Computer-aided drug screening method, system and equipment based on TRIM56 and STING

Abstract

The invention discloses a computer-aided drug screening method, system and equipment based on TRIM56 and STING, and discovers the interaction relationship that TRIM56 promotes adenovirus replication and transcription by promoting E1A to degrade STING for the first time. The invention provides a computer-aided drug screening method, system and equipment based on TRIM56 and STING and a computer readable storage medium for the field, provides an efficient, accurate and rapid screening method for research and development of new anti-adenovirus drugs, and has a very wide application prospect in the aspect of screening of the 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 equipment based on TRIM56 and STING. Background Art

[0002] Human adenovirus (HAdV) is a double-stranded DNA virus belonging to the genus Adenovirus of the family Adenoviridae. The virus is an icosahedral structure without an outer envelope. The genome is about 36kb long. The genomic DNA is combined with the viral structural protein to form the viral core, which is covered with a capsid. The capsid consists of 252 capsomeres, of which 240 are hexon proteins and 12 are penton proteins, with a diameter of about 70-90nm. Currently, there are 7 subgroups of adenoviruses, AG, and 67 different serotypes, of which 55 subtypes can infect humans and cause disease. The most common adenovirus infection in humans is respiratory infection and respiratory diseases. In addition, some adenoviruses can also cause systemic infections such as urinary and gastrointestinal infections.

[0003] The human adenoviruses associated with respiratory diseases mainly include subgenus B (HAdV-3, 7, 11, 14, 16, 21, 55), subgenus C (HAdV-1, 2, 5, 6) and subgenus E (HAdV-4). The types of adenovirus prevalent in my country mainly include type 1, type 3, type 4, type 5, type 7, type 11, type 14, type 40, type 41, and type 55, among which type 3 and type 5 adenovirus are the most prevalent. Studies have found that except for the hexon, fiber, and penton genes, the genomes of various types of adenovirus are highly conserved, and intraspecific recombination rarely occurs. This feature of adenovirus is conducive to overcoming the limitation of drugs on the specificity of adenovirus types. Antiviral drugs that usually target adenoviruses in later stages have broad-spectrum anti-adenovirus activity within the adenovirus species.

[0004] At present, there is no specific drug for adenovirus infection in clinical practice, and broad-spectrum antiviral therapy and immunotherapy are generally used. 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 anti-adenovirus activity in in vitro experiments and clinical settings, but the effects vary greatly and no satisfactory results have been shown. Therefore, this field urgently needs to develop anti-adenovirus drugs with high specificity, safety and bioavailability. Computer-aided drug design (CADD) is based on computer calculation and simulation technology. By learning the prior knowledge contained in massive drug data and mining the interaction relationship between target targets and candidate drugs, active drug molecules with drug-like properties can be quickly selected from millions of molecules. This greatly reduces the blindness of screening candidate drug molecules and improves research and development efficiency. Summary of the invention

[0005] In view of this, in view of the above technical problems existing in the current development of anti-adenovirus drugs, the purpose of the present invention is to provide a computer-assisted drug screening method, system and device based on TRIM56 and STING. Computer-assisted drug screening technology is used to screen drugs for treating and / or preventing adenovirus infection.

[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 TRIM56 and STING.

[0008] Further, the method comprises:

[0009] Obtain TRIM56 protein and E1A protein data;

[0010] 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;

[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 TRIM56 protein and the E1A protein complex comprises:

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

[0014] 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;

[0015] 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 E1A protein complex.

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

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

[0018] 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;

[0019] 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.

