Application of antiviral peptides in the preparation of drugs that inhibit human pathogenic RNA viruses

Abstract

This invention relates to the field of antiviral peptide drug technology, specifically to the application of antiviral peptides in the preparation of drugs that inhibit human pathogenic RNA viruses. The invention provides a peptide with antiviral efficacy, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2. This peptide inhibits viral replication by activating the MAVS-mediated innate immune signaling pathway and inducing the expression of antiviral cytokines such as IFNB1, IL6, IFNL2,3, and ISG15. This peptide exhibits significant inhibitory effects against various human pathogenic RNA viruses, including enteroviruses, alphaviruses, and orthomyxoviruses, suppressing viral load in the early stages of infection and maintaining a stable inhibitory effect during peak infection. This technical solution addresses the technical problem of the lack of effective antiviral peptide drugs targeting enteroviruses, alphaviruses, and orthomyxoviruses in the prior art, and has significant clinical value and market application prospects.

Description

Technical Field

[0001] This invention relates to the field of antiviral peptide drug technology, specifically to the application of antiviral peptides in the preparation of drugs that inhibit human pathogenic RNA viruses. Background Technology

[0002] Antiviral peptides (AVPs) are a class of small molecule peptides derived from natural sources or artificially designed to possess antiviral activity, and have become a research hotspot in the field of antiviral drug development in recent years. Compared with traditional chemically synthesized antiviral drugs, antiviral peptides have many unique advantages. The mechanisms of action of antiviral peptides are diverse, targeting multiple stages of the viral life cycle, including inhibiting viral entry into host cells, blocking viral genome release, interfering with viral replication and assembly, and regulating host immune responses. At the same time, antiviral peptides typically possess broad-spectrum activity, low cytotoxicity, rapid onset of action, and low likelihood of inducing drug resistance, making them ideal candidate drugs for combating viral diseases.

[0003] Despite significant progress in the research of antiviral peptides, antiviral peptide drugs targeting specific human pathogenic RNA viruses remain scarce. Enteroviruses ( Enterovirus Enteroviruses are significant pathogens that seriously threaten public health. Enterovirus A71 (EV-A71) and Enterovirus D68 (EV-D68) can cause serious illnesses such as hand-foot-and-mouth disease, meningitis, acute flaccid paralysis, and even fatal pneumonia, infecting hundreds of millions of people globally each year. However, there are currently no approved specific antiviral peptide drugs for the treatment of enterovirus infections. (Avira genus) Alphavirus Aviral viruses, including Chikungunya virus (CHIKV) and Eastern Equine Encephalitis Virus (EEEV), are important human pathogens that can cause fever, rash, joint disease, and even fatal encephalitis. However, there are currently no approved antiviral peptide drugs for the prevention and treatment of alphavirus infection. Although some studies have reported that compounds derived from certain natural products inhibit the alphavirus nsP2 protease, research on antiviral peptides targeting alphaviruses remains very limited. Orthomyxoviridae (… Orthomyxoviridae The H1N1 subtype of influenza A virus is one of the main pathogens causing seasonal influenza and global pandemics. Existing research indicates that certain antimicrobial peptides (such as LL-37 and lactoferrin-derived peptides) and synthetic peptide analogs possess inhibitory activity against the H1N1 virus in vitro, with their mechanisms of action primarily involving disruption of the viral envelope or blocking viral entry. However, compared to the emergence of drug-resistant viral strains, the development of novel anti-influenza peptide drugs remains lagging, and the in vivo efficacy and drug-likeness of most candidate peptides still need to be verified.

[0004] In summary, antiviral peptides theoretically possess the potential to inhibit various human pathogenic RNA viruses, including EV-A71, EV-D68, CHIKV, EEEV, and H1N1. However, current technologies lack antiviral peptide drugs that can be effectively applied clinically. Therefore, developing novel antiviral peptides targeting these viruses and exploring their applications in antiviral drug preparation has significant clinical value and urgent market demand. Summary of the Invention

[0005] The present invention aims to provide a polypeptide with antiviral efficacy to solve the technical problem of the lack of antiviral polypeptide drugs that can effectively target enteroviruses, alphaviruses and orthomyxoviruses in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A polypeptide with antiviral activity, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0007] Furthermore, the virus is an enterovirus, alphavirus, or orthomyxovirus.

[0008] Furthermore, the enterovirus is enterovirus A71 or enterovirus D68; the alphavirus is chikungunya virus or eastern equine encephalitis virus; and the orthomyxovirus is influenza A virus.

[0009] This protocol also provides the application of a peptide with antiviral efficacy in drugs that inhibit human pathogenic RNA viruses.

[0010] Furthermore, the drug also includes a pharmaceutically acceptable carrier, excipient, or adjuvant; the dosage form of the drug is a spray, nasal drops, injection, or lyophilized powder for injection.

[0011] Furthermore, the polypeptide achieves its antiviral effect through one or more of the following mechanisms: (a) Inducing type I and / or type III interferon responses in host cells; (b) Upward adjustment IFNB1 Gene, IL6 Gene, IFNL2 Gene, IFNL3 Genes and / or ISG15 Gene expression; (c) Regulation of interferon expression through IRF1, IRF3, MAVS and / or NF-κB transcription factors.

