Antiviral polypeptides targeting virus-host interactions and uses thereof

Abstract

The application discloses an antiviral polypeptide targeting virus-host interaction and application, the polypeptide is composed of 1-17 amino acids of ARF1, a transmembrane peptide TAT and an intermediate connecting peptide, and the amino acid sequence is shown as SEQ ID NO:1; the polypeptide provides a new strategy for the prevention and control of RNA viruses, and also provides a new theoretical basis for accelerating the research and development of broad-spectrum antiviral polypeptide small molecule drugs.

Description

Technical Field

[0001] This invention belongs to the field of antiviral peptide drug technology, specifically relating to an antiviral peptide that targets virus-host interaction and its application. Background Technology

[0002] The pathogen causing the novel coronavirus infection, SARS-CoV-2 (COVID-19), belongs to the family Coronaviridae, genus Betavirus, and is a typical positive-sense RNA virus. Currently, some effective and marketed direct-acting antiviral drugs for COVID-19 include inhibitors of 3-chymotrypsin-like cysteine ​​protease (3CLpro) and RNA-dependent RNA polymerase (RdRp) inhibitors. However, a specific drug for COVID-19 is still lacking. Furthermore, the COVID-19 virus mutates rapidly, highlighting the challenge of developing broad-spectrum antiviral drugs that are less likely to induce drug resistance.

[0003] Viruses depend on host cells for survival. Extensive interactions exist between viral and host proteins, which are crucial for viral infection, replication, transmission, and the host's resistance to viral infection. Targeting viral-interacting host molecules as therapeutic targets may offer durable, broad-spectrum treatment modalities that are less likely to induce drug resistance.

[0004] The novel coronavirus has four structural proteins: spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), and envelope protein (E protein). The M protein is the most abundant protein in coronavirus viral particles and is highly conserved among coronaviruses. The M protein can interact with other structural proteins and plays a crucial role in driving viral particle assembly and maturation. Our research shows that the host molecule ARF1 is a key factor in the replication of the novel coronavirus. ARF1 belongs to the highly conserved type I ARF family in eukaryotes, and ARF family members and upstream and downstream factors synergistically regulate intracellular vesicle structure and transport. Existing research has shown that ARF1 and its downstream effector molecules play important roles in the replication of several RNA viruses, such as poliovirus (PV), Coxsackievirus B3 (CVB3), Hepatitis C virus (HCV), dengue virus (DENV), classical swine fever virus (CSFV), influenza A virus (IAV), rotavirus (RV), and human enterovirus 71 (EV71). Summary of the Invention

[0005] The purpose of this invention is to provide an antiviral polypeptide that targets virus-host interactions, which can serve as a broad-spectrum antiviral drug and is less likely to induce drug resistance.

[0006] In view of this, the solution of the present invention is as follows:

[0007] The first objective of this invention is to provide an antiviral polypeptide that targets virus-host interactions, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0008] A second objective of this invention is to provide the application of the above-described antiviral peptides and / or their modified peptides in the preparation of drugs for inhibiting RNA viruses.

[0009] Preferably, the RNA virus includes SARS-CoV-2, human enterovirus 71, hepatitis C virus, and Coxsackievirus A16.

[0010] A third objective of this invention is to provide a nucleotide sequence encoding the antiviral polypeptide described above.

[0011] A fourth object of the present invention is to provide a recombinant vector carrying the above-described encoded nucleotide sequence.

[0012] A fifth object of the present invention is to provide recombinant microbial cells expressing the antiviral peptides described above.

[0013] Preferably, the microbial cells are prokaryotic cells or eukaryotic cells.

[0014] A sixth objective of this invention is to provide the use of the above-described nucleotide sequence, the recombinant vector, or the recombinant microbial cell in the preparation of a drug for inhibiting RNA viruses.

[0015] Preferably, the RNA virus includes SARS-CoV-2, human enterovirus 71, hepatitis C virus, and Coxsackievirus A16.