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

[0021] Furthermore, the method further comprises:

[0022] Obtain a system expressing STING;

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

[0024] Optionally, 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;

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

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

[0027] Further, the system comprises:

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

[0029] 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;

[0030] Drug screening unit: using computer-assisted drug screening methods to obtain candidate drugs targeting the binding site;

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

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

[0033] 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;

[0034] 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;

[0035] Optionally, the computer-assisted drug screening method comprises:

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

[0037] 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;

[0038] Performing molecular docking between the screened small molecule compounds and the E1A protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor E1A protein to obtain the score of each small molecule compound, and ranking them according to the score to obtain candidate drugs;

[0039] Optionally, the system further comprises a drug effectiveness verification unit: obtaining a system expressing STING; treating the system expressing STING with the screened small molecule compound to verify the effectiveness of the small molecule compound;

[0040] Optionally, 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;

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

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

[0043] Furthermore, the device comprises:

[0044] 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 the first aspect of the present invention is implemented.

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

[0046] Furthermore, the computer-readable storage medium stores a computer program, and when the computer program is executed by the processor, it implements the computer-assisted drug screening method based on TRIM56 and STING described in the first aspect of the present invention.

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

[0048] (1) a complex of TRIM56 protein and E1A protein, wherein 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 by the computer-assisted drug screening method based on TRIM56 and STING according to the first aspect of the present invention.

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

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

[0052] (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 anti-adenovirus.

[0053] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0054] The present invention discovers for the first time the interactive relationship that TRIM56 promotes the degradation of STING by E1A and thus promotes adenovirus replication and transcription. Based on this, the present invention provides a computer-assisted 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] 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.

[0056] Figure 1 : A schematic diagram of a computer-assisted drug screening method based on TRIM56 and STING provided in an embodiment of the present invention;

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

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

[0059] Figure 4: The result graph corresponding to the promotion of adenovirus replication by overexpression of TRIM56 provided in the embodiment of the present invention. (A) Mass spectrometry analysis results of 786-O cells overexpressing E1A and control cells. (B) A stably overexpressing TRIM56 A549 cell line was constructed, and the virus replicated in A549 cells overexpressing TRIM56. A549 cell overexpressing TRIM56 or a control A549 cell was infected with HAdV-C5 at an MOI of 5 or 20, and the supernatant was collected at a specific time point, and the virus titer was determined by TCID50 assay. ***, P<0.001; ****, P<0.0001. (C) The cell viability of A549 cells overexpressing TRIM56 was detected by CCK-8 assay, and the data are expressed as the mean ± standard deviation (SD) of three transfections. (D and E) Virus-infected cells were observed using a fluorescence microscope, scale bar, 200 μm, and virus-infected cells were subjected to Western blotting analysis using anti-GFP antibody. (F) siRNA was used to inhibit TRIM56 expression in A549 cells. After A549 cells were transfected with siRNA targeting TRIM56 or control siRNA for 48 h, cell lysates were collected and subjected to western blot analysis. (G) The effect of siRNA-mediated knockdown of TRIM56 in A549 cells on A549 cell viability was detected by CCK-8 assay. (H) Viral replication in A549 cells treated with siRNA targeting TRIM56. TRIM56 knockdown cells were infected with HAdV-C5 at MOIs of 5 and 20, and viral titers were detected 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: Result graphs corresponding to the promotion of adenoviral gene transcription by overexpression of TRIM56 provided in an embodiment of the present invention. (A, B and C) TRIM56-overexpressing A549 cells or control A549 cells were infected with HAdV-C5 at an MOI of 20, and the RNA levels of E1A, DBP and Hexon genomic transcripts were detected by RT-qPCR detection method at 24h and 48h after infection. The results are expressed as the mean ± SD of 3 independent experiments standardized according to 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 36h, and then infected with HAdV-C5 at an MOI of 20, and the RNA levels of E1A, DBP and Hexon genomic transcripts were detected by RT-qPCR detection method at the specified time points after infection. The results are expressed as the mean ± SD of 3 independent experiments normalized according to the results of GAPDH. *, 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 : The result graphs corresponding to the interaction between TRIM56 and adenovirus E1A provided in the embodiments of the present invention. (A) TRIM56-V5 was co-transfected with E1A in HAdV-C5 into HEK293T cells, and the cell lysates were immunoprecipitated (IP) using anti-E1A antibody (left figure), anti-V5 antibody (right figure) 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 figure), anti-TRIM56 antibody (right figure) or control IgG, and blotted as indicated. (C) E1A and TRIM56 were co-transfected into HeLa cells for 36 hours, and their colocalization 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 the construction of V5-tagged TRIM56 truncation mutants (Del R, Del BB, Del CC, Del N363). (E) E1A- and V5-tagged TRIM56 mutants were co-transfected into HEK293T cells, and cell lysates were immunoprecipitated using anti-E1A antibody and detected by Western blotting. At least three independent experiments were performed;

