Application of TREX1 protein non-substrate DNA binding interface as target spot in preparation of immunoregulation medicine

Abstract

The invention discloses application of a TREX1 protein non-substrate DNA binding interface as a target spot in preparation of an immunoregulation medicine, and relates to the technical field of biological medicines. Wherein the TREX1 protein is a human source TREX1 protein, and the non-substrate DNA binding interface comprises threonine at the 49th site of the TREX1 protein, arginine at the 211th site of the TREX1 protein, glutamine at the 213th site of the TREX1 protein and arginine at the 217th site of the TREX1 protein. The non-substrate DNA binding interface in the TREX1 is found for the first time, the key function of the non-substrate DNA binding interface in maintaining immune homeostasis and tumor immune escape is defined through mutation verification and functional analysis, and a new anti-tumor immune strategy with the non-substrate DNA binding interface as a target spot is provided. The discovery not only makes up the blank of TREX1 structure research, but also provides a new theoretical basis and a drug development direction for immune regulation molecule design.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the TREX1 protein non-substrate DNA binding interface as a target in the preparation of immunomodulatory drugs. Background Technology

[0002] TREX1 (Three Prime Repair Exonuclease 1) is a 3′→5′ exonuclease located on the endoplasmic reticulum membrane. It is an important negative regulator for maintaining intracellular nucleic acid homeostasis and inhibiting abnormal activation of innate immunity. Its main function is to clear abnormally sourced DNA from the cytoplasm, including reverse transcription products, damaged DNA fragments, or undegraded nucleic acid remnants. By degrading these DNAs, TREX1 prevents the continuous activation of cyclic GMP-AMP synthase (cGAS), thereby inhibiting the abnormal expression of type I interferon mediated by the cGAS-STING (Stimulator of Interferon Genes) signaling pathway.

[0003] Numerous studies have shown that TREX1 gene mutations lead to impaired cytoplasmic DNA clearance, thereby triggering chronic immune responses and interferon disorders. For example, TREX1 dysfunction is closely associated with autoimmune diseases such as Aicardi-Goutières syndrome (AGS) and systemic lupus erythematosus (SLE). In addition to autoimmunity, TREX1 also plays a crucial regulatory role in the tumor microenvironment; its overexpression or abnormal localization can suppress anti-tumor immune responses and promote tumor immune escape.

[0004] Although the catalytic activity of TREX1 and its role in immune homeostasis have been extensively studied, a systematic understanding of its specific interface and molecular mechanism of binding to DNA remains lacking. Existing crystal structure studies mainly focus on its catalytic core (DEDD nuclease domain), revealing its degradation mechanism of the 3′ end of DNA substrates. However, there is still no clear structural evidence or functional explanation for the DNA interaction interface outside the catalytic center, especially the binding mechanism with non-substrate DNA strands.

[0005] Therefore, existing technologies need to be improved and developed. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of the TREX1 protein non-substrate DNA binding interface as a target in the preparation of immunomodulatory drugs. The aim is to use the newly discovered non-substrate DNA binding interface (DNA B-site) in human TREX1 as a new target for immunomodulation, and to provide a new strategy for the treatment of TREX1-related diseases.

[0007] The technical solution of the present invention is as follows: In the first aspect, the application of the TREX1 protein non-substrate DNA binding interface as a target in the preparation of immunomodulatory drugs is provided, wherein the TREX1 protein is a human TREX1 protein with the UniProt number Q9NSU2; the non-substrate DNA binding interface includes: threonine at position 49, arginine at position 211, glutamine at position 213, and arginine at position 217 of the TREX1 protein.

[0008] In a preferred embodiment, the TREX1 protein non-substrate DNA binding interface is used as a target in the preparation of immunomodulatory drugs. The non-substrate DNA binding interface includes threonine at position 49 and glutamine at position 209 to arginine at position 217 of the TREX1 protein.

[0009] In a preferred embodiment, the TREX1 protein non-substrate DNA binding interface is used as a target in the preparation of immunomodulatory drugs, wherein the immunomodulatory drugs are selected from one or more of tumor immunomodulatory drugs, drugs for treating Ecardi-Goodtry syndrome, and drugs for treating systemic lupus erythematosus.

