A short peptide derivative based on the SIM site of the novel coronavirus spike protein and its uses

Abstract

This invention discloses a short peptide derivative based on the SIM site of the SARS-CoV-2 spike protein and its uses. The short peptide derivative includes cpSIM1, cpSIM1', cpSIM2, cpSIM3, and cpSIM4, and is composed of a membrane-penetrating peptide and a short peptide. The short peptide is based on SUMO modification of the SARS-CoV-2 Spike protein polypeptide at the SIM1, SIM1', SIM2, SIM3, and SIM4 sites; the membrane-penetrating peptide is attached to the N-terminus of the short peptide. The four short peptide derivatives cpSIM1, cpSIM1', cpSIM3, and cpSIM4 of this invention do not affect cell viability and are non-cytotoxic, and can promote the expression of the SARS-CoV-2 Spike protein on the cell surface and enhance immunogenicity. cpSIM2 exhibits high specificity and efficacy against SARS-CoV-2 infection, which has been verified in vivo and in vitro models. It can be used in the preparation of drugs against different mutant strains of SARS-CoV-2 and has important application value for targeted therapy of SARS-CoV-2.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a short peptide derivative based on the SIM site of the novel coronavirus spike protein and its uses. Background Technology

[0002] Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the pathogen of coronavirus disease 2019 (COVID-19), has become one of the most widespread and destructive viruses on record, posing a continued threat to public health due to its rapid evolution. SARS-CoV-2 is an enveloped, single-stranded, positive-sense RNA virus with a genome of approximately 29.9 kb. It has multiple open reading frames encoding four structural proteins: spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N), 16 non-structural proteins, and 11 accessory proteins. The spike protein plays a crucial role in host cell entry and includes two subunits, S1 and S2. The S1 subunit encodes the receptor-binding domain (RBD), responsible for binding to the ACE2 receptor, while the S2 subunit encodes the transmembrane portion of the Spike protein, responsible for viral-cell membrane fusion. The junction of S1 and S2 contains a polyarginine (polybasic) cleavage site, which can be recognized by the cellular protease furin. Cleavage at this site is crucial for Spike protein-mediated cell fusion and viral entry into human lung cells. In infected cells, the Spike protein undergoes furin cleavage during maturation through the Golgi and anti-Golgi networks, forming non-covalently linked S1 and S2 subunits. This allows S1 to interact more effectively with ACE2 and promotes S2 exposure, subsequent cleavage, and conformational rearrangement. Besides assembling into viral particles, Spike protein expression on the cell surface can also mediate intercellular viral transmission. Therefore, intracellular transport and cleavage of the Spike protein determine the mode of viral infection. Similar to other viral surface proteins, the Spike protein undergoes various post-translational modifications (PTMs), such as glycosylation, which affect its cleavage and integration into viral particles. However, the small ubiquitin-like modifier (SUMO) of viral surface proteins and its functional significance have not been reported.

[0003] SUMO modification is a dynamic and reversible post-translational modification of proteins that plays a crucial role in cellular responses to various stimuli. The human genome primarily encodes three isoforms of SUMO molecules: SUMO1, SUMO2, and SUMO3. Due to the 98% homology between SUMO2 and SUMO3, they are often collectively referred to as SUMO2 / 3. SUMO1 and SUMO2 / 3 modifications exhibit different physiological functions, with host and viral proteins showing a greater tendency to undergo SUMO2 / 3 modification under stress. Studies have shown that target proteins can undergo different types of SUMO modifications at different lysine (K) residues and can bind to other proteins through SUMO-interacting motifs (SIMs), providing an additional interaction platform for the recruitment of downstream effector proteins. Increasing evidence suggests that the SUMO modification system plays a critical role in the host's response to viral infection. Many viral proteins not only serve as substrates for SUMO modification but also regulate the host's SUMO pathway. Recent studies have highlighted the link between SUMOylation and SARS-CoV-2 infection. For example, TRIM28-mediated SUMOylation of the SARS-CoV-2 N protein enhances its oligomerization, RNA binding, and liquid-liquid phase separation capabilities. Furthermore, SUMO3 modification of ACE2 is thought to contribute to SARS-CoV-2 infection. Despite these findings, a comprehensive understanding of SUMO-modified proteins associated with SARS-CoV-2 infection remains to be established. The rapid spread and continuous evolution of SARS-CoV-2, particularly emerging variants such as BA.2.86 / JN.1 and KP.2, KP.3, pose increasing challenges to existing vaccines and treatments, urgently requiring new therapeutic strategies and drugs to address these challenges. Therefore, in-depth research into SARS-CoV-2 SUMOylation and its role in viral infection is of great significance for the development of new broad-spectrum antiviral drugs and vaccines. Summary of the Invention

[0004] To address the above technical problems, this invention provides a short peptide derivative based on the SIM sites of the SARS-CoV-2 spike protein and its uses. The short peptide derivative based on five different SIM sites (SIM1, SIM1', SIM2, SIM3, and SIM4) of the SARS-CoV-2 Spike protein in this invention is a stable polypeptide compound that can penetrate cell membranes and enter cells in vivo. It exhibits high specificity and efficacy against SARS-CoV-2 infection, or has the potential to promote the expression of the SARS-CoV-2 Spike protein on the cell surface and enhance its immunogenicity.

