Interfering peptide for inhibiting mitochondrial autophagy induced by coronavirus nucleocapsid protein and preparation method and application thereof

Abstract

The application belongs to the technical field of medicine preparation, and specifically discloses an interfering peptide for inhibiting mitochondrial autophagy induced by a coronavirus nucleocapsid protein, and a preparation method and application thereof. The interfering peptide (named ISQORbeta2) is composed of D-type amino acids, and the structure comprises: a polypeptide sequence as shown in SEQ ID NO:1; and 5-isothiocyanate fluorescein covalently connected to the N terminal of the polypeptide. The interfering peptide effectively inhibits mitochondrial autophagy activation, intracellular H2S accumulation, mitochondrial potential depolarization, and restores the expression of innate immune molecules by competitively blocking the interaction between IBV-N and SARS-CoV-2-N and sulfide quinone oxidoreductase, thereby resisting viral replication and reducing lung injury. The application solves the problems of the prior art, such as difficulty in blocking protein interaction, poor peptide stability, and inability to effectively restore the immune pathway, and has the advantages of high in-vitro and in-vivo stability, small toxic and side effects, and great potential for drug development.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a mitophagy-interfering peptide (named ISQORβ2) that inhibits the induction of coronavirus nucleocapsid proteins (including avian infectious bronchitis virus nucleocapsid protein IBV-N and severe acute respiratory syndrome coronavirus type 2 nucleocapsid protein SARS-CoV-2-N), its preparation method and application, especially for the preparation of antiviral drugs and drugs for restoring innate immunity. Background Technology

[0002] In viral pathology studies, the nucleocapsid protein (N protein) of coronaviruses such as IBV and SARS-CoV-2, by binding to thioquinone oxidoreductase (SQOR), leads to intracellular H2S accumulation, mitochondrial membrane depolarization, induces mitophagy, and downregulates innate immune molecules (such as ILF3 and MAVS), exacerbating viral replication and tissue damage. Existing strategies targeting the N protein mostly involve monoclonal antibodies or small molecule inhibitors, but these have the following drawbacks: (1) they are difficult to efficiently block protein-protein interface interactions because the N protein interacts with SQOR in a face-to-face binding manner; (2) they lack cell-penetrating ability, relying on vector delivery and introducing toxic side effects; and (3) they cannot fully restore the expression of immune molecules. Therefore, this invention proposes solutions to the above-mentioned technical problems. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a mitophagy-interfering peptide (hereinafter named ISQORβ2) that inhibits coronavirus nucleocapsid protein-induced mitophagy, its preparation method and application, thereby blocking the interaction between IBV-N (targeting amino acids 156-214) and SARS-CoV-2-N (targeting amino acids 181-246) and SQOR, inhibiting mitophagy and restoring the expression of innate immune pathways.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] In a first aspect, the present invention provides an ISQORβ2 peptide, which inhibits coronavirus nucleocapsid protein-induced mitophagy interference, composed of D-type amino acids, and its structure includes:

[0006] The polypeptide sequence shown in SEQ ID NO: 1 is: VKRKMRAAMTIGGSPPRRRGRRKKRG;

[0007] A functional group covalently attached to the N-terminus of the polypeptide.

[0008] Preferably, the functional groups include, but are not limited to, fluorescein 5-isothiocyanate.

[0009] Preferably, VKRKMRAAMTIGGS of SEQ ID NO:1 is a functional core domain targeting the coronavirus N protein; PPRRRGRRKKRG of SEQ ID NO:1 is a cell-penetrating domain derived from HIV-TAT; the functional core domain is derived from the overall reverse order and D-type amino acid conversion of the sequence SGGITMAARMKRKV (SEQ ID NO:3).

[0010] Preferably, the interfering peptide targets amino acid regions 181-246 of the SARS-CoV-2-N protein and amino acid regions 156-214 of the IBV-N protein, inhibiting the interaction between the coronavirus nucleocapsid protein (NP or N protein) and thioquinone oxidoreductase (SQOR) and the activation of mitophagy, thereby restoring the downregulation of NP-induced innate immune molecules.

[0011] In a second aspect, the present invention provides a method for preparing the interfering peptide as described in the first aspect, comprising the following steps:

[0012] Step 1: Through molecular docking and immunoprecipitation experiments, it was confirmed that the interaction region between SARS-CoV-2-N protein and SQOR is amino acid 181-246, and the interaction region between IBV-N protein and SQOR is amino acid 156-214; the interaction regions between SARS-CoV-2-N and IBV-N and SQOR are both in the 53-66 amino acid region of SQOR.

[0013] Step 2: Using the interaction region between the SARS-CoV-2-N protein and IBV-N protein and SQOR as described in Step 1 as the target, design and obtain a peptide that competitively binds to this region;

[0014] Step 3: Fuse the peptide designed in Step 2 with the HIV-TAT sequence to form a fusion sequence, thereby giving it membrane-penetrating activity;

[0015] Step 4: Convert the fusion sequence obtained in Step 3 into the reversed D-amino acid form (the entire sequence is reversed before subsequent D-amino acid synthesis), and fuse it with FITC fluorescent label to obtain the final amino acid sequence of the interfering peptide ISQORβ2.

[0016] Step 5: Synthesize the interfering peptide ISQORβ2 using D-type amino acids as raw materials, and purify it to achieve a peptide purity of ≥96.64%.

[0017] Preferably, in step 2, a polypeptide that competes with the region of interaction between SARS-CoV-2-N protein and IBV-N and SQOR is designed and obtained, and the amino acid sequence of the obtained polypeptide is SGGITMAARMKRKV (SEQ ID NO:3).