[0010] Secondly, the application of reagents that target and disrupt the non-substrate DNA binding interface of TREX1 protein in the preparation of immunomodulatory drugs is provided, wherein the TREX1 protein is a human TREX1 protein with UniProt number Q9NSU2; the non-substrate DNA binding interface includes: threonine at position 49, arginine at position 211, glutamine at position 213, and arginine at position 217 of the TREX1 protein.

[0011] In a preferred embodiment, the reagent that targets and disrupts the non-substrate DNA binding interface of the TREX1 protein is used in the preparation of immunomodulatory drugs. The non-substrate DNA binding interface includes threonine at position 49 and glutamine at position 209 to arginine at position 217 of the TREX1 protein.

[0012] In a preferred embodiment, the reagent that targets and disrupts the non-substrate DNA binding interface of the TREX1 protein is used in the preparation of immunomodulatory drugs, wherein the immunomodulatory drugs are selected from one or more of tumor immunomodulatory drugs, drugs for treating Ecardi-Goodtry syndrome, and drugs for treating systemic lupus erythematosus.

[0013] In a preferred embodiment, the reagent that targets and disrupts the non-substrate DNA binding interface of the TREX1 protein is selected from one or more of small molecules, nucleic acids, gene editing systems, proteins, and peptides when used in the preparation of immunomodulatory drugs.

[0014] Thirdly, an immunomodulatory drug is provided, the immunomodulatory drug comprising: a reagent that targets and disrupts the non-substrate DNA binding interface of the TREX1 protein; The TREX1 protein is a human TREX1 protein, with the UniProt number Q9NSU2; the non-substrate DNA binding interface includes threonine at position 49, arginine at position 211, glutamine at position 213, and arginine at position 217 of the TREX1 protein.

[0015] In a preferred embodiment, the non-substrate DNA binding interface of the immunomodulatory drug includes: threonine at position 49 and glutamine at position 209 to arginine at position 217 of the TREX1 protein.

[0016] In a preferred embodiment, the immunomodulatory drug further includes pharmaceutically acceptable excipients.

[0017] In a further preferred embodiment, the pharmaceutically acceptable excipients include one or more of the following: pharmaceutically acceptable excipients, additives, and adjuvants.

[0018] In a preferred embodiment, the immunomodulatory drug is administered via one or more of the following routes: oral, intravenous, intramuscular, and subcutaneous injection.

[0019] Beneficial effects: Compared with the prior art, the advantages of the present invention are as follows: (1) The non-substrate DNA binding interface (DNA B-site) in TREX1 was discovered for the first time, and a cooperative recognition model of DNA A-site (the classic substrate DNA binding interface reported before) and DNA B-site was proposed.

[0020] (2) Revealing a new mechanism of immune regulation of TREX1 non-enzymatic activity, providing a structural basis for explaining disease mutations.

[0021] (3) A strategy for anti-tumor immune regulation targeting DNA B-site is proposed, which can avoid the autoimmune side effects of traditional immunosuppressants.

[0022] (4) It reveals that B-site exists only in humans and primates, providing evidence for the evolutionary specialization of TREX1. Attached Figure Description

[0023] Figure 1This is a diagram illustrating the unique non-substrate DNA binding interface (DNA B-site) of hTREX1; where A is an overview of the hTREX1-DNA ternary complex structure, highlighting the DNA A-site and DNA B-site (submitted to the PDB database, not publicly available, PDB: 9L8C); B is a magnified cross-sectional view of the direct interaction between the DNA molecule and the DNA B-site residues of the TREX1 protein.

[0024] Figure 2 The diagram shows the results of TREX1 DNA B-site mutation affecting DNA degradation only within cGAS. A shows that TREX1 DNA B-site mutation prevents the formation of the TREX1-DNA higher-order complex. To characterize TREX1-DNA complex assembly, the TREX1 enzyme activity deletion variant H195A was introduced. B shows that the DNA degradation activity of TREX1 DNA B-site mutation is similar to that of wild-type TREX1. C shows that in the presence of cGAS-DNA phase separation, the DNA degradation activity of TREX1 DNA B-site mutation is lower than that of wild-type TREX1.