[0012] Furthermore, the polypeptide reduces the viral load in host organs in vivo; The host organ is selected from one or more of the following: brain, heart, lungs, liver, intestines, spleen, kidneys, and skeletal muscle. Preferably, the polypeptide significantly reduces viral load in the brain, heart, liver, lungs, intestines, and spleen; Preferably, the polypeptide significantly reduces viral load in the liver, lungs, and spleen; Preferably, the polypeptide significantly reduces viral load in the brain, liver, lungs, and intestines; Preferably, the polypeptide significantly reduces viral load in the brain, heart, liver, intestines, and spleen.

[0013] Furthermore, the human pathogenic RNA virus is selected from one or more of enterovirus A71, enterovirus D68, chikungunya virus, eastern equine encephalitis virus, or influenza A virus.

[0014] Furthermore, the polypeptide is used to inhibit initial viral replication by administering it in the early stages of viral infection; the polypeptide is also used to maintain a stable viral suppression effect by administering it at the peak of viral infection. Preferably, the early stage of viral infection is within 1 day after infection; the peak stage of viral infection is 4 days after infection.

[0015] This technical solution also provides a pharmaceutical composition for preventing or treating human pathogenic RNA virus infection or diseases caused by such infection, the pharmaceutical composition comprising a polypeptide with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2 and a pharmaceutically acceptable carrier.

[0016] The technical principle of this technical solution is as follows: The polypeptides provided by this invention, with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.2, do not rely on direct lysis and killing of free viral particles, nor do they target and inhibit the activity of viral-encoded proteases / replicates. Instead, they activate the host cell's innate immune response and induce the transcriptional expression of multiple antiviral cytokines, enabling the host cell to establish a long-lasting intracellular antiviral state, thereby broadly inhibiting the replication and proliferation of various RNA viruses. Specifically, after entering the host cell, the polypeptide can activate the MAVS (mitochondrial antiviral signaling protein)-mediated signaling pathway. MAVS is a key adaptor protein of the RIG-I-like receptor (RLR) pathway. The two antiviral polypeptides can also activate two signaling branches: IRF3 (interferon regulator 3) and NF-κB (nuclear factor κB). IRF3 mainly induces the expression of type I interferons (such as IFNB1), while NF-κB mainly induces the expression of pro-inflammatory factors (such as IL6). Furthermore, the above signals are integrated and amplified through IRF1 (interferon regulator 1), efficiently driving the transcription of type III interferons (such as IFNL2 and IFNL3). Type I and type III interferons induce the expression of hundreds of interferon-stimulated genes (ISGs), including ISG15, through the JAK-STAT signaling pathway, thereby establishing a broad-spectrum antiviral state within cells.

[0017] Experiments have shown that knockout MAVS Gene, IRF3 Gene, p65 Gene (NF-κB subunit) or IRF1 Genes can significantly reduce the peptide-induced [condition / effect]. IFNL2,3 The gene expression level was adjusted to inhibit various human pathogenic RNA viruses. In addition, in vivo experiments showed that the peptide could effectively inhibit the initial replication of the virus when administered at an early stage of viral infection (e.g., 1 day after infection) and maintain a stable inhibitory effect during the peak of viral infection (e.g., 4 days after infection). It also significantly reduced viral load in multiple organs, including the brain, heart, lungs, liver, intestines, and spleen.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) A novel antiviral polypeptide sequence is provided. The polypeptides with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.2 provided by this invention offer novel lead compounds for antiviral drug development, enriching the variety of antiviral polypeptides and providing a clear design template for subsequent structural optimization and drug development. The polypeptides of this invention establish an antiviral state by activating the host MAVS-mediated innate immune signaling pathway, inducing the expression of type I / III interferon and pro-inflammatory factors. This mechanism does not directly target viral proteins, therefore it is less prone to drug resistance due to viral mutations and has greater potential for sustained clinical use.

[0019] (2) Significant in vitro and in vivo efficacy In vitro experiments showed that the peptide of this invention could significantly induce [therapeutic effects] in A549 cells within 8 hours of treatment. IFNB1 Gene, IL6 Gene, IFNL2,3 Genes and ISG15 mRNA expression of the gene. In vivo experiments showed that the peptide of the present invention, when injected intraperitoneally at a dose of 10 mg / kg in a neonatal BALB / cAJcl mouse model, significantly reduced the viral load of EV-A71 and EV-D68 in multiple organs, including the brain, heart, lungs, liver, intestines, and spleen, and maintained a stable inhibitory effect in both the early stage (1 day) and the peak stage (4 days) of viral infection.

[0020] (3) Broad-spectrum antiviral activity The peptides of this invention exhibit significant inhibitory activity against a variety of human pathogenic RNA viruses, including enteroviruses (EV-A71, EV-D68), alphaviruses (CHIKV, EEEV), and orthomyxoviruses (H1N1). Compared with existing antiviral drugs that target only a single virus, the peptides of this invention have the advantage of broad-spectrum antiviral activity and are suitable for the prevention and treatment of various viral infections.

