Interfering peptide for inhibiting the interaction of interferon gene stimulator and its application

Abstract

The present invention relates to an interfering peptide that inhibits the interaction of interferon gene stimulator factors and its application, and belongs to the field of biomedicine technology. The present invention designs and synthesizes an interfering peptide that targets interferon gene stimulator factors. Specifically, the interfering peptide is designed to target the disulfide bond formation site region of interferon gene stimulator factors, which can specifically destroy the interaction of interferon gene stimulator factors, thereby avoiding the excessive activation of downstream signals caused by the high polymerization of interferon gene stimulator factors. The interfering peptide of the present invention destroys the intermolecular interaction mediated by disulfide bonds by binding to the action surface of the dimerization domain that mediates the oligomerization of interferon gene stimulator factors, thereby inhibiting the excessive activation of interferon gene stimulator factors caused by viral infection, thereby achieving the purpose of alleviating the excessive production of intracellular inflammatory factors that cause organ damage, thereby achieving effective treatment of clinical viral infections.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an interfering peptide for inhibiting the interaction of interferon gene stimulator factors and its application. Background Art

[0002] The stimulator of interferon genes (STING) protein, a key regulator of the immune response, plays a central role in maintaining immune homeostasis. When the body encounters a viral infection, such as the herpes simplex virus, the viral double-stranded DNA is recognized by cellular receptors, activating the STING protein. This activation, through a series of cascade reactions, recruits and activates the serine-threonine protein kinase (TBK1). TBK1 then phosphorylates interferon regulatory factor 3 (IRF3). Phosphorylated IRF3 dimerizes and translocates to the nucleus, initiating the transcription of cytokines such as type I interferons, thereby triggering the body's antiviral immune response. In inflammatory conditions, such as sterile inflammation caused by tissue damage, abnormal metabolites or damage-associated molecular patterns within cells can also activate STING, triggering a similar immune-inflammatory cascade.

[0003] However, the intense stimulation of the virus can also lead to overactivation of the STING protein, disrupting the body's immune balance and triggering an excessive inflammatory response. In dengue virus infection, the virus's intense stimulation of the immune system causes overactivation of the STING protein, leading to the sustained release of large amounts of inflammatory factors such as tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), triggering a cytokine storm. This leads to severe vascular leakage, organ dysfunction, and other symptoms in patients, significantly increasing their risk of death. In autoimmune inflammatory diseases such as systemic lupus erythematosus, the body's own production of nucleic acids abnormally activates the STING protein. Sustained overactivation causes a diffuse inflammatory response throughout multiple organ systems, causing extensive tissue damage, such as kidney involvement with proteinuria and joint inflammation leading to pain and deformities.

[0004] Currently, clinical interventions for the inflammatory response caused by excessive STING protein activation are very limited. Traditional immunosuppressants, such as glucocorticoids, can suppress the overall immune response to a certain extent. However, due to their lack of specificity, while suppressing excessive inflammatory responses, they also severely inhibit the body's normal immune defense functions, making patients more susceptible to various infectious diseases. Long-term use can also cause osteoporosis, dysglycemia and other serious side effects. While some monoclonal antibody drugs targeting inflammatory factors are currently under development, they can block the effects of specific inflammatory factors. However, due to the complexity of the inflammatory response, blocking a single inflammatory factor often cannot effectively control the cascade of inflammatory responses triggered by excessive STING protein activation, resulting in unsatisfactory therapeutic effects. Therefore, it is urgent to develop an interventional approach that can precisely target excessive STING protein activation, effectively inhibit excessive inflammatory responses, and have minimal side effects. Summary of the Invention

[0005] To this end, the technical problem to be solved by the present invention is to overcome the problem in the prior art of the lack of an intervention method that can accurately target the excessive activation of STING protein, effectively inhibit excessive inflammatory response and have few side effects.

