WO2023123722A1 - 一种抗冠状病毒的多肽、其衍生物及其应用 - Google Patents

一种抗冠状病毒的多肽、其衍生物及其应用 Download PDF

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WO2023123722A1
WO2023123722A1 PCT/CN2022/084473 CN2022084473W WO2023123722A1 WO 2023123722 A1 WO2023123722 A1 WO 2023123722A1 CN 2022084473 W CN2022084473 W CN 2022084473W WO 2023123722 A1 WO2023123722 A1 WO 2023123722A1
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polypeptide
cov
coronavirus
chol
sars
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PCT/CN2022/084473
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English (en)
French (fr)
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高福
曾少贵
王奇慧
唐洋明
陈健韬
仵丽丽
郑安琪
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中国科学院微生物研究所
深圳翰宇药业股份有限公司
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Publication of WO2023123722A1 publication Critical patent/WO2023123722A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biomedicine, in particular to polypeptides for preventing or treating coronaviruses, derivatives thereof, polynucleotides encoding polypeptides thereof, nucleic acid constructs comprising the polynucleotides, expression vectors comprising the nucleic acid constructs, Transformed cells and the above-mentioned pharmaceutical composition, and their application in the preparation of drugs or vaccines for preventing and/or treating the new coronavirus.
  • Coronavirus (English name: Coronavirus) is an RNA virus with an envelope and a linear single-stranded positive strand genome. It is a large class of viruses that exist widely in nature. Some of these coronaviruses can infect humans and cause disease. So far, there are seven coronaviruses that can infect humans, including SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1.
  • the SARS-CoV-2 virus is the pathogen of the new coronary pneumonia epidemic, and it is the third coronavirus that has caused human epidemics since the 21st century.
  • SARS-CoV-2 the pathogen of the new coronary pneumonia epidemic
  • 3 coronavirus the third coronavirus that has caused human epidemics since the 21st century.
  • vaccines and monoclonal antibody drugs against SARS-CoV-2 have been approved for emergency use, most studies have shown that they are not effective against the widely spread variant strains of the new coronavirus, such as Beta, Delta and Omicron. Therefore, it is urgent to develop broad-spectrum drugs or vaccines against the current epidemic and new mutant strains that will appear in the future.
  • the object of the present invention is to provide a polypeptide for preventing or treating coronavirus, a polynucleotide encoding it, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a transformed cells and the pharmaceutical composition comprising the above, and their application in the preparation of drugs or vaccines for preventing or treating coronavirus.
  • the present invention provides a polypeptide for anti-coronavirus, which is a polypeptide of any one of the following (1)-(8), wherein:
  • a polypeptide comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, added or inserted in any of the amino acid sequences of the polypeptide (1)-(6), and the polypeptide has an anti-coronavirus inhibitory activity; or
  • a polypeptide comprising at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94% of the amino acid sequence of any one of the polypeptides (1)-(6). % or 95% identical amino acid sequence, and the polypeptide has inhibitory activity against coronavirus.
  • sequence of the polypeptide is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14.
  • the present invention provides a polypeptide derivative for anti-coronavirus, wherein the polypeptide derivative is a derivative modified with cholesterol on the above-mentioned anti-coronavirus polypeptide.
  • the modification site of the cholesterol derivative is at the C-terminal, N-terminal or amino acid side chain of the polypeptide.
  • the modification site of the cholesterol modification is at the C-terminus of the polypeptide.
  • the derivative of cholesterol is connected through a linker or directly connected to the C-terminus of the polypeptide.
  • the cholesterol derivative is a derivative of cholesterol modified by cysteine and or PEG.
  • cholesterol derivative is selected from:
  • the R 2 is OH or NH 2 ;
  • n is any integer of 1-20, preferably any integer of 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20;
  • Said x is any integer of 1-6, such as 1, 2, 3, 4, 5, 6.
  • linker is -NH-PEGn-(CH 2 )x-CO-, (GSG)m, (GSGSG)m, (GSG)m-CONH-PEGn-(CH 2 )x-CO- or (GSGSG)m-CONH-PEGn-( CH2 )x-CO-;
  • n is any integer of 0-6, such as 0, 1, 2, 3, 4, 5 or 6;
  • n is any integer of 1-20, preferably any integer of 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20;
  • Said x is any integer of 1-6, such as 1, 2, 3, 4, 5, 6.
  • polypeptide derivative has the structural formulas shown in the following structure I, structure II and structure III:
  • polypeptide sequence refers to any one of the above (1)-(8) polypeptide sequences
  • polypeptide sequence is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14;
  • the R 1 is (GSG)m or (GSGSG)m, m is selected from any integer of 0-6; preferably m is 1 or 2;
  • the R 2 is OH or NH 2 ; preferably R 2 is NH 2 .
  • polypeptide derivative is selected from:
  • the present invention provides a polynucleotide sequence for encoding the polypeptide of the first aspect.
  • the present invention provides a vector comprising the polynucleotide sequence described in the third aspect.
  • the present invention provides a cell comprising the vector described in the fourth aspect of the present invention.
  • the present invention provides a pharmaceutical composition, which comprises the polypeptide provided in the first aspect and or the derivative provided in the second aspect, and a pharmaceutically acceptable carrier and/or excipient;
  • the pharmaceutical composition is in the form of inhalation, nasal or oral formulation or parenteral formulation;
  • the oral formulation is selected from tablets, capsules, granules, suspensions, pills and solutions;
  • the parenteral formulation is an injectable or bolus injectable formulation
  • the pharmaceutical composition is a vaccine composition.
  • the dosage form of the drug is tablet, capsule, drop pill, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, suppository or freeze-dried powder injection;
  • the drug is preferably administered in the following ways: injection administration, including subcutaneous injection, intravenous injection, intramuscular injection and intraperitoneal injection, intracistern injection or infusion, etc., cavity administration, such as rectal, vaginal and lingual administration Lower, respiratory tract administration, such as nasal cavity; mucosal administration, or topical administration;
  • the present invention provides a use of the above-mentioned polypeptide, polypeptide derivative, fusion protein or conjugate in the preparation of a medicament for inhibiting coronaviruses or treating and/or preventing diseases caused by coronaviruses.
  • the coronavirus is selected from original strains of SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 or variant strains thereof.
  • the coronavirus is selected from the original strain of SARS-CoV-2 and the mutant strain of SARS-CoV-2.
  • mutant strain of SARS-CoV-2 is selected from Alpha, Beta, Gamma, Delta, Omicron, Kappa, Lambda, Deltacron.
  • the disease caused by the coronavirus is selected from Novel Coronavirus Pneumonia (Corona Virus Disease 2019, COVID-19), Severe Acute Respiratory Syndrome, Middle East Respiratory Syndrome.
  • the present invention provides a method for inhibiting coronavirus in vitro, which includes the application of the above-mentioned polypeptide, polypeptide derivative, fusion protein or conjugate or pharmaceutical composition,
  • the coronavirus is an original strain of SARS-CoV-2 and/or a mutant strain of SARS-CoV-2;
  • mutant strains include Alpha, Beta, Gamma, Delta, Omicron.
