and development and can help scientific researchers quickly screen out compounds with potential drug efficacy.

[0119] The molecular docking method mainly focuses on spatial matching and energy matching. Spatial matching refers to the geometric shape complementarity between the drug molecule and the receptor protein, while energy matching refers to the minimization of the interaction between the drug molecule and the receptor protein. For the calculation of geometric matching, methods such as lattice calculation and fragment growth are usually used, while for energy calculation, methods such as simulated annealing and genetic algorithms are used.

[0120] According to the degree and method of simplification, the molecular docking method can be divided into rigid docking, semi-flexible docking, and flexible docking. In the rigid docking method, the conformations of the molecules participating in the docking do not change during the calculation, and only the spatial position and orientation of the molecules are changed. Semi-flexible docking allows partial conformations to change during the calculation. Flexible docking allows more conformational changes.

[0121] In one embodiment, the molecular libraries used in virtual drug screening mainly include the following:

[0122] ZINC: Contains more than 250 million purchasable compounds for small molecule virtual screening.

[0123] PubChem: Contains bioactive substances for small molecule virtual screening.

[0124] DrugBank: It contains drugs and small molecules and is used for drug design and discovery.

[0125] ChEMBL: It contains small molecules and is used for drug discovery and chemogenomics research.

[0126] ChemDB: It contains a large number of known small molecules and is used for chemogenomics research and drug discovery.

[0127] HMDB: It contains a large number of known small molecules and is used for chemogenomics research and drug discovery.

[0128] BindingDB: It contains a large number of known small molecules and is used for chemogenomics research and drug discovery.

[0129] SMPDB: It contains a large number of known small molecules and is used for chemogenomics research and drug discovery.

[0130] In addition, there are some commercial databases such as ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas-M, and Interbioscreen, etc. These databases are also often used for virtual drug screening.

[0131] In one embodiment, the method further includes:

[0132] Obtaining a system expressing STING;

[0133] Treating the system expressing STING with the screened small molecule compounds to verify the effectiveness of the small molecule compounds;

[0134] Optionally, the system expressing STING includes a cell system expressing STING, a subcellular system expressing STING, a tissue system expressing STING, a solution system expressing STING, an organ system expressing STING, or an animal system expressing STING;

[0135] Optionally, screening small molecule compounds with the promotion of STING expression as candidate drugs.

[0136] In one embodiment, the cell system expressing STING, the subcellular system expressing STING, the tissue system expressing STING, the solution system expressing STING, the organ system expressing STING, or the animal system expressing STING can be obtained through the conventional purchase channels well-known to those skilled in the art, or can be constructed through the conventional construction methods well-known to those skilled in the art.

[0137] In one embodiment, the animals included in the animal system expressing STING include, but are not limited to: animals such as mice, rats, guinea pigs, rabbits, pigs, chickens, pigeons, monkeys, and dogs.

[0138] Figure 2 FIG. is a schematic diagram of a computer-aided drug screening system based on TRIM56 and STING provided by an embodiment of the present invention. Specifically, the system includes:

[0139] Data acquisition unit: acquiring data of TRIM56 protein and E1A protein;

[0140] Site determination unit: selecting the spatial structure of the complex of the TRIM56 protein and the E1A protein, and determining the binding site of the complex of the TRIM56 protein and the E1A protein as the binding site of the target drug;

[0141] Drug screening unit: obtaining candidate drugs targeting the binding site by using a computer-aided drug screening method;

[0142] Optionally, the method for determining the binding site of the TRIM56 protein and the E1A protein includes:

[0143] Constructing n truncated expression mutants of TRIM56, where n is a natural number greater than or equal to 1;

[0144] Performing co-immunoprecipitation experiments based on the n truncated expression mutants to obtain that the N-terminal region of TRIM56 is the key region interacting with E1A;

[0145] The spatial conformation formed by the interaction of the N-terminal region of TRIM56 and E1A is the binding site of the complex of the TRIM56 protein and the E1A protein;

[0146] Optionally, the computer-aided drug screening method includes:

[0147] Obtaining the binding site of the complex of the TRIM56 protein and the E1A protein;