[0025] Figure 3 The graph shows the colocalization results of wild-type TREX1 and TREX1 DNA B-site mutant with cGAS condensates, indicating significant colocalization between wild-type TREX1 and cGAS condensates, while the degree of colocalization is reduced in DNA B-site mutants. Among them, A is the result of colocalization of TREX1 and cGAS as shown by fluorescence confocal microscopy; B is the statistical analysis based on graph A.

[0026] Figure 4 These are the results of TREX1 DNA B-site mutation validation; among them, A shows that expressing the TREX1 DNA B-site mutant in the HeLa TREX1 knockout cell line leads to upregulation of the type I interferon pathway (IFNβ); B shows that expressing the TREX1 DNA B-site mutant in the MCF10ATREX1 knockout cell line leads to significant upregulation of IFNβ; C shows that inoculating BALB / c mice with disease-associated TREX1 DNA B-site mutant (W210R) CT26 tumor cells significantly slows the growth of tumors carrying the W210R mutation.

[0027] Figure 5 It is based on the results of DNA B-site screening for mutation sites in autoimmune diseases and tumors, where magenta represents disease mutations on the DNA B-site. Detailed Implementation

[0028] This invention provides the application of the TREX1 protein non-substrate DNA binding interface as a target in the preparation of immunomodulatory drugs. To make the purpose, technical solution and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0029] Current research generally suggests that TREX1's DNA binding depends on the positively charged surface surrounding the catalytic center and the metal ion-assisted coordination structure. However, increasing evidence indicates that TREX1 may interact with DNA through multiple interfaces in cells, and its function depends not only on enzyme activity but also on structural conformation and spatial organization.

[0030] Furthermore, previous studies have suggested that TREX1 can form condensates and participate in phase separation to regulate the localization and activity of cGAS, but this mechanism cannot be explained by existing catalytic models. Therefore, it is speculated that TREX1 may have an unidentified DNA-binding interface in addition to its catalytic core, which is used to regulate its multivalent interactions with DNA.

[0031] Therefore, the shortcomings of the prior art include: (1) failure to recognize the non-substrate DNA binding interface of TREX1; (2) inability to explain the non-enzymatic activity-dependent immune regulation of TREX1; (3) lack of systematic comparison of the structural units specific to human TREX1; and (4) lack of structure-based targeted regulation strategies.

[0032] This invention addresses the technical problems in existing research that fail to explain TREX1's non-catalytic participation in immune regulation and the unclear differences in immune regulatory function between human TREX1 and other mammals. It proposes an innovative solution based on a newly discovered non-substrate DNA binding interface (DNA B-site) of human TREX1. Firstly, through crystal structure analysis of the human TREX1-DNA complex (submitted to the PDB database, not publicly available, PDB: 9L8C), a novel DNA binding interface—the DNA B-site—distinct from the classic catalytic substrate binding site (A-site)—was discovered and named. The crystal structure of the human TREX1-DNA complex shows that the TREX1 DNA B-site is a basic fragment (49, 209-217) composed of positively charged residues, and it forms a direct and stable interaction with the DNA phosphate backbone through four residues (threonine at position 49 (T49), arginine at position 211 (R211), glutamine at position 213 (Q213), and arginine at position 217 (R217)). Figure 1The presence of DNA B-site mutations, conserved only in humans and hominids, reveals the unique structural basis of human TREX1. Functional studies show that DNA B-site mutations disrupt TREX1's multivalent binding ability to DNA but do not affect its exonuclease activity, confirming that DNA B-sites play a non-catalytic role in TREX1 spatial organization and immune regulation. In the cGAS-DNA phase separation system, DNA B-site mutations significantly weaken TREX1's ability to compete for cGAS substrate DNA, leading to sustained activation of cGAS signaling. Cell and animal experiments further demonstrate that DNA B-site deficiency prevents TREX1 from effectively inhibiting the STING pathway, enhancing interferon responses and anti-tumor immunity. These experimental results reveal a novel non-enzymatic immune regulation mechanism mediated by TREX1 through DNA B-sites, providing a new structural basis and therapeutic strategy for targeting DNA B-sites to regulate tumor immunity.

[0033] Based on this, embodiments of the present invention provide the application of the TREX1 protein non-substrate DNA binding interface as a target in the preparation of immunomodulatory drugs, wherein the TREX1 protein is a human TREX1 protein, and its UniProt number is Q9NSU2; the non-substrate DNA binding interface includes: threonine at position 49 (T49), arginine at position 211 (R211), glutamine at position 213 (Q213), and arginine at position 217 (R217) of the TREX1 protein.