[0006] To address the above-mentioned technical problems, the present invention provides an interfering peptide that inhibits the interaction of stimulator of interferon genes and its use. Based on the basic functions of the STING protein and the mechanism by which its overactivation leads to immune inflammatory damage in the body, the present invention artificially designed and synthesized an interfering peptide targeting the STING protein. This interfering peptide binds to the active surface of the dimerization domain that mediates STING protein oligomerization, disrupting disulfide-mediated intermolecular interactions, thereby inhibiting the overactivation of the STING protein caused by viral infection, thereby alleviating organ damage caused by excessive production of cellular inflammatory factors, thereby effectively treating clinical conditions.

[0007] The first object of the present invention is to provide an interfering peptide that inhibits the interaction of STING proteins, wherein the amino acid sequence of the interfering peptide is as shown in SEQ ID NO.1.

[0008] Furthermore, the interfering peptide is a D-amino acid retroisomer. D-amino acids degrade more slowly in animals than natural L-amino acids. Modifying the interfering peptide into a D-amino acid retroisomer can give the interfering peptide good tolerability and therapeutic effect.

[0009] Furthermore, the interfering peptide inhibits the oligomerization of the STING protein.

[0010] The second object of the present invention is to provide a use of the above-mentioned interfering peptide in the preparation of drugs for preventing or treating viral infections.

[0011] Furthermore, the virus includes severe acute respiratory syndrome coronavirus 2.

[0012] Furthermore, the drug for preventing or treating viral infection alleviates inflammatory damage caused by viral infection, including but not limited to alveolar septal thickening, hemagglutination, and inflammatory cell infiltration.

[0013] A third object of the present invention is to provide a drug for inhibiting hemagglutination, comprising the aforementioned interfering peptide. The interfering peptide binds to stimulator of interferon genes, thereby inhibiting the elevated expression of coagulation factor III caused by overactivation of stimulator of interferon genes, thereby inhibiting hemagglutination.

[0014] A fourth object of the present invention is to provide a drug for inhibiting the phosphorylation of serine-threonine protein kinase, the drug comprising the above-mentioned interfering peptide. The interfering peptide inhibits the phosphorylation of serine-threonine protein kinase by the interferon gene stimulator by inhibiting the interaction of the interferon gene stimulator.

[0015] The fifth object of the present invention is to provide an immunosuppressive composition comprising the above-mentioned interfering peptide.

[0016] The sixth object of the present invention is to provide a use of the above-mentioned interfering peptide in the preparation of STING protein inhibitors.

[0017] Furthermore, the STING protein inhibitor inhibits the oligomerization of the STING protein.

[0018] Furthermore, the STING protein inhibitor inhibits the interaction between STING protein and serine-threonine protein kinase.

[0019] The seventh object of the present invention is to provide a drug for inhibiting the interaction of interferon gene stimulator, which comprises the above-mentioned interfering peptide.

[0020] The eighth object of the present invention is to provide an application of the above-mentioned interfering peptide in the preparation of an anti-inflammatory product, which inhibits the production of inflammatory factors, wherein the inflammatory factors are selected from one or more of interferon β1, interleukin-6, interferon regulatory factor 5, interferon regulatory factor 8, interferon-induced guanylate binding protein 2, tumor necrosis factor receptor superfamily member 12a, chemokine 2, chemokine CXC ligand 9, chemokine CXC motif 11 and resistin-like molecule α.

[0021] Beneficial effects of the present invention:

[0022] The interfering peptide targeting the STING protein described in the present invention effectively inhibits the oligomerization of the STING protein. Compared with traditional small molecule drugs, the interfering peptide of the present invention can effectively block the cross-linking of the STING protein through disulfide bonds; the interfering peptide targeting the STING protein contains a transmembrane sequence, which can directly allow the peptide segment to pass through the cell membrane and enter the cytoplasm to exert its effect, without the need for any carrier, thus avoiding the toxic side effects caused by the carrier; D-amino acids are degraded more slowly in the animal body than natural L-amino acids. Modifying the interfering peptide into a D-amino acid reverse (D-retroinverso, hereinafter referred to as "DRI") isomer makes the interfering peptide have good tolerability and therapeutic effect, so the DRI-modified interfering peptide targeting the STING protein is feasible for clinical trials; at the same time, the interfering peptide of the present invention is only a small peptide of 25 amino acids in length, has no immunogenicity or extremely low immunogenicity, and can avoid causing hypersensitivity reactions; in addition, the interfering peptide of the present invention can be directly obtained through existing mature polypeptide synthesis technology, and has the advantages of high purity, controllable quality, and great drug potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The diagram is a structural diagram of the STING protein and a schematic diagram of the target sequence of the interfering peptide, where SIP-III represents the interfering peptide, TM represents the transmembrane domain, CBD represents the cytoplasmic ligand binding domain, and CTT represents the C-terminal tail;