  • the applied method is to contact the polypeptide of the first aspect, the derivative provided by the second aspect and/or the pharmaceutical composition of the third aspect with the virus.
  • the present invention provides a method for preventing or treating a coronavirus infection in a subject or a disease caused by a coronavirus, which includes administering the above-mentioned polypeptide, polypeptide derivative, fusion protein or conjugate to the subject or pharmaceutical compositions,
  • the coronavirus is an original strain of SARS-CoV-2 and/or a mutant strain of SARS-CoV-2;
  • the mutant strain of SARS-CoV-2 includes Alpha, Beta, Gamma, Delta, Omicron, Kappa, Lambda, Deltacron.
  • the present invention provides the use of one of the above-mentioned polypeptides and polypeptide derivatives, fusion proteins or conjugates for the prevention or treatment of coronavirus infection or diseases caused by coronavirus in subjects,
  • the coronavirus is an original strain of SARS-CoV-2 and/or a mutant strain of SARS-CoV-2;
  • mutant strains include Alpha, Beta, Gamma, Delta, Omicron, Kappa, Lambda, Deltacron.
  • the present invention designs the polypeptide P3 based on the HR2 region of the S protein of the SARS-CoV-2 virus, and performs a series of transformations (including the addition or replacement of one or more amino acids) to obtain the polypeptide, and connects multiple and cholesterol modification to obtain peptide derivatives.
  • Peptides and their derivatives have a good inhibitory effect on the original SARS-CoV-2 strain and mutant strains Alpha, Beta, Gamma, Delta, and Omicron, which can be used as a preparation for the prevention or treatment of new crowns
  • a drug or vaccine against the virus has great potential for prevention or treatment.
  • Fig. 1 is the inhibitory effect of different polypeptides to SARS-CoV-2 original strain pseudovirus in embodiment 2;
  • Fig. 2 is the inhibitory effect of different polypeptides to SARS-CoV-2 mutant strain Omicron pseudovirus in embodiment 2;
  • Fig. 3 is the inhibitory effect of polypeptide P3-2 on new crown original strain and variant strain Alpha, Beta, Gamma, Delta, Omicron pseudovirus in embodiment 3;
  • Fig. 4 is the inhibitory effect of polypeptide P3-2-GS-Chol on the original strain of new crown and mutant strain Alpha, Beta, Gamma, Delta, Omicron pseudovirus in embodiment 3;
  • Fig. 5 is the inhibitory effect of polypeptide P3-2-PEG-Chol on the original strain of new crown and mutant strain Alpha, Beta, Gamma, Delta, Omicron pseudovirus in embodiment 3;
  • Fig. 6 is the inhibitory effect of polypeptide P3-2-GS-PEG-Chol on the original strain of new crown and mutant strain Alpha, Beta, Gamma, Delta, Omicron pseudovirus in embodiment 3;
  • Figure 7 is the inhibitory effect of the polypeptide P3-1-GS-PEG-Chol in Example 3 on the original strain of the new coronavirus and the mutant strain Alpha, Beta, Gamma, Delta, Omicron pseudovirus.
  • Fig. 8 is the fake of the original strain (SARS-CoV-2 Prototype) of the truncated polypeptide after polypeptide P3-2-GS-Chol and P3-2-GS-PEG-Chol knock out amino acids one by one from the N-terminus in Example 4 Virus inhibitory effect;
  • Fig. 9 is the inhibitory effect of the truncated polypeptide after the amino acids of polypeptides P3-2-GS-Chol and P3-2-GS-PEG-Chol are knocked out one by one from the N-terminal in Example 4 to the pseudovirus of the mutant strain Delta;
  • Figure 10 is the inhibitory effect of the truncated polypeptides after the amino acids of the polypeptides P3-2-GS-Chol and P3-2-GS-PEG-Chol are knocked out one by one from the N-terminus in Example 4 to the pseudovirus of the mutant strain Omicron;
  • Figure 11 is the inhibitory effect of polypeptide P3-2-GS-chol on Vero cells on the live virus of the original strain of SARS-CoV-2 and its variant strains in Example 5;
  • Figure 12 is the inhibitory effect of the polypeptide P3-2-PEG-chol on Vero cells on the live virus of the original strain of SARS-CoV-2 and its mutant strains in Example 5;
  • Figure 13 is the inhibitory effect of the polypeptide P3-2-GS-PEG-chol on Vero cells on the original strain of SARS-CoV-2 and its variant strain live virus in Example 5;
  • Figure 14 is the inhibitory effect of the polypeptide P3-2-GS-chol on Vero E6 cells on the original strain of SARS-CoV-2 and its variant strain live virus in embodiment 6;
  • Figure 15 is the inhibitory effect of the polypeptide P3-2-PEG-chol on Vero E6 cells on the original strain of SARS-CoV-2 and its variant strain live virus in Example 6;
  • Figure 16 is the inhibitory effect of the polypeptide P3-2-GS-PEG-chol in Example 6 on the live virus of the original strain of SARS-CoV-2 and its mutant strains on Vero E6 cells.
  • the coronavirus spike (S) protein plays an important role in mediating the virus invasion process and is divided into two subunits, S1 and S2; the S1 subunit is responsible for recognizing the receptor, and the S2 subunit mediates the interaction between the viral envelope and the host cell membrane. membrane fusion.
  • the heptapeptide repeat sequences HR1 and HR2 in S2 play a role in membrane fusion by forming a six-helix bundle structure. Studies have shown that adding exogenous HR1 or HR2 polypeptides can inhibit the formation of the HR1 and HR2 six-helix bundle structure of the virus itself, thereby inhibiting membrane fusion. process.
  • the purpose of the present invention is to provide a group of polypeptide entry inhibitors that can inhibit the infection of SARS-CoV-2 and similar SARS viruses. Binding to the HR1 region of the protein interferes with the formation of the virus's own six-helix, thereby inhibiting the fusion infection process of the virus.
  • the present invention designs a polypeptide P3 based on the HR2 region of the S protein of the SARS-CoV-2 virus, and performs a series of modifications (including the addition or substitution of one or more amino acids) to obtain polypeptides P3-1 and P3-2 , P3-3, and modify the polypeptides P3-1, P3-2, and P3-3 with linkers and cholesterol, so that their cholesterol-modified polypeptide derivatives have strong
  • the inhibitory effect can be used as a drug or vaccine for the prevention or treatment of new coronavirus.
  • polypeptides of the invention have inhibitory activity against SARS-CoV-2, and the inhibitory activity of some polypeptides has reached an unexpected high level, especially the inhibition of the new crown mutant strain Omicron has achieved a very good inhibitory effect.
  • polypeptide may be a polypeptide comprising SEQ ID NO: 1-6 or a variant thereof.
  • polypeptide may include substitution, addition, deletion or insertion of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1, 2 or 3.
