CN113663073A

CN113663073A - Application of targeted S protein palmitoylation polypeptide in preparation of broad-spectrum anti-coronavirus drugs

Info

Publication number: CN113663073A
Application number: CN202110954956.5A
Authority: CN
Inventors: 马春红; 武专昌; 王鑫; 梁晓红; 高立芬; 张召英; 任彩月; 李春阳; 傅振东
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2021-08-19
Filing date: 2021-08-19
Publication date: 2021-11-19
Anticipated expiration: 2041-08-19
Also published as: CN113663073B

Abstract

The invention particularly relates to application of a targeted S protein palmitoylation polypeptide in preparation of a broad-spectrum anti-coronavirus medicament. Exploring the coronavirus infection regulation and control mechanism such as SARS-CoV-2, etc. is helpful to cut off the initial step and spread of virus infected cells, and providing a new coronary pneumonia treatment medicine with definite curative effect. The research result of the invention proves that CRD is a functional domain necessary for maintaining coronavirus S protein induced membrane fusion and virus infectivity, the key function of the core Cys and Cys core functional region in maintaining membrane fusion and virus infectivity is defined, and a targeting polypeptide is designed aiming at the region.

Description

Application of targeted S protein palmitoylation polypeptide in preparation of broad-spectrum anti-coronavirus drugs

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to a Cysteine-rich domain (CRD) inhibitor shared by S protein intracellular segments of coronaviruses, antagonistic polypeptide targeting S protein mediated membrane fusion, nucleic acid encoding the polypeptide, an expression vector, a host cell, and application of a pharmaceutical composition containing the polypeptide in preparation of an anti-coronavirus infection drug.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

A coronavirus is an RNA virus with an envelope and a linear single positive strand genome. There are currently 7 kinds of human-infecting coronaviruses, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV-1 (causing severe acute respiratory syndrome), MERS-CoV (causing middle east respiratory syndrome), and SARS-CoV-2 (causing novel coronavirus pneumonia COVID-19), in which SARS-CoV-1, MERS-CoV, and SARS-CoV-2 have seriously harmed human health, and SARS-CoV-2 is still constantly mutated. Therefore, the infection regulation mechanism of the coronavirus is deeply explored, and a new intervention target is searched, so that the method has very important significance for limiting the infection and the transmission of the virus.

After the coronavirus S protein is combined with a receptor, the S protein is cut into S1 subunits and S2 subunits by host protease, the S2 subunit initiates fusion of a virus envelope and a cell membrane and a subsequent cell entering process, the initial step of virus infection of a cell is completed, and the capacity of inducing membrane fusion is a key factor for determining a coronavirus cell entering path and virus infection efficiency. However, the mechanism of fusion of virus and target cell membrane is less understood at present, and the research on the regulation mechanism of S protein induced membrane fusion and the development of drugs for inhibiting virus-cell membrane fusion have important significance for controlling the infection and spread of SARS-CoV-2. The fusion of the viral envelope and the envelope is a complex continuous process, after the S protein is cut, the conformation of the S2 subunit is changed, the FP structural domain is exposed and embedded in the host cell membrane, and the coiled-coil hydrophobic structures of HR1 and HR2 interact to form a six-helix bundle (6-HB) fusion core, so that the space distance between the viral envelope and the cell membrane is shortened, and the occurrence of membrane fusion is promoted. The polypeptide membrane fusion inhibitor EK1C4 based on the targeting 6-HB structure designed by Zingiber officinale Kimura/Luolu team can effectively inhibit the membrane fusion ability and virus infection induced by various coronavirus S proteins such as SARS-CoV-1, MERS-CoV and SARS-CoV-2. There are studies showing that palmitoylation modification of SARS-CoV-1S protein can promote cell fusion, and the newly published studies of the inventors also found that palmitoylation modification of the intracellular cysteine-rich region (Cys-rich domain, CRD) of SARS-CoV-2S protein is necessary for membrane fusion and viral infection, suggesting that targeted palmitoylation is a potential target for the development of anti-coronavirus drugs. Whether CRD is a conserved functional domain for regulating coronavirus S protein mediated membrane fusion and virus infection is not clear at present, and whether CRD can be used as a universal intervention target of broad-spectrum coronavirus has not been reported so far.

Disclosure of Invention

Aiming at the research background, the invention defines that the S protein CRD is used as a conservative functional domain for regulating and controlling different coronavirus infection and inducing membrane fusion, designs a transmembrane polypeptide simulating the CRD to competitively inhibit palmitoylation modification and inhibit membrane fusion induced by the S protein and coronavirus replication, and has very positive significance for theoretical research and clinical treatment of infection regulation and control and pathogenic mechanism of the coronavirus.