[0021] (4) It has good safety profile and has the potential to be developed into a drug. Peptide drugs exhibit good biocompatibility and low cytotoxicity, with degradation products being amino acids. Their metabolic clearance pathways in vivo are clearly defined, resulting in a low risk of toxic side effects. No significant adverse reactions were observed in animals at effective doses of the peptides of this invention, demonstrating promising drug development prospects. Currently, there are no approved antiviral peptide drugs for treating viral infections such as EV-A71, EV-D68, CHIKV, and EEEV, and antiviral peptide drugs targeting H1N1 are extremely limited. This invention fills this technological gap and has significant clinical value and market application prospects. Attached Figure Description

[0022] Figure 1This is a schematic diagram of the genome structure of Chikungunya virus CHIKV (TAT-CHIKV) expressing the TAT protein and Eastern Equine Encephalitis Virus EEEV (TAT-EEEV) expressing the TAT protein, as described in Example 2.

[0023] Figure 2 Representative images of eGFP signal detection used in Example 3 to evaluate the antiviral (EV-A71) efficacy of the peptide using a multi-channel virus reporter system (MOCK indicates uninfected control; (-) indicates negative control; rupintrivir is the positive control).

[0024] Figure 3 This is a statistical graph of the relative inhibition rate for evaluating the antiviral (EV-A71) effect of the peptide using a multi-channel virus reporter system in Example 3 (Rupintrivir was the positive control; statistical results are expressed as "mean ± SD", n=3).

[0025] Figure 4 Representative images of eGFP signal detection used in Example 3 to evaluate the antiviral (EV-D68) efficacy of the peptide using a multi-channel virus reporter system ((-) indicates a negative control; rupintrivir is the positive control).

[0026] Figure 5 Representative images of eGFP signal detection used in Example 3 to evaluate the antiviral (CHIKV and EEEV) efficacy of peptides using a multi-channel virus reporter system (MOCK indicates uninfected control; (-) indicates negative control; rupintrivir is the positive control).

[0027] Figure 6 The experimental verification results of the peptide in Example 4 on the in vitro inhibition of viral proliferation activity (H1N1 PR8 virus) show the relative level of viral RNA in the culture supernatant after viral infection of cells; (-) indicates negative control; A is the first antiviral peptide; B is the second antiviral peptide; the statistical results are expressed as "mean ± SD", n=3; **** indicates that compared with the corresponding negative control group, p<0.0001).

[0028] Figure 7The statistical results of the in vivo inhibition of viral activity by the first antiviral peptide (SEQ ID NO.1) in Example 5 are shown (the relative levels of viral RNA in different parts of the experimental animals are displayed; A is the blank control; B is the first antiviral peptide; the statistical results are expressed as "mean ± SD", n=4; * indicates p < 0.05 compared with the corresponding negative control group; ** indicates p < 0.01 compared with the corresponding negative control group; *** indicates p < 0.001 compared with the corresponding negative control group; **** indicates p < 0.0001 compared with the corresponding negative control group).

[0029] Figure 8 The statistical results of the in vivo inhibition of viral activity by the second antiviral peptide (SEQ ID NO.2) in Example 5 are shown (the relative levels of viral RNA in different parts of the experimental animals are displayed; A is the blank control; B is the second antiviral peptide; the statistical results are expressed as "mean ± SD", n=4; * indicates p < 0.05 compared with the corresponding negative control group; ** indicates p < 0.01 compared with the corresponding negative control group; *** indicates p < 0.001 compared with the corresponding negative control group; **** indicates p < 0.0001 compared with the corresponding negative control group).

[0030] Figure 9 The results of the study on the mechanism of action of the peptides in Example 6 (A: Effects of different antiviral peptides on the expression of cytokines in A549 cells; B: Effects of different antiviral peptides on the expression of cytokines in different gene knockout cells) IFNL2,3 Effects of cytokine expression; MOCK blank control; ① first antiviral peptide; ② second antiviral peptide; ③ antiviral peptide VSV; Statistical results are expressed as mean ± SD, n=3). Detailed Implementation

[0031] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0032] Example 1: Antiviral peptide information In this scheme, the antiviral peptide used as a drug to inhibit human pathogenic RNA viruses is either a first antiviral peptide or a second antiviral peptide. The sequence of the first antiviral peptide is shown in SEQ ID NO.1, and the sequence of the second antiviral peptide is shown in SEQ ID NO.2. SEQ ID NO.1 is specifically: GIKCRFCCGCCTPGICGVCCRF; SEQ ID NO.2 is specifically: CNIAPASIVSRNIVYTRAQPNQDIA. Both of the above antiviral peptides can be obtained through conventional chemical synthesis methods using existing technology, and can be commissioned to a biotechnology company.

[0033] Example 2: Multi-channel virus reporting system The efficacy of both antiviral peptides was studied using a multi-channel virus reporter system previously developed by the team. More specifically, this technical solution employs the prior patented "Multi-channel Virus Reporter System Based on TAT Trans-activation" for drug screening (CN120485138A, A Multi-channel Virus Reporter System Based on TAT Trans-activation), and the entire contents of this patent (CN120485138A) are incorporated into this patent document.