  • Amino acid addition refers to the addition of amino acids to the amino acid sequence, such as the C-terminal or N-terminal of SEQ ID NO: 1, 2 or 3, as long as the polypeptide has inhibitory activity against coronavirus.
  • Amino acid substitution refers to the replacement of an amino acid residue at a certain position in an amino acid sequence, such as a sequence of SEQ ID NO: 1, 2 or 3, by other amino acid residues, as long as the polypeptide has inhibitory activity against coronavirus.
  • Amino acid insertion refers to the insertion of amino acid residues at an appropriate position in an amino acid sequence such as SEQ ID NO: 1, 2 or 3, and the inserted amino acid residues can also be adjacent to each other in whole or in part, or the inserted amino acids do not differ from each other. Neighbor, as long as the polypeptide has inhibitory activity against coronavirus.
  • Amino acid deletion means that 1, 2 or 3 or more amino acids can be deleted from the amino acid sequence, such as the sequence of SEQ ID NO: 1, 2 or 3, as long as the polypeptide has inhibitory activity against coronavirus.
  • a polypeptide or polypeptide derivative of the invention may be produced synthetically or a polypeptide of the invention may be expressed by a cell.
  • polypeptides of the invention can be synthesized by chemical means.
  • polypeptides of the invention can be expressed in recombinant cells.
  • the type of cell is not limited, for example, the cell may be eukaryotic or prokaryotic.
  • Eukaryotic cells may be fungal cells, such as yeast cells, or insect cells or mammalian cells, such as mouse cells.
  • Prokaryotic cells may be bacterial cells, such as E. coli cells.
  • Cholesterol modification of the polypeptide of the present invention can be carried out at the C-terminus of the polypeptide, such that the polypeptide is linked to a cholesterol moiety at the C-terminus.
  • the connection may be a direct connection or may be connected via a linker.
  • the cholesterol modification method of the polypeptide in the present invention is a well-known method in the prior art.
  • the linker can be (GSG)m or (GSGSG)m, where m can be any integer, such as 1, 2, 3, 4, 5, 6, etc.
  • Linkers can be PEGylated.
  • the means of PEGylation are known to those skilled in the art.
  • PEGylated linker refers to a linker to which one or more PEGs are appended.
  • the PEGylated linker may be PEGylated (GSG)n or (GSGSG)n.
  • n can be any integer, such as 1, 2, 3, 4, 5, 6, and so on.
  • PEGm means that the number of repeating units of polyethylene glycol is m.
  • m can be any integer such as 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20.
  • a coronavirus can be any kind of coronavirus.
  • the coronavirus is an original strain Prototype or a mutant strain Alpha, Beta, Gamma, Delta, Omicron, Kappa, Lambda, Deltacron.
  • polypeptide or polypeptide derivative of the present invention can be prepared into medicine or pharmaceutical composition, such pharmaceutical composition is in the form of inhalation, nasal cavity preparation, oral preparation or parenteral preparation;
  • the oral formulation is selected from tablets, capsules, granules, suspensions, pills and solutions;
  • the parenteral formulation is an injectable or bolus injectable formulation
  • the pharmaceutical composition is a vaccine composition
  • Such medicines can be tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, suppositories or freeze-dried powder injections.
  • These drugs or pharmaceutical compositions can be applied by various administration methods, such as injection administration, including subcutaneous injection, intravenous injection, intramuscular injection and intraperitoneal injection, intracisternal injection or infusion, etc., cavity administration, such as rectal, vaginal and Sublingual, respiratory, eg nasal; mucosal, or topical.
  • the invention provides a method for preventing or treating a coronavirus infection or a disease caused by a coronavirus in a subject, which comprises administering the polypeptide or polypeptide derivative of the invention.
  • the present invention also provides a polypeptide or a polypeptide derivative used in preventing or treating a coronavirus infection or a disease caused by a coronavirus in a subject.
  • the present invention also relates to a method for preventing or treating a coronavirus infection in a subject or a disease caused by a coronavirus, which comprises administering the composition of the polypeptide or polypeptide derivative of the present invention to the subject/patient,
  • coronavirus is an original strain Prototype or a mutant strain Alpha, Beta, Gamma, Delta, Omicron, Kappa, Lambda, Deltacron.
  • the present invention also relates to polypeptides or polypeptide derivatives or compositions for preventing or treating coronavirus infections in subjects or diseases caused by coronaviruses, preferably, wherein the original coronavirus strain Prototype or mutant virus Strains Alpha, Beta, Gamma, Delta, Omicron, Kappa, Lambda, Deltacron.
  • the present invention also relates to compositions, pharmaceutical compositions, and kits for preventing or treating polypeptides or polypeptide derivatives in subjects/patients, preferably, wherein the coronavirus is an original strain Prototype or a mutant virus Strains Alpha, Beta, Gamma, Delta, Omicron, Kappa, Lambda, Deltacron.
  • the present invention designs the polypeptide P3 based on the HR2 region of the SARS-CoV-2 S protein, and performs a series of modifications to the P3 polypeptide (including the addition or substitution of one or more amino acids), and the modified polypeptides are named P3- 1. P3-2, P3-3, and modifying the polypeptides P3-1, P3-2, and P3-3 with linkers and cholesterol to obtain corresponding polypeptide derivatives, which are directly synthesized by methods routinely used in the art.
  • Eukaryotic system protein pCAGGS expression vector (provided by Suzhou Jinweizhi Company).
  • HEK293T ACE2 cells purchased from Suzhou Jinweizhi Company.
  • the inventor designed the polypeptide P3 by comparing the homology of HR1 and HR2 sequences in the human infection coronavirus protein, and carried out a series of modifications to the P3 polypeptide (including the addition or substitution of one or more amino acids).
  • the polypeptides were named P3-1, P3-2, and P3-3, respectively, and a series of linkers and cholesterol modifications were performed on the polypeptides P3-1, P3-2, and P3-3 to obtain the corresponding polypeptide derivatives, which were obtained in Table 1.
  • the specific sequences and structures of these polypeptides and polypeptide derivatives are shown. It was synthesized and purified by Shenzhen Hanyu Pharmaceutical Co., Ltd., and its purity is >95%.
  • the original SARS-CoV-2 strain of the present invention and its mutated strain pseudovirus are packaged by the inventor's laboratory (see Example 2).
  • HEK293T cells Spread HEK293T cells in a 10cm cell culture dish, so that the cell confluency density reaches about 80% the next day.
  • Toxification Add the pseudovirus packaging skeleton virus G*VSV-delG (purchased from Wuhan Privy Brain Science and Technology Co., Ltd.) to the above transfected HEK293T cells, incubate at 37°C for 2h, and change the culture medium (containing 10% FBS) DMEM medium), and VSV-G antibody (hybridoma cells expressing this antibody were purchased from ATCC cell bank) was added, and culture was continued for 30 h in the incubator.