Based on the above purpose, the invention provides the following technical scheme:

the invention proves that a Cys-enriched domain (CRD) of the S protein is a conserved functional domain of coronavirus, determines the cell fusion and pseudovirus infection capacity induced by the S protein, and mutation analysis reveals the core Cys composition rule of CRD playing a role. Furthermore, the invention designs a transmembrane polypeptide targeting a novel coronavirus pneumonia COVID-19CRD, and the polypeptide treatment can obviously inhibit S protein mediated cell fusion and pseudovirus infection capacity. The polypeptides are also effective in inhibiting viral replication and virus-induced cell death in a model of natural infection with HCoV-229E virus.

Therefore, in a first aspect of the present invention, there is provided a use of an inhibitor of the intracellular domain CRD of the S protein of coronavirus for the preparation of a medicament for the treatment of a coronavirus related disease.

It is understood that the CRD domain provided by the present invention belongs to a region with high conservation of function regulation in coronavirus, which means that corresponding inhibitors can be designed for CRD domain to achieve anti-coronavirus effect for different coronaviruses. The inhibitor designed aiming at the CRD structural domain is expected to be applied as a broad-spectrum anti-coronavirus drug.

Furthermore, the invention takes SARS-CoV-2 as a research model, screens the core region of CRD, and defines the key region of SARS-CoV-2 virus for starting membrane fusion, wherein, Cys 1241 is necessary for inducing membrane fusion, further the analysis of the reversion mutation finds that Cys 1235-1247 is the minimum functional unit for maintaining CRD function, and Cys 1247 is equivalent to Cys 1248, 1250, 1251 and 1253 in maintaining S-induced cell fusion, and the sites constitute the core functional region of Cys 1241, which has important significance for maintaining S protein-induced membrane fusion. And the invention also discovers a similar Cys distribution rule by analyzing the sequences of other coronavirus CRD domains, thereby providing a more accurate action site for the design of the inhibitor.

In the technical scheme of the first aspect, the inhibitor comprises the following three design ideas:

(1) substances which inhibit the expression of the above-mentioned regions or have a shearing or mutating action on said regions are exemplified by: interfering RNA, Cas9 nuclease based on CRISPR system, Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or fokl nucleases (RFNs), etc.;

(2) a substance that inhibits palmitoylation of said region; as is well known in the art, palmitoylation modification is a reversible protein lipid modification mode, and is specifically represented by covalent modification of 16-carbon palmitoyl to a sulfhydryl group of a protein cysteine through a thioester bond, and a component for blocking the covalent modification is one of the inhibitors disclosed by the invention;

(3) substances of the type with high affinity to the CRD domain block the domain palmitoylation modification by competitive inhibition.

The invention compares CRD structural sequences of various coronaviruses, finds that the CRD region of the coronaviruses of the same subgroup has higher homology, shows that the conservative property of the region is higher, and an inhibitor designed aiming at the CRD region is expected to have an inhibiting effect on various coronaviruses and has higher research value. The first aspect of the present invention provides a solution for inhibition of the upstream signaling pathway in response to a membrane fusion process initiated by the CRD domain of the S protein. The active binding site in the region is screened to be used as an exogenous polypeptide supplement, and the fusion of virus S protein and cell membrane is hopefully prevented through competitive inhibition so as to block the membrane fusion process.

In a second aspect of the invention, there is provided a novel coronavirus S protein palmitoylation and membrane fusion mediating antagonist polypeptide, which polypeptide:

(1) is a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs 1-7;

(2) a derivative polypeptide formed by adding, substituting or deleting one or more amino acids to or from the amino acid sequence of the polypeptide in (1), wherein the derivative polypeptide has the same or basically the same function as the polypeptide shown in SEQ ID NO. 8;

(3) a derivative polypeptide obtained by modifying the polypeptide of (1) or (2).

Based on the above thought, the antagonist polypeptide for S protein palmitoylation and mediated membrane fusion provided by the second aspect of the invention targets the CRD region of the S protein, and can effectively inhibit S protein-induced membrane fusion and coronavirus replication.

In addition to the polypeptides described above, nucleic acids encoding the polypeptides of the second aspect, expression vectors and host cells comprising the expression vectors are well within the skill of the art in light of the routine availability of the polypeptides, and are also within the scope of the present invention.

Thirdly, the invention verifies that the polypeptide can inhibit the S protein mediated membrane fusion process and the infection of SARS-CoV-2 pseudo virus, which means that the polypeptide or the pharmaceutical composition containing the polypeptide can be applied to the treatment of novel coronary pneumonia and the development of anti-novel coronary pneumonia drugs.

