An interfering peptide for inhibiting SARS-CoV-2-induced cell senescence, and its preparation method and application

Abstract

The present invention discloses an interfering peptide for inhibiting SARS-CoV-2-induced cell senescence, and a preparation method and application thereof. The method is to use the 175-246 amino acid region of SARS-CoV-2 NP to screen and design an interfering peptide segment through a T7 phage display library, and fuse it with HIV-TAT; the interfering peptide segment fused with HIV-TAT is modified to a D-type amino acid retroisomer to obtain the amino acid sequence of the final interfering peptide NMIP-Ⅲ; and the interfering peptide NMIP-Ⅲ is synthesized using D-type amino acids as raw materials. The interfering peptide NMIP-Ⅲ of the present invention can interact with the 175-246 amino acid region of SARS-CoV-2 NP, inhibit the interaction between NP and mitochondrial fusion protein MFN1 / 2 and the activation of the NF-κB pathway, thereby inhibiting the expression of SASP induced by NP, thereby inhibiting SARS-CoV-2-induced cell senescence.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug preparation, and specifically relates to an interfering peptide for inhibiting SARS-CoV-2-induced cell senescence, and a preparation method and application thereof. Background Art

[0002] The new coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) belongs to the genus Coronavirus (CoV), and its basic structure is composed of M protein, S protein, N protein, E protein, genomic single-stranded RNA and envelope. N protein (NP) is the core component of the virus particle. The SARS-CoV-2 N protein is 419 amino acids in length and mainly consists of the N-terminal RNA binding domain and the C-terminal dimerization domain as well as other sequences connecting the two domains. The dimerization domain can mediate the formation of homodimers of the N protein. The N protein contains multiple relatively conservative positive charge distribution regions that bind to RNA. It binds to the viral genomic RNA and packages the RNA into a ribonucleocapsid (RNP) complex.

[0003] A large number of case reports have shown that many COVID-19 patients will have obvious sequelae after recovery. Their physical fitness indicators such as vital capacity and maximum oxygen uptake are significantly lower than before infection. Studies at the subcellular level have shown that the new crown can induce mitochondrial dysfunction, autophagy, and changes in morphology and quality. SARS-CoV-2 M protein, ORF9c and other proteins interact with the electron transport chain and inner and outer membrane proteins of mitochondria, damage mitochondrial metabolism and control mitochondrial quality and morphology. Mitochondrial damage can induce an increase in mitochondrial reactive oxygen species (mROS) and leakage of mitochondrial DNA (mtDNA), which in turn activates immune responses through multiple signaling pathways such as cGAS-STING, leading to inflammatory storms, cell senescence and apoptosis.

[0004] The latest research shows that infection with SARS-CoV-2 can induce cellular senescence. Cells in the lung, liver, and kidney tissues of COVID-19 patients and in vitro cell lines infected with SARS-CoV-2 highly express the senescence marker p16. INK4a , p21 WAF1 / Cip1, as well as senescence-associated secretory phenotype (SASP)-related proteins such as IL-1A, CXCL10, IL-6, and IL-8, and both β-gal staining and lipofuscin staining were positive. This finding is consistent with previous reports that elderly COVID-19 patients are more likely to experience severe symptoms and have a higher mortality rate. It is speculated that the SASP expressed by senescent cells enhances the inflammatory response. Summary of the invention

[0005] Based on this, the present invention provides an interfering peptide (hereinafter named NMIP-Ⅲ) for inhibiting SARS-CoV-2-induced cell senescence, and a preparation method and pharmaceutical use thereof. The interfering peptide can interact with the intrinsically disordered Region 2 (IDR2) domain of SARS-CoV-2 NP (the 175-246 amino acid region of the N protein), inhibit the interaction between the N protein and the mitochondrial fusion protein MFN1 / 2 and the activation of the NF-κB pathway, thereby inhibiting the expression of SASP induced by NP, thereby achieving the purpose of inhibiting SARS-CoV-2-induced cell senescence, and contributing to the preparation of drugs for resisting SARS-CoV-2-induced cell senescence.

[0006] In order to solve the above technical problems, the present invention is implemented through the following technical solutions: An interfering peptide that inhibits SARS-CoV-2-induced cell senescence, composed of a D-type amino acid sequence shown by TSLCVLSVVIPPRRRQRRKKRG, and having a molecular weight of 2606.14 Da.

[0007] Furthermore, the interfering peptide can interact with the 175-246 amino acid region of SARS-CoV-2 NP by targeting the SARS-CoV-2 N protein, inhibiting the interaction between the N protein and the mitochondrial fusion protein MFN1 / 2 and the activation of the NF-κB pathway, thereby inhibiting the expression of NP-induced aging markers and aging-related secretory phenotype-related proteins, thereby inhibiting SARS-CoV-2-induced cell senescence.