  • G*VSV-delG purchased from Wuhan Privy Brain Science and Technology Co., Ltd.
  • Toxin collection collect the supernatant and centrifuge at 3000rpm for 10min, filter through a 0.45 ⁇ m sterile filter in an ultra-clean workbench to remove cell debris, aliquot, and store in a -80°C refrigerator.
  • the original SARS-CoV-2 strain SARS-CoV-2prototype
  • mutant strains Alpha (B.1.1.7)
  • Beta B.1.351
  • Gamma P.1
  • Delta B.1.617.2
  • Omicron BA.1
  • the purpose of this embodiment is to measure the inhibitory effect of the polypeptide in Table 1 on the pseudoviruses of the original strain (SARS-CoV-2 Prototype) and the mutant strain Omicron (BA.1).
  • HEK293T ACE2 cells (blank control group, cells not infected with virus); HEK293T ACE2 cells + original strain or mutant strain pseudovirus + DMEM medium (negative control group, cells infected with virus, but not treated with polypeptide) ; HEK293T ACE2 cells + original strain or variant strain pseudovirus + polypeptide P3 (P3 treatment group); HEK293T ACE2 cells + original strain or variant strain pseudovirus + P3-1 (P3-1 treatment group), HEK 293T ACE2 cells + original strain or variant strain pseudovirus + P3-2 (P3-2 treatment group); HEK 293T ACE2 cells + original strain or variant strain pseudovirus + P3-3 (P3-3 treatment group); HEK 293T ACE2 cells + original strain or variant strain pseudovirus + P3-1-GS-Chol (P3-1-GS-Chol treatment group); HEK 293T ACE2 cells + original strain or variant strain pseudovirus + P3 -2-GS-C
  • Polypeptide stock solution preparation Each polypeptide in Table 1 was first prepared with DMSO to make 10 mM stock solution, and then diluted to 20 ⁇ M with DMEM medium containing 10% FBS as stock solution for further gradient dilution.
  • Preparation of polypeptide gradient dilutions Dilute the above-mentioned 20 ⁇ M polypeptide stock solutions by 2 times with DMEM medium containing 10% FBS, and dilute 9 gradients (respectively 10 ⁇ M, 5 ⁇ M, 2.5 ⁇ M, 1.25 ⁇ M, 0.625 ⁇ M, 0.3125 ⁇ M, 0.156 ⁇ M, 0.078 ⁇ M, 0.039 ⁇ M), each gradient has 3 replicate wells, 50 ⁇ L per well.
  • Determination of the amount of pseudovirus Quantify the pseudovirus stock solution of the original strain or mutant strain of SARS-CoV-2 and a series of dilutions on HEK 293T ACE2 cells, and use the dilution when 1000 FFUs appear as the evaluation of the inhibitory effect of the polypeptide The amount of virus used (dilution factor is between 6 and 20 times).
  • step c Carefully discard the 96-well cell culture plate in step a, add the polypeptide-pseudovirus mixture (100 ⁇ L/well) in step b, and continue culturing in the incubator for 15h-24h.
  • ND means that in this experiment, the half effective concentration (EC 50 ) for inhibiting the strain could not be detected within the designed concentration range of the polypeptide.
  • the inhibitory effects of P3-1-PEG-Chol, P3-2-PEG-Chol, and P3-3-PEG-Chol on the original strain Prototype and the mutant strain Omicron were significantly better than their corresponding unmodified P3-1 , P3-2, and P3-3, among which the inhibitory effect of P3-2-PEG-Chol has been greatly improved.
  • the EC50 of the two experiments against the original strain Prototype were 0.2113 ⁇ M and 0.1955 ⁇ M, respectively, and it was against the mutant strain Omicron
  • the EC50 of the two experiments were 0.004168 ⁇ M and 0.01631 ⁇ M respectively;
  • P3-1-GS-PEG-Chol, P3-2-GS-PEG-Chol, P3-3-GS-PEG-Chol are superior to the corresponding unmodified P3 for the original strain Prototype and the mutant strain Omicron -1, P3-2, P3-3, among them, the inhibitory effect of P3-2-GS-PEG-Chol has been greatly improved, and the EC50 of the two experiments against the original strain Prototype were 0.1535 ⁇ M and 0.0454 ⁇ M, respectively.
  • the EC50 of the two experiments of the mutant strain Omicron were 0.007021 ⁇ M and 0.007624 ⁇ M respectively;
  • P3-2-GS-Chol, P3-2-PEG-Chol, and P3-2-GS-PEG-Chol had the best inhibitory effects on the original strain Prototype and the mutant strain Omicron, especially for the mutant strain Omicron
  • the inhibitory effect of the P3-2 derivative is remarkable, and the repeatability of the two experimental results of the P3-2 derivative shows that it has the most stable inhibitory ability to the virus.
  • the purpose of this embodiment is to determine the effect of polypeptide P3-2, P3-2-GS-Chol, P3-2-PEG-Chol, P3-2-GS-PEG-Chol, P3-1-GS-PEG-Chol on the original strain (SARS-CoV-2Prototype) and mutant strains (Alpha(B.1.1.7), Beta(B.1.351), Gamma(P.1), Delta(B.1.617.2) and Omicron(BA.1) ) of the pseudovirus inhibitory effect.
  • HEK293T ACE2 cells (blank control group, cells not infected with virus); HEK293T ACE2 cells + original virus strain or mutant strain pseudovirus + DMEM medium (negative control group, cells infected with virus, but not treated with polypeptide) ; HEK293T ACE2 cells + original strain or variant strain pseudovirus + polypeptide P3-2 (P3-2 treatment group); HEK293T ACE2 cells + original strain or variant strain pseudovirus + P3-2-GS-Chol (P3 -2-GS-Chol treatment group); HEK293T ACE2 cells + original strain or mutant strain pseudovirus + P3-2-PEG-Chol (P3-2-PEG-Chol treatment group); HEK293T ACE2 cells + original strain Or mutant strain pseudovirus + P3-2-GS-PEG-Chol (P3-2-GS-PEG-Chol treatment group); HEK 293T ACE2 cells + original strain or mutant strain pseudovirus + P3-1-GS - PEG-
  • Polypeptide stock solution preparation Each polypeptide in Table 1 was first prepared with DMSO to make 10 mM stock solution, and then diluted to 20 ⁇ M with DMEM medium containing 10% FBS, as stock solution for further gradient dilution.
  • Preparation of polypeptide gradient dilutions Dilute the above-mentioned 20 ⁇ M polypeptide stock solutions by 2 times with DMEM medium containing 10% FBS, and dilute 9 gradients in total (10 ⁇ M, 5 ⁇ M, 2.5 ⁇ M, 1.25 ⁇ M, 0.625 ⁇ M, respectively). ⁇ M, 0.3125 ⁇ M, 0.156 ⁇ M, 0.078 ⁇ M, 0.039 ⁇ M), each gradient has 3 replicate wells, 50 ⁇ L per well.