[0024] Figure 2 is a schematic diagram of the molecular weight of the synthesized interfering peptide detected by mass spectrometry (MS);

[0025] Figure 3 Schematic diagram of the purity of the synthesized interfering peptide detected by high performance liquid chromatography (HPLC);

[0026] Figure 4 The figure shows the results of Western Blot detection of STING protein dimerization and downstream signal transduction activation after treatment with interfering peptides, where "+" indicates the addition of interfering peptides and "-" indicates the absence of interfering peptides.

[0027] Figure 5 This is the result of immunofluorescence detection of the effect of interfering peptide treatment on the liquid-liquid phase separation ability of STING protein;

[0028] Figure 6 This is a schematic diagram of qPCR experiments detecting the mRNA expression of inflammatory factors in the spleen, liver, and lung tissues of ACE2 transgenic mice treated with interfering peptides and infected with SARS-CoV-2, where fold change is used to represent the difference in gene expression under different conditions;

[0029] Figure 7Schematic diagram of hematoxylin-eosin (HE) staining to detect lung inflammation damage in mice treated with interfering peptides and infected with SARS-CoV-2;

[0030] Figure 8 This is a diagram showing the weight of mice treated with interfering peptides and infected with SARS-CoV-2 using statistical experiments;

[0031] Figure 9 Schematic diagram of immunofluorescence assay to detect the aggregation of STING protein in the lungs of ACE2 transgenic mice treated with interfering peptides and mice infected with SARS-CoV-2. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0033] Example 1

[0034] like Figure 1 As shown, the design idea of ​​the interfering peptide drug in this embodiment is: first, an interfering peptide segment is designed to target amino acids 286-298 near the disulfide bond formation site in the STING protein molecule. The amino acid sequence of this segment is IDELTRCFLKAQE, and the natural amino acids are L-type. A D-type short peptide is designed to specifically disrupt the interaction between STING proteins to prevent them from forming high polymers and causing excessive downstream signal activation.

[0035] Secondly, to promote cellular uptake of the interfering peptide, the interfering peptide was designed to be fused with a membrane-penetrating sequence (HIV-TAT). HIV-TAT is a hydrophilic sequence with the amino acid sequence GRKKRRQRRRPP, which enables the peptide to cross the cell membrane in an energy-independent manner for cellular uptake.

[0036] DRI modification of the peptide can improve its stability and efficacy in cell and animal studies. The entire interfering peptide was modified into its retromer, resulting in the amino acid sequence of IDELTRCFLKAQEPPRRRQRRKKRG (SEQ ID NO. 1). This peptide was synthesized by Jier Biochemical (Shanghai) Co., Ltd. using D-amino acids.

[0037] like Figure 2 As shown, the molecular weight of the synthesized interfering peptide was identified as 3138.64 Da by matrix-assisted laser desorption / time-of-flight mass spectrometry liquid chromatography-mass spectrometry system.

[0038] like Figure 3As shown, high performance liquid chromatography (HPLC) was performed using an Inertsil ODS-SP liquid chromatography column (Shimadzu, 4.6 mm × 250 mm) as the stationary phase, and mobile phase A (100% nitrile, 0.1% trifluoroacetic acid) and mobile phase B (100% ultrapure water, 0.1% trifluoroacetic acid) for gradient elution. The purity of the product was greater than 98% as determined by HPLC.