[0018] Preferably, in step 3, the amino acid sequence of HIV-TAT is GRKKRRGRRRPP (SEQ ID NO: 2).

[0019] The amino acid sequence of the fusion sequence is GRKKRRGRRRPPSGGITMAARMKRKV (SEQ ID NO: 4).

[0020] Preferably, in step 3, the amino acids after HIV-TAT is fused with the obtained polypeptide are L-type natural amino acids.

[0021] Preferably, in step 4, the interfering peptide ISQORβ2 structure comprises a polypeptide sequence as shown in VKRKMRAAMTIGGSPPRRRGRRKKRG (SEQ ID NO: 1) and 5-FITC (5-isothiocyanate fluorescein) covalently linked to the N-terminus of the polypeptide, wherein the 5-FITC is linked to the first amino acid residue via a 6-aminohexanoic acid (Acp) linker group; forming the complete structure 5-FITC-(Acp)-VKRKMRAAMTIGGSPPRRRGRRKKRG. This modification endows the peptide with intracellular localization tracking function, but does not affect its binding activity with the target protein (SARS-CoV-2-N / IBV-N); its amino acids are D-type amino acids.

[0022] In this invention, the artificially synthesized D-amino acid retroisomer not only has a structure similar to that of L-amino acids, but also endows the interfering peptide with new chemical properties, improving the stability of the interfering peptide ISQORβ2 in vitro and in vivo. In addition, HIV-TAT (Human Immunodeficiency Virus Transactivator of Transcription) is a hydrophilic sequence, and its fusion with the interfering peptide can enable the interfering peptide ISQORβ2 to be absorbed by the cell by crossing the cell membrane in an energy-independent manner.

[0023] Preferably, the purification in step 5 is performed using high performance liquid chromatography (HPLC) gradient elution.

[0024] In a third aspect, the present invention provides the use of the interfering peptide as described in the first aspect in the preparation of a drug for inhibiting coronavirus-induced lesions, characterized in that the interfering peptide is used to block the interaction between IBV-N or SARS-CoV-2-N and SQOR, thereby inhibiting mitophagy, restoring the expression of innate immune molecules, or treating coronavirus-related lung injury.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. The interfering peptide ISQORβ2 targeting SARS-CoV-2-N and IBV-N proteins prepared in this invention significantly inhibits the activation of SARS-CoV-2-N and IBV-N protein-mediated mitophagy and innate immune pathways.

[0027] 2. The interfering peptide ISQORβ2 targeting the 181-246 amino acid region of the SARS-CoV-2-N protein prepared in this invention can effectively block the interaction between SARS-CoV-2-N and IBV-N proteins and SQOR. Protein-protein interactions are usually face-to-face interactions, and compared with traditional small molecule drugs, peptide drugs can more effectively block protein-protein interactions.

[0028] 3. The interfering peptide ISQORβ2 targeting SARS-CoV-2-N and IBV-N proteins prepared in this invention contains the HIV-TAT sequence, which allows the peptide to directly cross the cell membrane and enter the cytoplasm to exert its effect without any carrier, thus avoiding the toxicity and side effects caused by the carrier.

[0029] 4. The interfering peptide ISQORβ2 targeting SARS-CoV-2-N and IBV-N proteins prepared in this invention can be directly obtained through existing mature peptide synthesis technology. It has the characteristics of high purity, almost no need for purification, controllable quality, and great potential for drug development. Attached Figure Description

[0030] Figure 1 This diagram illustrates the interaction between the LKR domain and SQOR in the SARS-CoV-2-N protein (top) and the IBV-N protein (bottom).

[0031] Figure 2 This is a schematic diagram showing the molecular weight of the synthesized interfering peptide ISQORβ2 as determined by mass spectrometry.

[0032] Figure 3 This is a schematic diagram showing the purity of the synthesized interfering peptide ISQORβ2 as determined by HPLC.

[0033] Figure 4The figure shows the experimental results of laser confocal analysis of the intracellular colocalization of the interfering peptide ISQORβ2 and the coronavirus nucleocapsid protein (NP). The left two columns are the IBV-N group, and the right two columns are the SARS-CoV-2N group.

[0034] Figure 5 The figure shows the experimental results of immunoprecipitation analysis of the interfering peptide ISQORβ2 inhibiting the interaction between SQOR and NP in cells. The left side is IBV-N and the right side is SARS-CoV-2-N.

[0035] Figure 6 To restore SQOR function affected by NP by detecting the interfering peptide ISQORβ2 through enzyme activity assay.

[0036] Figure 7 To detect the reduction of intracellular H2S accumulation by the interfering peptide ISQORβ2 using flow cytometry.

[0037] Figure 8 To detect the inhibition of NP-induced mitochondrial membrane potential depolarization by the interfering peptide ISQORβ2 by flow cytometry; left: blank control, middle: SP+IBV-N protein particle, right: interfering peptide ISQORβ2+IBV-N protein particle.

[0038] Figure 9 Western blotting was used to detect the effect of the interfering peptide ISQORβ2 on inhibiting NP-induced mitophagy in cells. The left image shows cells transfected with the IBV-N protein plasmid, and the right image shows cells transfected with the SARS-CoV-2-N plasmid.

[0039] Figure 10 Figure 1 shows the results of qPCR detection of the downregulation of the classic innate immune molecule IFNB1 in cells by the interfering peptide ISQORβ2; left is IBV-N, right is SARS-CoV-2-N.