[0016] Compared with the prior art, the beneficial effects of the present invention include, but are not limited to:

[0017] The antiviral peptide provided by this invention is composed of 1-17 amino acids of ARF1 fused with a membrane-penetrating peptide TAT and an intermediate linker peptide. The peptide is less likely to induce drug resistance in RNA viruses, providing a new strategy for virus control and also providing a new theoretical basis for accelerating the development of broad-spectrum antiviral peptide small molecule drugs. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This invention provides an inhibitory effect of the antiviral peptide p17 on SARS-CoV-2 and its mutant strains. Specifically: A represents the cytotoxicity of p17 against Huh-7-ACE2 cells as detected by the CCK8 assay; B and C represent the anti-SARS-CoV-2 effect of p17 in Huh-7-ACE2 cells: the relative RNA content of SARS-CoV-2 in cells (B) and the copy number of viral particles outside the cells (C) after treatment with different concentrations of p17; D represents the cytotoxicity experiment of the control peptide TAT in Huh-7-ACE2 cells; E represents the lack of anti-SARS-CoV-2 activity of the control peptide TAT in Huh-7-ACE2 cells; FH represents the inhibitory effect of p17 on SARS-CoV-2 mutant strains: the relative viral RNA content of the SARS-CoV-2 mutant strains Delta (F), Omicron BA.4 (G), and BA.5 (H) in cells after treatment with different concentrations of p17; IK represents the lack of inhibitory effect of the control peptide TAT on any SARS-CoV-2 mutant strain.

[0020] Figure 2 The present invention provides an inhibitory effect of the antiviral peptide p17 on HCV, EV71 and CA16; wherein: AC is the relative RNA content of HCV (A), EV71 (B) and CA16 in cells after treatment with different concentrations of p17; DF is the control peptide TAT, which has no activity against HCV, EV71 and CA16. Detailed Implementation

[0021] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described in this specification are only for explaining this invention and are not intended to limit this invention.

[0022] Our research revealed that the SARS-CoV-2 M protein directly promotes viral replication by binding to the host molecule ARF1. Therefore, we further elucidated the fine-grained binding region of the M protein targeting ARF1. Based on this, we designed an ARF1-mimicking peptide that both interferes with the normal function of ARF1 and disrupts the interaction between M and the host ARF1, thereby inhibiting viral replication and reducing the likelihood of drug resistance. This requires adaptive mutations in both the viral M protein and the host ARF1, without affecting the M-ARF1 interaction.

[0023] In one embodiment, an antiviral polypeptide targeting virus-host interaction is provided, the amino acid sequence of which is shown in SEQ ID NO: 1 and named p17, as shown in Table 1.

[0024] Table 1:

[0025]

[0026] The peptide is composed of amino acids 1-17 of ARF1, with the sequence MGNIFANLFKGLFGKKE, acetylated at the N-terminus and amidated at the C-terminus to improve peptide stability. It is fused with a membrane-penetrating peptide TAT (amino acid sequence YGRKKRRQRRR) and an intermediate linker peptide GSG. The fusion of the membrane-penetrating peptide facilitates better entry into cells; it does not possess antiviral activity on its own. Previously, there were no reports of antiviral activity from ARF1 (1-17) related peptides. In addition to SARS-CoV-2, we have also found that this peptide has broad-spectrum antiviral activity and can therefore be used to prepare drugs or formulations that inhibit RNA viruses.

[0027] The N-terminal amino group and C-terminal carboxyl group of the polypeptide, as well as the amino acid side chain groups, may not be modified, or may be modified without substantially affecting the activity of the polypeptide of the present invention, such as forming a "pharmaceuticalally acceptable ester". Modification of the N-terminal amino group includes, but is not limited to, deamination, N-lower alkyl, N-dilower alkyl and N-acyl modification, and modification of the C-terminal carboxyl group includes, but is not limited to, amide, lower alkylamide, dialkylamide and lower alkyl ester modification.

[0028] In another embodiment, the polypeptide can be used to prepare drugs that inhibit the activity of RNA viruses, including but not limited to drugs that inhibit RNA viruses such as SARS-CoV-2, human enterovirus 71, hepatitis C virus, and Coxsackievirus A16.

[0029] In one embodiment, when the polypeptide is used to prepare a drug for inhibiting the novel coronavirus, the half-maximal inhibitory concentration (IC50) is 1.285 μM, i.e., IC50. 50 =1.285μM.

[0030] Those skilled in the art should know that the antiviral peptide can be expressed using commonly used expression vectors and transformation hosts, including but not limited to the nucleotide sequence encoding the peptide, the recombinant vector carrying the nucleotide sequence, and the recombinant microbial cell expressing the peptide. The selection is not limited to these, and both the expression vector and the host that can achieve the expression of the antiviral peptide are within the scope of protection of this invention.