[0062] Figure 7 : The result graph corresponding to the promotion of E1A protein stability by TRIM56 provided in the embodiment of the present invention. (A) HEK293T cells were co-transfected with E1A and TRIM56-V5 (TRIM56-V5 was gradually increased), and the cell lysates were immunoblotted with anti-E1A antibody and anti-V5 antibody. (B) Grayscale 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 hours after infection. Western blotting was performed with mouse anti-E1A antibody and rabbit anti-TRIM56 antibody, and at least three independent experiments were performed;

[0063] Figure 8 : The result graph corresponding to the TRIM56 provided in the embodiment of the present invention promoting the degradation of STING by E1A. (A) E1A of HAdV-C5 was co-transfected with STING plasmid with Flag tag into HEK293T cells, and the cell lysate was immunoprecipitated with anti-E1A antibody and control IgG, and blotted as indicated. (B) TRIM56-V5 was co-transfected with STING-Flag into HEK293T cells. Cell lysate was immunoprecipitated with anti-Flag antibody or control IgG, and blotted as indicated. (C) HEK293T cells were co-transfected with STING-Flag and TRIM56-V5 was gradually increased. Cell lysate was immunoblotted with the specified antibodies. (D) HEK293T cells were co-transfected with STING-Flag and E1A (gradually increasing E1A), and cell lysate was immunoblotted with the specified 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, and cell lysates were collected and analyzed by IB using the indicated antibodies. (G) Gray value calculation of three independent experiments in (F). ****, P<0.0001. At least three independent experiments were performed. DETAILED DESCRIPTION

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Figure 1 1 is a flow chart of a computer-assisted drug screening method based on TRIM56 and STING provided by an embodiment of the present invention. Specifically, the method comprises 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, comprising a RING domain, a B-box domain and a coiled-coil domain. As a TRIM family member that mostly functions as an E3 ubiquitinase, the conserved RING domain of TRIM56 confers E3 ubiquitin ligase activity. The Gene ID of the TRIM56 (tripartite motif containing 56 [Homo sapiens (human)]) in NCBI is 81844, and detailed information of the above gene (including sequence information, etc.) can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / . At present, there is no relevant research or report on the application of TRIM56 in screening anti-adenovirus drugs.

[0070] In one embodiment, the present invention demonstrates that TRIM56 overexpression 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). Combined with the results of mass spectrometry analysis, we speculated that TRIM56 may regulate HAdV infection.

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

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

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

[0074]

[0075] In one embodiment, the present invention demonstrates that TRIM56 overexpression promotes adenoviral gene transcription. TCID50 detection found that TRIM56 can increase the viral titer during HAdV-C5 infection. Therefore, we tested whether TRIM56 can promote the transcription and genome replication of HAdV-C5. HAdV-C5 infected TRIM56 overexpressing and control A549 cell lines. The genomic transcripts of E1A, Hexon and DBP of HAdV-C5 were detected by qPCR. Compared with control cells, the levels of E1A, Hexon and DBP in TRIM56 overexpressing cells were significantly increased at 24 or 48h after infection ( Figure 5 A, B, C). In addition, the results of real-time quantitative PCR showed that the expression level of TRIM56 in the TRIM56-overexpressing cell line 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 control siRNA-treated A549 cells, the sequences of which are shown in Table 1, and then infected with HAdV-C5 virus. The results showed that the transcription levels of E1A, Hexon, and DBP in HAdV-C5-infected cells treated with TRIM56-specific siRNA were significantly reduced compared with A549 cells treated with control siRNA ( Figure 5 E, F, G). Real-time PCR results showed that the expression of TRIM56 in A549 cells treated with siRNA targeting TRIM56 was decreased compared with that in A549 cells treated with control siRNA ( Figure 5 H). Together, these results suggest that TRIM56 overexpression promotes transcription of the adenoviral genome, whereas TRIM56 knockdown does not.