The beneficial effects of one or more technical schemes are as follows:

1. the new coronavirus pneumonia is currently the most serious "public health event of international concern" and "major crisis". The infection regulation and control mechanism of the virus is defined, which has important significance for controlling the virus transmission and developing effective treatment drugs. The research result of the invention defines the key structural domain of the coronavirus initiating membrane fusion process, and the domain has higher homology in different coronaviruses and is expected to realize the broad-spectrum virus inhibition effect. The research result defines a way for infecting normal cells by coronavirus, and promotes the progress of related research.

2. According to the invention, the screening confirms the key region of the coronavirus initiation membrane fusion, the existence of the Cys core functional domain is defined, and a defined target is provided for inhibiting the initiation of the virus membrane fusion.

3. The invention further provides a polypeptide for competitively inhibiting CRD-initiated membrane fusion. In vitro experiments, the polypeptide can effectively inhibit the fusion process of pseudovirus and cell membranes, is expected to block the infection of novel coronavirus pneumonia to organisms, and is applied to development of anti-novel coronavirus pneumonia medicines.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1SARS-CoV-2S protein palmitoylation is essential for cell fusion and viral infection;

wherein, in figure 1A, the ZDHHC5/GOLGA7 complex regulates S protein palmitoylation modification;

FIG. 1B S shows that the Cys-rich region CRD of protein is a critical region for palmitoylation modification;

FIG. 1C CRD palmitoylation modification site mutation results in a substantial reduction in the ability of a new pseudocoronary virus to infect;

FIG. 1D CRD palmitoylation modification site mutation results in a substantial reduction in cell fusion capacity;

FIG. 1E ZDHHC5 knockdown inhibits S protein palmitoylation modification and its ability to mediate cell fusion and pseudoviral infection;

FIG. 1F palmitoylation inhibitor 2-BP treatment inhibited S protein palmitoylation modification and its mediated cell fusion and pseudoviral infectivity.

FIG. 2 shows the results of the verification that coronavirus S protein CRD maintains S protein induced membrane fusion and virus infectivity;

wherein, FIG. 2A is the comparison and analysis of the sequence of coronavirus S protein CRD;

FIG. 2B is a comparison of the ability of wild-type and CRD mutants of the S proteins of 5 kinds of coronavirus infecting human to induce cell fusion;

FIG. 2C is an analysis of the ability of wild-type and CRD mutants of the S proteins of 5 coronaviruses infecting humans to mediate pseudoviral infection.

FIG. 3 is a graph showing the results of the rule verification of the functional Cys composition of the coronavirus S protein CRD;

wherein, FIG. 3A is the fluorescence detection result diagram of the mutation of SARS-CoV-2S protein CRD and the ability of inducing cell fusion;

FIG. 3B is the result of comparative analysis of the mutation of SARS-CoV-2S protein CRD and the ability to induce cell fusion;

FIG. 3C is a comparative analysis of the ability of the CRD mutation of SARS-CoV-1S protein to induce cell fusion;

FIG. 3D is a comparative analysis of CRD mutation of MERS S protein and ability to induce cell fusion;

FIG. 3E analysis of the ability of different mutants of SARS-CoV-2, SARS-CoV-1, MERS virus S protein CRD to mediate pseudovirus infection.

FIG. 4 is a diagram showing the results of verification that SARS-CoV-2S protein CRD polypeptide inhibits cell fusion and pseudovirus infectivity;

wherein, FIG. 4A shows the design strategy and sequence of the membrane-penetrating polypeptide targeting SARS-CoV-2S protein CRD;

FIG. 4B shows CRD transmembrane polypeptide inhibiting early cell fusion;

FIG. 4C shows the formation of syncytia with CRD transmembrane polypeptide inhibiting late fusion;

FIG. 4D shows that CRD transmembrane polypeptide inhibits SARS-CoV-2 pseudovirus infectivity.

FIG. 5 shows that HCoV-229E S protein CRD polypeptide inhibits viral replication and virus-induced cytopathic effects;

wherein, FIG. 5A shows the design strategy and sequence of the cell-penetrating polypeptide targeting HCoV-229E S protein CRD;

FIG. 5B is a cytotoxicity assay for CRD transmembrane polypeptides;

FIG. 5C shows that CRD transmembrane polypeptide inhibits HCoV-229E viral replication;

FIG. 5D shows that CRD-penetrating polypeptide inhibits HCoV-229E virus-induced cytopathic effect.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, studies have shown that palmitoylation of the CRD region of the SARS-CoV-2S protein is present, a change which may be related to the process of viral infection of cells; the design experiment of the invention verifies that the CRD region of the S protein is the key field for maintaining the S protein to induce membrane fusion and virus infectivity, and further, the invention confirms the core region for starting the membrane fusion process in the CRD structure, and the inhibition of the core region can effectively inhibit the virus infection process. The invention also designs corresponding transmembrane polypeptide, which is used as an antagonist of the CRD core region and is expected to be applied to novel medicaments for treating coronavirus pneumonia.