[0034] Specifically, this invention has found that threonine at position 49 (T49), arginine at position 211 (R211), glutamine at position 213 (Q213), and arginine at position 217 (R217) in the TREX1 protein non-substrate DNA binding interface are key amino acid residues that can form a direct and stable interaction with the DNA phosphate backbone. Therefore, these four amino acid residues are key factors for the target and can be used to screen reagents that target and disrupt the TREX1 protein non-substrate DNA binding interface, thereby applying them to the preparation of immunomodulatory drugs.

[0035] In some embodiments, the TREX1 protein non-substrate DNA binding interface is used as a target in the preparation of immunomodulatory drugs. The non-substrate DNA binding interface includes: threonine at position 49 (T49) and glutamine at position 209 to arginine at position 217 (Q209-R217) of the TREX1 protein.

[0036] Specifically, this invention has found that the TREX1 protein non-substrate DNA binding interface is a basic fragment (T 49, Q209-R217) composed of positively charged residues. Therefore, the amino acid residues constituting this non-substrate DNA binding interface may mediate immune regulation mechanisms and can be used to screen reagents that target and disrupt the TREX1 protein non-substrate DNA binding interface, thereby being applied to the preparation of immunomodulatory drugs.

[0037] In some embodiments, the TREX1 protein non-substrate DNA binding interface is used as a target in the preparation of immunomodulatory drugs, wherein the immunomodulatory drugs are selected from one or more of tumor immunomodulatory drugs, Aicardi–Goutières Syndrome (AGS) treatment drugs, and Systemic Lupus Erythematosus (SLE) treatment drugs.

[0038] Based on the same inventive concept, embodiments of the present invention provide the application of a reagent that targets and disrupts the non-substrate DNA binding interface of TREX1 protein in the preparation of immunomodulatory drugs, wherein the TREX1 protein is a human TREX1 protein, and its UniProt number is Q9NSU2; the non-substrate DNA binding interface includes: threonine at position 49, arginine at position 211, glutamine at position 213, and arginine at position 217 of the TREX1 protein.

[0039] Specifically, the TREX1 DNA B-site disclosed in this invention is a novel target for regulating innate immunity. Therapeutic measures aimed at disrupting or inhibiting B-site function include, for example, nucleic acids / vectors encoding TREX1 DNA B-site inactivation mutations (such as W210R), small molecules / peptides / antibodies that inhibit the interaction between TREX1 DNA B-site and DNA, or corresponding drug delivery compositions. These can be used to enhance tumor immunogenicity and promote type I IFN-mediated anti-tumor responses. In the future, AI molecular screening and structural optimization can be combined to design specific TREX1 DNA B-site small molecule inhibitors or peptide modulators to achieve a "regulatory immunosuppression" strategy.

[0040] Mutation analysis revealed multiple autoimmune and tumor-related mutations in the TREX1 DNA B-site region, suggesting that this site may have biomarker value in disease development and treatment response. Future research could further establish TREX1 DNA B-site mutant tumor or immune disease models for drug screening and mechanism validation.

[0041] Furthermore, TREX1's participation in phase separation may be driven by DNA B-site-mediated multivalent binding to DNA. This finding not only has applications in understanding the immune regulatory mechanisms of TREX1 but also provides a paradigm for studying the phase separation behavior of other nuclease proteins.

[0042] In some embodiments, the reagent that targets and disrupts the non-substrate DNA binding interface of the TREX1 protein is used in the preparation of immunomodulatory drugs, wherein the non-substrate DNA binding interface includes threonine at position 49 and glutamine at positions 209 to arginine at positions 217 of the TREX1 protein.

[0043] In some embodiments, the reagent that targets and disrupts the non-substrate DNA binding interface of the TREX1 protein is used in the preparation of immunomodulatory drugs, wherein the immunomodulatory drugs are selected from one or more of tumor immunomodulatory drugs, drugs for treating Ecardi-Goodtry syndrome, and drugs for treating systemic lupus erythematosus.