[0005] The first objective of this invention is to provide a short peptide derivative of the SIM site of the novel coronavirus spike protein, the short peptide derivative comprising cpSIM1, cpSIM1', cpSIM2, cpSIM3, and cpSIM4, the short peptide derivative being composed of a membrane-penetrating peptide and a short peptide; the short peptide being a short peptide based on SUMO modification of the SARS-CoV-2 Spike protein polypeptide at the SIM1, SIM1', SIM2, SIM3, and SIM4 sites; the membrane-penetrating peptide being attached to the N-terminus of the short peptide.

[0006] In some embodiments of the present invention, the amino acid sequences of the short peptides at the SIM1, SIM1', SIM2, SIM3 and SIM4 sites are as shown in SEQ ID No. 1-4, respectively.

[0007] The chemical structural formulas of the short peptides at SIM1, SIM1', SIM2, SIM3, and SIM4 sites are as follows:

[0008] Short peptide structure of SIM1 site:

[0009]

[0010] Short peptide structure at SIM1' site:

[0011]

[0012] Short peptide structure at SIM2 site:

[0013]

[0014] Short peptide structure at SIM3 site:

[0015]

[0016] Short peptide structure at SIM4 site:

[0017]

[0018] In some embodiments of the present invention, the amino acid sequence of the membrane-penetrating peptide is shown in SEQ ID No. 5.

[0019] In some embodiments of the present invention, the amino acid sequences of cpSIM1, cpSIM1', cpSIM2, cpSIM3, and cpSIM4 are as shown in SEQ ID Nos. 6-9, respectively. The short peptide derivatives are prepared by solid-phase synthesis.

[0020] A second object of the present invention is to provide a composition comprising a short peptide derivative of the SIM site of the novel coronavirus spike protein.

[0021] A third object of the present invention is to provide an antiviral drug comprising a short peptide derivative of the SIM site of the novel coronavirus spike protein or the composition thereof. Further, the short peptide derivative of the SIM site of the novel coronavirus spike protein or the composition thereof comprises cpSIM2.

[0022] In some embodiments of the present invention, the virus includes the novel coronavirus SARS-CoV-2 or SARS-CoV.

[0023] Furthermore, the short peptide derivatives (such as cpSIM2) can inhibit the increase in the viral copy number of SARS-CoV-2.

[0024] A fourth object of the present invention is to provide a viral vaccine adjuvant comprising a short peptide derivative of the SIM site of the novel coronavirus spike protein or the composition thereof. Further, the short peptide derivative of the SIM site of the novel coronavirus spike protein or the composition thereof comprises cpSIM1, cpSIM1', cpSIM3, or cpSIM4.

[0025] In some embodiments of the present invention, the virus includes the novel coronavirus SARS-CoV-2 or SARS-CoV.

[0026] In some embodiments of the present invention, the short peptide derivative promotes the expression of SARS-CoV-2 Spike protein on the cell surface and enhances immunogenicity.

[0027] In this invention, SARS-CoV-2 infection triggers a comprehensive remodeling of the host SUMO modification network, significantly affecting the localization of SUMO1 and SUMO2 / 3. SUMO2 modification of the SARS-CoV-2 Spike protein inhibits its cleavage, thereby preventing virus-mediated intercellular transmission. This invention, through a series of experiments, identified the sites of SUMO modification and interaction in the SARS-CoV-2 Spike protein, and screened for peptides associated with SIM1, SIM1', SIM2, SIM3, and SIM4 sites. These peptides can competitively inhibit the interaction of the Spike protein with other host proteins, promoting or inhibiting Spike protein expression, thereby playing a role in preventing or combating SARS-CoV-2 virus infection.

[0028] The technical solution of the present invention has the following advantages compared with the prior art:

[0029] This invention designs multiple peptide compounds, cpSIM1, cpSIM1', cpSIM2, cpSIM3, and cpSIM4, derived from the SIM sites of the SARS-CoV-2 Spike protein. The peptide sequences and lengths are determined using the Spike protein sequence and specific SUMO interaction sites. The effects of different peptide compounds on the infection and replication of different SARS-CoV-2 mutant strains are compared using reverse transcription-quantitative PCR, immunoblotting, and immunofluorescence analysis. Furthermore, the potential preventive effects of cpSIM1 / 1' / 3 / 4 on SARS-CoV-2 infection and the therapeutic effect of cpSIM2 on SARS-CoV-2 infection are verified using the hACE2 transgenic mouse model, providing a new strategy for the prevention and control of SARS-CoV-2 and its mutant strains.

[0030] This invention provides five short peptide derivatives based on the SIM site of the SARS-CoV-2 Spike protein: cpSIM1, cpSIM1', cpSIM2, cpSIM3, and cpSIM4. Among these, cpSIM1, cpSIM1', cpSIM3, and cpSIM4 do not affect cell viability and are non-cytotoxic, promoting the expression of the SARS-CoV-2 Spike protein on the cell surface and enhancing immunogenicity. cpSIM2 exhibits high specificity and efficacy against SARS-CoV-2 infection, validated in both in vivo and in vitro models, laying the foundation for the treatment of SARS-CoV-2 infection. Attached Figure Description

[0031] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0032] Figure 1 In Example 1 of this invention, SUMO2 modification of the SARS-CoV-2 Spike protein resulted in perinuclear punctate localization of SUMO2; A) SARS-CoV-2 Spike protein underwent SUMO modification; B) Exogenous CoV-2 Spike protein underwent SUMO modification; C) SUMO2 gene knockout affected the perinuclear punctate localization of Spike protein; D) SUMO2 KO increased the cleavage and modified forms of Spike protein.