The invention provides an application of an inhibitor of an intracellular segment CRD structural domain of a coronavirus S protein in preparing a medicine for resisting coronavirus related diseases.

The coronavirus is BcoV-1, MHV, HcoV-OC43, HcoV-HKU1, Pi-Bat CoV-HKU5, Ty-BatCoV-HKU4, MERS-CoV, BatCoV-Rm1, BatSL-CoV-Rs3367, BatSL-CoV-WIV1, SL-CoV-WIV16, SARS-CoV-1, SARS-CoV-2, Ro-Bat CoV HKU5, HcoV-229E, HcoV-NL63, TEGV, PRCV, FCoV-C1Je, PEDV, PDCoV, PorCoV-HKU15, BuCoV-HKU11, CMV-HKU 21, ThCoV-HKU12 or IBV, etc.

The present invention analyzes the sequence of the currently known coronavirus CRD domain to find a similar Cys composition rule (refer to fig. 2), specifically:

the sequence of the CRD structural domain of the BcoV-1 virus is as follows: CCCTGCGASCFKKCGNCC, respectively;

the sequence of the MHV virus CRD domain is: CCCTGCGSCCFKKCGNCC, respectively;

the sequence of the CRD domain of the Hcov-OC43 virus is as follows: CCCTGCGTSCFKKCGGCC, respectively;

the sequence of the CRD domain of the Hcov-HKU1 virus is as follows: CCCTGCGSACFSKCHNCC, respectively;

the sequence of the CRD domain of the Pi-Bat CoV-HKU5 virus is as follows:

CCTGCGTSCLGKLKCNRCC；

the sequence of the CRD domain of the Ty-BatCoV-HKU4 virus is as follows:

CCTGCGTSCLGKMKCKNCC；

the sequence of the MERS-CoV virus CRD structural domain is as follows: CCTGCGTNCMGKLKCNRCC, respectively;

the sequence of the CRD domain of the BatCoV-Rm1 virus is as follows:

CCMTSCCSCLKGACSCGSCC；

the sequence of the CRD domain of the BatSL-CoV-Rs3367 virus is as follows:

CCMTSCCSCLKGACSCGSCC；

the sequence of the CRD domain of the BatSL-CoV-WIV1 virus is as follows:

CCMTSCCSCLKGACSCGSCC；

the sequence of CRD domain of SL-CoV-WIV16 virus is as follows:

CCMTSCCSCLKGACSCGSCC；

the sequence of the CRD structural domain of the SARS-CoV-1 virus is as follows:

CCMTSCCSCLKGACSCGSCC；

the sequence of the CRD structural domain of the SARS-CoV-2 virus is as follows:

CCMTSCCSCLKGCCSCGSCC；

the sequence of the CRD domain of the Ro-Bat CoV HKU5 virus is as follows:

CMTNCCSCFKGMCDCRRCC；

the sequence of the CRD domain of the Hcov-229E virus is as follows:

CCCSTGCCGFFSCFASSIRGCC；

the sequence of the CRD domain of the Hcov-NL63 virus is:

CCLSTGCCGCCNCLTSSMRGCCDC；

the sequence of the CRD domain of the TEGV virus is as follows: CCCSTGCCGCIGCLGSCC, respectively;

the sequence of the PRCV virus CRD domain is as follows: CCCSTGCCGCIGCLGSCCHSIC, respectively;

the sequence of the CRD domain of the FCoV-C1Je virus is as follows:

CCLSTGCCGCFGCLGSCCHSLC；

the sequence of the CRD domain of the PEDV virus is as follows: CCISTGCCGCCGCCGACFSGCC, respectively;

the sequence of the CRD structural domain of the PDCoV virus is as follows: CTGCCGGCFGCCGGC, respectively;

the sequence of the CRD domain of the PorCoV-HKU15 virus is as follows: CTGCCGGCFGCCGGC, respectively;

the sequence of the CRD domain of the BuCoV-HKU11 virus is as follows: CTGCCGSCFGCCGGC, respectively;

the CMCoV-HKU21 virus CRD domain has the sequence: CTGCCGGCFGCFGGCC, respectively;

the sequence of the CRD domain of the ThCoV-HKU12 virus is: CTGCCGGCFGCCGGC, respectively;

the IBV virus CRD domain sequence is as follows: CCGCCCGC.

The inhibitor designed aiming at any section of the structural domain is expected to be applied to the preparation of anti-coronavirus medicines;

in the above technical solution, the inhibitor comprises:

(2) a substance that inhibits palmitoylation of said region; as is well known in the art, palmitoylation modification is a reversible protein lipid modification mode, and is specifically represented by covalent modification of a 16-carbon palmitate to a sulfhydryl group of a protein cysteine through a thioester bond, and a component for blocking the covalent modification is one of the inhibitors disclosed by the invention;

And the CRD region has high conservation, which means that the person skilled in the art can conclude that the region is possibly present in the later discovered coronavirus, and an inhibitor designed for the CRD domain of the coronavirus is expected to have an inhibitory effect on the later-appearing coronavirus.