[0039] Example 2

[0040] 1. Experimental Materials

[0041] The interfering peptide prepared in Example 1, 2'3'-cyclic guanosine monophosphate (2'3'-cGAMP), fetal bovine serum, modified dextrose minimal essential medium (DMEM medium), penicillin / streptomycin solution (from Gibco), ATCC-derived human monocytic leukemia cell line (THP-1 cell line), STING protein antibody, serine-threonine protein kinase antibody (TBK1 antibody), phosphorylated serine-threonine protein kinase antibody (p-TBK1 antibody), phosphorylated interferon regulatory factor 3 antibody (p-IRF3 antibody), interferon regulatory factor 3 antibody (IRF3 antibody), actin antibody (Actin antibody) and related secondary antibodies (from CST).

[0042] 2. Experimental methods

[0043] THP-1 cells were plated in 12-well plates. When the cell density reached 90%, cells were treated with 100 μM interfering peptide in four wells for 0, 2, 4, and 6 hours, respectively. The cells were then stimulated with 2'3'-cGAMP. After 2 hours, cells were harvested and Western blotting was performed to analyze STING protein oligomerization and phosphorylation of key downstream kinases, TBK1 and IRF3.

[0044] 3. Experimental results

[0045] like Figure 4 As shown, the oligomeric state of the STING protein plays a core role in its activation and downstream signal transduction, and is an important link in regulating the immune response. However, excessive activation of the STING protein will lead to excessive production of cellular inflammatory factors, thereby causing tissue and organ damage. In this example, after treating cells with interfering peptides, it was found that almost no oligomerization of the STING protein was observed in the non-reducing gel electrophoresis diagram, indicating that the interfering peptide can significantly inhibit the 2'3'-cGAMP-induced STING protein oligomerization. At the same time, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the phosphorylation levels of TBK1 and IRF3 were significantly reduced 2-6 hours after the addition of the interfering peptides, indicating that the interfering peptides can significantly inhibit the phosphorylation of downstream key protein kinases TBK1 and transcription factors IRF3.

[0046] Example 3

[0047] 1. Experimental Materials

[0048] Interfering peptides prepared in Example 1, bovine serum albumin (BSA), 2'3'-cGAMP, 4% paraformaldehyde / PBS, 0.1% polyethylene glycol octylphenyl ether (TritonX-100), fluorescein isothiocyanate-labeled rabbit anti-sheep immunoglobulin G, rhodamine fluorescein-labeled goat anti-mouse immunoglobulin G, STING protein (from CST), TBK1 antibody (from Santacruz), and 4',6-diamidino-2-phenylindole (DAPI).

[0049] 2. Experimental methods

[0050] The control group was treated with 100 μM BSA (i.e. Figure 5 In the experimental group, cells were treated with 100 μM interfering peptide for 2 and 6 hours, respectively, and stimulated with 1 μg / mL 2'3'-cGAMP. After 1 hour, cells were harvested, fixed with 4% paraformaldehyde / PBS, and perforated. Antibodies to STING protein and TBK1 were then added and incubated overnight. Fluorescein isothiocyanate-conjugated rabbit anti-goat immunoglobulin G and rhodamine fluorescein-conjugated goat anti-mouse immunoglobulin G were then added and incubated at room temperature for 1 hour. The slides were then mounted and observed under a confocal microscope.

[0051] 3. Experimental results

[0052] like Figure 5 As shown, in the control group, STING protein underwent strong liquid-liquid phase separation (LLPS) in cells after stimulation with 2'3'-cGAMP, forming spherical spots and colocalizing with TBK1 in aggregates. However, after 2 hours of treatment with 100 μM interfering peptide, STING protein's ability to form spherical spots was weakened, and colocalization with TBK1 aggregates decreased. 6 hours of treatment with 100 μM interfering peptide completely inhibited STING protein's liquid-liquid phase separation, and colocalization with TBK1 aggregates was significantly lower than in the control group and the 2-hour treatment group. These results indicate that the interfering peptide significantly inhibits the interaction between STING protein and TBK1 kinase, providing strong support for the inhibition of downstream signal transduction.