[0031] In the following examples, the polypeptide p17, with the amino acid sequence shown in SEQ ID NO: 1, was synthesized under a commissioned process, and the Huh7-ACE2, Huh7.5.1, and RD cells were all commercially available. Unless otherwise specified, the techniques used in the examples are conventional methods well known to those skilled in the art.

[0032] Example 1: Cytotoxicity detection of p17

[0033] Huh7-ACE2 cells were seeded in 96-well plates. The next day, different concentrations of p17, TAT, and a water control were added to the cells. Blank control wells containing culture medium and CCK-8 solution, but without cells or peptides, were also included. After 24 hours of culture, CCK-8 solution was added to the cell culture medium at a 1 / 10 volume ratio (e.g., 10 μL to 100 μL of cell culture medium) to assess cell viability. Cells were cultured for another 45 minutes to 2 hours, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula:

[0034] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%, where:

[0035] As: Absorbance of experimental wells (including cells, culture medium, CCK-8 solution and peptide solution);

[0036] Ac: Absorbance of control wells (including cells, culture medium, CCK-8 solution, and water);

[0037] Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drugs).

[0038] like Figure 1 As shown, the results indicate that the minimum drug concentration required for p17 to kill 50% of cells is greater than 30 μM, i.e., CC. 50 >30μM ( Figure 1 A) The minimum concentration of TAT required to kill 50% of cells is greater than 50 μM, i.e., CC. 50 >50μM ( Figure 1 D).

[0039] Example 2 p17 Antiviral activity detection

[0040] In this embodiment, Huh7-ACE2 cells were selected for the detection of SARS-CoV-2, Huh7.5.1 cells were selected for the detection of HCV, and RD cells were selected for the detection of CA16 and EV71.

[0041] Huh7-ACE2, Huh7.5.1, and RD cells were seeded in 24-well plates. The following day, different concentrations of p17, control peptide TAT, and a water-based control (control check, CK) were added to the cells. After 2 hours of treatment, virus (multiple of infection 0.01-0.05) was added, and the cells were cultured for another 24 hours. Cell supernatants were collected, and cells were lysed using Trizol. The supernatants were heated at 65°C for 30 minutes, and nucleic acid was extracted using a micro-viral RNA extraction kit. The Trizol-lysed cells were used for subsequent extraction of total intracellular RNA. After reverse transcription, the viral nucleic acid content was detected by quantitative PCR using different virus-specific primers. The viral copy number in the supernatant was determined using a standard curve method (a standard curve was created using a serially diluted SARS-CoV-2 N expression plasmid). Intracellular nucleic acid quantification was performed using the comparative CT value method (with the cellular GAPDH gene used as an internal control).

[0042] like Figure 1-2 As shown, the results indicate that treatment with the control peptide TAT had no inhibitory effect on the replication of SARS-CoV-2 and its mutant strains, EV71, HCV, and CA16. Figure 1 E, 1I-K, 2D-F). p17 showed a strong dose-dependent antagonistic effect against the original SARS-CoV-2 strain, with a half-maximal inhibitory concentration (IC50) of 1.285 μM. 50=1.285μM ( Figure 1 B). Calculations showed that p17 had a selection index (SI: CC50 / IC50) greater than 24 against the original SARS-CoV-2 strain (the higher the SI, the lower the concentration required to inhibit the pathogen and the less harm it causes to host cells). p17 also showed good antiviral effects against SARS-CoV-2 mutant strains (Delta, as well as Omicron BA.4 and BA.5). Figure 1 FH). In addition, p17 also has antiviral effects against EV71, HCV, and CA16. Figure 2 AC) showed broad-spectrum antiviral activity.

[0043] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. An antiviral polypeptide targeting virus-host interaction, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The use of the antiviral polypeptide of claim 1 in the preparation of drugs that inhibit RNA viruses; wherein the RNA virus includes SARS-CoV-2, human enterovirus 71, hepatitis C virus, or Coxsackievirus A16.

3. The nucleic acid encoding the antiviral polypeptide of claim 1.

4. A recombinant vector carrying the nucleic acid of claim 3.

5. Recombinant microbial cells expressing the antiviral polypeptide of claim 1.

6. The recombinant microbial cell according to claim 5, characterized in that, The microbial cells are either prokaryotic or eukaryotic cells.

7. The use of the nucleic acid of claim 3, the recombinant vector of claim 4, or the recombinant microbial cell of claim 5 in the preparation of a drug for inhibiting RNA viruses, wherein the RNA virus includes SARS-CoV-2, human enterovirus 71, hepatitis C virus, or Coxsackievirus A16.

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