In the further technical scheme provided by the invention, the coronavirus is one of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV-1, MERS-CoV and SARS-CoV-2.

Preferably, the coronavirus related disease includes, but is not limited to, a respiratory infection, a gastrointestinal infection, tracheitis, or a nervous system disease.

In the research process of the invention, the core functional region consisting of Cys exists in the CRD domain of coronavirus, and similar Cys arrangement rules exist in the CRD domain of coronavirus as can be seen by combining figure 1. The present inventors have conducted studies on the core Cys region, taking the CRD region of the novel coronavirus S protein as an example, and found that Cys 1241 is essential for the ability to induce membrane fusion, that Cys 1235-1247 is the minimum functional unit for maintaining the CRD function, and that Cys 1247 and Cys 1248, 1250, 1251, and 1253 are equivalent in maintaining the ability to induce cell fusion. Based on the above findings, the present invention specifically provides a novel coronavirus inhibitor which acts on Cys in the 1235-1247 region or Cys at

positions

1247, 1248, 1250, 1251 and 1253; or, the site of action is Cys 1241.

In a second aspect of the invention, there is provided a novel coronavirus S protein palmitoylation and membrane fusion mediated antagonist polypeptide, which polypeptide is as follows:

The polypeptides of the amino acid sequences shown in SEQ ID NO. 27-33 are CRD core Cys regions of SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1 respectively, and the viruses are human-infectable coronavirus types, so that the polypeptide designed aiming at the key region of the virus membrane fusion is expected to realize the treatment effect on the new corona, and in the preferable scheme of the technical scheme (1), the polypeptide is the polypeptide of the amino acid sequence shown in SEQ ID NO. 8.

Preferably, in the above technical scheme (2), the "addition, substitution or deletion of one or more amino acids" refers to a polypeptide sequence which can still inhibit palmitoylation of coronavirus S protein after one or more amino acids are added, substituted or deleted from the polypeptide of the amino acid sequence shown in SEQ ID NO. 27; said "one or more amino acids" is 1 to 7 amino acids, preferably 1 to 6 amino acids; more preferably, the polypeptide is formed by substitution, deletion or addition of 1-3 amino acid residues and has the derivative polypeptide of the invention.

In addition, the polypeptides of the present invention also broadly include polypeptides derived by chemical or genetic modification. The modification method comprises polyethylene glycol (PEG), streptavidin, various molecules, such as biotin, radioactive isotopes, fluorescent agents, enzymes, cytotoxic substances, antitumor agents and the like, and also comprises a derivative polypeptide formed by connecting the polypeptide shown in SEQ ID NO. 27 and a functional polypeptide, wherein the functional group comprises but is not limited to targeting peptide, cell-penetrating peptide and the like. The modification of the functional group can be carried out by a known method, such as solid phase synthesis or liquid phase synthesis.

In a further aspect, the invention provides an embodiment of the polypeptide modified cell-penetrating peptide, wherein in this series of embodiments, the cell-penetrating peptide is one of H7R8, HIV-I TAT (48-60), TAT (47-57), TAT (48-57), and R9-TAT. In a specific example, the cell-penetrating peptide is CPPtat and the amino acid sequence is YGRKKRRQRRR. The sequence of the modified cell-penetrating peptide is shown in SEQ ID NO. 9.

In a third aspect of the invention, there is provided a nucleic acid sequence encoding a polypeptide according to the second aspect.

The nucleic acid sequence comprises a nucleic acid sequence which, due to codon degeneracy, is capable of being translated to a polypeptide or peptide derived according to the first aspect.

The encoding nucleic acid may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

In a fourth aspect of the invention, there is provided an expression vector comprising a nucleic acid sequence according to the third aspect.

Additionally, the coding nucleic acid sequence of the third aspect may be inserted into the expression vector. Specific examples of "expression vectors" include: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Those skilled in the art can use well-known methods for constructing expression vectors containing the DNA sequences encoding the proteins of the present invention and appropriate transcription/translation control signals, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or kanamycin or ampicillin resistance for E.coli.

In a fifth aspect, the present invention provides a host cell comprising an expression vector according to the fourth aspect or a host cell having integrated in its genome a nucleic acid encoding a polypeptide according to the third aspect.

Preferably, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells. In specific examples, the strains include, but are not limited to: coli (e.coli), corynebacterium glutamicum (corynebacterium glutamicum), Hafnia alvei (Hafnia alvei), Bacillus subtilis (Bacillus subtilis). More preferably, the strain is Escherichia coli.