[0034] A multi-channel viral reporter system based on TAT transactivation includes reporter cells and an activating virus. The activating virus infects reporter cells, and by adding GCV, it is converted into a toxic product by TK, thus enabling inhibitory screening and efficacy evaluation. Flow cytometry and fluorescence imaging are used to observe GFP signals, enabling single-cell monitoring. Detection of Gluc fluorescence allows for monitoring of the overall cell population. This study utilizes the transactivation of TAT, which specifically binds to the transactivation response region element in the LTR promoter, thereby regulating gene transcription and expression. Linking several reporter genes after the LTR promoter, placing them in the same coding frame and subjecting them to LTR regulation, overcomes the limitations of traditional single-channel screening with reporter viruses.

[0035] The preparation method of reporter cells is detailed in "Example 2: Construction of reporter cell line" in patent CN120485138A, which involves transfecting A549 cells (human lung adenocarcinoma epithelial cell line) with reporter plasmids. These cells contain three reporter genes: TK (thymidine kinase), GFP (green fluorescent protein), and Gluc (gaussian luciferase). The expression of these reporter genes is regulated by LTRs (long terminal repeats). LTRs contain promoter and enhancer regions. TAT (trans-activator of transcription) proteins exert their function by binding to a specific sequence within the LTR (called TAR, trans-activation response element), thereby transactivating gene expression.

[0036] This study investigated the effects of antiviral peptides on enteroviruses A71 / D68 (EV-A71 / D68), chikungunya virus (CHIKV), eastern equine encephalitis virus (EEEV), and orthomyxovirus (H1N1 PR8). Therefore, activated viruses representing these different viruses were constructed for a multi-channel virus reporting system. Four types of activated viruses were developed, and their details are as follows: Human enterovirus A71 expressing TAT protein (TAT-EV-A71): This is the virus obtained in "Example 1: Construction of Activated Virus (Human Enterovirus A71 Expressing TAT Protein)" in prior patent CN120485138A. TAT-EV-A71 represents EV-A71 (Enterovirus A71). EV-A71 virus is a single-stranded positive-sense RNA virus with a genome of approximately 7.4 kb, containing a 5' untranslated region (5'UTR), an open reading frame (ORF), and a 3' untranslated region (3'UTR). The ORF encodes a multimeric precursor protein, which is cleaved by viral proteases to form a structural protein (P1, which sequentially includes VP4, VP3, VP2, and VP1) and non-structural proteins (P2 and P3). In this technical solution, a TAT sequence is inserted into the EV-A71 viral genome, with the TAT sequence inserted in the middle of the VP4 sequence. Specifically, after the "atgggc" sequence inserted at position VP4, the TAT sequence is followed by the T2A sequence (cleavage peptide sequence), and the T2A sequence is immediately followed by the VP4 sequence. The structure and specific sequence of the plasmid corresponding to the activated virus TAT-EV-A71 can be found in the prior patent CN120485138A, and will not be repeated here. Following the method described in the prior patent, the plasmid can be transcribed, translated, and packaged into the TAT-EV-A71 virus.

[0037] Human enterovirus D68 expressing TAT protein (TAT-EV-D68): This virus was obtained in "Example 1: Screening of antiviral drugs using a multi-channel viral reporter system based on TAT transactivation" in prior patent CN121081473A (Application of Ensetvir in the Preparation of Anti-Enterovirus Drugs). TAT-EV-D68 represents EV-D68 (Enterovirus D68), a member of the Enterovirus genus of the Picornaviridae family, approximately 7.3 kb in length (slightly shorter than EV-A71's 7.4 kb). The EV-D68 genome can be divided into three core regions from the 5' end to the 3' end: the 5' untranslated region (5'UTR), the open reading frame (ORF), and the 3' untranslated region (3'UTR). The EV-D68 open reading frame (ORF) encodes structural proteins (P1, including VP4, VP3, VP2, and VP1 in sequence) and non-structural proteins (P2 and P3). The structural protein P1 of EV-D68 has the same sequence as that of EV-A71; however, there are certain sequence differences between the non-structural proteins of EV-D68 and EV-A71. Therefore, the insertion site of TAT in the EV-D68 genome is the same as that in the EV-A71 genome, which can effectively achieve the expression of the TAT protein in the TAT-EV-D68 virus, thus achieving the screening purpose. The structure and specific sequence of the plasmid corresponding to the activated virus TAT-EV-D68 can be found in prior patent CN121081473A, and will not be elaborated upon here.

[0038] Chikungunya virus CHIKV expressing TAT protein (TAT-CHIKV): TAT-CHIKV represents Chikungunya virus CHIKV, and its expression of the TAT protein is to adapt to the "multi-channel virus reporter system based on TAT trans-activation" constructed by the applicant in a previous patent. Referring to the preparation methods of EV-A71-TAT and TAT-EV-D68 activated viruses in the prior patent, a nucleotide fragment of TAT is inserted into the Chikungunya virus CHIKV genome to obtain the TAT-CHIKV activated virus. Its structural diagram is shown below. Figure 1 .