[0008] A method for preparing an interfering peptide for inhibiting SARS-CoV-2-induced cell senescence, comprising 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 mitochondrial fusion-related protein MFN1 / 2 is the 175-246 amino acid region; Step 2) Targeting the 175-246 amino acid region of the SARS-CoV-2 N protein, the amino acid sequence of a peptide that interacts with this region was obtained by panning and designing a random 10-peptide T7 phage display library; Step 3) Fusing the designed peptide with HIV-TAT to make it have membrane-penetrating activity; Step 4) synthesizing the polypeptide fused with HIV-TAT in the form of reverse order of D-type amino acids to obtain the amino acid sequence of the final interfering peptide NMIP-Ⅲ; Step 5) D-amino acids are used as raw materials to synthesize the interfering peptide NMIP-Ⅲ, and the purity of the peptide segment is more than 99%.

[0009] Furthermore, in step 1), the interaction surface is amino acids 175-246 of the SARS-CoV-2 N protein.

[0010] Furthermore, in step 2), the amino acid region 175-246 of the SARS-CoV-2 N protein was used for panning of a 10-peptide random T7 phage display library.

[0011] Furthermore, the amino acid sequence of the polypeptide obtained by panning using the T7 phage display library is IVVSLVCLST.

[0012] Furthermore, in step 3), the amino acid sequence of HIV-TAT is GRKKRRQRRRPP, and the amino acid sequence after fusion with the polypeptide obtained by panning using the T7 phage display library is GRKKRRQRRRPPIVVSLVCLST.

[0013] Furthermore, the amino acids after the HIV-TAT is fused with the polypeptide obtained by panning using the T7 phage display library are L-type natural amino acids.

[0014] Furthermore, in step 4), the amino acid sequence of the interfering peptide NMIP-III is TSLCVLSVVIPPRRRQRRKKRG, the molecular weight is 2606.14 Da, and its amino acids are D-type amino acids.

[0015] The artificially synthesized D-type amino acid reverse isomers not only have a structure similar to that of L-type amino acids, but also can give the interfering peptide new chemical properties, thereby improving the stability of the interfering peptide NMIP-Ⅲ in vitro and in vivo. In addition, HIV-TAT is a hydrophilic sequence, which, after fusion with the interfering peptide segment, can enable the interfering peptide NMIP-Ⅲ to have the ability to cross the cell membrane in an energy-independent manner and be absorbed by the cell.

[0016] Application of an interfering peptide for inhibiting SARS-CoV-2-induced cell senescence in the preparation of a drug for resisting SARS-CoV-2-induced cell senescence.

[0017] The beneficial effects of the present invention are: 1. The interfering peptide NMIP-Ⅲ targeting SARS-CoV-2 N protein prepared by the present invention significantly inhibited SARS-CoV-2 N protein-mediated mitochondrial dysfunction, activation of NF-κB pathway and expression of SASP-like factors, and inhibited SARS-CoV-2-induced cell senescence.

[0018] 2. The interfering peptide NMIP-Ⅲ targeting the 175-246 amino acid region of the SARS-CoV-2 N protein prepared by the present invention can effectively block the interaction between the SARS-CoV-2 N protein and the mitochondrial fusion-related protein MFN1 / 2. The interaction between proteins is usually a face-to-face interaction. Compared with traditional small molecule drugs, peptide drugs can more effectively block the interaction between proteins, and experiments have found that the peptide drugs also have a significant inhibitory effect on the N protein of the latest mutant strain JN.1 of SARS-CoV-2.

[0019] 3. The interfering peptide NMIP-Ⅲ targeting SARS-CoV-2 N protein prepared by the present invention contains the HIV-TAT 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 and side effects caused by the carrier.

[0020] 4. The interfering peptide NMIP-Ⅲ targeting SARS-CoV-2 N protein prepared by the present invention can be directly obtained through existing mature peptide synthesis technology, has the characteristics of high purity, almost no need for purification, controllable quality, and great potential for drug development.

[0021] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. The specific implementation of the present invention is given in detail by the following embodiments and their accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 This is a structural diagram of the interaction between the SARS-CoV-2 NP-IDR2 domain and NMIP-Ⅲ.

[0023] Figure 2Schematic diagram of the molecular weight of the synthetic interfering peptide NMIP-Ⅲ detected by mass spectrometry.

[0024] Figure 3 Schematic diagram of using HPLC to detect the purity of the synthesized interfering peptide NMIP-Ⅲ.

[0025] Figure 4 This is a diagram of the experimental results showing that the use of the interfering peptide NMIP-Ⅲ can significantly inhibit the interaction between overexpressed MFN1 / 2 and NP in cells through co-immunoprecipitation analysis.