Wherein, the transformation of the host cell with the recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If necessary, transformation is also carried outCan be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

In a sixth aspect of the invention, there is provided a pharmaceutical composition comprising the polypeptide or the derived peptide of the second aspect, and a pharmaceutically acceptable excipient.

In the pharmaceutical composition, the polypeptide or the derivative peptide is used as an active ingredient of the pharmaceutical composition. In the pharmaceutical composition, other active ingredients for inhibiting or assisting in the treatment of the novel coronavirus pneumonia may be further included, such as antiviral drugs (e.g., α -interferon, ribavirin, lopinavir, ritonavir, chloroquine phosphate or arbidol), anti-infective drugs, expectorants (e.g., ambroxol), hepatoprotective drugs, and the like.

In a seventh aspect of the invention, there is provided a medicament for treating novel coronavirus pneumonia, the medicament comprising the polypeptide of the second aspect and/or the pharmaceutical composition of the sixth aspect.

In the above-mentioned medicament for treating the novel coronavirus pneumonia, the polypeptide, derivative or the pharmaceutical composition should be in an effective dose, and the dose of the medicament is determined based on the conventional manner in the art.

The administration mode of the medicament is preferably as follows: oral, aerosol inhalation, nasal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion. Considering the drug, in which the polypeptide is used as an active ingredient, the drug is preferably a lyophilized powder or a liquid preparation; the freeze-dried powder is dissolved by buffer solution or water for injection and then used, and the liquid preparation is real solution, colloid, microparticle, emulsion or suspension.

In an eighth aspect of the present invention, there is provided a method for treating a novel coronary pneumonia, the method comprising administering to an individual in need thereof a polypeptide of the second aspect or a medicament for treating a novel coronary pneumonia as described in the seventh aspect.

In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the technical solution of the present invention will be described in detail below with reference to specific examples and comparative examples.

Example 1

1) CRD region palmitoylation of the SARS-CoV-2S protein is essential for cell fusion and viral infection

The SARS-CoV-2S protein has palmitoylation modification, and the complex of ZDHHC5 and GOLGA7 regulates the S protein palmitoylation modification (A). The CSS-Palm tool predicts that the 15 th Cys and C terminal CRD structural domain of the N terminal of the S protein can be potential palmitoylation modification sites, HEK293T cells are transfected by mutant expression plasmids of wild type S and potential palmitoylation modification sites, and the S protein is detected by ABE test, and the CRD structural domain is found to be a key region (B) of S protein palmitoylation modification. S-WT and S-delta C-Pal SARS-CoV-2 pseudo virus infection experiments show that CRD region palmitoylation modification site mutation causes great reduction of pseudo virus infection capacity (C), and further mechanism exploration shows that CRD region palmitoylation modification site mutation causes S protein-mediated cell fusion capacity loss (D), which indicates that CRD palmitoylation modification is necessary for SARS-CoV-2 cell fusion and virus infection. The shRNA knockdown of ZDHHC5 expression (E) or treatment with palmitoylation inhibitor 2-BP (F) significantly inhibits the ability of cell fusion and pseudoviral infection while down-regulating the S protein palmitoylation modification level.

2) Coronavirus S protein CRD is essential for maintaining S protein induced membrane fusion and virus infectivity

The intracellular domain of the S protein of the coronavirus subgroups α, β, γ and δ contains a Cys-rich domain (CRD) and the CRD homology of the same subgroup coronavirus is higher (a). After a Cys mutation (S-mCRD) of a CRD structure is found by using a fusion fluorescence imaging model of an S/GFP co-expression HEK293T cell and a Huh7 cell, the capability of S protein for mediating membrane fusion is almost completely lost (B), and the capability of pseudovirus infection is greatly reduced (C), which indicates that the CRD is a functional domain necessary for maintaining coronavirus S protein-mediated membrane fusion and virus infectivity.

3) Law of composition of functional Cys in S protein CRD of coronavirus

The presence of 7-10 Cys in the coronavirus S protein CRD, the presence or absence of Cys redundancy and the core Cys region are unknown. Firstly, on the basis of SARS-CoV-2, 10 Cys point mutations and cell fusion ability analysis are carried out, and the Cys 1241 is found to be necessary for inducing membrane fusion ability. Further back mutation analysis found that 1235-1247Cys was the smallest functional unit that maintained CRD function, and Cys 1247 and Cys 1248, 1250, 1251, 1253 were equivalent in maintaining S-induced cell fusion capacity, indicating that there was a functional wobble for the last Cys 1 (a and B). Similarly, in mutational analysis of SARS-CoV-1 and MERS S S proteins, it was also found that there are 1 core Cys and several adjacent Cys that together constitute the core region where the Cys core domain maintains the ability of S protein to induce membrane fusion (C and D).