[0033] Figure 2This document describes the identification of SUMO sites and SIM motifs of the SARS-CoV-2 Spike protein in Example 2 of the present invention; A) Schematic diagram of GPS-SUMO prediction of the conservation of SUMO sites and SIM motifs of the Spike protein; B) Three-dimensional spatial distribution of SUMO sites and SIM motifs of the Spike protein; C) Effect of SUMO site and SIM motif mutations on Spike protein splicing; D) Effect of SUMO site and SIM motif mutations on Spike protein SUMOylation; E) Schematic diagram of the types of SUMO modifications occurring at SUMOylation sites and SIM motifs of the Spike protein.

[0034] Figure 3 The purity of different short peptides synthesized from Spike was determined by HPLC in Example 3 of this invention; wherein, A) cpSIM1; B) cpSIM1'; C) cpSIM2; D) cpSIM3; E) cpSIM4.

[0035] Figure 4 This is an example of the effects of different short peptide compounds of Spike on the cell fusion and replication of SARS-CoV-2 in Example 4 of the present invention; A) Schematic diagram of different short peptide compounds of Spike; B) Effect of different short peptide compounds of Spike on cell morphology after CoV-2 infection; C) Effect of different short peptide compounds of Spike on CoV-2 replication; D) Effect of different short peptide compounds of Spike on the expression level of Spike protein in cells after CoV-2 infection; D) cpSIM2 inhibits the replication of CoV-2 VOC strain in A549-hACE2 and Calu3 cell lines, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0036] Figure 5 Example 5 of this invention demonstrates that cpSIM2 effectively inhibits SARS-CoV-2 replication in mice; A) Schematic diagram of CoV-2 infection and cpSIM2 treatment in mice; B) Viral load in mouse lung tissue detected by reverse transcription-quantitative PCR targeting the CoV-2N gene. ****p<0.0001 indicates statistical significance; C) Immunohistochemical staining of lung tissue with hematoxylin and eosin (H&E) and antibodies against the CoV-2N protein was used to assess the degree of pathological damage and viral load in lung tissue. The following figure is a representative magnified image. D) Quantitative results of the N-positive cell rate in lung tissue. Detailed Implementation

[0037] 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 and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0038] Example 1: SUMO2 modification of SARS-CoV-2 Spike protein inhibits Spike protein cleavage

[0039] 1. SUMO2 modification of the SARS-CoV-2 Spike protein, which involves SUMO modification, leads to the perinuclear punctate localization of SUMO2.

[0040] To verify whether the SARS-CoV-2 Spike protein undergoes SUMO modification, A549-hACE2 cells infected with the original SARS-CoV-2 strain SH01 were collected. Denaturing immunoprecipitation was performed using endogenous SUMO1 and SUMO2 / 3 antibodies, and Western blot analysis was conducted using Spike antibodies. The results showed that Spike could be captured by SUMO1 and SUMO2 / 3 antibodies, indicating that Spike undergoes SUMO modification. Figure 1 A). Furthermore, N proteins known to undergo SUMO modification were detected, and the results showed effective immunoprecipitation of N protein SUMO1 and SUMO2 / 3 antibodies, indicating the reliability of the detection method. Figure 1 A).

[0041] Viral infection is a complex process involving multiple viral proteins. To exclude interference from other viral proteins, SARS-CoV-2 Spike was co-expressed with HA (a small fragment of influenza virus hemagglutinin protein, Zhang Y, Dong Z, Gu F, et al. Degradation of TRIM32 is induced by RTA for Kaposi's sarcoma-associated herpesvirus lytic replication. J Virol. 2024 Jun 13; 98(6):e0000524. doi:10.1128 / jvi.00005-24. PubMed PMID:38717113; PubMed Central PMCID:PMCPMC11237441.eng.) labeled SUMO1, SUMO2, and SUMO3 in 293T cells, and Spike was detected by co-immunoprecipitation. The results showed that Spike interacted with SUMO1, SUMO2, and SUMO3, and overexpression of SUMO increased the expression level of Spike (Zhang Y, Dong Z, Gu F, et al. Degradation of TRIM32 is induced by RTA for Kaposi's sarcoma-associated herpesvirus lytic replication. J Virol. 2024 Jun 13; 98(6):e0000524. doi:10.1128 / jvi.00005-24. PubMed PMID:38717113; PubMed Central PMCID:PMCPMC11237441.eng.). Figure 1 B).

[0042] 2. SUMO2 modification of the SARS-CoV-2 Spike protein leads to perinuclear punctate localization of SUMO2.

[0043] To investigate the effect of SUMO modification on the function of the SARS-CoV-2 Spike protein, A549-hACE2 SUMO1, SUMO2, and SUMO3 knockout cell lines were constructed using the CRISPR-Cas9 system. These cell lines were infected with the original SARS-CoV-2 strain SH01, and the effects of different SUMO molecules on Spike protein expression and localization were further examined by immunofluorescence and Western blotting. Results are as follows: Figure 1 C shows that SARS-CoV-2 infection leads to a perinuclear punctate distribution of SUMO2 / 3, and Spike protein co-localizes with SUMO2 / 3 in the perinuclear region. Compared with A549-hACE2 wild-type cells, SUMO2 knockout blocked the perinuclear punctate distribution of SUMO2 / 3 and led to a decrease in Spike protein abundance. Figure 1 C) indicates that SUMO2 modification of the SARS-CoV-2 Spike protein leads to perinuclear punctate localization of SUMO2.