[0039] Eastern equine encephalitis virus (EEEV) expressing TAT protein (TAT-EEEV): TAT-EEEV represents Eastern equine encephalitis virus (EEEV), and its expression of the TAT protein is to adapt to the "multi-channel virus reporter system based on TAT trans-activation" constructed by the applicant in a previous patent. Referring to the preparation methods of EV-A71-TAT and TAT-EV-D68 activated viruses in the prior patent, a nucleotide fragment of TAT is inserted into the EEEV genome to obtain the TAT-EEEV activated virus. Its structural diagram is shown below. Figure 1 .

[0040] Influenza A virus H1N1 PR8 expressing TAT protein (TAT-H1N1 PR8): TAT-H1N1 PR8 represents orthomyxovirus H1N1 PR8, and its expression of TAT protein is to adapt to the "multi-channel virus reporter system based on TAT trans-activation" constructed by the applicant in previous patents. Referring to the preparation methods of EV-A71-TAT and TAT-EV-D68 activated viruses in prior patents, the TAT nucleotide fragment is inserted into the genome of influenza A virus H1N1 PR8 to obtain the TAT-H1N1PR8 activated virus.

[0041] The preparation method of the activated virus used as the tool virus for evaluating the efficacy of this scheme has been disclosed in a prior patent. Based on this prior patent, those skilled in the art can prepare the desired type of activated virus simply by inserting a TAT element into the viral genome. The activated viruses used in this study, such as TAT-EV-A71 (including the aforementioned TAT-EV-A71, TAT-EV-D68, TAT-CHIKV, TAT-EEEV, and TAT-H1N1 PR8), only have a TAT expression element inserted into the wild-type viral backbone, completely preserving all replication and infection-related functional genes of EV-A71 and other viruses. Previous preliminary experiments have shown that, through multiple verifications using viral proliferation kinetics, cellular pathogenic damage, and drug dose-inhibition curves, at the same infection dose, the cell infection efficiency, viral replication and release levels, and response efficacy to small molecule / peptide drugs of activated viruses such as TAT-EV-A71 are completely consistent with those of wild-type viruses such as EV-A71, with no statistically significant differences. Therefore, activated viruses such as TAT-EV-A71 (including the aforementioned five activated viruses) can accurately represent wild-type viruses such as EV-A71 (including wild-type viruses of EV-A71, EV-D68, CHIKV, EEEV and H1N1 PR8 species) and can be used for antiviral peptide screening and validation, visual monitoring of viral replication levels and quantitative evaluation of drug efficacy.

[0042] Example 3: Evaluation of the antiviral efficacy of peptides using a multi-channel virus reporting system (1) Detection method On day -1, reporter cells (reporter cells containing three reporter genes) were seeded in DMEM medium (96-well plate) without antibiotics at a density of approximately 60% and cultured overnight.

[0043] On day 0, add the first antiviral peptide (unless otherwise specified, final concentration is 2.5 μM) or the second antiviral peptide (unless otherwise specified, final concentration is 2.5 μM) or the antiviral drug rupintrivir (unless otherwise specified, final concentration is 2.5 μM). Then, add EV-A71, EV-D68, CHIKV, EEEV, and H1N1 PR8 expressing TAT protein to activate the virus (TAT-EV-A71, TAT-EV-D68, TAT-CHIKV, TAT-EEEV, TAT-H1N1PR8) at an MOI ≈ 0.1 to induce viral infection.

[0044] On day 2, 48 hours after activating the virus-infected reporter cells, intracellular eGFP signals were detected using a Zeiss LSM 980 confocal microscope. Images were acquired and processed using ZEN imaging software under consistent parameters. In addition to observing eGFP signals, ganciclovir (GCV) was added to a final concentration of 200 μM 48 hours after activation. GCV activates the reporter cell suicide system (tk / GCV); wells where the viral life cycle was inhibited had a higher cell survival rate, and vice versa.

[0045] On day 3, 24 hours later, the CellTiter-Glo device and reagent kit were used for detection and analysis to quantify the number of surviving cells in the well plate. The higher the number of surviving cells, the stronger the antiviral effect of the drug.

[0046] (2) Detection results for enterovirus EV-A71 For enterovirus EV-A71, the eGFP signal detection results are as follows: Figure 2As shown in the figure. MOCK represents the uninfected control, inoculated with reporter cells only, without the addition of virus or antiviral drugs; (-) represents the negative control, inoculated with reporter cells only and with the addition of virus, without the addition of antiviral drugs; the concentrations of the first antiviral peptide were 0.5 μM and 2.5 μM; the concentrations of the second antiviral peptide were 0.5 μM and 2.5 μM; the concentrations of rupintrivir were 0.5 μM and 2.5 μM. This study used the TAT trans-activated reporter system to evaluate the efficacy of the two antiviral peptides. This multi-channel viral reporter system can rapidly and intuitively evaluate the efficacy of antiviral drugs. The results showed that the negative control group (-) had a strong fluorescence signal, indicating that virus activation can effectively infect reporter cells; compared with the negative control group (-), both the first and second antiviral peptides significantly reduced the eGFP fluorescence signal in reporter cells at both concentrations, indicating that the viral level was inhibited by the peptides. Furthermore, the fluorescence intensity of the two polypeptide groups was weaker than that of the positive control drug rupintrivir, indicating that the two polypeptides have strong antiviral activity and their inhibitory effect is better than that of rupintrivir.