Furthermore, a pseudoviral infection test also finds that the mRD and the core Cys mutation cause the great reduction of the virus infection capacity, and the Cys core functional domain recovery mutation can save the virus infectivity (E), thereby further confirming the key role of the core Cys and the Cys core functional domain in the maintenance of membrane fusion and virus infectivity.

4) CRD cell-penetrating peptide for inhibiting SARS-CoV-2S protein mediated cell fusion and pseudo virus infection

Based on the important role of CRD in maintaining coronavirus membrane fusion and pseudovirus infection, the CRD transmembrane polypeptide of the targeting S protein is further designed to competitively inhibit the function of CRD. Fusing S-CRD wild-type polypeptide and mutant (C → A) polypeptide with cell-penetrating peptide (CPPtat: YGRKKRRQRRR), respectively: YGRKKRRQRRRLCCMTSCCSCLKGCCSCGSCCK, wild type transmembrane polypeptide SARS-CoV-2-S-CRD-WT; the mutant transmembrane polypeptide SARS-CoV-2-S-CRD-Mut: YGRKKRRQRRRLAAMTSAASALKGAASAGSAAK(A). The polypeptide treatment can inhibit the early fusion (B) and the late syncytia formation (C) of the S/GFP co-expressed HEK293T cell and Huh7 cell in a dose-dependent manner, and further researches show that the polypeptide treatment can also significantly inhibit the infection (D) of SARS-CoV-2 pseudovirus, which indicates the potential application value of the targeted S-CRD transmembrane polypeptide.

Targeting polypeptides designed according to the sequence SEQ ID NO 1-26 also have similar technical effects.

5) CRD-penetrating peptides inhibit replication of HCoV-229E virus

Synthesis of wild-type and mutant transmembrane polypeptides targeting the S protein CRD of HCoV-229E virus (A): wild-type transmembrane polypeptide 229E-S-CRD-WT: YGRKKRRQRRRLCCCSTGCCGFFSCF; mutant transmembrane polypeptide 229E-S-CRD-Mut: YGRKKRRQRRRLAAASTGAAGFFSAF. Huh7 cells treated with 20. mu.M polypeptide were not significantly cytotoxic (B). After HCoV-229E virus infected Huh7 cells for 6h, the polypeptide was treated for 48h at different concentrations, and the polypeptide was found to significantly reduce the virus titer in the supernatant and the viral RNA level (C) in the cells in a dose-dependent manner. The cytopathic effect caused by the virus infection after 4D leads to almost complete cell death, the cells in the 5-20 mu M polypeptide-treated group are in good condition, and the 229E-induced cell death (D) can be still remarkably inhibited by 1 mu M polypeptide treatment. The results show that the polypeptide targeting HCoV-229E virus S protein CRD can obviously inhibit the replication and pathological changes of 229E virus, and indicate that the targeting S-CRD membrane-penetrating polypeptide has good application value.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong university

<120> application of targeted S protein palmitoylation polypeptide in preparation of broad-spectrum anti-coronavirus drugs

<130> 2010

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> PRT

<213> Artificial sequence

<400> 1

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys

1 5 10 15

Gly Ser Cys Cys

20

<210> 2

<211> 20

<212> PRT

<213> Artificial sequence

<400> 2

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys

1 5 10 15

Gly Ser Cys Cys

20

<210> 3

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<212> PRT

<213> Artificial sequence

<400> 3

Cys Cys Thr Gly Cys Gly Thr Asn Cys Met Gly Lys Leu Lys Cys Asn

1 5 10 15

Arg Cys Cys

<210> 4

<211> 22

<212> PRT

<213> Artificial sequence

<400> 4

Cys Cys Cys Ser Thr Gly Cys Cys Gly Phe Phe Ser Cys Phe Ala Ser

1 5 10 15

Ser Ile Arg Gly Cys Cys

20

<210> 5

<211> 22

<212> PRT

<213> Artificial sequence

<400> 5

Cys Cys Leu Ser Thr Gly Cys Cys Gly Cys Cys Asn Cys Leu Thr Ser

1 5 10 15

Ser Met Arg Gly Cys Cys

20

<210> 6

<211> 18

<212> PRT

<213> Artificial sequence

<400> 6

Cys Cys Cys Thr Gly Cys Gly Thr Ser Cys Phe Lys Lys Cys Gly Gly

1 5 10 15

Cys Cys

<210> 7

<211> 18

<212> PRT

<213> Artificial sequence

<400> 7

Cys Cys Cys Thr Gly Cys Gly Ser Ala Cys Phe Ser Lys Cys His Asn

1 5 10 15

Cys Cys

<210> 8

<211> 20

<212> PRT

<213> Artificial sequence

<400> 8

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys

1 5 10 15

Gly Ser Cys Cys

20

<210> 9

<211> 33

<212> PRT

<213> Artificial sequence

<400> 9

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Cys Cys Met Thr

1 5 10 15

Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys

20 25 30

Lys

Claims

1. The application of coronavirus S protein intracellular segment CRD structural domain inhibitor in preparing medicine for resisting coronavirus related diseases;