[0026] Figure 5 This is the experimental result showing that NMIP-Ⅲ can significantly inhibit NP-induced mitochondrial content leakage through Western Blot detection after separation of mitochondria and cytoplasm (left) and live cell staining detection through laser confocal microscopy (right).

[0027] Figure 6 This is an experimental result showing that using laser confocal microscopy to stain live cells, it was found that treating cells transfected with NP-CFP with the interfering peptide NMIP-Ⅲ could inhibit NP-induced mitochondrial condensation around the nucleus.

[0028] Figure 7 This is a diagram of the experimental results showing that the use of the interfering peptide NMIP-Ⅲ can inhibit the increase of ROS induced by NP through flow cytometry detection.

[0029] Figure 8 This is the experimental result showing that the interference peptide NMIP-III treatment inhibited NP-induced A549 cell growth slowing and rescued cell proliferation.

[0030] Fig. 9 This is the experimental result showing that the interfering peptide NMIP-III can inhibit NP-induced RIP1 phase separation.

[0031] Fig.10 The experimental results of qPCR detection (left) and WB detection (right) show that NMIP-III can inhibit the increase of SASP-like factors in PM cells induced by NP-VSV infection.

[0032] Fig.11 This is a graph showing the experimental results of β-gal staining to detect that NMIP-III can inhibit NP-VSV-induced cell senescence.

[0033] Fig.12 The experimental results of qPCR detection (left) and WB detection (right) of mouse spleen, liver and lung showed that NMIP-III can inhibit the increase of SASP-like factors in mouse spleen, liver and lung induced by NP-VSV.

[0034] Fig.13This is the experimental result showing that qPCR detected that NMIP-III can inhibit the SASP induced by the latest mutant strain of SARS-CoV-2, JN.1 NP.

[0035] Fig.14 This is a diagram showing the experimental results of WB detection that NMIP-III can inhibit JN.1 NP-induced mitochondrial content leakage.

[0036] Fig.15 This is a diagram of the experimental results showing that co-immunoprecipitation detection of NMIP-III can inhibit the interaction between JN.1 NP and MFN1 / 2 induced by NP. DETAILED DESCRIPTION

[0037] The inventors of the present invention screened all SARS-CoV-2 encoded proteins in their previous work and found that N protein (NP) can significantly induce the cell senescence marker p16 INK4a , p21 WAF1 / Cip1 , as well as SASP factors such as IL-1A, CXCL10, IL-6, and IL-8, significantly enhanced β-gal staining and inhibited cell proliferation, indicating that SARS-CoV-2 NP plays an important role in inducing cell senescence. The inventors of the present invention determined through molecular docking and immunoprecipitation that the N protein interacts with the mitochondrial fusion-related protein MFN1 / 2 through its IDR2 region, causing abnormal mitochondrial morphology, leakage of mitochondrial contents, and a significant increase in ROS, which further activates the NF-κB pathway, leading to an increase in the expression of inflammatory SASP factors, while the lack of the IDR2 region of NP can completely inhibit its effect of inducing cell senescence.

[0038] Since protein-protein interactions have a large action area, a single small molecule may not be able to effectively interfere. It is very effective to use macromolecular drugs such as polypeptides (interfering peptides) with similar action surfaces to interfere with protein-protein interactions. In order to inhibit the effect of NP-induced cell senescence, according to the molecular mechanism of SARS-CoV-2 NP-induced cell senescence, the inventors of the present invention screened out NMIP-Ⅲ, a polypeptide drug that can specifically bind to the NP IDR2 region (175-246 amino acid region) through T7 phage library panning. Subsequently, NMIP-Ⅲ was artificially synthesized, and after being added to cells, it can directly bind to the NPIDR2 region (175-246 amino acid region) through the cell membrane, blocking its interaction with MFN1 / 2, inhibiting NP-induced mitochondrial aggregation, content leakage, ROS elevation, NF-κB pathway activation, and SASP-like factor expression, ultimately achieving the purpose of inhibiting SARS-CoV-2 infection-induced cell senescence, alleviating its clinical symptoms, and preventing new crown sequelae.

[0039] The method for preparing an interfering peptide for inhibiting SARS-CoV-2-induced cell senescence described in the present invention specifically comprises the following steps: Step 1) Through molecular docking and co-immunoprecipitation experiments, the interaction surface between SARS-CoV-2 N protein and mitochondrial fusion-related protein MFN1 / 2 was confirmed, and the interaction surface was the 175-246 amino acid region of SARS-CoV-2 N protein.

[0040] Step 2) Targeting the 175-246 amino acid region of the SARS-CoV-2 N protein, the amino acid sequence of a polypeptide that interacts with this region was obtained by panning and designing a random 10-peptide T7 phage display library, and its amino acid sequence was IVVSLVCLST.