[0053] Example 4

[0054] 1. Experimental Materials

[0055] ACE2 transgenic mice (transgenic mice stably expressing human angiotensin-converting enzyme 2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the interfering peptide prepared in Example 1, guanidine isothiocyanate-phenol (Trizol, TAKARA), and a qPCR kit. Table 1 lists the primers required for qPCR (the required primers were synthesized by Youkang Biotechnology). The inflammatory factors detected included interferon β1 (IFN-β1), interleukin-6 (IL-6), interferon regulatory factor 5 (Irf5), interferon regulatory factor 8 (Irf8), interferon-induced guanylate binding protein 2 (Gbp2), tumor necrosis factor receptor superfamily member 12A (Tnfrsf12a), chemokine 2 (Ccl2), chemokine CXC ligand 9 (Cxcl9), chemokine CXC motif ligand 11 (Cxcl11), resistin-like molecule α (Retnla), and coagulation factor III (F3).

[0056] Table 1 Primers required for qPCR

[0057]

[0058] 2. Experimental methods

[0059] The interfering peptide was dissolved in sterile phosphate-buffered saline (PBS) to a concentration of 1 mg / mL. ACE2 transgenic mice were divided into three groups, each with six mice. Groups 1 and 2 were injected with 0.5 mL of sterile PBS as controls, while group 3 was injected with 0.5 mg of the interfering peptide. One hour later, mice in groups 2 and 3 were anesthetized and intranasally inoculated with SARS-CoV-2 virus, with each mouse receiving approximately 1×10 5 TCID 50 ) SARS-CoV-2 virus. 16 hours after virus infection, spleen, liver, and lung tissues of mice were collected and total RNA was extracted using the guanidine isothiocyanate-phenol method (Trizol method). After reverse transcription, the expression of inflammatory factor mRNA in spleen, liver, and lung tissues was detected by qPCR. The statistical analysis of the results was expressed as "mean ± standard deviation" (mean ± SEM) and compared using analysis of variance (ANOVA). p <0.05 was considered a significant difference. p <0.01 indicates extremely significant difference.

[0060] 3. Experimental results

[0061] like Figure 6As shown in the results, the content of cellular inflammatory factors in ACE2 mice in the SARS-CoV-2+SIP-III group was significantly lower than that in the ACE2 mice in the SARS-CoV-2+PBS group, indicating that the interfering peptide can significantly reduce the transcription of cellular inflammatory factor mRNA induced by SARS-CoV-2 in the tissues of ACE2 transgenic mice.

[0062] Example 5

[0063] 1. Experimental Materials

[0064] ACE2 transgenic mice, SARS-CoV-2, the interfering peptide prepared in Example 1, and materials and reagents related to tissue fixation, embedding, and HE staining (provided by Shanghai Sangon Biotechnology Co., Ltd.).

[0065] 2. Experimental methods

[0066] Sterile PBS was used to dissolve the interfering peptide to a concentration of 1 mg / mL. ACE2 transgenic mice were divided into three groups. The first group was injected with only 0.5 mL of sterile PBS, and the second group was injected with 0.5 mL of sterile PBS. One hour later, 1×10 5 TCID 50 The third group was injected with 0.5 mg of interfering peptide, and 1×10 5 TCID 50 SARS-CoV-2. 24 hours after virus infection, mouse lung tissue was collected and fixed in 4% paraformaldehyde / PBS. Paraffin sections of the lung tissue were prepared, and HE staining was used to detect lesions in the mouse lungs.

[0067] 3. Experimental results

[0068] like Figure 7 As shown, the interfering peptide significantly reduced lung lesions in ACE2 transgenic mice induced by SARS-CoV-2 infection. Lung tissue morphology in uninfected mice (PBS group) was normal, with clear alveoli and fine septa. However, the SARS-CoV-2 + PBS group showed significant thickening of the alveolar septa, with localized viral hemagglutination and inflammatory cell infiltration, demonstrating a significant inflammatory response. In the SARS-CoV-2 + SIP-III group, alveolar septa thickening was less pronounced, and hemagglutination and inflammatory cell infiltration were significantly less than in the SARS-CoV-2 + PBS group.

[0069] Example 6

[0070] 1. Experimental Materials

[0071] ACE2 transgenic mice, SARS-CoV-2, the interfering peptide prepared in Example 1, etc.