the coronavirus is BcoV-1, MHV, HcoV-OC43, HcoV-HKU1, Pi-Bat CoV-HKU5, Ty-BatCoV-HKU4, MERS-CoV, BatCoV-Rm1, BatSL-CoV-Rs3367, BatSL-CoV-WIV1, SL-CoV-WIV16, SARS-CoV-1, SARS-CoV-2, Ro-Bat CoV HKU5, HcoV-229E, HcoV-NL63, TEGV, PRCV, FCoV-C1Je, PEDV, PDCoV, PorCoV-HKU15, BuCoV-HKU11, CMV-HKU 21, ThCoV-HKU12 or IBV; preferably, the inhibitor comprises:

(1) substances inhibiting the expression of amino acids in the region or having a shearing and amino acid mutation effect on the region, further being interfering RNA, Cas9 nuclease based on CRISPR system, zinc finger nuclease, transcription activator-like effector nuclease or FokI nuclease;

(2) a substance that inhibits the extent of palmitoylation in said region;

(3) a substance having a high affinity for the CRD domain.

2. Use of an inhibitor of the intracellular domain CRD of the S protein of a coronavirus according to claim 1, wherein the coronavirus includes but is not limited to one of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV-1, MERS-CoV, SARS-CoV-2;

or, the coronavirus related diseases include, but are not limited to, respiratory system infections, gastrointestinal infections, tracheitis, nervous system diseases;

or the action region of the coronavirus S protein intracellular CRD domain inhibitor is further 1235-1247Cys, or the Cys at position 1247 and the Cys at positions 1248, 1250, 1251 and 1253; or, Cys position 1241.

3. An antagonistic polypeptide of palmitoylation of the S protein of a novel coronavirus and mediated membrane fusion, said polypeptide comprising:

4. An antagonist polypeptide for palmitoylation of the S protein of the novel coronavirus and mediating membrane fusion according to claim 3, wherein the antagonist polypeptide has a modification of a cell-penetrating peptide selected from the group consisting of H7R8, HIV-I TAT (48-60), TAT (47-57), TAT (48-57), R9-TAT;

preferably, the cell-penetrating peptide is CPPtat, and the modified cell-penetrating peptide has a cell-penetrating polypeptide sequence shown in SEQ ID NO. 9.

5. A nucleic acid sequence encoding the novel coronavirus S protein palmitoylation and antagonist polypeptide mediating membrane fusion of claim 3 or 4;

preferably, the nucleic acid sequence comprises a nucleic acid sequence which, due to codon degeneracy, is capable of being translated into a polypeptide or a derivatized peptide according to claim 3 or 4;

preferably, the coding nucleic acid includes a DNA form and an RNA form; the form of DNA includes cDNA, genomic DNA or artificially synthesized DNA; DNA includes single-stranded or double-stranded, as well as coding or non-coding strands.

6. An expression vector comprising the nucleic acid sequence of claim 5; wherein the coding nucleic acid sequence of claim 4 is insertable into the expression vector;

preferably, the "expression vector" includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors.

7. A host cell comprising the expression vector of claim 6 or a genome thereof into which the coding nucleic acid of claim 5 has been integrated;

preferably, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells;

further, the strains include, but are not limited to: escherichia coli (e.coli), corynebacterium glutamicum (corynebacterium glutamicum), Hafnia alvei (Hafnia alvei), Bacillus subtilis (Bacillus subtilis);

specifically, the strain is escherichia coli.

8. A pharmaceutical composition comprising an antagonist polypeptide of claim 3 or 4 and a pharmaceutically acceptable excipient;

preferably, the antagonist polypeptide is used as an active ingredient of a pharmaceutical composition;

preferably, the pharmaceutical composition further comprises other active ingredients for inhibiting or assisting in treating the novel coronavirus pneumonia, such as antiviral drugs, anti-infective drugs, phlegm-eliminating drugs or liver-protecting drugs.

9. A medicament for treating a novel coronavirus pneumonia, said medicament comprising an antagonist polypeptide according to claim 3 or 4 and/or a pharmaceutical composition according to claim 8.

10. A method of treating a novel form of coronary pneumonia, comprising administering to a subject in need thereof an antagonist polypeptide of claim 3 or 4 or a medicament of claim 9 for treating a novel form of coronary pneumonia.