[0044] 3. SUMO2 modification can inhibit the cleavage of SARS-CoV-2 Spike protein.

[0045] Since the Spike protein requires cleavage to form its active conformation for viral transmission, Western blotting was used to assess the expression of SARS-CoV-2 Spike protein in wild-type A549-hACE2 cells and cells with different SUMO knockouts after infection. The results showed that SUMO1 knockout led to an increase in Spike protein trimer and a decrease in cleavage products, while SUMO2 knockout led to an increase in Spike cleavage and a decrease in trimer. Figure 1 D). These results indicate that SUMO2 modification can inhibit the cleavage of the Spike protein.

[0046] The experimental steps involved in this embodiment are as follows:

[0047] 1. SARS-CoV-2 cell infection model:

[0048] 1) Prepare well-grown cells and pre-culture them at 37°C and 5% CO2 for 24 hours to achieve a density of 90%, according to experimental requirements.

[0049] 2) Dilute the SARS-CoV-2 virus solution to the required multiple of infection (MOI) using DMEM medium with 2% FBS, discard the original culture medium, add the virus solution, add 100 μl of virus dilution to each well, and place in a cell culture incubator.

[0050] 3) After incubating in a cell culture incubator for 24 hours, collect the cells and supernatant samples and perform subsequent tests as needed.

[0051] 2. Preparation for cell sample lysis:

[0052] 1) Collect an appropriate amount of cells, centrifuge at 2500 rpm for 3 min at room temperature, and discard the culture medium.

[0053] 2) Wash twice with pre-cooled PBS, centrifuge at 2500 rpm for 3 min at room temperature, and discard the PBS.

[0054] 3) After transfecting cells for 48 hours or infecting cells for 24 hours, discard the supernatant, wash twice with PBS, add trypsin for 5 minutes, add serum-containing culture medium to neutralize the trypsin, repeatedly pipette the cells, transfer the cell suspension to a new centrifuge tube, and count the cells. The total viable cell count should be approximately 3 × 10⁻⁶. 7 The sample.

[0055] 4) Denaturation step (denaturing immunoprecipitation proprietary step): Add 180 μL of protein lysis buffer RIPA (1% NP-40, 2 mM EDTA, 150 mM NaCl, 50 mM Tris (pH 7.6), 1 mM PMSF, 1 mM NEM, 1 g / mL Aprotinin, 1 g / mL leupeptin and 1 g / mL pepstatin), and 20 μL of 20% SDS solution. Mix the cell sample by pipetting and incubate at 100°C for 10 min.

[0056] 5) Cool to room temperature, add 1.8 mL of RIPA protein lysis buffer containing protease inhibitors, lyse on ice for 30 min, and shake to mix every 5 min.

[0057] 6) The fully lysed cells were centrifuged at 14,500 rpm for 5 min at 4°C.

[0058] 7) Transfer the supernatant after centrifugation to a new EP tube, which is the whole cell lysis buffer. Measure the protein concentration according to the Coomassie Brilliant Blue G-250 method.

[0059] 8) Aliquot the protein samples according to the loading amount, add 6x protein loading, and boil in a 100℃ metal bath for 5 minutes until the protein denatures.

[0060] 9) Protein samples can be frozen at -80℃ for later use or directly subjected to immunoprecipitation experiments.

[0061] 2. Immunoprecipitation:

[0062] 1) Protein A / G pretreatment: 30 μL each of Protein A and G, washed with TBS, centrifuged at 1000 g for 30 s at 4 °C, repeated 3 times.

[0063] 2) Remove the protein supernatant from the cell lysis in the previous step:

[0064] a) IP group: Add an appropriate amount of negative control (Control IgG) and an appropriate amount of washed Protein A / G premixed agarose beads, incubate at 4°C with thorough rotation for 30 min. -1 •h, eliminates nonspecific background.

[0065] b) Input group: Take 2.5%-5% whole cell lysate as the input group, add 6x protein loading, boil at 100℃ for 10 min, and store at -20℃ for later use.

[0066] 3) Antibody incubation:

[0067] a) After IgG incubation, centrifuge at 1000g for 30 seconds at 4°C, then allow to stand at room temperature for 10 minutes to allow the IgG antibody and its bound proteins to precipitate slowly. Both the supernatant and the precipitate are retained after centrifugation; the precipitate serves as the IgG negative control group.

[0068] b) Supernatant: Transfer the supernatant to a new EP tube, add an appropriate volume of primary antibody according to the antibody instructions, and incubate at 4°C with slow rotation overnight (8-12h) to facilitate the full binding of the antibody and the target protein.

[0069] c) Precipitation (IgG negative control group): Add 1 mL TBS to resuspend, centrifuge at 1000g for 30 s at 4℃, repeat three times; add 20 μL TBS to resuspend, then add 6× protein loading to cook the protein, cook for 10 min, and store at -20℃.

[0070] 4) Sample protein denaturation:

[0071] a) The next day, add 30 μL of Protein A / G premixed agarose beads to the supernatant and continue to incubate at 4°C with slow rotation for 3 hours to allow the supernatant to fully combine with the beads.