[0072] 2. Experimental methods

[0073] Sterile PBS was used to dissolve the interfering peptide to a concentration of 1 mg / mL. ACE2 transgenic mice were divided into three groups. The first group was injected with only 0.5 mL of sterile PBS, and the second group was injected with 0.5 mL of sterile PBS. One hour later, 1×10 5 TCID 50 The third group was injected with 0.5 mg of interfering peptide, and 1×10 5 TCID 50 SARS-CoV-2. After 24 hours of SARS-CoV-2 infection, the mice were observed and weighed every day for changes in body weight. Statistics were completed 7 days after infection.

[0074] 3. Experimental results

[0075] like Figure 8 As shown, the interfering peptide can significantly alleviate the weight loss of ACE2 transgenic mice caused by SARS-CoV-2 infection. The weight of mice not infected with the virus (PBS group) gradually increased within a week, while the weight of mice in the SARS-CoV-2+PBS group gradually decreased. The SARS-CoV-2+SIP-III group alleviated the weight loss of mice to a certain extent.

[0076] Example 7

[0077] 1. Experimental Materials

[0078] ACE2 transgenic mice, SARS-CoV-2, the interfering peptide prepared in Example 1, paraformaldehyde and other tissue fixation and embedding-related materials and reagents were all domestically produced, rabbit anti-STING protein (from CST), DAPI, and fluorescein isothiocyanate-labeled rabbit anti-sheep immunoglobulin G.

[0079] 2. Experimental methods

[0080] Sterile PBS was used to dissolve the interfering peptide to a concentration of 1 mg / mL. ACE2 transgenic mice were divided into three groups. The first group was injected with only 0.5 mL of sterile PBS, and the second group was injected with 0.5 mL of sterile PBS. One hour later, 1×10 5 TCID 50 The third group was injected with 0.5 mg of interfering peptide, and 1×10 5 TCID 50SARS-CoV-2. 24 hours after SARS-CoV-2 infection, mouse lung tissue was collected and fixed in 4% paraformaldehyde / PBS, and paraffin sections were prepared. Immunofluorescence was used to detect the expression of STING protein in the mouse lungs.

[0081] 3. Experimental results

[0082] like Figure 9 As shown in , in this example, the aggregation of STING protein was detected by a fluorescent secondary antibody coupled to fluorescein isothiocyanate. There was almost no aggregation fluorescence signal in the lung tissue of mice not infected with the virus (PBS group), while there was a strong STING aggregation fluorescence signal in the lung tissue of mice in the SARS-CoV-2+PBS group. Only a weak STING protein aggregation signal was detected in the lung tissue of mice in the SARS-CoV-2+SIP-III group.

[0083] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of an interfering peptide for inhibiting the interaction of interferon gene stimulator factors in the preparation of a drug for preventing or treating viral infection, characterized in that: The amino acid sequence of the interfering peptide is shown in SEQ ID NO.1, and the virus is severe acute respiratory syndrome coronavirus 2.

2. An interfering peptide that inhibits the interaction of interferon gene stimulator, characterized in that: The amino acid sequence of the interfering peptide is shown in SEQ ID NO.

1.

3. The interfering peptide according to claim 2, characterized in that The interfering peptide is a D-amino acid retroisomer.

4. A drug for inhibiting hemagglutination, characterized in that: The drug comprises the interfering peptide according to claim 2 or 3, and the hemagglutination is caused by severe acute respiratory syndrome coronavirus 2 infection.

5. An immunosuppressive composition, characterized in that The immunosuppressive composition comprises the interfering peptide according to claim 2 or 3.

6. A drug for inhibiting the interaction of interferon gene stimulator, characterized in that: The drug comprises the interfering peptide according to claim 2 or 3.

7. Use of the interfering peptide according to claim 2 or 3 in the preparation of anti-inflammatory products, characterized in that: The anti-inflammatory product inhibits the production of inflammatory factors, wherein the inflammatory factors are selected from one or more of interferon β1, interleukin-6, interferon regulatory factor 5, interferon regulatory factor 8, interferon-induced guanylate binding protein 2, tumor necrosis factor receptor superfamily member 12a, chemokine 2, chemokine CXC ligand 9, chemokine CXC motif 11 and resistin-like molecule α.

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