[0040] Figure 11 TCID50 was tested on recombinant VSV expressing IBV-N (VSV-IBV-N) or recombinant VSV expressing SARS-CoV-2-N (VSV-SARS-CoV-2-N); the left figure shows the VSV-IBV-N group, and the right figure shows the VSV-SARS-CoV-2-N group.

[0041] Figure 12This image shows the results of hematoxylin-eosin (HE) staining and immunohistochemical experiments, demonstrating that treatment with the interfering peptide ISQORβ2 in mice / chicks infected with recombinant VSV-NP can inhibit lung lesions induced by recombinant VSV-NP infection. The left two columns represent the chicken VSV-IBV-N infection model; the right two columns represent the mouse VSV-SARS-CoV-2-N infection model.

[0042] Figure 13 The figure shows the results of enhancing the expression of classical innate immune molecules mRNA in mouse and chicken lung tissues by the interfering peptide ISQORβ2. The upper part (chicken VSV-IBV-N infection model): AD represents the relative mRNA expression levels of Gallus Ifnb1, Gallus Cxcli2, Gallus Cxcli1, and Gallus Ifit5 in chicken lung tissue infected with VSV-IBV-N, respectively. The lower part (mouse VSV-SARS-CoV-2N infection model): EH represents the relative mRNA expression levels of Mouse Ifnb1, Mouse Cxcl10, Mouse Cxcl15, and Mouse Ifit1 in mouse lung tissue infected with VSV-IBV-N, respectively. Detailed Implementation

[0043] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0044] Example 1: Molecular targeting design of the interfering peptide ISQORβ2

[0045] The design of the interfering peptide ISQORβ2 includes the following steps:

[0046] Step 1: Through molecular docking and immunoprecipitation experiments, it was confirmed that the interaction region between SARS-CoV-2-N protein and SQOR is amino acid 181-246, and the interaction region between IBV-N protein and SQOR is amino acid 156-214. The interaction regions of both SARS-CoV-2-N and IBV-N with SQOR are both in the 53-66 amino acid region of SQOR. (See...) Figure 1 ;

[0047] Step 2: Using the interaction region between the SARS-CoV-2-N protein and IBV-N protein and SQOR described in Step 1 as the target, design and obtain a polypeptide that competes with this region for binding. The amino acid sequence of the obtained polypeptide is SGGITMAARMKRKV (SEQ ID NO: 3).

[0048] Step 3: The peptide designed in Step 2 is fused with the HIV-TAT sequence to form a fusion sequence, which gives it membrane-penetrating activity; the amino acid sequence of the HIV-TAT is GRKKRRGRRRPP (SEQ ID NO: 2), and the amino acid sequence of the fusion sequence is GRKKRRGRRRPPSGGITMAARMKRKV (SEQ ID NO: 4).

[0049] Step 4: Convert the fusion sequence obtained in Step 3 into a D-type amino acid reverse order and fuse it with a FITC fluorescent label to obtain the final amino acid sequence of the interfering peptide ISQORβ2; the structure of the interfering peptide ISQORβ2 includes a polypeptide sequence as shown in VKRKMRAAMTIGGSPPRRRGRRKKRG (SEQ ID NO: 1) and a 5-fluorescein isothiocyanate (5-FITC) covalently linked to the N-terminus of the polypeptide, wherein the 5-FITC is linked to the first amino acid residue by a 6-aminohexanoic acid (Acp) linker group; forming the complete structure 5-FITC-(Acp)-VKRKMRAAMTIGGSPPRRRGRRKKRG.

[0050] Example 2: Synthesis and Detection of ISQORβ2

[0051] (1) Synthesis: Solid-phase synthesis was adopted, using D-type amino acids as raw materials (supplier: Jier Biochemical (Shanghai) Co., Ltd.). The synthesis sequence FITC-VKRKMRAAMTIGGSPPRRRGRRKKRG was synthesized. The synthesis of the interfering peptide ISQORβ2 and the detection of its molecular weight and purity were entrusted to Qiangyao Biotechnology.

[0052] (2) Molecular weight determination: Analyzed using an Agilent-6125B liquid chromatography-mass spectrometry system, molecular weight 3510.01 Da (see...) Figure 2 ).

[0053] (3) Purity determination: HPLC was performed using an Inertsil ODS-SP column (Shimadzu, 4.6 mm × 250 mm) as the stationary phase, with gradient elution using mobile phases A (acetonitrile containing 0.1% trifluoroacetic acid) and B (ultrapure water containing 0.1% trifluoroacetic acid) (gradient program shown in Table 1). The purity was 96.64% (see Table 1). Figure 3 ).

[0054] Table 1: HPLC gradient elution program

[0055] Time (min) mobile phase A proportion (%) mobile phase B proportion (%) 0.0 22 78 20 47 53 20.1 100 0

[0056] (4) Biological verification: as attached Figures 4-13 In cell models (such as transfected human lung epithelial cells) and animal models (VSV-NP infected mice), ISQORβ2 was found to inhibit NP-induced autophagy, restore the expression of immune molecules, and enhance anti-replication efficacy.

[0057] 4.1 Laser confocal analysis revealed that the interfering peptide ISQORβ2 significantly binds to NP.