[0041] Step 3) The polypeptide obtained by panning the T7 phage display library is fused with HIV-TAT to make it have transmembrane activity. The HIV-TAT is a hydrophilic sequence with an amino acid sequence of GRKKRRQRRRPP, which can make the peptide cross the cell membrane in an energy-independent manner and be absorbed by the cell. The amino acid sequence of HIV-TAT after fusion with the polypeptide is GRKKRRQRRRPPIVVSLVCLST, and its amino acids are L-type natural amino acids.

[0042] Step 4) The polypeptide fused with HIV-TAT is synthesized in the form of D-type amino acid reverse order to obtain the amino acid sequence of the final interfering peptide NMIP-Ⅲ, whose amino acid sequence is TSLCVLSVVIPPRRRQRRKKRG and molecular weight is 2606.14Da.

[0043] Step 5) The interfering peptide NMIP-Ⅲ is synthesized using D-amino acids as raw materials, and the purity of the peptide segment is more than 99%. The artificially synthesized D-amino acid reverse isomers not only have a structure similar to that of L-amino acids, but also can give the interfering peptide new chemical properties and improve the stability of the interfering peptide NMIP-Ⅲ in vitro and in vivo.

[0044] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention will be described in detail with reference to the accompanying drawings and in combination with embodiments. The descriptions made herein are used to provide a further understanding of the present invention and constitute a part of this application. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. Example 1

[0045] Synthesis and detection of the interfering peptide drug NIP-V.

[0046] The amino acid sequence of the interfering peptide drug NMIP-III designed by the present invention is TSLCVLSVVI, and the targeting sequence is as follows Figure 1 As shown, the D-amino acids were used as raw materials and synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0047] See also Figure 2 As shown, the molecular weight of the synthesized interfering peptide drug NMIP-III was identified as 2606.20 by Agilent-6125B LC-MS system (Agilent Technologies). HPLC used an Inertsil ODS-SP liquid chromatography column (Shimadzu, 4.6 mm × 250 mm) as the stationary phase, and used mobile phase A (100% acetonitrile, 0.1% trifluoroacetic acid) and mobile phase B (100% ultrapure water, 0.1% trifluoroacetic acid) for gradient elution, see Figure 3 As shown in Table 1, the purity was greater than 95% as determined by HPLC.

[0048] Table 1 Example 2

[0049] Immunoprecipitation analysis found that the use of interfering peptide NMIP-Ⅲ could significantly inhibit the interaction between overexpressed MFN1 / 2 and NP in cells.

[0050] 1. Experimental Materials HEK293T cells (ATCC source), NMIP-III interfering peptide drug prepared in Example 1, rabbit anti-Myc-Tag antibody, rabbit anti-Flag-Tag antibody and related secondary antibodies, Flag-beads.

[0051] 2. Test methods Sterile PBS was used to dissolve the NMIP-Ⅲ interfering peptide drug to a concentration of 1 mg / mL. NP-Myc and MFN1 / 2-Flag plasmids were co-transfected into 4 groups of HEK293T cells with the same cell density, and PBS, NMIP-Ⅰ, NMIP-Ⅱ, and NMIP-Ⅲ were added at a ratio of 1:1000, respectively. After 24 hours, the cells were collected for immunoprecipitation detection.

[0052] 3. Experimental results See also Figure 4 As shown, the interfering peptide NMIP-Ⅲ can significantly inhibit the interaction between overexpressed MFN1 / 2 and NP in cells.

[0053] The developed bands in the control group with PBS and the experimental group with NMIP-Ⅰ and NMIP-Ⅱ were darker in color, indicating that there was a strong interaction between MFN1 / 2 and NP and that NMIP-Ⅰ and NMIP-Ⅱ could not inhibit the interaction between MFN1 / 2 and NP. The developed bands in the experimental group with NMIP-Ⅲ were lighter in color, indicating that NMIP-Ⅲ could significantly inhibit the interaction between overexpressed MFN1 / 2 and NP in cells. Example 3

[0054] Western Blot and laser confocal detection showed that NMIP-Ⅲ could significantly inhibit NP-induced mitochondrial content leakage.

[0055] 1. Experimental Materials A549 cells (from ATCC), HeLa cells (from ATCC), laser confocal microscope, NMIP-Ⅲ interfering peptide drug prepared in Example 1, mouse anti-Tubulin antibody, rabbit anti-COX Ⅳ antibody (abclonal), rabbit anti-Myc-Tag antibody, Tom20 antibody (proteintech).