[0072] b) After 3 hours, centrifuge at 1000g for 30 seconds at 4°C and discard the supernatant. Wash the precipitate three times with pre-cooled 1mL TBS, centrifuging at 1000g for 30 seconds each time at 4°C. Resuspend in 20μL TBS, add 4μL of 6×protein loading solution to cook the protein, and store as the IP group at -20°C.

[0073] 5) Western blot analysis of proteins: Input group, IgG group, and IP group were detected together.

[0074] 9. Denaturing immunoprecipitation:

[0075] 1) Preparation of common cell proteins: Same as the previous immunoprecipitation procedure.

[0076] 2) Protein A / G pretreatment: Same as the previous immunoprecipitation procedure.

[0077] 3) Obtaining whole-cell lysate:

[0078] a) For each sample, add 200 μL of protein lysis buffer RIPA (containing the required protease inhibitors PMSF, NEM, aprotitin, leupeptin, and pepstin) and a mixed solution containing 2% SDS, and shake the cell sample to mix.

[0079] b) Place the shaken and mixed sample in a 100°C water bath for 10 min to allow the protein to denature.

[0080] c) After 10 minutes, remove the sample and wait for it to cool to room temperature. Do not place it directly on ice.

[0081] d) After the sample has returned to room temperature, add 1.8 mL of RIPA lysis buffer containing protease inhibitors (containing the required protease inhibitors PMSF, NEM, aprotitin, leupeptin and pepstin), place on ice at 4°C for lysis for 30 min, and shake to mix every 5 min.

[0082] 4) The remaining procedures are the same as those for immunoprecipitation.

[0083] 3. Procedure for Western blotting protein immunoblotting:

[0084] 1) SDS-PAGE electrophoresis: Add the above denatured protein samples to an appropriate concentration of SDS-PAGE gel for gel electrophoresis separation.

[0085] 2) Transfer: A 0.45 μm NC membrane was immersed in 1X NC membrane transfer equilibration buffer and transferred using GenScript eBlot. TM The L1 rapid wet transfer apparatus uses a standard procedure for film transfer, which takes approximately 10 minutes.

[0086] 3) Blocking: Place the NC membrane in 5% skim milk powder (prepared with PBS) and incubate at room temperature for 1 hour.

[0087] 4) Primary antibody incubation: Wash 3 times with TBST at room temperature for 5 minutes each time, add the primary antibody diluted in an appropriate ratio, and incubate overnight at 4°C.

[0088] 5) Secondary antibody incubation: Wash 3 times with TBST at room temperature, 5 min each time, using the corresponding species' fluorescent secondary antibody 800.

[0089] 6) Incubate at room temperature in the dark for 1 hour; wash 3 times with TBST at room temperature, 5 minutes each time.

[0090] 7) Development: Develop using an Odyssey dual-color imager.

[0091] 4. Immunofluorescence

[0092] 1) Fix the cell slides with 4% paraformaldehyde for 20 min, then wash three times with PBS.

[0093] 2) Dilute the primary antibody in Blocking Buffer according to the recommended ratio in the instructions, vortex to mix, and centrifuge at 14000 rpm for 2 min. Add 50 μL of the diluted primary antibody to the sealing film, gently place the slide face down on the droplet, and incubate at room temperature for 2 h.

[0094] 3) Transfer the slide face up into the well plate and wash three times with Blocking Buffer.

[0095] 4) Dilute the secondary antibody in Blocking Buffer according to the recommended ratio in the instructions, vortex to mix, and centrifuge at 14000 rpm for 2 min. Add 50 μL of the diluted secondary antibody solution to the sealing film, gently place the slide face down on the droplet, and incubate at room temperature in the dark for 1 h.

[0096] 5) Transfer the slide face up into the well plate and wash three times with Blocking Buffer.

[0097] 6) Dilute DAPI in PBS at a ratio of 1:1000, vortex to mix, centrifuge at 4000 rpm for 2 min, add 1 mL of DAPI dilution solution to each well, incubate in the dark for 3 min, wash three times with PBS, and air dry the slides in the dark in sequence.

[0098] 7) Add 20 μL of resin to the glass slide, place the slide face down on the glass slide, and apply the sealing agent around the slide to seal it.

[0099] 5. Construction of recombinant plasmids:

[0100] 1) Primer design: Primers were designed based on the target sequence obtained from NCBI query. The primer sequences are shown in the table below.

[0101]

[0102]

[0103]

[0104] 2) PCR amplification of the target fragment: obtaining the reverse transcription product; obtaining and using existing plasmids. Max DNAPolymerase (R045A, Takara),

[0105] PCR amplification system preparation

[0106]

[0107] PCR amplification reaction procedure

[0108]

[0109] 3) Enzyme digestion:

[0110] Preparation of enzyme digestion system

[0111]

[0112] Enzyme digestion reaction procedure

[0113] 37℃ water bath 2h

[0114] 4) Ligation sequencing: Ligation was performed using the Beyotime Seamless Cloning Kit (D7010M). For specific precautions, please refer to the product manual.

[0115] 6. Construction of Short hairpin RNA (ShRNA) plasmid

[0116] 1. Primer design: Design the target gene sequence according to the following method. The primers are shown in the table below.

[0117] (1) Review relevant literature on the target gene

[0118] (2) Website search: https: / / zlab.bio / guide-design-resources

[0119]

[0120] 2. Synthesize primer sequences:

[0121] (1) Prepare a 20 μL primer sequence synthesis system

[0122] Preparation of the Oligos system

[0123]

[0124] (2) Annealing program: In the PCR instrument, set the gradient cooling program: 95℃ for 30s, 72℃ for 2min, 37℃ for 2min, 25℃ for 2min.