[0058] Experimental methods: H1299 cells were seeded in 35 mm glass-bottom cell culture dishes and transfected with the plasmids or treated with the drugs as described below. Specifically, 1 million H1299 cells were seeded into four 35 mm glass-bottom cell culture dishes. After 6 hours, the cells adhered to the dish. Sp (control peptide) was added to two dishes and ISQORβ2 (20 μM) was added to two other dishes. The sp and ISQORβ2 dishes were transfected with the flag-IBV-N plasmid, and the flag-SARS-CoV-2-N plasmid was transfected into the sp and ISQORβ2 dishes, respectively. The dishes were then incubated for 24 hours. The structure of sp includes a polypeptide sequence as shown in TSNRSSNRSRPPRRRGRRKKRG (SEQ ID NO: 5) and 5-FITC (5-isothiocyanate fluorescein) covalently linked to the N-terminus of the polypeptide, wherein the 5-FITC is linked to the first amino acid residue by a 6-aminohexanoic acid (Acp) linker group; forming the complete structure 5-FITC-(Acp)-TSNRSSNRSRPPRRRGRRKKRG.

[0059] The structure of the interfering peptide ISQORβ2 comprises a polypeptide sequence as shown in VKRKMRAAMTIGGSPPRRRGRRKKRG (SEQ ID NO: 1) and a 5-fluorescein isothiocyanate (5-FITC) covalently linked to the N-terminus of the polypeptide, wherein the 5-FITC is linked to the first amino acid residue via a 6-aminohexanoic acid (Acp) linker group; forming the complete structure 5-FITC-(Acp)-VKRKMRAAMTIGGSPPRRRGRRKKRG, where sp and ISQORβ2 refer to the amino acid sequence shown herein.

[0060] Twenty-four hours later, cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and then blocked with 5% bovine serum albumin (BSA) for 30 minutes at room temperature. After washing three times with PBS, cells were incubated overnight at 4°C with primary antibody (flag-tagged primary antibody, 1:100 dilution), and then incubated for 1 hour at room temperature with Alexa Fluor 594 red fluorescently labeled secondary antibody (1:200 dilution). Nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI) at room temperature for 10 minutes. Images were acquired and processed using a ZEISS confocal immunofluorescence microscope and a ZEN system (ZEISS, Oberkochen, Germany). Results are shown below. Figure 4 , Figure 4 The figure shows the experimental results of laser confocal microscopy analysis, which found that the interfering peptide ISQORβ2 can significantly bind to NP. Laser confocal microscopy observation shows that FITC-labeled ISQORβ2 (green) and overexpressed NP protein (red) are colocalized in cells (yellow), suggesting that the two may interact.

[0061] 4.2 Immunoprecipitation analysis revealed that the use of the interfering peptide ISQORβ2 significantly inhibited the interaction between SQOR and NP in cells.

[0062] Experimental groups: IBV-N group and SARS-CoV-2-N group were set up.

[0063] IBV-N experimental grouping: Groups 1 and 2 were treated with SP, and Group 3 was treated with ISQORβ2 at a dose of 20 μM. Six hours later, Group 1 was transfected with the empty flag antibody for 24 hours, Group 2 with Flag-ibv-N for 24 hours, and Group 3 with Flag-IBV-N for 24 hours. After extraction with RIPA lysis buffer (Beyotime, P0013B), intraperitoneal (IP) experiments were performed using the flag primary antibody, as detailed in the experimental procedure.

[0064] The design logic for the SARS-CoV-2-N group is the same, except that the transfection plasmid is replaced with Flag-SARS-CoV-2-N, which is used to compare whether the interaction between the two NP proteins (IBV-N, SARS-CoV-2-N) and SQOR is inhibited by ISQORβ2.

[0065] Experimental Methods: H1299 cells were inoculated into cell culture plates at a density of 1 million cells per group. Cell samples were obtained after grouping and processing as described above. Total protein from the cell samples was extracted using RIPA lysis buffer (Beyotime, P0013B) containing a protease inhibitor mixture (Sangon Biotech, A003587), and protein concentration was determined using a BCA protein quantification kit (ThermoScientific, A55864). Then, equal amounts of protein from each sample were subjected to SDS-PAGE electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010). The PVDF membrane was then blocked at room temperature with rapid blocking buffer (Epizyme Biomedical, PS108) for 90 minutes, incubated overnight at 4°C with primary antibody (Flag primary antibody, 1:1000 dilution), and then incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-labeled antibody (1:10000 dilution). Protein bands were visualized using an AI680 imaging system (GE Health Care, Chicago, USA) and incubated overnight with primary antibodies against SQOR, IBV-N, and IBV-M proteins. Results are as follows: Figure 5 As shown. Figure 5 The figure shows the experimental results of immunoprecipitation analysis, which revealed that the interfering peptide ISQORβ2 can significantly inhibit the interaction between overexpressed SQOR and NP in cells, indicating that ISQORβ2 inhibits SQOR-NP interaction.

[0066] like Figure 5 As shown, Input (total protein, verifying the basis of protein expression): The expression level of SQOR protein was consistent in all groups (excluding the influence of "changes in SQOR expression itself" on the interaction); In groups 2 and 3, the Flag-IBV-N / SARS-CoV-2-N bands were clear and had similar intensities (proving that NP protein was overexpressed and that ISQORβ2 did not affect the expression level of NP); In group 1 (empty vector), the Flag band was extremely weak (no NP expression, consistent with the expectation of the negative control).

[0067] IP: Flag (co-precipitated product, directly reflecting the interaction): Group 1 (empty vector): Both Flag and SQOR bands are extremely weak (proving that the Flag antibody does not specifically bind, and the IP background is low); Group 2 (SP+NP): The Flag (NP) band is strong, and the SQOR band is significant (indicating that NP and SQOR have a stable interaction); Group 3 (ISQORβ2+NP): The intensity of the Flag (NP) band is similar to that of Group 2 (consistent IP efficiency), but the SQOR band is significantly weakened (directly proving that ISQORβ2 inhibits the interaction between the two).