  • Determination of the amount of pseudovirus Quantify the pseudovirus stock solution of the original strain or mutant strain of SARS-CoV-2 and a series of dilutions on HEK 293T ACE2 cells, and use the dilution when 1000 FFUs appear as the evaluation of the inhibitory effect of the polypeptide The amount of virus used (the dilution factor is between 6 and 20 times).
  • step c Carefully discard the 96-well cell culture plate in step a, add the polypeptide-pseudovirus mixture (100 ⁇ L/well) in step b, and continue culturing in the incubator for 15h-24h.
  • ND means that in this experiment, the half effective concentration (EC 50 ) for inhibiting the strain could not be detected within the designed concentration range of the polypeptide.
  • the modified derivatives P3-2-GS-Chol, P3-2-PEG-Chol, and P3-2-GS-PEG-Chol of the polypeptide P3-2 not only inhibit the original strain Prototype and mutation of the new coronavirus
  • the strain Omicron has a good inhibitory effect, and also has good data effects on other mutant strains Alpha, Beta, Gamma, and Delta, and the inhibition test results on the original strain and the mutant strain Omicron are reproducible;
  • the derivatives P3-2-GS-Chol, P3-2-PEG-Chol, P3-2-GS-PEG of the polypeptide P3-2 -Chol has a better inhibitory effect on the original strain Prototype and mutant strains Alpha, Beta, Gamma, Delta, and Omicron;
  • This embodiment aims to evaluate the effect of truncated polypeptides after polypeptides P3-2-GS-Chol and P3-2-GS-PEG-Chol have knocked out amino acids one by one from the N-terminus on the original strain (SARS-CoV-2 Prototype) and mutant viruses. Inhibitory effect of pseudoviruses of strains (Delta (B.1.617.2) and Omicron (BA.1)).
  • amino acid sequences of the truncated polypeptides after P3-2-GS-Chol and P3-2-GS-PEG-Chol are knocked out one by one from the N-terminus are shown in Table 4 and Table 5.
  • P3-2-GS-Chol-1 and P3-2-GS-PEG-Chol-1 are extremely unstable in the purification process of synthetic preparation, so they will not continue to participate in the detection of the original strain of SARS-CoV-2 and its Evaluation experiment of the inhibitory effect of mutant strain pseudovirus.
  • HEK 293T ACE2 cells (blank control group, cells not infected with virus); HEK 293T ACE2 cells + original virus strain or mutant strain pseudovirus + DMEM medium (negative control group, cells infected with virus, but not treated with peptide treatment); HEK 293T ACE2 cells + original strain or mutant strain pseudovirus + P3-2-GS-PEG-Chol (P3-2-GS-PEG-Chol treatment group); HEK 293T ACE2 cells + original strain or Mutant strain pseudovirus + polypeptide P3-2-GS-PEG-Chol-2 (P3-2-GS-PEG-Chol-2 treatment group); HEK 293T ACE2 cells + original strain or mutant strain pseudovirus + P3 -2-GS-PEG-Chol-3 (P3-2-GS-PEG-Chol-3 treatment group); HEK 293T ACE2 cells + original strain or variant strain pseudovirus + P3-2-GS-Chol-2 (P3-2-GS-Chol-2 treatment group); HEK
  • Polypeptide stock solution preparation Each polypeptide in Table 4 and Table 5 was prepared into 10 mM stock solution with DMSO, and then diluted to 20 ⁇ M with DMEM medium containing 10% FBS, as stock solution for further gradient dilution.
  • Preparation of polypeptide gradient dilutions use 10% FBS-containing DMEM medium to dilute the above-mentioned 20 ⁇ M polypeptide stock solutions by 3 times, and dilute 9 gradients (respectively 6.67, 2.22, 0.74, 0.25, 0.082, 0.027 , 0.009, 0.003, 0.001 ⁇ M), each gradient has 3 replicate wells, 50 ⁇ L per well.
  • Determination of the amount of pseudovirus Quantify the pseudovirus stock solution of the original strain or mutant strain of SARS-CoV-2 and a series of dilutions on HEK 293T ACE2 cells, and use the dilution when 1000 FFUs appear as the evaluation of the inhibitory effect of the polypeptide The amount of virus used (dilution factor is between 6 and 20 times).
  • step c Carefully discard the 96-well cell culture plate in step a, add the polypeptide-pseudovirus mixture (100 ⁇ L/well) in step b, and continue culturing in the incubator for 15h-24h.
  • the truncated polypeptides P3-2-GS-Chol-1 and P3-2-GS-PEG-Chol of P3-2-GS-Chol and P3-2-GS-PEG-Chol were synthesized by solid phase -1, but the stability of these two polypeptides is poor, high-purity samples cannot be obtained through purification, and cannot be used in pseudovirus neutralization tests to evaluate the effect of virus inhibition.
  • This embodiment aims at measuring polypeptide P3-2-GS-Chol, P3-2-PEG-Chol, P3-2-GS-PEG-Chol in Table 1 to original virus strain (SARS-CoV-2WH01) on Vero cell And the inhibitory effect of live virus of mutant strains Delta (B.1.617.2) and Omicron (BA.1).
  • Vero cells (blank control group, cells not infected with virus); Vero cells + original strain or mutant strain live virus + DMEM medium (negative control group, cells infected with virus, but not treated with polypeptide); Vero cells Cells + original strain or variant strain live virus + P3-2-GS-Chol (P3-2-GS-Chol treatment group); Vero cells + original strain or variant strain live virus + P3-2-PEG- Chol (P3-2-PEG-Chol treatment group); Vero cells + original strain or mutant strain live virus + P3-2-GS-PEG-Chol (P3-2-GS-PEG-Chol treatment group).
  • Polypeptide stock solution preparation Determination in Table 1 Peptides P3-2, P3-2-GS-Chol, P3-2-PEG-Chol, P3-2-GS-PEG-Chol in Table 1 were first prepared with DMSO into a 5mM mother solution, The mother solution was then diluted to 5 ⁇ M with DMEM medium containing 2% FBS, and used as a stock solution for further serial dilution.
  • Peptides P3-2-GS-Chol and P3-2-PEG-Chol were 3-fold diluted from 5 ⁇ M to the three strains, with a total of 11 gradients; -2 Prototype and Delta strains were diluted 3 times from 5 ⁇ M, Omicron was diluted 5 times from 5 ⁇ M, a total of 11 gradients; 4 replicate wells for each gradient, 50 ⁇ l per well.
  • TCID50 Determination of the dosage of live virus: According to TCID50 calculation, respectively dilute the virus liquid of SARS-CoV-2WH01, Delta and Omicron to 2000TCID50/mL, each virus liquid requires a total of 9.6mL (prepared as 12mL). DMEM (containing 2% FBS) was used for dilution.
  • P3-2-GS-Chol, P3-2-PEG-Chol and P3-2-GS-PEG-Chol are effective against the live virus of the new coronavirus on Vero cells, especially for Omicron. good.