[0044] In a preferred embodiment, the reagent that targets and disrupts the non-substrate DNA binding interface of the TREX1 protein is selected from one or more of small molecules, nucleic acids, gene editing systems, proteins, and peptides when used in the preparation of immunomodulatory drugs.

[0045] Based on the same inventive concept, this invention provides an immunomodulatory drug, which includes: a reagent that targets and disrupts the non-substrate DNA binding interface of the TREX1 protein; The TREX1 protein is a human TREX1 protein, with the UniProt number Q9NSU2; the non-substrate DNA binding interface includes threonine at position 49, arginine at position 211, glutamine at position 213, and arginine at position 217 of the TREX1 protein.

[0046] In some embodiments, the non-substrate DNA binding interface of the immunomodulatory drug includes threonine at position 49 and glutamine at position 209 through arginine at position 217 of the TREX1 protein.

[0047] In some embodiments, the immunomodulatory drug further includes pharmaceutically acceptable excipients.

[0048] In some more specific embodiments, the pharmaceutically acceptable excipients include one or more of the following: pharmaceutically acceptable excipients, additives, and adjuvants.

[0049] In some embodiments, the route of administration of the immunomodulatory drug includes one or more combinations of oral, intravenous, intramuscular, and subcutaneous injection.

[0050] The invention will now be further described with reference to the accompanying drawings.

[0051] The inventors first performed sequence alignment analysis using hTREX1 on NCBI to obtain TREX-like protein sequences. After synthesizing sequences from different species, the proteins were expressed and purified in *E. coli*. High-purity proteins were then crystallized and screened. TREX1-like proteins were mixed with dsDNA at a 1:1.25 molar ratio and incubated to form a protein-DNA mixture. This mixture was then suspended dropwise onto 96-well plates at a 1:1 volume ratio under different crystallization conditions and incubated at 16°C. After obtaining preliminary crystals, experimental samples were prepared using the same method, and further crystal optimization was performed on 15-well plates to obtain high-quality protein crystals. Diffraction data of the protein crystals were collected at the Shanghai Synchrotron Radiation Facility, and electron cloud density was obtained after preliminary processing with an HKL2000. The structural model was then optimized using COOT and Phenix software to obtain high-resolution crystal structures of the TREX protein-DNA complex. By screening the TREX1-DNA complex structures of different species (including the South Asian wild scad TREX1 (Labeo rohita TREX1, LrTREX1), the Peruvian poison frog TREX1 (Ranitomeya imitator TREX1, RiTREX1), the human TREX1 mutant K66R, and the human TREX1 mutant K160R), it was found that hTREX1 exhibits an additional DNA-binding interface. Using wild-type hTREX1 (corresponding to protein database UniProt ID Q9NSU2) and the supercrystalline hTREX1 K66R mutant, and with optimized crystallization conditions, various structures of the hTREX1-DNA complex were obtained. The inventors discovered that in some of these structures, hTREX1 possesses a non-conserved interface for binding to non-substrate DNA, which is distinct from and completely separate from its classic DNA-binding site. Figure 1 (A). The newly discovered non-substrate DNA binding interface is characterized by a basic fragment mainly composed of positively charged residues, which interacts directly with the DNA phosphate backbone through four key residues (T49, R211, Q213, and R217). Figure 1 (B). Given that TREX1 has a classic DNA-binding interface and uses DNA as a substrate for degradation (Nat. Commun. 2022; 13(4277. https: / / doi.org / 10.1038 / s41467-022-32055-z); This invention discovers a novel non-substrate DNA binding interface that exists only in humans and hominids. Therefore, in this invention, the classic substrate DNA binding interface is designated as "DNA A-site," and the newly discovered non-substrate DNA binding interface is designated as "DNA B-site."