[0056] 2. Test methods Western Blot detection: Empty vector was transfected into the cells of the control group, and NP was transfected into the two experimental groups respectively. NMIP-Ⅲ was added into one group at 1:1000, and PBS was added into the other group. After 24 hours, the cells were harvested to separate the cytoplasm and mitochondria, and the contents of COX Ⅳ in the cytoplasm and mitochondria of each group were detected by WB.

[0057] Laser confocal detection: Cox8A-GFP and mCherry were co-transfected in the control group, and Cox8A-GFP and NP-mCherry were co-transfected in the two experimental groups. NMIP-Ⅲ was added to one group at a ratio of 1:1000, and PBS was added to the other group. After 24 hours, the cells were moved to a laser confocal microscope for observation.

[0058] 3. Experimental results See also Figure 5As shown in the figure, COX Ⅳ and COX8A are important components of the mitochondrial electron transport chain, and the leakage of mitochondrial contents can be simulated by COX Ⅳ and COX8A. Mitochondrial content leakage can lead to increased ROS, inflammatory response, etc., which further leads to the activation of the NF-κB pathway, the increase of SASP-like factors, and ultimately cell senescence. In the WB detection of isolated cytoplasm, NP significantly caused COX Ⅳ in the cytoplasm. After the addition of NMIP-Ⅲ, COX Ⅳ in the cytoplasm was significantly reduced, indicating that NMIP-Ⅲ can significantly inhibit the leakage of mitochondrial contents induced by NP. In order to further detect the leakage of mitochondrial contents, we combined the COX8A mitochondrial localization sequence with GFP. When the mitochondrial contents leaked, COX8A-GFP would also leak from the mitochondria to the cytoplasm, and green diffusion could be seen in the cytoplasm under a laser confocal microscope. In the laser confocal microscopy detection, green fluorescence diffusion was obviously seen in the cytoplasm of cells expressing NP, indicating that mitochondrial contents leaked into the cytoplasm. After the addition of NMIP-Ⅲ, the green fluorescence diffusion in the cytoplasm decreased, indicating that NMIP-Ⅲ could significantly inhibit the leakage of mitochondrial contents induced by NP. Example 4

[0059] Laser confocal microscopy revealed that treatment of cells transfected with NP-CFP with the interfering peptide NMIP-Ⅲ could inhibit NP-induced mitochondrial aggregation around the nucleus.

[0060] 1. Experimental Materials HeLa cells (sourced from ATCC), laser confocal microscope, NMIP-Ⅲ interfering peptide drug prepared in Example 1, Mito-Traker mitochondrial green fluorescent dye.

[0061] 2. Test methods CFP was transfected in the control group, and NP-CFP was transfected in the two experimental groups. NMIP-Ⅲ was added at a dilution of 1:1000 in one group and PBS was added in the other group. Mito-Traker mitochondrial green fluorescent dye (100 nM) was added to the cells 24 hours later. After incubation at 37 degrees for 30 minutes, the cells were moved to a laser confocal microscope for observation.

[0062] 3. Experimental results See also Figure 6 As shown, in cells transfected with NP, mitochondria were observed to obviously condense around the nucleus. After the addition of NMIP-Ⅲ interfering peptide drugs, NP-induced mitochondrial condensation was inhibited. Example 5

[0063] Flow cytometry detection found that the use of interfering peptide NMIP-Ⅲ could inhibit the increase of ROS induced by NP.

[0064] 1. Experimental Materials Flow cytometer, HEK293T cells (ATCC source), ROS staining kit (Biyuntian), and NMIP-Ⅲ interfering peptide drug prepared in Example 1.

[0065] 2. Experimental methods Empty vector mCherrry was transfected into cells of the control group, and NP-mCherrry was transfected into cells of the two experimental groups. NMIP-Ⅲ was added at a dilution of 1:1000 into one of the groups, and PBS was added into the other group. After 24 hours, cells were collected for ROS staining, and the ROS content in each group was detected by flow cytometry.

[0066] 3. Experimental results See also Figure 7 As shown, ROS is a key factor in activating innate immune responses, cell senescence and apoptosis. Flow cytometry showed that in cells transfected with NP-mCherry, the fluorescence intensity of ROS staining increased significantly. After adding NMIP-Ⅲ to the cells, the fluorescence intensity of ROS staining induced by NP-mCherry was inhibited, indicating that the interfering peptide NMIP-Ⅲ can inhibit the increase of ROS induced by NP. Example 6

[0067] NMIP-III treatment inhibited NP-induced growth reduction of A549 cells.

[0068] 1. Experimental Materials Cell counter, A549 cells (from ATCC), NMIP-Ⅲ interfering peptide drug prepared in Example 1.