  • This embodiment aims at measuring polypeptide P3-2-GS-Chol, P3-2-PEG-Chol, P3-2-GS-PEG-Chol in Table 1 on Vero E6 cell to original virus strain (SARS-CoV-2WH01 ) and mutant strains Delta (B.1.617.2) and Omicron (BA.1) live virus inhibitory effect.
  • Vero E6 cells (blank control group, cells not infected with virus); Vero E6 cells + original virus strain or mutant strain live virus + DMEM medium (negative control group, cells infected with virus, but not treated with polypeptide) ; Vero E6 cells + original strain or variant strain live virus + P3-2-GS-Chol (P3-2-GS-Chol treatment group); Vero E6 cells + original strain or variant strain live virus + P3- 2-PEG-Chol (P3-2-PEG-Chol treatment group); Vero E6 cells + original strain or mutant strain live virus + P3-2-GS-PEG-Chol (P3-2-GS-PEG-Chol treatment group).
  • Polypeptide stock solution preparation Determination in Table 1 Peptides P3-2, P3-2-GS-Chol, P3-2-PEG-Chol, P3-2-GS-PEG-Chol in Table 1 were first prepared with DMSO into a 5mM mother solution, The mother solution was then diluted to 5 ⁇ M with DMEM medium containing 2% FBS, and used as a stock solution for further serial dilution.
  • Peptides P3-2-GS-Chol and P3-2-PEG-Chol were 3-fold diluted from 5 ⁇ M to the three strains, with a total of 11 gradients; -2 Prototype and Delta strains were diluted 3 times from 5 ⁇ M, Omicron was diluted 5 times from 5 ⁇ M, a total of 11 gradients; 4 replicate wells for each gradient, 50 ⁇ l per well.
  • TCID50 Determination of the dosage of live virus: According to TCID50 calculation, respectively dilute the virus liquid of SARS-CoV-2WH01, Delta and Omicron to 2000TCID50/mL, each virus liquid requires a total of 9.6mL (prepared as 12mL). DMEM (containing 2% FBS) was used for dilution.
  • ND means that in this experiment, the half effective concentration (EC 50 ) for inhibiting the strain could not be detected within the designed concentration range of the polypeptide.
  • CPE In the live virus neutralization test of P3-2-GS-PEG-Chol, CPE could be observed in the 48-hour experimental group for the new coronavirus Delta variant on Vero E6 cells, and the calculated EC50 was 0.1208 ⁇ M.
  • CPE can be observed in the 48-hour experimental group, and the calculated EC50 is 0.0403 ⁇ M.
  • CPE can be observed in the experimental group of 48, and the calculated EC 50 is 0.000028 ⁇ M.
  • P3-2-GS-Chol and P3-2-PEG-Chol are effective against live virus variants of the new coronavirus on Vero E6, especially against Omicron;
  • P3-2-GS- PEG-Chol is effective against both the original strain and the mutant strain of the live virus of the new coronavirus, especially for Omicron, which has the best inhibitory effect.
  • the polypeptides of the present invention and their derivatives inhibit virus invasion by inhibiting the membrane fusion process mediated by the S protein, and are suitable for coronaviruses, especially SARS-CoV-2 Prevention or treatment of diseases caused by original strains and mutant strains.
  • the polypeptide of the present invention and its derivatives have a significant inhibitory effect on the original SARS-CoV-2 strain and mutant strains Alpha, Beta, Gamma, Delta, Omicron, especially the mutant strain Omicron, and have potential wide application value.

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Abstract

提供一种用于抗冠状病毒的多肽，并在此基础上提供了上述多肽的含胆固醇的衍生物，这些多肽衍生物对冠状病毒，尤其是SARS-CoV-2原始毒株以及变异毒株产生意料不到的抑制效果，可作为制备预防或治疗新冠病毒的药物或疫苗，具有极大的预防或治疗潜力。