[0077] In one embodiment, the E1A (adenovirus early region 1) refers to the adenovirus E1A gene. E1A plays a vital 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 changing 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, causing the E2F transcription factor to separate from the pRb protein, thereby activating E2F to mediate the transcription of other early transcription genes (E1B, E2, E3, E4), and initiate the replication of viral DNA.

[0078] In one embodiment, the adenovirus refers to human adenovirus (HAdV), which belongs to the genus Mastadenovirus of the mammalian adenovirus family (Adenoviridae). Adenovirus infection is relatively common in clinical practice, and the organ systems where infection often occurs are the cornea and respiratory tract. Adenovirus infection is self-limiting, but for some special populations such as people with low immunity, children, etc., its infection can lead to more serious consequences. Since the adenovirus was first isolated, there are seven species of adenovirus AG that have been identified, with more than 100 serotypes, of which 52 are human adenoviruses, named AdVl-AdV52, respectively, and divided into six subgroups A, B, C, D, E and F. With the continuous development of molecular biology technology and the continuous exploration of adenovirus, the structure of adenovirus and its genome replication mechanism have been studied more clearly. Human adenovirus infection is widespread worldwide and induces a variety of inflammatory diseases in the human body.

[0079] S102: 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;

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

[0081] Constructing 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, the immunoprecipitation experiment showed that the N-terminal region of TRIM56 is the key region for interaction with E1A;

[0083] 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 E1A protein complex.

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

[0085] We co-transfected E1A and TRIM56 into HeLa cells and observed by confocal microscopy that the two proteins were mainly co-localized in the cytoplasm ( Figure 6 C). These results indicate that TRIM56 is associated with E1A of HAdV-C5. Next, we constructed TRIM56 truncation mutants (Del R, Del BB, Del CC, Del N363) with V5 tags ( Figure 6 D) to locate the interaction region between TRIM56 and E1A. Immunoprecipitation experiments found that Del N363 had little effect on the interaction between TRIM56 and E1A. On the contrary, interaction only occurs when other domains co-exist ( 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 tested the effect of TRIM56 on the stability of HAdV-C5 E1A protein through a dose-dependent experiment. The results showed that the protein stability of E1A increased with the increase of TRIM56 expression ( Figure 7 A). In addition, the grayscale value shows that the expression of E1A increases to the maximum value, at which time the expression of TRIM56 is 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, TRIM56 overexpression promoted the replication of HAdV-C5 and produced more E1A viral protein, especially at 48h after infection ( Figure 7 C).

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

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

[0091] In one embodiment, STING is activated when a DNA virus infects cells and transmits signals to induce the production of downstream interferon. Studies have shown that the oncogenes E7 and E1A of the DNA tumor virus 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 a Flag-tagged STING plasmid into HEK293T cells for a co-IP experiment. The results confirmed that there was a specific interaction between E1A of HAdV-C5 and STING ( Figure 8 A). Similarly, we performed co-immunoprecipitation experiments to determine the interaction between TRIM56 and STING. The results showed that TRIM56 interacted with STING ( Figure 8 B).