[0069] 2. Experimental methods A549 cells stably expressing NP were obtained by lentiviral packaging. One group of A549 and two groups of A549 stably expressing NP were plated in 6-well plates at the same cell density. NMIP-Ⅲ was added to one group of A549 stably expressing NP at a dilution of 1:1000, and the same volume of PBS was added to the other group. Cell counts were performed every other day for a total of three days.

[0070] 3. Experimental results See also Figure 8 As shown in Table 2, senescent cells will show cell cycle arrest. The growth rate of A549 cells stably expressing NP was significantly lower than that of the control group. Adding NMIP-Ⅲ cells to A549 cells stably expressing NP restored the growth rate, indicating that the interfering peptide NMIP-III treatment inhibited the NP-induced growth slowing of A549 cells and rescued cell proliferation.

[0071] Table 2 Example 7

[0072] NMIP-III can inhibit NP-induced RIP1 phase separation.

[0073] 1. Experimental Materials HeLa cells (from ATCC), laser confocal microscope, and the NMIP-Ⅲ interfering peptide drug prepared in Example 1.

[0074] 2. Experimental methods The control group was co-transfected with RIP1-mCherry and GFP, and the two experimental groups were co-transfected with RIP1-mCherry and NP-GFP, respectively. NMIP-Ⅲ interfering peptide drug was added to one group at a ratio of 1:1000, and an equal volume of PBS was added to the other group. After 24 hours, the samples were collected and observed under a laser confocal microscope.

[0075] 3. Experimental results See also Fig. 9 As shown in the figure, RIP1 is an important ROS sensing protein. RIP1 is rapidly activated after sensing ROS and forms a RIP1-RIP3 complex with RIP3, thereby activating the downstream NF-κB pathway and inducing high expression of SASP-like factors. In the experimental group co-transfected with RIP1-mCherry and NP-GFP, it was found that RIP1 was significantly aggregated in a dot-like manner, indicating that NP induced RIP1 phase separation. After the addition of NMIP-Ⅲ interfering peptide drugs, the dot-like aggregation of RIP1 disappeared, indicating that NMIP-III can inhibit NP-induced RIP1 phase separation. Example 8

[0076] NMIP-III can inhibit the increase of SASP-like factors in PM cells induced by NP-VSV infection.

[0077] 1. Experimental Materials PM cells (mouse peritoneal macrophages), NMIP-Ⅲ interfering peptide drug prepared in Example 1, mouse anti-Tubulin antibody, rabbit anti-P16INK4A antibody (abclonal), rabbit anti-P21WAF1 antibody (cell signaling technology), mouse anti-NP antibody (abclonal) and corresponding secondary antibodies.

[0078] 2. Experimental methods qPCR detection: PM cells in the control group were infected with VSV-GFP virus, and the two experimental groups were infected with VSV-NP. One group was added with NMIP-Ⅲ interfering peptide drug at a dilution of 1:1000, and the other group was added with an equal amount of PBS. Cells were collected 24 hours after virus infection, and RNA was extracted for qPCR detection of the mRNA expression levels of P16INK4A, P21WAF1, IL-1β, and IL-6.

[0079] WB detection: PM cells in the control group were infected with VSV-GFP virus, and the two experimental groups were infected with VSV-NP. NMIP-Ⅲ interfering peptide drugs were added to one group at a ratio of 1:1000, and an equal amount of PBS was added to the other group. Cells were collected 24 hours after virus infection, and proteins were extracted for WB detection of the expression levels of P16INK4A and P21WAF1.

[0080] 3. Experimental results See also Fig.10 As shown in Table 3, the expression levels of SASP factors such as P16INK4A, P21WAF1, IL-1β, and IL-6 mRNA and the expression of P16INK4A and P21WAF1 proteins in PM cells infected with VSV-NP virus were significantly increased. After the addition of NMIP-Ⅲ interfering peptide drugs, the inducing ability of NP on SASP factors in PM cells was inhibited, indicating that NMIP-III can inhibit the increase of SASP factors in PM cells induced by NP-VSV infection.

[0081] Table 3 Example 9

[0082] NMIP can inhibit NP-VSV-induced cell senescence.

[0083] 1. Experimental Materials β-gal staining kit (Biyuntian), PM cells (mouse peritoneal macrophages), NMIP-Ⅲ interfering peptide drug prepared in Example 1, ordinary optical microscope.

[0084] 2. Experimental methods PM cells in the control group were infected with VSV-GFP virus, and the two experimental groups were infected with VSV-NP. NMIP-Ⅲ interfering peptide drugs were added to one group at a ratio of 1:1000, and an equal amount of PBS was added to the other group. β-gal staining was performed 24 hours after virus infection, and the cells were observed under a microscope after staining.