[0125] (3) Dilute the above annealed fragments with ddH2O at a ratio of 1:200.

[0126] 3. Enzyme digestion: Linearize the LentiCRISPR vector with restriction endonuclease BsmBI and digest at 55℃ for 3 hours.

[0127] Preparation of enzyme digestion system

[0128]

[0129] 4. Connection

[0130] For specific precautions regarding the use of Beyotime's ultra-fast T4 DNA ligase (D7009M), please refer to the product instructions.

[0131] 5. Transformation: Using competent Stbl3 cells.

[0132] 6. Lentiviral packaging and purification:

[0133] 1) Cell preparation: Each batch of revived HEK293T cells should be passaged no more than 20 times. The virus can be packaged starting from the third generation after revival.

[0134] 2) Prepare plasmids: Total plasmid amount 40μg (target plasmid 14μg, VSVG: 5μg, Plet-CMV-GFP-Hygro: 21μg).

[0135] 3) Prepare for transfection: Use product (Polyethylenimine Linear (PEI) MW40000 (rapid lysis)) or Lipofectamine TM Transfection was performed at 3000.

[0136] 4) Collect supernatant: Collect cell supernatant in 50mL centrifuge tubes at 24h and 48h after medium change.

[0137] 5) Concentrate the supernatant: Centrifuge the cell supernatant at 2000 rpm for 5 min and filter it using a 0.45 μm filter.

[0138] 6) Centrifuge at 10000g at 4℃ for 18h, with an acceleration of 5 and a deceleration of 0.

[0139] 7) Virus preservation: Remove the virus tube from the centrifuge, carefully aspirate the supernatant, leaving approximately 1.5 mL of supernatant. Mix thoroughly by pipetting, aliquot into 3 tubes, and freeze at -80°C. Label with the virus name, date, and batch number.

[0140] 7. Construction of knockout cell lines:

[0141] 1) Determine the optimal antibiotic screening concentration: Before lentiviral infection, first determine the optimal drug screening concentration for killing the target cells.

[0142] 2) Cell plating: For adherent cells, prepare 500 μL of 0.5 × 10⁻⁶ cells one day in advance. 6 The cells to be infected were seeded in a 24-well plate and transfected the next day.

[0143] 3) Prepare the virus: Take the virus out of the -80℃ freezer in advance and place it on ice to wait for it to thaw.

[0144] 4) Viral infection: Take out the cells plated the day before and add the prepared virus drop by drop.

[0145] 5) Supplement the culture medium: After 24h and 48h, replenish the medium in time and divide the wells.

[0146] 6) Cell screening: Cell screening begins 72 hours after viral infection. The culture medium is replaced with one containing 1 μg / mL puromycin, and the cells are screened in a cell culture incubator for 48 hours.

[0147] 7) Monoclonal screening: Digest and count the selected cells. Dilute 80-100 cells in 10 ml of culture medium and seed the cell suspension into a 96-well plate at 100 μL / well. Culture for about 2 weeks. Once the monoclonal cells have grown to about 50% of the wells, digest them and transfer them to 24-well plates for expansion culture.

[0148] 8) Cell identification: Cells picked into 24-well plates were allowed to grow to confluence, then digested. Half were transferred to 6-well plates, and the other half were used to extract DNA for PCR identification. PCR identification confirmed the knockout cells, which were then expanded, preserved, and subjected to Western blotting.

[0149] Example 2: Identification of SUMO-modified specific sites and SIM motifs in SARS-CoV-2 Spike

[0150] To identify specific SUMOylation sites on the SARS-CoV-2 Spike protein, the Spike sequence was scanned using the GPS-SUMO tool (http: / / sumo.biocuckoo.cn / advanced.php). Multiple SUMO modification sites and potential SUMO-interacting motifs (SIMs) were identified, which corresponded to the homologous sequence valine (V) / isoleucine (I) / leucine (L)-XV / I / LV / I / L motif. Among these motifs, two adjacent SIM motifs were identified, named SIM1 and SIM1'. Comparison of the conservation of these sites between SARS-CoV and SARS-CoV-2 VOC variants revealed that Spike protein SUMO modification sites and SIMs were highly conserved among SARS-CoV-2 variants, even those located in the highly variable S1 subunit's N-terminal domain (NTD) and receptor-binding domain (RBD). Figure 2A). Conversely, the conservation of these sites is inconsistent between SARS-CoV and SARS-CoV-2: in SARS-CoV, sites 462 and 933 are not lysine (K) residues, and the linker sequences of SIM1 and SIM1' are also different from those in SARS-CoV-2. This suggests that SUMO modification sites and SIMs on the Spike protein play an important role in the evolution of SARS-CoV-2. Further projection of these sites onto the three-dimensional structure of the Spike protein extracellular domain, including its closed, open, and fused conformations, revealed that all identifiable SUMOylation sites are located on the surface of the three-dimensional structure, with the opening of the RBD further exposing sites 986 and 462, while SIM1 and SIM1' maintain spatial proximity, and only SIM2 is located inside the RBD domain. Figure 2 B).