[0047] The relative inhibition rate was detected using the aforementioned GCV-activated reporter cell suicide system (tk / GCV). The experimental results are shown in [link to results]. Figure 3 This quantitative study is based on the tk / GCV suicide selection pathway of the TAT multichannel reporter system: During normal viral replication, TAT is expressed in large quantities, acting on the LTR of reporter cells and initiating tk gene expression, converting GCV into toxic products, leading to mass cell death. If the virus is inhibited with drugs, TAT expression is reduced, tk gene expression decreases, GCV toxicity is reduced, and more cells survive. Therefore, the greater the number of surviving cells, the stronger the viral inhibition, and the higher the relative inhibition percentage.

[0048] The experimental groups were as follows: MOCK uninfected control group (reporter cells only, no virus or antiviral drugs), negative control group (reporter cells only, virus added, no antiviral drugs), and drug-treated group (reporter cells + activated virus + first antiviral peptide / second antiviral peptide / rupintrivir). The experimental procedure was followed as described above. For signal detection, CellTiter-Glo (CTG) was used, which is an ATP bioluminescent cell viability assay kit: stable ATP exists within live cells, a lysis reagent releases intracellular ATP, and luciferase generates a luminescent signal using ATP as a substrate; the luminescence value (RLU) is strictly linearly positively correlated with the total number of live cells; the stronger the luminescence, the more live cells. The RLU signals of each group were measured, and the relative cell viability was calculated based on the ATP luminescence RLU values. Relative cell viability in the drug-treated group = RLU signal intensity in the drug-treated group / RLU signal intensity in the uninfected MOCK control group; The relative cell survival rate of the negative control group = RLU signal intensity of the negative control group / RLU signal intensity of the uninfected MOCK control group.

[0049] Relative inhibition rate (%) = (Relative cell survival rate of drug-treated group - Relative cell survival rate of negative control group) / (Relative cell survival rate of uninfected MOCK control group - Relative cell survival rate of negative control group) × 100%; In the MOCK uninfected control group, the relative survival rate of cells was 100%. In the formula for relative inhibition rate (%), the denominator represents the maximum salvageable cell survival in the system, corresponding to 100% viral inhibition; the molecule represents the increase in cell survival brought about by the drug, and after normalization, the relative inhibition effect in the range of 0-100% is obtained. If the peptide slightly promotes cell proliferation, the inhibition rate will be >100% (for example, the result of the first antiviral peptide in this protocol at 2.5 μM), which is a normal phenomenon.

[0050] according to Figure 3 The experimental results show that this study used the TAT multichannel viral reporter system based on tk / GCV suicide screening. Cell viability was quantified and the relative viral inhibition rate was calculated using CellTiter-Glo chemiluminescence immunoassay. The in vitro antiviral activity of the positive control drug rupintrivir, the first antiviral peptide, and the second antiviral peptide was compared at two concentration gradients of 0.5 μM and 2.5 μM. At the low concentration of 0.5 μM, rupintrivir showed only a weak viral inhibitory effect, with a relative inhibition rate of about 10%, while both peptides showed strong inhibitory activity of nearly 90-95% (approximately 95% for the first antiviral peptide and approximately 90% for the second antiviral peptide), significantly better than the positive control drug at the same concentration. After increasing the drug concentration to the experimental standard of 2.5 μM, the inhibitory activity of rupintrivir was significantly improved, with a relative inhibition rate of about 55%, but still much lower than that of the two peptides. The first antiviral peptide showed almost complete inhibition of the virus, exhibiting the best antiviral effect. The second antiviral peptide had a relative inhibition rate of about 82%, weaker than the first antiviral peptide but still significantly stronger than rupintrivir. Overall activity patterns showed that rupinxivir exhibited a significant dose-dependent effect, with increased concentration enhancing its inhibitory capacity. The first antiviral peptide nearly completely inhibited the virus at low concentrations, with further enhanced inhibition at higher concentrations. The second antiviral peptide achieved a relatively ideal viral inhibition effect at 0.5 μM. The combined results from both concentrations confirm that the in vitro antiviral activity of both self-developed antiviral peptides is significantly superior to that of the positive control drug rupinxivir, making them promising drugs against enterovirus EV-A71.

[0051] (3) Detection results for enterovirus EV-D68 For enterovirus EV-D68, the experimental results are as follows: Figure 4 As shown. (-) indicates the negative control, inoculated only with reporter cells and added virus, without antiviral drugs; the concentration of the first antiviral peptide was 2.5 μM; the concentration of the second antiviral peptide was 2.5 μM; the concentration of rupintrivir was 2.5 μM. The experimental results show that the first and second antiviral peptides can significantly inhibit enterovirus EV-D68.