[0085] 3. Experimental results See also Fig.11As shown, when cells are senescent, they will express β-galactosidase (β-gal), which has high enzymatic activity at pH 6.0. With X-Gal as a substrate, a dark blue product will be generated under the catalysis of aging-specific β-galactosidase. Therefore, the strength of β-gal can be used to detect whether the cells are senescent. After PM was infected with VSV-NP, it was found under a microscope that the β-gal staining effect was significantly enhanced. After the addition of NMIP-Ⅲ interfering peptide drugs, the enhancement effect of NP on β-gal staining was weakened, indicating that NMIP can inhibit NP-VSV-induced cell senescence. Example 10

[0086] NMIP-III can inhibit the increase of SASP-like factors in the spleen, liver and lungs of mice induced by NP-VSV.

[0087] 1. Experimental Materials C57BL / 6J mice, NMIP-Ⅲ interfering peptide drug prepared in Example 1 2. Experimental methods The mice in the control group were injected intraperitoneally with 5×10 8 PFU of VSV-GFP, two experimental groups of mice were intraperitoneally injected with 5×108 PFU of VSV-NP, one group was injected with NMIP-Ⅲ interfering peptide drugs at the same time as the virus injection, and the spleen, liver and lungs of the mice were taken out 24 hours later, and RNA and protein were extracted respectively to detect SASP factors.

[0088] 3. Experimental results See also Fig.12 As shown in Table 4, qPCR detection found that the expression levels of P16, P21, IL-6, and IL-1β in the spleen, liver, and lungs of mice infected with VSV-NP were significantly increased, while the increase of NP-induced SASP-like factors in the spleen, liver, and lungs of mice injected with NMIP-Ⅲ interfering peptide drugs was inhibited. The WB detection results were the same as those of qPCR, indicating that NMIP-III can inhibit the increase of SASP-like factors in the spleen, liver, and lungs of mice induced by NP-VSV.

[0089] Table 4 Embodiment 11

[0090] NMIP-III can inhibit the SASP induced by the latest mutant strain of SARS-CoV-2, JN.1 NP.

[0091] 1. Experimental Materials A549 cells (ATCC source), NMIP-Ⅲ interfering peptide drug prepared in Example 1.

[0092] 2. Experimental methods The control group cells were transfected with empty plasmid, and the experimental groups were transfected with NP and JN.1 NP, respectively. NMIP-Ⅲ (10 μM) was added to one of the NP and JN.1 NP groups, and an equal amount of PBS was added to the other group. After 24 hours, samples were collected and RNA was extracted to detect SASP factors by qPCR.

[0093] 3. Experimental results See also Fig.13 As shown in Table 5, qPCR detection found that the expression levels of P16, P21, IL-1β, IL-8, and TNFα in A549 cells transfected with NP and JN.1 NP were significantly increased, while the increase of SASP-like factors induced by NP and JN.1 NP was inhibited in A549 cells to which NMIP-Ⅲ interfering peptide drugs were added.

[0094] Table 5 Example 12

[0095] NMIP-III can inhibit JN.1 NP-induced mitochondrial content leakage.

[0096] 1. Experimental Materials A549 cells (ATCC source), NMIP-Ⅲ interfering peptide drug prepared in Example 1, mouse anti-Tubulin antibody, rabbit anti-COX Ⅳ antibody (abclonal), rabbit anti-Myc-Tag antibody, Tom20 antibody (proteintech).

[0097] 2. Experimental methods The control group cells were transfected with empty plasmid, and the experimental groups were transfected with NP and JN.1 NP, respectively. NMIP-Ⅲ (10 μM) was added to one of the NP and JN.1 NP groups, and an equal amount of PBS was added to the other group. After 24 hours, the samples were collected to separate the cytoplasm and mitochondria, and the proteins were detected by WB.

[0098] 3. Experimental results See also Fig.14 As shown, WB detection found that the COX IV content in the cytoplasm increased significantly after transfection of NP or JN.1 NP, indicating that the mitochondrial protein leaked into the cytoplasm, while the COX IV content in the cytoplasm after the addition of NMIP-Ⅲ interfering peptide drug decreased, indicating that the leakage of mitochondrial contents induced by NP or JN.1 NP was inhibited. Embodiment 13

[0099] NMIP-III can inhibit the interaction between JN.1 NP and MFN1 / 2 induced by NP.

[0100] 1. Experimental Materials HEK293T cells (ATCC source), NMIP-III interfering peptide drug prepared in Example 1, rabbit anti-Myc-Tag antibody, rabbit anti-Flag-Tag antibody and related secondary antibodies, Flag-beads.

[0101] 2. Test methods HEK293T cells were co-transfected with MFN1-Flag plasmid and NP-Myc, JN.1 NP-Myc plasmid ( Fig.15 Left); MFN2-Flag plasmid and NP-Myc, JN.1 NP-Myc plasmid ( Fig.15 Right), NMIP-Ⅲ (10 μM) was added, and cells were collected 24 h later for co-immunoprecipitation detection.