[0092] Considering the relationship between E1A, TRIM56 and STING, we first investigated whether TRIM56 regulates E1A by affecting the protein expression of STING. Dose-dependent experiments showed that the protein stability of STING was not affected by the increase of TRIM56 ( Figure 8 C) However, dose-dependent experiments found that as the level of E1A viral protein increased, the stability of STING protein 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 E1A of HAdV-C5 can significantly degrade STING, while TRIM56 does not affect the stability of STING.

[0093] In addition, Western blot analysis showed that E1A degraded STING, and TRIM56 could significantly increase the expression of E1A, indirectly enhancing the degradation of STING by E1A protein and playing an antagonistic role in 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 demonstrate the interactive relationship that TRIM56 promotes the degradation of STING by E1A and thus promotes adenovirus replication and transcription. Based on this, the present invention develops for the first time a computer-assisted drug screening method based on TRIM56 and STING.

[0095] S103: using a computer-assisted drug screening method to obtain a candidate drug targeting the binding site;

[0096] In one embodiment, the computer-assisted drug screening method comprises:

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

[0098] 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;

[0099] 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.

[0100] In one embodiment, the small molecule compound includes 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 has no particular limitation on the specific type of the small molecule compound, and any small molecule compound that may be used in the computer-assisted 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 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).

[0102] In the process of molecular docking, affinity depends on the interaction between molecules and receptors, including hydrogen bonds, van der Waals forces, electrostatic interactions, etc. These interactions jointly determine the binding mode between molecules and receptors, thus affecting affinity.

[0103] In order to evaluate affinity, some scoring systems or scoring methods are usually used to quantify the interaction between molecules and receptors. These scoring methods are based on different algorithms and physical models, which can reflect the binding energy, interaction type and affinity between molecules and receptors.

[0104] 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.

[0105] 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.

[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 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.

[0107] 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.

[0108] 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.

[0109] 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.

[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, 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.

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

[0112] 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.

[0113] Next, a large number of small molecule compounds are virtually screened using computer-aided drug design methods. In this process, the binding ability of small molecule compounds to target proteins will be predicted and evaluated. Generally, small molecules with low binding energy and good shape complementarity will be selected as potential drug candidates.

[0114] Finally, the screened candidate drugs are further experimentally verified to confirm their interaction with the target protein and their biological activity.

[0115] In one embodiment, in drug virtual screening, the binding site refers to the region 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.

[0116] Once the binding site is determined, the computer-aided drug design method can be used to dock the compounds in the small molecule compound library with the target protein. During the docking process, the computer simulates the interaction between the compound and the binding site and evaluates their binding ability. According to the docking results, small molecule compounds with strong binding ability to the target protein can be screened as potential drug candidates. The determination of drug virtual screening binding sites is crucial to the success of drug design. Therefore, when determining the binding site, it is necessary to consider a variety of factors, such as the structural characteristics of the target protein, the binding mode of known ligands, etc. At the same time, in the virtual screening process, the binding site also needs to be reasonably processed and optimized to improve the accuracy and efficiency of the screening.

[0117] 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.

[0118] 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 their binding mode and affinity. This method is widely used in the early stages of drug development and can help researchers quickly screen out compounds with potential efficacy.

[0119] Molecular docking methods mainly focus on spatial matching and energy matching. Spatial matching refers to the geometric complementarity between the drug molecule and the receptor protein, while energy matching refers to the minimization of the interaction between the drug molecule and the receptor protein. For the calculation of geometric matching, grid point calculation, fragment growth and other methods are usually used, while energy calculation uses simulated annealing, genetic algorithm and other methods.

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

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

[0122] ZINC: contains more than 250 million commercially available compounds for small molecule virtual screening.

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

[0124] DrugBank: Contains drugs and small molecules for drug design and discovery.

[0125] ChEMBL: Contains small molecules and is used for drug discovery and chemical genomics research.

[0126] ChemDB: Contains a large number of known small molecules for chemical genomics research and drug discovery.