[0052] (4) Detection results for CHIKV and EEEV. The experimental results for CHIKV and EEEV are as follows: Figure 5 As shown in the diagram. MOCK represents the uninfected control, inoculated with reporter cells only, without the addition of virus or antiviral drugs; (-) represents the negative control, inoculated with reporter cells only and with the addition of virus, without the addition of antiviral drugs; the concentration of the first antiviral peptide was 2.5 μM; the concentration of the second antiviral peptide was 2.5 μM. The experimental results show that the first and second antiviral peptides can significantly inhibit CHIKV and EEEV.

[0053] Example 4: Experimental verification of the in vitro inhibitory effect of peptides on viral activity On day -1, A549 was inoculated in DMEM medium (96-well plate) without antibiotics at a density of approximately 60% and cultured overnight.

[0054] On day 0, add either the first antiviral peptide (final concentration 2.5 μM) or the second antiviral peptide (final concentration 2.5 μM), and then add wild-type H1N1 PR8 virus at an MOI of ≈0.1 or 0.01 to infect the virus.

[0055] On day 2, 48 hours after virus infection of cells, cell culture supernatant (containing progeny influenza virus particles released from cells) was collected. Total viral RNA was extracted from the supernatant samples and purified to obtain viral nucleic acid. Viral load was then quantitatively detected by qRT-PCR, with the viral RNA copy number of the drug-free negative control group (-) at the same MOI set as the baseline value of 1.0. The relative viral RNA levels (copy counts) in the supernatant between the two peptide treatment groups were calculated.

[0056] The experimental results for orthomyxovirus H1N1 PR8 are as follows: Figure 6 As shown: (-) indicates the negative control, inoculated only with reporter cells and added virus, without the addition of antiviral drugs; the concentration of the first antiviral peptide was 2.5 μM; the concentration of the second antiviral peptide was 2.5 μM. The experimental results show that the first and second antiviral peptides significantly reduced the viral copy number in the supernatant and effectively inhibited viral proliferation.

[0057] This study quantitatively detected the H1N1 PR8 viral RNA level in cell supernatants. Using the viral RNA abundance of the drug-free negative control group at the same MOI as a baseline of 1.0, the antiviral efficacy of 2.5 μM first and second antiviral peptides was verified at two infection doses: MOI=0.1 and MOI=0.01. The results showed that at both viral infection doses, the relative viral RNA level in the cell supernatant after peptide treatment was significantly lower than that in the negative control group. Under the high viral load condition of MOI=0.1, both the first and second antiviral peptides reduced the viral RNA level in the supernatant to a relatively low level. More specifically, under the condition of MOI=0.1, the relative H1N1 PR8 viral RNA level in the supernatant of the first antiviral peptide was 0.58±0.05, and the relative H1N1 PR8 viral RNA level in the supernatant of the second antiviral peptide was 0.21±0.01. At the low infection dose of MOI=0.01, the inhibitory effect of both peptides was further enhanced, and the viral copy number in the supernatant of the group treated with the second antiviral peptide was almost completely inhibited. More specifically, under the condition of MOI=0.01, the relative RNA level of H1N1 PR8 virus in the supernatant of the first antiviral peptide was 0.07±0.01, and the relative RNA level of H1N1 PR8 virus in the supernatant of the second antiviral peptide was 0.002±0.001.

[0058] The above results confirm that both antiviral peptides can effectively inhibit the proliferation and progeny virus release of influenza A H1N1 PR8, and the second antiviral peptide exhibits superior in vitro anti-H1N1 activity compared to the first antiviral peptide. Furthermore, both peptides showed significant antiviral effects at both high and low viral infection doses, suggesting their potential for broad-spectrum adaptation to different viral infection intensities. Under high viral load (MOI=0.1), the second antiviral peptide reduced the relative viral RNA level to 0.21, with an inhibition efficiency approximately 2.76 times that of the first antiviral peptide (0.58). At low infection doses (MOI=0.01), while the first antiviral peptide showed some enhancement, a residual effect remained (0.07), whereas the second antiviral peptide further reduced the viral RNA level to 0.002, only 1 / 35th that of the first antiviral peptide, approaching the background level of the detection technology. The fold difference, ranging from 2.76 times at high MOI to 35 times at low MOI, spanning an order of magnitude, indicates a significant enhancement in the inhibitory effect of the second antiviral peptide at lower viral loads, exceeding the range reasonably expected based on the performance of the first antiviral peptide. This result suggests that the second antiviral peptide may not simply slow viral replication, but rather more thoroughly eliminate the virus under low viral load conditions, making it worthy of further investigation in antiviral drug development.

[0059] Based on the experimental results against viruses such as EV-D68 mentioned above, it is demonstrated that this series of peptides possesses broad-spectrum antiviral activity against enteroviruses, influenza A viruses, and the like.