[0102] 3. Experimental results See also Fig.15 As shown, the interfering peptide NMIP-Ⅲ can significantly inhibit the interaction between overexpressed MFN1 / 2 and JN.1 NP in cells.

[0103] The WB bands of JN.1 NP with MFN1 and MFN2 were darker, indicating that JN.1 had a strong interaction with MFN1 / 2. After the addition of NMIP-Ⅲ, the WB bands became lighter, indicating that the interaction between JN.1 and MFN1 / 2 was inhibited.

[0104] In summary, the interfering peptide NMIP-Ⅲ provided by the present invention can interact with the IDR2 domain of SARS-CoV-2 NP, inhibit the interaction between N protein and mitochondrial fusion protein MFN1 / 2 and the activation of NF-κB pathway, thereby inhibiting NP-induced SASP expression, thereby achieving the purpose of inhibiting SARS-CoV-2-induced cell senescence. Therefore, the interfering peptide drug NMIP-Ⅲ provided by the present invention is also expected to be used to prepare drugs against SARS-CoV-2-induced cell senescence.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An interfering peptide for inhibiting SARS-CoV-2-induced cell senescence, characterized in that: It is composed of the D-type amino acid sequence shown by TSLCVLSVVIPPRRRQRRKKRG and has a molecular weight of 2606.14 Da.

2. The interfering peptide according to claim 1, characterized in that The interfering peptide targets the SARS-CoV-2 N protein and can interact with the 175-246 amino acid region of the SARS-CoV-2 N protein, inhibiting the interaction between the N protein and the mitochondrial fusion protein MFN1 / 2 and the activation of the NF-κB pathway, thereby inhibiting the expression of NP-induced aging markers and aging-related secretory phenotype-related proteins, thereby inhibiting SARS-CoV-2-induced cell senescence.

3. A method for preparing an interfering peptide as claimed in claim 1, characterized in that: include: Step 1) Through molecular docking and immunoprecipitation experiments, it was confirmed that the interaction region between SARS-CoV-2 N protein and mitochondrial fusion-related protein MFN1 / 2 is the 175-246 amino acid region; Step 2) Targeting the 175-246 amino acid region of the SARS-CoV-2 N protein, the amino acid sequence of a peptide that interacts with this region was obtained by panning and designing a random 10-peptide T7 phage display library; Step 3) fusing the designed polypeptide with HIV-TAT to make it have membrane-penetrating activity; Step 4) synthesizing the polypeptide fused with HIV-TAT in the form of reverse order of D-type amino acids to obtain the amino acid sequence of the final interfering peptide NMIP-Ⅲ; Step 5) Using D-amino acids as raw materials to synthesize the interfering peptide NMIP-Ⅲ.

4. The method for preparing the interfering peptide according to claim 3, characterized in that: In step 1), the interaction surface is amino acids 175-246 of the SARS-CoV-2 N protein.

5. The method for preparing an interfering peptide for inhibiting SARS-CoV-2-induced cell senescence according to claim 3, characterized in that: In step 2), the amino acid region 175-246 of the SARS-CoV-2 N protein was used for panning with a 10-peptide random T7 phage display library.

6. The method for preparing an interfering peptide for inhibiting SARS-CoV-2-induced cell senescence according to claim 5, characterized in that: The amino acid sequence of the polypeptide obtained by panning using the T7 phage display library is IVVSLVCLST.

7. The method for preparing an interfering peptide for inhibiting SARS-CoV-2-induced cell senescence according to claim 6, characterized in that: In step 3), the amino acid sequence of HIV-TAT is GRKKRRQRRRPP, and the amino acid sequence after fusion with the polypeptide obtained by panning using the T7 phage display library is GRKKRRQRRRPPIVVSLVCLST.

8. The method for preparing an interfering peptide for inhibiting SARS-CoV-2-induced cell senescence according to claim 7, characterized in that: The amino acids after the HIV-TAT is fused with the polypeptide obtained by panning using the T7 phage display library are L-type natural amino acids.

9. The method for preparing an interfering peptide for inhibiting SARS-CoV-2-induced cell senescence according to claim 3, characterized in that: In step 4), the amino acid sequence of the interfering peptide NMIP-III is TSLCVLSVVIPPRRRQRRKKRG, the molecular weight is 2606.14 Da, and its amino acids are D-type amino acids.

10. Use of an interfering peptide as described in any one of claims 1 to 2 or an interfering peptide prepared by the method for preparing an interfering peptide for inhibiting SARS-CoV-2-induced cell senescence as described in any one of claims 2 to 9 in the preparation of a drug for resisting SARS-CoV-2-induced cell senescence.

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