[0052] This binding pattern of the TREX1 DNA B-site, which is not conserved across species, is a special surface on which human TREX1 evolved to assist DNA binding. Further experimental evidence also confirms that this special DNA B-site is crucial for hTREX1 to recognize DNA: EMSA experiments showed that mutations in key amino acids (T49E / R211E / Q213E / R217E) in the DNA B-site cause the disappearance of the original higher-order oligomeric complex between TREX1 and DNA. Figure 2 While the TREX1 DNA A-site mutant exhibits a high molecular weight (A), the low molecular weight complex (a 2:2 complex formed by the binding of the TREX1 DNA A-site to DNA) remains. Surprisingly, in in vitro nuclease activity assays, the TREX1 DNA B-site mutant showed nucleic acid degradation capabilities comparable to the wild type. Figure 2 (Middle B), suggesting that DNA binding at the DNA B-site may not directly participate in nuclease activity. To investigate the degradation efficiency of TREX1 DNA B-site dysfunction on DNA within cGAS condensates, the inventors added TREX1 protein to the cGAS-DNA phase separation system. Data showed that wild-type TREX1 could degrade approximately 80% of cGAS-bound DNA, while the TREX1 DNA B-site mutant protein only digested 40% (Middle B). Figure 2 (C). The above results suggest that the DNA B-site of TREX1 may be an important reason for constructing TREX1-DNA multivalent interactions, enabling TREX1 to compete with cGAS DNA in the form of a higher-order complex to exert its immunomodulatory function.

[0053] Multivalent interactions are the theoretical basis and condition for phase separation, and biomolecules undergoing phase separation often form microscopic "droplets" in vitro and in cells. Research published by the inventors' research group (Mol. Cell. 2021; 81(739–755. https: / / doi.org / 10.1016 / j.molcel.2021.01.024), TREX1 regulates the number of intracellular cGAS-DNA droplets in a phase-separated manner, thereby affecting cGAS signal transduction. Therefore, this invention further investigated whether the binding of TREX1 to cGAS-DNA condensates in cells requires its DNA B-site. By stimulating cGAS condensate formation with transfected DNA, the inventors found that ~48% of the condensates were positive for both TREX1 and cGAS, while the proportion of TREX1 DNA B-site mutations co-localized with cGAS decreased to ~33%. Figure 3 (A and B in the middle). These experimental results preliminarily demonstrate that hTREX1 can compete for and remove DNA inside cGAS condensates via DNA B-sites in a non-enzymatic phase separation manner.

[0054] Studies have shown that expressing the TREX1 DNA B-site mutant in HeLa TREX1 knockout cell lines significantly weakens TREX1's DNA binding capacity and exonuclease activity, leading to upregulation of the type I interferon pathway (IFNβ). Figure 4 (A). Meanwhile, expression of the TREX1 B-site mutant in the MCF10A TREX1 knockout cell line also resulted in a significant upregulation of IFNβ ( Figure 4 The presence of B indicates that this mechanism is not specific to a single cell line. Further functional validation showed that inoculating BALB / c mice with disease-associated TREX1 DNA B-site mutation (W210R) CT26 tumor cells significantly slowed tumor growth (see [reference needed]). Figure 4 (C) Directly demonstrated that intervention with DNA B-sites can enhance anti-tumor immune responses and inhibit tumor growth. Therefore, DNA B-sites hold promise as a potential target for tumor therapy and may circumvent the autoimmune risks posed by widespread inhibition of TREX1.

[0055] Based on the structurally discovered TREX1 DNA B-site sequence (T49, Q209-R217) according to this invention, disease-related site mutations can be summarized using databases. Analysis revealed six autoimmune disease-related sites occurring on the DNA B-site. Figure 5 Furthermore, four tumor-associated mutations were found to occur on the TREX1 DNA B-site. Figure 5The impact of point mutations in TREX1 on tumor development remains unclear. Therefore, the inventors' future research will focus on validating these disease-related mutations through in vitro biochemical, immunological signaling, and tumor growth assays.

[0056] In summary, the DNA B-site structure revealed in this invention provides new structural evidence for the non-catalytic immune regulation of TREX1. Through mutation verification and functional analysis, this invention clarifies the crucial role of DNA B-sites in maintaining immune homeostasis and tumor immune escape, and proposes a novel anti-tumor immune strategy targeting DNA B-sites. This discovery not only fills a gap in TREX1 structural research but also provides a new theoretical basis and direction for the design of immune regulatory molecules and drug development.

[0057] From an overall conceptual perspective, the core innovation of this invention lies in identifying a newly discovered DNA-binding interface (DNA B-site) in human TREX1 and revealing its non-catalytic immune regulatory function. Therefore, the applications and extensions of this invention include, but are not limited to: (1) By identifying DNA-binding proteins or nucleases other than TREX1, molecular alternatives with similar "non-catalytic DNA binding-immune regulation" functions can be sought. For example, by screening DNA metabolism-related proteins such as TREX2, RNASEH2, or SAMHD1, a similar "structural interface-mediated non-catalytic immune regulation model" can be constructed to avoid the direct use of TREX1.