[0068] 4.3 Enzyme activity detection

[0069] Experimental grouping: Three groups were set up. Groups 1 and 2 were given sp, and group 3 was given ISQORβ2 at a dose of 20uM. After 6 hours, group 1 was transfected with flag empty vector for 24 hours, group 2 was transfected with Flag-IBV-N for 24 hours, and group 3 was transfected with Flag-IBV-N for 24 hours.

[0070] Experimental Methods: H1299 cells were inoculated into cell culture plates, with 1 million cells per group. After grouping and processing as described above, cell samples were obtained. Intracellular SQOR enzyme activity was detected using an assay kit (Yuanxin Biological, YX-191715H). Results are as follows: Figure 6 As shown. Figure 6 The study found that using the interfering peptide ISQORβ2 could restore SQOR function affected by NP, as determined by enzyme activity assay.

[0071] 4.4 Flow cytometry analysis revealed intracellular H2S accumulation using the interfering peptide ISQORβ2.

[0072] Experimental grouping: Three groups were set up. Groups 1 and 2 were given sp, and group 3 was given ISQORβ2 at a dose of 20uM. After 6 hours, group 1 was transfected with Flag empty vector for 24 hours, group 2 was transfected with flag-ibv-n for 24 hours, and group 3 was transfected with Flag-IBV-N for 24 hours.

[0073] Experimental Methods: H1299 cells were inoculated into cell culture plates at a density of 1 million cells per group. Cell samples were obtained after the above-described grouping process. Intracellular H2S content was detected using a colorimetric method with an H2S assay kit (Solarbio, BC2050) and flow cytometry using the H2S fluorescent probe SSP4 (Dojindo, SB10), according to the manufacturer's instructions. Absorbance was measured using a spectrophotometer (Tecan Spark, Männedorf, Switzerland), while intracellular fluorescence intensity was detected by flow cytometry using FACSuite (BD Biosciences, San Diego, USA). Results are as follows: Figure 7 As shown. Figure 7 To detect intracellular H2S accumulation using the interfering peptide ISQORβ2 via flow cytometry.

[0074] 4.5 Flow cytometry analysis revealed that the interfering peptide ISQORβ2 could inhibit NP-induced mitochondrial membrane potential depolarization.

[0075] JC-1 is an ideal fluorescent probe widely used for detecting mitochondrial membrane potential (ΔΨm). It can detect the membrane potential of cells, tissues, or purified mitochondria. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix, forming J-aggregates that produce red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix; in this case, JC-1 exists as a monomer and produces green fluorescence. This allows for convenient detection of changes in mitochondrial membrane potential by observing the change in fluorescence color. The relative ratio of red to green fluorescence is commonly used to measure the proportion of mitochondrial depolarization.

[0076] H1299 cells were inoculated into cell culture plates at a density of 1 million cells per group. Experimental treatments were as follows: Groups 1 and 2 received sp. (sp.), and Group 3 received ISQORβ2 at a dose of 20 μM. Six hours later, Group 1 was transfected with the empty flag vector for 24 hours, Group 2 with Flag-IBV-N for 24 hours, and Group 3 with Flag-IBV-N for 24 hours. Live cells were then digested with trypsin to detect JC-1 levels. Intracellular JC-1 levels were measured using a JC-1 assay kit (Solarbio, C2006) via flow cytometry. Results are as follows: Figure 8 As shown. Figure 8 Flow cytometry analysis revealed that the interfering peptide ISQORβ2 inhibited NP-induced mitochondrial membrane potential depolarization. The left image shows a blank control, the middle image shows transfection with IBV-N protein particles, and the right image shows transfection with IBV-N protein particles combined with the interfering peptide ISQORβ2.

[0077] 4.6 Western blot analysis revealed that the interfering peptide ISQORβ2 could inhibit NP-induced mitophagy in cells.

[0078] Experimental groups: IBV-N group and SARS-CoV-2-N group were set up.

[0079] The Ibv-n experimental groupings were as follows: Groups 1 and 2 were treated with SP, and Group 3 was treated with ISQORβ2 at a dose of 20 μM. Six hours later, Group 1 was transfected with Flag-IBV-n empty vector for 24 hours, Group 2 with Flag-IBV-n for 24 hours, and Group 3 with Flag-IBV-N for 24 hours. After extraction with RIPA lysis buffer (Beyotime, P0013B), intraperitoneal (IP) experiments were performed using the Flag primary antibody, as detailed in the experimental procedure.

[0080] The design logic for the SARS-CoV-2-N group is the same, except that the transfection plasmid is replaced with Flag-SARS-CoV-2-N.