[0151] To investigate the function of these sites, predicted SUMOylation sites were mutated by replacing lysine (K) with arginine (R) and replacing residues on SIMs with alanine (A), respectively. These mutants were named Spike mSIM1 / 2 / 3 / 4. These mutants were then transfected into 293T wild-type (WT) and SUMOs knockout cell lines. Immunoblot analysis showed that mutations at K129 and K1269 significantly enhanced Spike protein cleavage. In the WT cell group, both K129R and K1269R mutants were present in their cleaved form, while in the SUMO2 knockout cell group, the K1269R mutant partially reverted to its original size (S0). In contrast, all other mutants inhibited Spike protein cleavage, maintaining the S0 form (S0). Figure 2 C). These mutants were then co-expressed with HA-labeled SUMO1, SUMO2, and SUMO3 in 293T cells, and their SUMO modification status was examined using a co-immunoprecipitation method. The results showed that mutations at single sites did not completely block Spike SUMO modification, but certain sites exhibited a preference for specific SUMO modifications. For example, K462R reduced SUMO1 modification, while mSIM1 reduced both SUMO1 and SUMO3 modification. Figure 2 D). Spike proteins exhibit diverse SUMO modification patterns, with specific modification types at each site... Figure 2 The summary in E shows that these findings demonstrate that SUMOylation sites and SIMs on the Spike protein are involved in regulating its cleavage and indicate a potential antiviral mechanism.

[0152] Example 3: Synthesis of short peptides at SARS-CoV-2 Spike SUMO-modified sites

[0153] This embodiment designs corresponding short peptide derivatives for treating SARS-CoV-2, cpSIM1, cpSIM1', cpSIM2, cpSIM3, and cpSIM4, based on the short peptides modified with SUM O at the SARS-CoV-2 Spike protein sites screened in Example 2. The short peptide derivatives consist of the aforementioned short peptides and a leading transmembrane peptide. The amino acid sequences of the short peptides at the SIM1, SIM1', SIM2, SIM3, and SIM4 sites are as follows: SEQ ID No. 1: KTQSLLIVNNATN, SEQ ID No. 2: NATNVVIKVCEFQ, SEQ ID No. 3: QPYRVVVLSFELL, SEQ ID No. 4: INASVVNIQKEID, SEQ ID No. 5: DSEPVLKGVKLHY. The leading transmembrane peptide is attached to the N-terminus of the aforementioned short peptides, and its amino acid sequence is as shown in SEQ ID No. 6: YGR KKRRQRRR. The short peptides at SIM1, SIM1', SIM2, SIM3, and SIM4 sites correspond to the short peptide derivatives cpSIM1, cpSIM1', cpSIM2, cpSIM3, and cpSIM4, respectively. Therefore, the amino acid sequences of the short peptide derivatives cpSIM1, cpSIM1', cpSIM2, cpSIM3, and cpSIM4 are as follows: SEQ ID No. 7: YGRKKRRQRRRKTQSLLIVNNATN, SEQ ID No. 8: YGRKKRRQRRRNATNVVIKVCEFQ, SEQ ID No. 9: YGRKKRRQRRRQPYRV VVLSFELL, SEQ ID No. 10: YGRKKRRQRRRINASVVNIQKEID, SEQ ID No. 11: YGRKKRRQRRRDSEPVLKGVKLHY.

[0154] The above-mentioned polypeptide sequences were all prepared by solid-phase synthesis, and the purity of the polypeptides was detected by HPLC. Figure 3 As shown in Tables AH and 2A-2H, the chromatograms show that the purity of all five polypeptides is greater than 95%.

[0155] Table 2A. HPLC detection parameters for cpSIM1

[0156]

[0157] Table 2B. HPLC detection parameters of cpSIM1

[0158]

[0159] Table 2. C.cpSIM2 HPLC detection parameters

[0160]

[0161]

[0162] Table 2. HPLC detection parameters for D.cpSIM3

[0163]

[0164] Table 2E.cpSIM4 HPLC detection parameters

[0165]

[0166] Example 4: Broad-spectrum inhibitory effect of cpSIM2 on SARS-CoV-2 infection and cell fusion

[0167] A549-hACE2 cells were infected with SARS-CoV-2 (SH01), and treated with the above five peptide drugs one hour later. Figure 4 A). High-content scanning analysis showed that cpSIM1, cpSIM1', cpSIM3, and cpSIM4 all increased SARS-CoV-2 infection rates in a dose-dependent manner. Microscopic imaging revealed that treatment with these four drugs promoted the formation and shedding of numerous syncytia in infected cells, while the uninfected control group did not show this effect, ruling out the possibility of drug toxicity. Conversely, cpSIM2 significantly inhibited SARS-CoV-2 infection in a dose-dependent manner. Figure 4 B). Reverse transcription-quantitative PCR results further confirmed that cpSIM2 treatment could inhibit intracellular viral replication and viral release in a dose-dependent manner, with 20 μM cpSIM2 reducing the amount of virus released from the supernatant by 100-fold. Figure 4 C). Immunoblot (IB) assay results showed that cpSIM2 treatment significantly reduced the expression level of Spike protein. Figure 4 D). To evaluate the broad-spectrum antiviral potential of cpSIM2, A549-hACE2 and Calu3 cells (a human lung cell line naturally infected with SARS-CoV-2) were infected with SARS-CoV-2 Delta and Omicron mutants and treated with cpSIM2. The results showed that cpSIM2 treatment significantly inhibited intracellular replication and viral release of the SARS-CoV-2 mutants, and also exhibited a significant antiviral effect in Calu3 cells. Figure 4 E).