[0058] (2) Expansion of DNA binding interface (DNA B-site): A. Structural deformation direction: “interface reconstruction” or “charge substitution design” can be carried out in the region near the key residues (T49, R211, Q213, R217) of DNA B-site, and similar DNA binding effects can be achieved by introducing other positively charged amino acids or peptides; B. Functional substitution direction: DNA B-site can be targeted directly, but similar immune regulation functions can be achieved by regulating the oligomerization or subcellular localization of TREX1 (e.g., ER membrane anchoring sequence mutation, N-terminal modification); C. Drug design change direction: At the drug development level, small molecules or peptides can be designed to act on the spatial conformation between DNA A-site and DNA, indirectly affecting the function of DNA B-site, without directly interacting with DNA B-site.

[0059] (3) Functional variations of extended ideas: A. Extend the regulatory ideas of DNA B-site to phase separation regulatory proteins (such as cGAS, IFI16, DDX41) and achieve fine regulation of immune signals by changing their DNA binding dynamics; B. Use artificial fusion proteins (such as fusing TREX1 DNA B-site fragments with inactive exonucleases or DNA binding modules) to reconstruct non-catalytic complexes with immunomodulatory functions.

[0060] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. The application of the TREX1 protein non-substrate DNA binding interface as a target in the preparation of immunomodulatory drugs, characterized in that, The TREX1 protein is a human TREX1 protein, with the UniProt number Q9NSU2; the non-substrate DNA binding interface includes: threonine at position 49, arginine at position 211, glutamine at position 213, and arginine at position 217 of the TREX1 protein.

2. The application according to claim 1, characterized in that, The non-substrate DNA binding interface includes threonine at position 49 and glutamine at position 209 through arginine at position 217 of the TREX1 protein.

3. The application according to claim 1, characterized in that, The immunomodulatory drug is selected from one or more of the following: tumor immunomodulatory drugs, drugs for treating Ecardi-Goodtry syndrome, and drugs for treating systemic lupus erythematosus.

4. The application of reagents that target and disrupt the non-substrate DNA binding interface of TREX1 protein in the preparation of immunomodulatory drugs, characterized in that, The TREX1 protein is a human TREX1 protein, with the UniProt number Q9NSU2; the non-substrate DNA binding interface includes: threonine at position 49, arginine at position 211, glutamine at position 213, and arginine at position 217 of the TREX1 protein.

5. The application according to claim 4, characterized in that, The non-substrate DNA binding interface includes threonine at position 49 and glutamine at position 209 through arginine at position 217 of the TREX1 protein.

6. The application according to claim 4, characterized in that, The immunomodulatory drug is selected from one or more of the following: tumor immunomodulatory drugs, drugs for treating Ecardi-Goodtry syndrome, and drugs for treating systemic lupus erythematosus. The reagent that targets and disrupts the non-substrate DNA binding interface of the TREX1 protein is selected from one or more of small molecules, nucleic acids, gene editing systems, proteins, and peptides.

7. An immunomodulatory drug, characterized in that, The immunomodulatory drugs include: reagents that target and disrupt the non-substrate DNA binding interface of the TREX1 protein; The TREX1 protein is a human TREX1 protein, with the UniProt number Q9NSU2; the non-substrate DNA binding interface includes threonine at position 49, arginine at position 211, glutamine at position 213, and arginine at position 217 of the TREX1 protein.

8. The immunomodulatory drug according to claim 7, characterized in that, The non-substrate DNA binding interface includes threonine at position 49 and glutamine at position 209 through arginine at position 217 of the TREX1 protein.

9. The immunomodulatory drug according to claim 7, characterized in that, The immunomodulatory drugs also include: pharmaceutically acceptable excipients; The routes of administration for the immunomodulatory drugs include one or more combinations of oral, intravenous, intramuscular, and subcutaneous injection.

10. The immunomodulatory drug according to claim 9, characterized in that, The pharmaceutically acceptable excipients include one or more of the following: pharmaceutically acceptable excipients, additives, and adjuvants.

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