[0081] Experimental Methods: H1299 cells were inoculated into cell culture plates at a density of 1 million cells per group. Cell samples were obtained after grouping and processing as described above. Total protein from the cell samples was extracted using RIPA lysis buffer (Beyotime, P0013B) containing a protease inhibitor mixture (Sangon Biotech, A003587), and protein concentration was determined using a BCA protein quantification kit (ThermoScientific, A55864). Then, equal amounts of protein from each sample were subjected to SDS-PAGE electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010). The PVDF membrane was then blocked at room temperature with rapid blocking buffer (Epizyme Biomedical, PS108) for 90 minutes, incubated overnight at 4°C with primary antibody (1:1000 dilution), and then incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-labeled antibody (1:10000 dilution). Protein bands were visualized using an AI680 imaging system (GE Health Care, Chicago, USA). Incubation was performed overnight with primary antibodies against LC3, SQSTM1, PINK1, PARKIN, TOM20, COX4, ILF3, MAVS, and FLAG proteins. Results are as follows: Figure 9 As shown. Figure 9 Western blotting analysis revealed that the interfering peptide ISQORβ2 inhibited NP-induced mitophagy in cells; the left image shows transfection with the ibv-n protein plasmid, and the right image shows the addition of the interfering peptide ISQORβ2 after transfection with the SARS-CoV-2-N plasmid. Figure 9 It is known that the interfering peptide ISQORβ2 can inhibit NP-induced mitophagy in cells.

[0082] 4.7 qPCR detection revealed that the use of the interfering peptide ISQORβ2 could restore the downregulation of classic innate immune molecules in cells.

[0083] Experimental groups: IBV-N group and SARS-CoV-2-N group were set up.

[0084] IBV-N experimental grouping: Four experimental groups were prepared. Groups 1, 2, and 3 were treated with sp, and group 4 was treated with ISQORβ2 at a dose of 20 uM. After 6 hours, groups 1 and 2 were transfected with the flag blank plasmid, and groups 3 and 4 were transfected with the Flag-IBV-N plasmid. After 12 hours in an incubator, groups 2, 3, and 4 were transfected with Poly(I:C) (Polyinosinic-polycytidylic acid). RNA samples were collected after 8 hours.

[0085] The design logic for the SARS-CoV-2-N group is the same, except that Flag-IBV-N is replaced with Flag-SARS-CoV-2-N, and everything else remains the same.

[0086] Experimental Methods: H1299 cells were inoculated into cell culture plates at a density of 1 million cells per group. Cell samples were obtained after the above-described grouping process. Total RNA was extracted from the cell samples using an RNA-Quick purification kit (Yishan, RN001). Quantitative real-time PCR (RT-qPCR) was performed according to the instructions for HiScript II reverse transcriptase SuperMix with gDNA remover (Vazyme, RR047A) and TB Green PremixEx Taq™ II (Takara, RR420A) to determine the relative expression levels of the corresponding genes. ACTB was used as an internal control. RT-qPCR primers are shown in Table 2. The relative fold change of the target gene was calculated using 2 - ΔΔCt (ΔCt = ΔCttarget – ΔCtACTB, ΔΔCt = ΔCt – ΔCtcontrol). IFNB RT-qPCR primers were used for detection. Results are as follows: Figure 10 As shown. Figure 10 The image shows the experimental results of qPCR detection, which showed that the interfering peptide ISQORβ2 can restore the downregulation of classic innate immune molecules in cells; the left is IBV-N, and the right is SARS-CoV-2-N.

[0087] Table 2 Primers used for reverse transcription quantitative polymerase chain reaction (RT-qPCR)

[0088] Gene Upstream primer sequence (5'-3') Downstream primer sequence (5'-3') Human IFNB1 CTATTGTTGAGAACCTCCTGG (SEQ ID NO: 6) TCGGAGGTAACCTGTAAGTC (SEQ ID NO: 7) Human ACTB CACCAACTGGGACGACAT (SEQ ID NO: 8) ACAGCCTGGATAGCAACG (SEQ ID NO: 9)

[0089] Depend on Figure 10 It is known that the interfering peptide ISQORβ2 can restore the downregulation of classic innate immune molecules in cells.

[0090] 4.8 TCID50 Detection

[0091] VSV is a model virus. By inserting the IBV protein and SARS-CoV-2-N protein into the VSV vector, recombinant VSV (VSV-IBV-N) and recombinant VSV (VSV-SARS-CoV-2-N) were obtained. The construction of recombinant VSV (VSV-IBV-N) and recombinant VSV (VSV-SARS-CoV-2-N) was entrusted to Gemma Corporation.

[0092] Vero E6 cells were infected with recombinant VSV: Recombinant VSV was added to serum-free Vero E6 cells at 1×10^6 pfu for 3 hours. After washing twice with PBS, medium containing 2% fetal bovine serum was added. Cells were harvested 48 hours after infection and subjected to three freeze-thaw cycles. After centrifugation at 12,000 × g for 10 minutes at 4°C, the supernatant was analyzed for TCID50 (50% tissue cell infection dose). The virus suspension was diluted 10-fold with DMEM containing 2% FBS and added to fresh Vero E6 cells in 96-well plates, simultaneously treated with SP or ISQORβ2. Each dilution was tested eight times. Immunofluorescence analysis of cells was performed using the indicated antibody 48 hours after infection. Viral titers were determined by observing infected cells under a fluorescence microscope (OLYMPUS, U-RFL-T), and TCID50 was calculated at 0.1 mL using the Reed-Muench method. Results are as follows. Figure 11 As shown. Figure 11 TCID50 was used to construct recombinant VSV expressing IBV-N (VSV-IBV-N) or recombinant VSV expressing SARS-CoV-2-N (VSV-SARS-CoV-2-N), and it was found that viral replication was significantly blocked by ISQORβ2 in a dose-dependent manner.

[0093] 4.9 Hematoxylin-eosin (HE) staining and immunohistochemical experiments

[0094] The two groups used 8 seven-day-old chicks, divided into two groups of 4. The chicks were infected with 50 μL of VSV-IBV-N (1×10^8 pfu) by nasal and ocular drops. Three days before infection, the animals were pre-inoculated nasally with interfering peptide ISQORβ2 or random peptide SP (30 mg / kg, 50 μL volume), and then infected with the virus on the third day according to the above method.