Description

一种抗冠状病毒的多肽、其衍生物及其应用 技术领域
本发明涉及生物医药领域，具体涉及用于预防或治疗冠状病毒的多肽、其衍生物、编码其多肽的多核苷酸、包含该多核苷酸的核酸构建体、包含该核酸构建体的表达载体、转化的细胞以及包含上述的药物组合物，以及它们在制备预防和/或治疗新冠病毒的药物或疫苗中的应用。
背景技术
冠状病毒(英文名：Coronavirus)是一种具有囊膜、基因组为线性单股正链的RNA病毒，是自然界广泛存在的一大类病毒。其中一些冠状病毒会感染人类并引起疾病，目前为止能够感染人的冠状病毒有七种，包括SARS-CoV、MERS-CoV、SARS-CoV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1。
SARS-CoV-2病毒为新冠肺炎疫情的病原体，是21世纪以来引起人类疫情的第三种冠状病毒。目前，虽然已有多种针对SARS-CoV-2的疫苗和单克隆抗体药物获得紧急使用许可，但是多数研究表明它们对当下广泛传播的新冠病毒变异毒株，例如Beta、Delta和Omicron等，表现出下降的保护效果，因而亟待开发针对目前流行以及未来出现的新的变异毒株的广谱药物或疫苗。
目前已报道有多种新冠相关冠状病毒(属于sarbecovirus)，如蝙蝠源RaTG13、RmYN02、ZC45、ZXC21等，穿山甲源GX/P2V/2017和GD/1/2019，研究表明RaTG13、GX/P2V/2017、GD/1/2019等也存在感染人的潜力，因而也亟需研发针对这些相关冠状病毒的药物或疫苗，以应对将来可能出现的新的疫情。
发明内容
针对现有技术的不足，本发明的目的在于提供用于预防或治疗冠状病毒的多肽、编码其的多核苷酸、包含该多核苷酸的核酸构建体、包含该核酸构建体的表达载体、转化的细胞以及包含上述的药物组合物，以及它们在制备预防或治疗冠状病毒的药物或疫苗中的应用。
第一方面，本发明提供一种用于抗冠状病毒的多肽，其为以下(1)-(8)任一所述序列的多肽，其中：
(1)多肽，其包含以SEQ ID NO.1所示的氨基酸序列；
(2)多肽，其包含以SEQ ID NO.2所示的氨基酸序列；
(3)多肽，其包含以SEQ ID NO.3所示的氨基酸序列；
(4)多肽，其包含以SEQ ID NO.12所示的氨基酸序列；
(5)多肽，其包含以SEQ ID NO.13所示的氨基酸序列；
(6)多肽，其包含以SEQ ID NO.14所示的氨基酸序列；
(7)多肽，其包含在所述多肽(1)-(6)任一的氨基酸序列中取代、缺失、添加或插入1个或多个氨基酸残基的氨基酸序列，且该多肽具有针对冠状病毒的抑制活性；或
(8)多肽，其包含与所述多肽(1)-(6)任一的氨基酸序列具有至少60％、65％、70％、75％、80％、85％、90％、93％、94％或95％同一性的氨基酸序列，且该多肽具有针对冠状病毒的抑制活性。
优选地，所述多肽的序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.12、SEQ ID NO.13或SEQ ID NO.14。
第二方面，本发明提供一种用于抗冠状病毒的多肽衍生物，所述多肽衍生物为上述抗冠状病毒的多肽上修饰有胆固醇的衍生物。
进一步地，所述胆固醇的衍生物的修饰位点在多肽的C端、N端或多肽氨基酸碱基侧链上。优选地，所述胆固醇修饰的修饰位点在所述多肽的C端。
进一步地，所述胆固醇的衍生物通过连接子连接或者直接连接在所述多肽的C端。
进一步地，所述胆固醇的衍生物为胆固醇经过半胱氨酸和或PEG修饰的衍生物。
进一步地，所述胆固醇衍生物选自
Figure PCTCN2022084473-appb-000001
Figure PCTCN2022084473-appb-000002
所述R 2为OH或者NH 2；
所述n为1-20的任意整数，优选为2-6的任意整数，例如为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20；
所述x为1-6的任意整数，例如为1、2、3、4、5、6。
进一步地，所述连接子为-NH-PEGn-(CH 2)x-CO-、(GSG)m、(GSGSG)m、 (GSG)m-CONH-PEGn-(CH 2)x-CO-或(GSGSG)m-CONH-PEGn-(CH 2)x-CO-；
其中m为0-6的任意整数，例如为0、1、2、3、4、5或6；
所述n为1-20的任意整数，优选为2-6的任意整数，例如为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20；
所述x为1-6的任意整数，例如为1、2、3、4、5、6。
进一步地，所述多肽衍生物具有如下结构I、结构II及结构III所示的结构式：
Figure PCTCN2022084473-appb-000003
其中，所述多肽序列是指上述(1)-(8)任一所述多肽序列；
优选地，所述多肽序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.12、SEQ ID NO.13或SEQ ID NO.14；
所述R 1为(GSG)m或(GSGSG)m，m选自0-6的任意整数；优选地m为1或2；
所述R 2为OH或者NH 2；优选地R 2为NH 2。
进一步地，所述多肽衍生物选自：
Figure PCTCN2022084473-appb-000004
Figure PCTCN2022084473-appb-000005
Figure PCTCN2022084473-appb-000006
Figure PCTCN2022084473-appb-000007
Figure PCTCN2022084473-appb-000008
第三方面，本发明提供了一种多核苷酸序列，所述多核苷酸序列用于编码第一方面的多肽。
第四方面，本发明提供了一种载体，所述载体中包含第三方面所述的多核苷酸序列。
第五方面，本发明提供了一种细胞，所述细胞中包含本发明第四方面所述的载体。
第六方面，本发明提供了一种药用组合物，其包含第一方面提供的多肽和或第二方面提供的衍生物，以及药学上可接受的载体和/或赋形剂；
在一个实施例中，所述药物组合物为吸入剂、鼻腔用制剂或口服制剂或胃肠外制剂的形式；
优选的，所述口服制剂选自片剂、胶囊剂、颗粒剂、混悬剂、丸剂和溶液剂；
优选地，所述胃肠外制剂为可注射或可推注制剂；
更优选地，所述药物组合物为疫苗组合物。
优选地，其中所述药物的剂型是片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、栓剂或冻干粉针剂；
优选地，其中所述药物优选通过以下方式给药：注射给药，包括皮下注射、静脉注射、肌肉注射和腹腔注射、脑池内注射或灌输等，腔道给药，如经直肠、阴道和舌下，呼吸道给药，如经鼻腔；粘膜给药，或者表面给药；
第七方面，本发明提供一种上述多肽、多肽衍生物、融合蛋白或缀合物在制备药物中的用途，所述药物用于抑制冠状病毒或治疗和/或预防冠状病毒引起的疾病。
优选地，其中所述冠状病毒选自SARS-CoV、MERS-CoV、SARS-CoV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1的原始毒株或其变异毒株。
优选地，所述冠状病毒选自SARS-CoV-2原始毒株、SARS-CoV-2变异毒株。
进一步优选地，所述SARS-CoV-2变异毒株选自Alpha、Beta、Gamma、Delta、Omicron、Kappa、Lambda、Deltacron。
优选地，冠状病毒引起的疾病选自新型冠状病毒肺炎(Corona Virus Disease 2019，COVID-19)、严重急性呼吸道综合症、中东呼吸综合症。
第八方面，本发明提供一种在体外抑制冠状病毒的方法，其包括应用上述多肽、多肽衍生物、融合蛋白或缀合物或药物组合物，
优选地，其中所述冠状病毒为SARS-CoV-2原始毒株和/或SARS-CoV-2变异毒株；
进一步优选地，所述变异毒株包括Alpha、Beta、Gamma、Delta、Omicron。