[0060] Example 5: Experimental verification of the in vivo inhibition of viral activity by peptides All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with relevant guidelines. Newborn BALB / cAJcl mice (1-2 days old) were purchased from Solarbio (Shanghai) Co., Ltd. Mice were intraperitoneally injected with EV-A71 (1×10⁻⁶). 6 Gene copy number / mL, TAT-EV-A71 or EV-D68 (1×10⁻⁶) as described in Example 2. 6 Gene copy number / mL, TAT-EV-D68 as described in Example 2), or simultaneous intraperitoneal injection of the first antiviral peptide (10 mg / kg) or the second antiviral peptide (10 mg / kg), with DMSO as a blank control (Vehicle), 4 mice per group. Mice were euthanized on day 1 or 4 post-inoculation (1 dpi or 4 dpi), and multiple organs including brain, heart, lung, liver, spleen, kidney, and skeletal muscle were collected. Total RNA was extracted from each organ using a rapid RNA extraction kit (Accurate Biotechnology), and viral RNA levels were quantified by RT-qPCR according to the methods described below. See detailed experimental results. Figure 7 and Figure 8 With the viral RNA level (copy count) in the brain at 1 dpi as 1, the relative viral RNA levels in other treatment groups were calculated.

[0061] All organs and both viruses showed a consistent trend: the viral load in the blank control group at 4 dpi was much higher than that in the control group at 1 dpi, indicating that EV-A71 and EV-D68 proliferated rapidly in mice over time, with the peak viral load occurring on day 4 of infection.

[0062] First antiviral polypeptide ( Figure 7 This product targets EV-A71 virus, reducing viral load in the brain, heart, liver, lungs, intestines, and spleen at both 1 and 4 days post-infection (dpi). Its potent target organs are the liver, lungs, heart, and intestines, showing the greatest inhibitory effect and significant reduction in viral load. It also targets EV-D68 virus, reducing viral load to some extent in the brain, heart, liver, lungs, intestines, and spleen at both 1 and 4 dpi, with particularly strong inhibitory effects in the intestines, lungs, and heart. Using the first antiviral peptide, early intervention at 1 dpi can suppress initial viral replication, and the inhibitory effect remains stable at the peak at 4 dpi.

[0063] Second antiviral polypeptide ( Figure 8It exhibits antiviral activity against EV-A71 virus in all organs, significantly inhibiting the virus in the brain, liver, lungs, and intestines. Against EV-D68 virus, its dominant sites of viral inhibition are the brain, heart, liver, intestines, and spleen.

[0064] Example 6: Study on the mechanism of action of peptides A549 cells were seeded in DMEM medium (96-well plates) without antibiotics, at a density of approximately 60%, and cultured overnight. A first antiviral peptide (final concentration 2.5 μM), a second antiviral peptide (final concentration 2.5 μM), or interferon-activated control vesicular stomatitis virus (VSV) (MOI=0.01) was added. After 8 hours of treatment, the ability of the two peptides to induce cytokine responses in A549 cells was assessed (mRNA levels were measured). Cytokines included... IFNB1, IL6, IFNL2,3 as well as ISG15 Using the untreated control group MOCK as 1.0, the relative expression levels of cytokine mRNA in the corresponding drug-treated groups were calculated. Details regarding cytokines are as follows: interferons include... IFNB1 (β-interferon, type I interferon) and IFNL2,3 (λ2, λ3 interferon, type III interferon); pro-inflammatory factors are IL6 (Interleukin-6) and ISG15 (Interferon stimulates gene 15). Experimental results ( Figure 9 A) It was observed that each peptide induced significant [acidity / induction] 8 hours after treatment. IFNB1, IL6, IFNL2,3 as well as ISG15 mRNA response.

[0065] Experimental studies were conducted using gene knockout cell lines, following the same experimental methods as above, to investigate the effects of different antiviral peptides on different cell lines. IFNL2,3 Cytokine expression. Different cell lines included: wild-type A549 cells (Ctrl). IRF1 Gene knockout A549 cells ( IRF1 KO ), IRF3 Gene knockout A549 cells ( IRF3 KO ), P65 Gene knockout A549 cells ( P65 KO ), MAVS Gene knockout A549 cells ( MAVS KO The establishment of gene knockout cell lines is a conventional method in existing technology and is not within the scope of this patent. IRF1 is interferon regulatory factor 1, IRF3 is interferon regulatory factor 3, p65 is the NF-κB subunit, and MAVS is a mitochondrial antiviral signaling protein. See detailed experimental results. Figure 9 B, using wild-type A549 cells in the absence of drug application. IFNL2,3The mRNA level of cytokines was 1.0 (data not shown in...). Figure 9 (B) Calculate the other experimental groups IFNL2,3 Relative expression levels of cytokines (fold change). The experimental results show that knocking out any one of the transcription factors, especially... IRF1 Both significantly reduced the effect of peptide stimulation on A549 cells. IFNL2,3 The levels of these two antiviral peptides are such that they can activate cytokine responses in a variety of host cells, which forms the basis of their antiviral efficacy.

[0066] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The application of a polypeptide with antiviral activity in the preparation of drugs that inhibit human pathogenic RNA viruses, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.2; the human pathogenic RNA virus is selected from one or more of enterovirus A71, enterovirus D68, chikungunya virus, eastern equine encephalitis virus, or influenza A virus.

2. The application of the polypeptide with antiviral activity according to claim 1 in the preparation of a drug for inhibiting human pathogenic RNA viruses, characterized in that, The drug also includes a pharmaceutically acceptable carrier, excipient, or adjuvant; the dosage form of the drug is a spray, nasal drops, injection, or lyophilized powder for injection.

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