[0095] Eight 14-day-old BALB / c mice were used in both VSV-SARS-CoV-2-N groups, divided into two groups of four. The mice were infected with 50 μL of VSV-SARS-CoV-2-N (5 × 10^8 pfu) via nasal or ocular instillation. Three days prior to infection, the animals were pre-inoculated intranasally with either the interfering peptide ISQORβ2 or the random peptide SP (30 mg / kg, 50 μL volume). Infection with the virus was then performed on the third day using the same method. Both the chicks and BALB / c mice were purchased from the Zhejiang University animal platform.

[0096] Seven days after infection, mice and chicks were anesthetized with dry ice, dissected, and then immersed in formalin before being sent to Frix Medical Systems for section staining: lung tissue samples were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Lung tissue sections were excised from the paraffin blocks. For H&E staining, lung tissue sections were treated with a hematoxylin-eosin staining kit and observed under an optical microscope. Immunohistochemistry was performed using the original antibody (1:100 dilution), including TOM20, ILF3, and FLAG, followed by secondary antibody (HRP-labeled antibody, 1:500). For immunofluorescence detection, cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI). Image acquisition and processing were performed using a Leica fluorescence microscope and a Leica LAS X microscopy system (Leica, Weitzler, Germany). Results are as follows. Figure 12 As shown. Figure 12 This image shows the results of hematoxylin-eosin (HE) staining and immunohistochemical experiments, demonstrating that treatment with the interfering peptide ISQORβ2 in mice / chicks infected with recombinant VSV-NP can inhibit lung lesions induced by recombinant VSV-NP infection. The left two columns represent the chicken VSV-IBV-N infection model; the right two columns represent the mouse VSV-SARS-CoV-2-N infection model.

[0097] Depend on Figure 12 It can be seen that the SP group showed severe pathological damage to the lung tissue (alveolar collapse, inflammatory cell infiltration) and weak TOM20 signal (due to increased mitochondrial degradation); the ISQORβ2 group showed significantly reduced pathological damage to the lung tissue and enhanced TOM20 signal (due to increased mitochondrial retention), indicating that ISQORβ2 can alleviate NP-mediated viral lung injury.

[0098] 4.10 Recombinant VSV-NP infection of mouse and chicken lung tissue

[0099] Experimental Groups:

[0100] The VSV-IBV-N group used 12 seven-day-old chicks, divided into 3 groups of 4 chicks each. The first group was the control group, while the second and third groups were infected with the virus. The chicks were infected with 50 μl of VSV-IBV-N (1×10^8 p.fu) via nasal or ocular drops. Three days before infection, the animals were pre-inoculated nasally with either the interfering peptide ISQORβ2 (third group) or the random peptide SP (second group) (30 mg / kg, 50 μL volume). At the same time, a control group (first group) that was not infected with VSV-IBV-N but was pre-inoculated with the random peptide SP was set up. Then, on the third day, the chicks were infected with the virus in the same way as above.

[0101] The VSV-SARS-CoV-2-N group used 12 14-day-old BALB / c mice, divided into 3 groups of 4 mice each. Group 1 served as the control group, while groups 2 and 3 were infected with the virus. Mice were infected with 50 μL of VSV-SARS-CoV-2-N (5 × 10^8 pfu) via nasal or ocular drops. Three days prior to infection, the animals were pre-inoculated intranasally with either the interfering peptide ISQORβ2 (group 3) or the random peptide SP (group 2) (30 mg / kg, 50 μL volume). A control group (group 1) was also included, consisting of mice that were not infected with VSV-IBV-N but were pre-inoculated with the random peptide SP. The mice were then infected with the virus on the third day using the same method. Both the chicks and BALB / c mice were purchased from the Zhejiang University animal platform.

[0102] Seven days after infection, mice and chicks were anesthetized with dry ice, and lung tissue samples were collected after dissection. Total RNA was extracted from the lung tissue samples using an RNA-Quick purification kit (Yishan, RN001). Quantitative real-time PCR (RT-qPCR) was performed according to the instructions of HiScript II reverse transcriptase SuperMix with gDNA remover (Vazyme, RR047A) and TB Green Premix Ex Taq™ II (Takara, RR420A) to determine the relative expression levels of the corresponding genes. ACTB was used as an internal control. RT-qPCR primers are shown in Table 3. The relative fold change of the target gene was calculated using 2 - ΔΔCt (ΔCt = ΔCttarget – ΔCtACTB, ΔΔCt = ΔCt – ΔCtcontrol). Detection was performed using IFNB RT-qPCR primers. Results are shown below. Figure 13 As shown. Figure 13 The figure shows the results of how the interfering peptide ISQORβ2 enhances the expression of classical innate immune molecules mRNA in mouse and chicken lung tissues infected with recombinant VSV-NP. In the figure, AD represents the relative expression levels of Gallus Ifnb1, Gallus Cxcli2, Gallus Cxcli1, and Gallus Ifit5 mRNAs in chicken lung tissues infected with VSV-IBV-N, respectively; EH represents the relative expression levels of Mouse Ifnb1, Mouse Cxcl10, Mouse Cxcl15, and MouseIfit1 mRNAs in mouse lung tissues infected with VSV-IBV-N, respectively.