[0168] The experimental steps involved in this embodiment are as follows:

[0169] 1. SARS-CoV-2 cell infection and peptide therapy model:

[0170] 1) Prepare 100 μL of cell suspension in a 96-well plate and culture it for 24 hours (at 37°C and 5% CO2) as required for the experiment.

[0171] 2) Dilute the SARS-CoV-2 virus solution to the required multiple of infection (MOI) using DMEM medium with 2% FBS, discard the liquid in the culture plate, add 100 μL of virus dilution to each well, and incubate in a cell culture incubator for 1 hour.

[0172] 3) Discard the virus solution in the culture plate, wash three times with PBS, and add PBS or different concentrations of peptide drugs.

[0173] 5) After incubating in a cell culture incubator for 18 hours, collect the cells and supernatant samples and perform subsequent tests as needed.

[0174] 2. Quantitative determination of viral copy number:

[0175] 1) RNA was extracted from cells and supernatant using TRIzol reagent (Thermo Fisher). Refer to the instruction manual for detailed instructions.

[0176] 2) Quantification of viral copy number: Reverse transcription polymerase chain reaction (RT-PCR) was performed using the HiScript II One Step RT-qPCR SYBR Green Kit (Q221-01, Vazyme) on a CFX Connect real-time system (Bio-Rad). The Q-PCR primers are shown in Table 1 below:

[0177] Table 1

[0178]

[0179] Example 5: Short peptide cpSIM2 inhibits SARS-CoV-2 viral replication in vivo.

[0180] To further verify the antiviral effect of cpSIM2 in vivo, an infection model of 6-8 week old K18-hACE2C57BL / 6 transgenic mice was used. In short, 1×10⁻⁶ mice were used... ^4 Mice were intranasally infected with PFU containing SARS-CoV-2 Omicron BA5.2, and intraperitoneally injected with PBS, cpControl (cpCtrl), or cpSIM2 (dose 25 mg / kg) at 2, 24, 48, and 72 hours post-infection. Figure 5A). On day 5 post-infection, all mice were dissected, and lung tissue was collected for further analysis. qPCR results showed that, compared with the PBS-treated group and the cpCtrl-treated group, cpSIM2 treatment significantly inhibited SARS-CoV-2 replication in the mouse lungs. Figure 5 B). Hematoxylin and eosin (H&E) staining showed that SARS-CoV-2 infection caused severe lung damage in mice treated with PBS and cpCtrl, while cpSIM2 treatment antagonized lung damage at minimal levels. Figure 5 C). Immunohistochemical staining of the N protein showed a significant reduction in viral distribution in the lungs of cpSIM2-treated mice. Figure 5 C and Figure 5 D).

[0181] The experimental steps involved in this embodiment are as follows:

[0182] 1. K18-hACE2 mouse SARS-CoV-2 infection treatment model:

[0183] 1) Prepare mice: Purchase female K18-hACE2-2A-CreERT2 C57BL / 6J mice aged 6-8 weeks and acclimatize them for 7 days in a biosafety level 3 (BSL-3) laboratory at Fudan University (Shanghai), China. Weigh and record the mice one day before inoculation.

[0184] 2) Mouse infection: 1 × 10⁻⁶ mmol / L was administered intranasally to each mouse. ^4 PFU SARS-CoV-2OmicronBA.5.2 strain.

[0185] 3) Drug treatment: After infection, mice were injected intraperitoneally daily with 25 mg / kg of PBS, or cpCtrl or cpSIM diluted in PBS, and their weight was measured.

[0186] 4) Dissecting mice: On the 5th day after infection, the mice were euthanized and lung tissue was collected by dissecting them.

[0187] 2. Hematoxylin and eosin staining:

[0188] Mouse lungs were fixed in 4% formaldehyde. The experiment was performed by Severyl Laboratories, and the experimental procedures included: paraffin embedding, sectioning, hematoxylin staining, eosin staining, dehydration, and mounting. Tissue sections were imaged using a WS-10 Zhiyue panoramic scanner.

[0189] 3. Immunohistochemical staining and analysis:

[0190] Immunohistochemical staining of lung tissue sections from each group of mice was performed using N protein antibody (A18797, Abclonal, dilution 1:200). The experiment was performed by Sewell Pharmaceuticals, and the procedures included: dewaxing of sections, antigen retrieval, treatment with 3% hydrogen peroxide, serum blocking, overnight incubation with primary antibody at 4°C, incubation with secondary antibody at room temperature, staining, hematoxylin staining of nuclei, dehydration, permeabilization, mounting, and microscopic examination. Images of the stained tissue sections were captured using a WS-10 Zhiyue panoramic scanner, and the immunohistochemical positivity rate was calculated using ImageJ software.

[0191] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A short peptide derivative based on the SIM site of the novel coronavirus spike protein, characterized in that, The short peptide derivative is composed of cpSIM2, the amino acid sequence of which is shown in SEQ ID No.

9.

2. A composition, characterized in that, Includes the short peptide derivative based on the SIM site of the novel coronavirus spike protein as described in claim 1.

3. An antiviral drug, characterized in that, Includes the short peptide derivative based on the SIM site of the novel coronavirus spike protein as described in claim 1 or the composition as described in claim 2.

4. The antiviral drug according to claim 3, characterized in that, The virus in question includes the novel coronavirus SARS-CoV-2.

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