[0148] Screening for small molecule compounds with similar structures in a molecular database based on the spatial structure of the binding site of the complex of the TRIM56 protein and the E1A protein;

[0149] Performing molecular docking of the screened small molecule compounds with the E1A protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor E1A protein to obtain the scores of each small molecule compound, and sorting according to the scores to obtain candidate drugs;

[0150] Optionally, the system further includes a drug efficacy verification unit: obtaining a system expressing STING; treating the system expressing STING with the screened small molecule compound to verify the efficacy of the small molecule compound;

[0151] Optionally, the system expressing STING includes a cell system expressing STING, a subcellular system expressing STING, a tissue system expressing STING, a solution system expressing STING, an organ system expressing STING, or an animal system expressing STING;

[0152] Optionally, screen small molecule compounds with the ability to promote STING expression as candidate drugs.

[0153] Figure 3 It is a schematic diagram of a computer-aided drug screening device based on TRIM56 and STING provided by an embodiment of the present invention. Specifically, the device includes:

[0154] A memory and a processor, 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-aided drug screening method based on TRIM56 and STING as described above in the present invention is implemented.

[0155] An embodiment of the present invention also provides a computer-readable storage medium, on which a computer program is stored, and when the computer program is executed by a processor, the computer-aided drug screening method based on TRIM56 and STING as described above in the present invention is implemented.

[0156] An embodiment of the present invention also provides any of the following products:

[0157] (1) A complex of TRIM56 protein and E1A protein, and the complex of TRIM56 protein and E1A protein is the complex of TRIM56 protein and E1A protein as described above;

[0158] (2) A drug screened according to the computer-aided drug screening method based on TRIM56 and STING as described above.

[0159] An embodiment of the present invention also provides any of the following applications:

[0160] (1) An application of a protein complex, and the complex is the complex of TRIM56 protein and E1A protein as described above, and the application includes: the application of the complex in regulating adenovirus replication and / or transcription;

[0161] (2) An application of a drug, and the drug is the drug as described above, and the application includes: the application of the drug in anti-adenovirus.

[0162] The verification results of this verification example show that allocating fixed weights for indications can moderately improve the performance of this method compared to the default settings.

[0163] 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 foregoing method embodiments and will not be elaborated herein.

[0164] In several embodiments provided in this application, it should be understood that the disclosed systems, devices, and methods can be implemented in other ways. For example, the device embodiments described above are merely illustrative. For example, the division of the units is only a logical function division, and there can be other division methods in 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. Another point is that the displayed or discussed couplings or direct couplings or communication connections to each other can be through some interfaces, and the indirect couplings or communication connections of the devices or units can be in electrical, mechanical, or other forms.

[0165] The units described as separate components may or may not be physically separated, and the components displayed as units may or may not be physical units, that is, they can be located in one place or distributed to multiple network units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0166] In addition, in each embodiment of the present invention, the functional units can be integrated into one processing unit, or each unit can exist physically alone, or two or more units can be integrated into one unit. The above-mentioned integrated units can be implemented in the form of hardware or in the form of software functional units.

[0167] Those 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 relevant hardware through a program, and the program can be stored in a computer-readable storage medium. The storage medium can include: read-only memory (ROM, Read Only Memory), random access memory (RAM, Random Access Memory), magnetic disk or optical disk, etc.

[0168] Those of ordinary skill in the art can understand that all or part of the steps in implementing the methods of the above embodiments can be completed by instructing relevant hardware through a program, and the program can be stored in a computer-readable storage medium. The above-mentioned storage medium can be read-only memory, magnetic disk or optical disk, etc.