[0103] Table 3 Primers used for reverse transcription quantitative polymerase chain reaction (RT-qPCR)

[0104] Gene Upstream primer sequence (5'-3') Downstream primer sequence (5'-3') Mouse Ifnb1 CTCAACTGCTCTCAACACAC (SEQ ID NO: 10) TAAAGGTTGCCTTGTTGCAACC (SEQ ID NO: 11) Mouse Cxcl10 CGTTTAACAGGCACTCGAGAA (SEQ ID NO: 12) GGCAATGATCTCAACACGTG (SEQ ID NO: 13) Mouse Cxcl15 TTATAGGCAAACGAAAATGTGGATGG (SEQ ID NO: 14) TACATGTTCACAGTCCTCTTTGG (SEQ ID NO: 15) Mouse Ifit1 GTCACTTCACATGGAAGCTG (SEQ ID NO: 16) AGGCTTCTCTTTGTTGCTCAG (SEQ ID NO: 17) Mouse Actb CTACCTCATGAAGATCCTGACC (SEQ ID NO:18) CACAGCTTCTCTTTGATGTCAC (SEQ ID NO: 19) Gallus Ifnb1 CACCAAGGTTATGAACAACTGC (SEQ ID NO: 20) CTGCTTGCTTCTTGTCCTTG (SEQ ID NO: 21) Gallus Cxcli1 ATGAACGGCAAGCTTGGAGCT (SEQ ID NO: 22) GCCATAAGTGCCTTTACGATCAG (SEQ ID NO: 23) Gallus Cxcli2 TGGCTCTTCTCCTGATCTCAATG (SEQ ID NO: 24) GCACTGGCATCGGAGTTC(SEQ ID NO: 25) Gallus Ifit5 CAAACAAGCTGAAGCACTCC(SEQ ID NO: 26) GAAGGTGAGATCTGTTTTCCAG(SEQ ID NO: 27) Gallus Actb CATCACCATTGGCAATGAGAGG(SEQ ID NO: 28) GATTCATCGTACTCCTGCTTGC(SEQ ID NO: 29)

[0105] Experimental results demonstrate that the interfering peptide ISQORβ2 can enhance the mRNA expression of classical innate immune molecules in the lung tissues of mice and chickens infected with recombinant VSV-NP.

[0106] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A peptide that inhibits coronavirus nucleocapsid protein-induced mitophagy interference, characterized in that, It is composed of D-type amino acids, and its structure includes: The polypeptide sequence shown in SEQ ID NO: 1 is: VKRKMRAAMTIGGSPPRRRGRRKKRG; A functional group covalently attached to the N-terminus of the polypeptide; The functional group includes fluorescein 5-isothiocyanate.

2. The peptide for inhibiting mitochondrial autophagy interference induced by coronavirus nucleocapsid protein according to claim 1, characterized in that, The interfering peptide targets amino acid regions 181-246 of the SARS-CoV-2-N protein and amino acid regions 156-214 of the IBV-N protein, inhibiting the interaction between coronavirus nucleocapsid protein and thioquinone oxidoreductase and the activation of mitophagy, thereby restoring the downregulation of innate immune molecules induced by coronavirus nucleocapsid protein.

3. A method for preparing the interfering peptide as described in any one of claims 1-2, characterized in that, Includes the following steps: Step 1: Through molecular docking and immunoprecipitation experiments, it was confirmed that the interaction region between SARS-CoV-2-N protein and thioquinone oxidoreductase is the amino acid region of position 181-246, and the interaction region between IBV-N protein and thioquinone oxidoreductase is the amino acid region of position 156-214. Step 2: Using the interaction region between the SARS-CoV-2-N protein and IBV-N protein and thioquinone oxidoreductase described in Step 1 as the target, design and obtain a polypeptide that competes with this region for binding. Step 3: Fuse the peptide obtained in Step 2 with the HIV-TAT sequence to form a fusion sequence; Step 4: Convert the fusion sequence obtained in Step 3 into a reverse D-type amino acid sequence and fuse it with a functional group to obtain the final amino acid sequence of the interfering peptide ISQORβ2. Step 5: Synthesize the interfering peptide ISQORβ2 using D-type amino acids as raw materials, and then purify it.

4. The preparation method according to claim 3, characterized in that, In step 2, a polypeptide that competes with the region of interaction between SARS-CoV-2-N protein and IBV-N and thioquinone oxidoreductase was designed and obtained, with the amino acid sequence of the obtained polypeptide as shown in SEQ ID NO:

3.

5. The preparation method according to claim 3, characterized in that, In step 3, the amino acid sequence of the HIV-TAT is shown in SEQ ID NO:

2. The amino acid sequence of the fusion sequence is shown in SEQ ID NO:

4.

6. The preparation method according to claim 3, characterized in that, In step 3, the amino acids obtained after HIV-TAT is fused with the obtained polypeptide are L-type natural amino acids.

7. The preparation method according to claim 3, characterized in that, In step 4, the structure of the interfering peptide ISQORβ2 comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 and a functional group covalently linked to the N-terminus of the polypeptide; its amino acids are D-type amino acids.

8. The preparation method according to claim 3, characterized in that, The purification in step 5 was performed using high performance liquid chromatography gradient elution.

9. The use of the interfering peptide as described in claim 1 in the preparation of a lesion drug for inhibiting coronavirus infection, characterized in that, The coronaviruses are IBV-N and SARS-CoV-2-N; the interfering peptide is used to block the interaction between IBV-N or SARS-CoV-2-N and thioquinone oxidoreductase, thereby inhibiting mitophagy, restoring the expression of innate immune molecules, or treating coronavirus-related lung injury.

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