[0127] HMDB: contains a large number of known small molecules for use in chemical genomics research and drug discovery.

[0128] BindingDB: Contains a large number of known small molecules for chemical genomics research and drug discovery.

[0129] SMPDB: Contains a large number of known small molecules for chemical genomics 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, which are also often used for drug virtual screening.

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

[0132] Obtain a system expressing STING;

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

[0134] Optionally, 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;

[0135] Optionally, small molecule compounds that promote STING expression are screened as candidate drugs.

[0136] In one embodiment, the STING-expressing cell system, STING-expressing subcellular system, STING-expressing tissue system, STING-expressing solution system, STING-expressing organ system or STING-expressing animal system can be obtained through conventional purchasing routes known to those skilled in the art, or can be constructed and obtained through conventional construction methods 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, mice, rats, guinea pigs, rabbits, pigs, chickens, pigeons, monkeys, dogs and other animals.

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

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

[0140] 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;

[0141] Drug screening unit: using computer-assisted drug screening methods to obtain candidate drugs targeting the binding site;

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

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

[0144] 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;

[0145] 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;

[0146] Optionally, the computer-assisted drug screening method comprises:

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

[0148] 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;

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

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

[0151] Optionally, 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;

[0152] Optionally, small molecule compounds that promote STING expression are screened as candidate drugs.

[0153] Figure 3 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, 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 as described above of the present invention is implemented.

[0155] 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 TRIM56 and STING as described above in the present invention is implemented.

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

[0157] (1) a complex of TRIM56 protein and E1A protein, wherein 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 by the computer-assisted drug screening method based on TRIM56 and STING as described above.

[0159] The embodiments of the present invention also provide any of the following applications:

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

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

[0162] 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.

[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 aforementioned method embodiments and will not be repeated here.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] The above is a detailed introduction to a computer device provided by the present invention. For a person skilled in the art, according to the concept of the embodiments of the present invention, there may be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A 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; A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

2. The computer-aided drug screening method based on TRIM56 and STING according to claim 1, characterized in that: 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 E1A protein complex.

3. The computer-assisted drug screening method based on TRIM56 and STING according to claim 1, characterized in that: 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 small molecule compounds obtained by screening are subjected to molecular docking with the E1A protein to calculate the affinity or binding energy of the small molecule compounds targeting 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.

4. The computer-aided drug screening method based on TRIM56 and STING according to claim 3, 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.

5. The computer-aided drug screening method based on TRIM56 and STING according to claim 3, characterized in that: The method further comprises: Obtain a system expressing STING; Treating the STING-expressing system with the screened small molecule compound to verify the effectiveness of the small molecule compound; Optionally, 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; Optionally, small molecule compounds that promote STING expression are screened 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; Optionally, 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; Optionally, 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; Performing molecular docking between the screened small molecule compounds and the E1A protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor E1A protein to obtain the score of each small molecule compound, and ranking them according to the score to obtain candidate drugs; Optionally, the system further comprises a drug effectiveness verification unit: obtaining a system expressing STING; treating the system expressing STING with the screened small molecule compound to verify the effectiveness of the small molecule compound; Optionally, 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; Optionally, small molecule compounds that promote STING expression are screened as candidate drugs.

7. 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.

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

9. Any of the following products: (1) A complex of TRIM56 protein and E1A protein, characterized in that: The TRIM56 protein and E1A protein complex is the TRIM56 protein and E1A protein complex described in claim 1; (2) A drug screened by the computer-assisted drug screening method based on TRIM56 and STING according to any one of claims 1 to 5.

10. Any of the following applications: (1) An application of a protein complex, characterized in that: The complex is the TRIM56 protein and E1A protein complex as described in claim 9, and the application includes: application of the complex in regulating adenovirus replication and / or transcription; (2) A use of a drug, characterized in that the drug is the drug described in claim 9, and the use includes: use of the drug in anti-adenovirus.

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