[0169] The above has introduced in detail a computer device provided by the present invention. For those of ordinary skill in the art, according to the idea of the embodiments of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A computer-assisted drug screening method based on TRIM56 and STING, characterized in that: The method comprises: Obtain TRIM56 protein and E1A protein data; Selecting the spatial structure of the TRIM56 protein and E1A protein complex, and determining the binding site of the TRIM56 protein and E1A protein complex as the binding site of the targeted drug; Using a computer-assisted drug screening method to obtain candidate drugs targeting the binding site; The method for determining the binding site of the TRIM56 protein and the E1A protein complex comprises: Constructing n truncated expression mutants of TRIM56, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants, the immunoprecipitation experiment showed that the N-terminal region of TRIM56 is the key region for interaction with E1A; The spatial conformation formed by the interaction between the N-terminal region of TRIM56 and the E1A is the binding site of the TRIM56 protein and the E1A protein complex; The computer-assisted drug screening method comprises: Obtain the binding site of TRIM56 protein and E1A protein complex; Based on the spatial structure of the binding site of the TRIM56 protein and the E1A protein complex, small molecule compounds with similar structures are screened in a molecular database; The screened small molecule compounds are subjected to molecular docking with the E1A protein to calculate the affinity or binding energy of the small molecule compounds for the receptor E1A protein to obtain the score of each small molecule compound, and the candidate drugs are obtained by ranking according to the score.

2. The computer-aided drug screening method based on TRIM56 and STING according to claim 1, characterized in that: The small molecule compound includes siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic small molecule compound.

3. The computer-assisted drug screening method based on TRIM56 and STING according to claim 1, characterized in that: The method further comprises: Obtain a system expressing STING; The screened small molecule compound is used to treat the STING-expressing system to verify the effectiveness of the small molecule compound.

4. The computer-aided drug screening method based on TRIM56 and STING according to claim 3, characterized in that: The STING-expressing system includes a STING-expressing cell system, a STING-expressing subcellular system, a STING-expressing tissue system, a STING-expressing solution system, a STING-expressing organ system or a STING-expressing animal system.

5. The computer-aided drug screening method based on TRIM56 and STING according to claim 3, characterized in that: Screen small molecule compounds that promote STING expression as candidate drugs.

6. A computer-aided drug screening system based on TRIM56 and STING, characterized in that: The system comprises: Data acquisition unit: obtain TRIM56 protein and E1A protein data; A site determination unit: selecting the spatial structure of the TRIM56 protein and E1A protein complex, and determining the binding site of the TRIM56 protein and E1A protein complex as the binding site of the targeted drug; Drug screening unit: using computer-assisted drug screening methods to obtain candidate drugs targeting the binding site; The method for determining the binding site of the TRIM56 protein and the E1A protein complex comprises: Constructing n truncated expression mutants of TRIM56, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants, the immunoprecipitation experiment showed that the N-terminal region of TRIM56 is the key region for interaction with E1A; The spatial conformation formed by the interaction between the N-terminal region of TRIM56 and the E1A is the binding site of the TRIM56 protein and the E1A protein complex; The computer-assisted drug screening method comprises: Obtain the binding site of TRIM56 protein and E1A protein complex; Based on the spatial structure of the binding site of the TRIM56 protein and the E1A protein complex, small molecule compounds with similar structures are screened in a molecular database; The screened small molecule compounds are subjected to molecular docking with the E1A protein to calculate the affinity or binding energy of the small molecule compounds for the receptor E1A protein to obtain the score of each small molecule compound, and the candidate drugs are obtained by ranking according to the score.

7. The computer-aided drug screening system based on TRIM56 and STING according to claim 6, characterized in that: The system also includes a drug effectiveness verification unit: obtaining a system expressing STING; and treating the system expressing STING with the screened small molecule compound to verify the effectiveness of the small molecule compound.

8. The computer-aided drug screening system based on TRIM56 and STING according to claim 7, characterized in that: The STING-expressing system includes a STING-expressing cell system, a STING-expressing subcellular system, a STING-expressing tissue system, a STING-expressing solution system, a STING-expressing organ system or a STING-expressing animal system.

9. The computer-aided drug screening system based on TRIM56 and STING according to claim 7, characterized in that: Screen small molecule compounds that promote STING expression as candidate drugs.

10. A computer-aided drug screening device based on TRIM56 and STING, 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 TRIM56 and STING described in any one of claims 1-5 is implemented.

11. 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 TRIM56 and STING described in any one of claims 1 to 5 is implemented.

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