进一步地，应用的方法为将第一方面的多肽、第二方面提供的衍生物和/或第三方面的药用组合物与病毒进行接触。
第九方面，本发明提供一种预防或治疗受试者中的冠状病毒感染或由冠状病毒引起的疾 病的方法，其包括向受试者施用上述多肽、多肽衍生物、融合蛋白或缀合物或药物组合物，
优选地，其中所述冠状病毒为SARS-CoV-2原始毒株和/或SARS-CoV-2变异毒株；
进一步优选地，所述SARS-CoV-2变异毒株包括Alpha、Beta、Gamma、Delta、Omicron、Kappa、Lambda、Deltacron。
第十方面，本发明提供使用一种上述多肽以及多肽衍生物、融合蛋白或缀合物用于预防或治疗受试者中的冠状病毒感染或由冠状病毒引起的疾病，
优选地，其中所述冠状病毒为SARS-CoV-2原始毒株和/或SARS-CoV-2变异毒株；
进一步优选地，所述变异毒株包括Alpha、Beta、Gamma、Delta、Omicron、Kappa、Lambda、Deltacron。
本发明的有益效果为：
本发明基于SARS-CoV-2病毒的S蛋白的HR2区域，设计出多肽P3，并对其进行一系列改造(包括一个或多个氨基酸的增加或替换)，得到多肽，并对多进行连接子和胆固醇修饰得到多肽衍生物，多肽及其衍生物对SARS-CoV-2原始毒株及以及变异毒株Alpha、Beta、Gamma、Delta、Omicron产生很好的抑制效果，可作为制备预防或治疗新冠病毒的药物或疫苗，具有极大的预防或治疗潜力。
附图说明
图1为实施例2中不同多肽对SARS-CoV-2原始毒株假病毒的抑制效果；
图2为实施例2中不同多肽对SARS-CoV-2变异毒株Omicron假病毒的抑制效果；
图3为实施例3中多肽P3-2对新冠原始毒株和变异毒株Alpha、Beta、Gamma、Delta、Omicron假病毒的抑制效果；
图4为实施例3中多肽P3-2-GS-Chol对新冠原始毒株和变异毒株Alpha、Beta、Gamma、Delta、Omicron假病毒的抑制效果；
图5为实施例3中多肽P3-2-PEG-Chol对新冠原始毒株和变异毒株Alpha、Beta、Gamma、Delta、Omicron假病毒的抑制效果；
图6为实施例3中多肽P3-2-GS-PEG-Chol对新冠原始毒株和变异毒株Alpha、Beta、Gamma、Delta、Omicron假病毒的抑制效果；
图7为实施例3中多肽P3-1-GS-PEG-Chol对新冠原始毒株和变异毒株Alpha、Beta、Gamma、Delta、Omicron假病毒的抑制效果。
图8为实施例4中多肽P3-2-GS-Chol和P3-2-GS-PEG-Chol从N端逐个敲除氨基酸后的截短多肽对原始毒株(SARS-CoV-2Prototype)的假病毒的抑制效果；
图9为实施例4中多肽P3-2-GS-Chol和P3-2-GS-PEG-Chol从N端逐个敲除氨基酸后的截短多肽对变异毒株Delta的假病毒的抑制效果；
图10为实施例4中多肽P3-2-GS-Chol和P3-2-GS-PEG-Chol从N端逐个敲除氨基酸后的截短多肽对变异毒株Omicron的假病毒的抑制效果；
图11为实施例5中多肽P3-2-GS-chol在Vero细胞上对SARS-CoV-2原始毒株及其变异毒株活病毒抑制效果；
图12为实施例5中多肽P3-2-PEG-chol在Vero细胞上对SARS-CoV-2原始毒株及其变异毒株活病毒抑制效果；
图13为实施例5中多肽P3-2-GS-PEG-chol在Vero细胞上对SARS-CoV-2原始毒株及其变异毒株活病毒抑制效果；
图14为实施例6中多肽P3-2-GS-chol在Vero E6细胞上对SARS-CoV-2原始毒株及其变异毒株活病毒抑制效果；
图15为实施例6中多肽P3-2-PEG-chol在Vero E6细胞上对SARS-CoV-2原始毒株及其变异毒株活病毒抑制效果；
图16为实施例6中多肽P3-2-GS-PEG-chol在Vero E6细胞上对SARS-CoV-2原始毒株及其变异毒株活病毒抑制效果。
具体实施方式
冠状病毒刺突(S)蛋白在介导病毒入侵过程中发挥着重要作用，分为S1和S2两个亚基；其中S1亚基负责识别受体，S2亚基介导病毒囊膜与宿主细胞膜的膜融合。S2中的七肽重复序列HR1和HR2通过形成六螺旋束结构发挥膜融合功能，研究表明加入外源的HR1或HR2多肽能够抑制病毒本身的HR1和HR2六螺旋束结构的形成，进而抑制膜融合过程。
本发明的目的是提供一组可对SARS-CoV-2及类SARS病毒感染具有抑制功能的多肽类进入抑制剂，所述的多肽类进入抑制剂就是与SARS-CoV-2及类SARS病毒S2蛋白中的HR1区域结合，干扰病毒自身六螺旋的形成过程，从而抑制病毒的融合感染过程。
本发明基于SARS-CoV-2病毒的S蛋白的HR2区域，设计出多肽P3，并对其进行一系列改造(包括一个或多个氨基酸的增加或替换)，得到多肽P3-1、P3-2、P3-3，并对多肽P3-1、P3-2、P3-3进行连接子和胆固醇修饰，使得其胆固醇修饰多肽衍生物对SARS-CoV-2原始毒株及变异毒株Omicron具有较强的抑制效果，可作为制备预防或治疗新冠病毒的药物或疫苗。
该组多肽的具体序列如表1所示，或详见于多肽序列表中。该发明的多肽对SARS-CoV-2具有抑制活性，有些多肽的抑制活性达到了预料不到的高水平，尤其是对新冠变异毒株Omicron的抑制达到了很好的抑制的效果。
在本文中，多肽可以是包含SEQ ID NO:1-6的多肽或其变体。
例如，多肽可以包括以SEQ ID NO:1、2或3所示的氨基酸序列中取代、添加、缺失或插入1个或多个氨基酸。
氨基酸添加指在氨基酸序列，例如SEQ ID NO:1、2或3的C端或N端添加氨基酸，只要多肽具有针对冠状病毒的抑制活性。
氨基酸取代指在氨基酸序列，例如SEQ ID NO:1、2或3的序列的某个位置的某个氨基酸残基被其他氨基酸残基替代，只要多肽具有针对冠状病毒的抑制活性。
氨基酸插入指在氨基酸序列例如SEQ ID NO:1、2或3的序列的适当位置插入氨基酸残基，插入的氨基酸残基也可以全部或部分彼此相邻，或插入的氨基酸之间都不彼此相邻，只要多肽具有针对冠状病毒的抑制活性。
氨基酸缺失指可以从氨基酸序列，例如SEQ ID NO:1、2或3的序列中删除1、2或3个以上氨基酸，只要多肽具有针对冠状病毒的抑制活性。
本发明的多肽或多肽衍生物可以是合成生成的或者本发明的多肽可以是细胞表达的。例如，可以通过化学手段合成本发明的多肽。或者，可以在重组细胞中表达本发明的多肽。细胞的类型不受限制，例如细胞可以是真核细胞或者原核细胞。真核细胞可以是真菌细胞，例如酵母细胞，或者昆虫细胞或哺乳动物细胞，例如小鼠细胞。原核细胞可以是细菌细胞，例如大肠杆菌细胞。
本发明的多肽的胆固醇修饰可以在多肽的C端进行，使得多肽在C端与胆固醇部分连接。该连接可以是直接连接或者可以通过连接子连接。本发明中多肽进行胆固醇修饰的方法是现有技术中公知的方法。
连接子的种类和长度可以有所变化。例如，连接子可以是(GSG)m或(GSGSG)m，其中m可以是任何整数，例如1、2、3、4、5、6等等。
连接子可以是PEG化的。PEG化修饰的手段对于本领域技术人员是已知的。如本文中使用，术语PEG化连接子是指连接子附加有一个或者更多个PEG。在本文中，PEG化连接子可以是PEG化的(GSG)n或(GSGSG)n。n可以是任何整数，例如1、2、3、4、5、6等等。
如本文中使用，“PEGm”表示聚乙二醇重复单元的数目为m。m可以是任何整数，例如1、2、3、4、5、6、8、10、12、14、16、18、20。
如本文中使用，冠状病毒可以是任何种类的冠状病毒。优选地，所述冠状病毒是原始毒株Prototype或变异毒株Alpha、Beta、Gamma、Delta、Omicron、Kappa、Lambda、Deltacron。
本发明的多肽或多肽衍生物可以制备成药物或药物组合物，此类药物组合物为吸入剂、鼻腔用制剂或口服制剂或胃肠外制剂的形式；
优选地，优选地，所述口服制剂选自片剂、胶囊剂、颗粒剂、混悬剂、丸剂和溶液剂；
优选地，所述胃肠外制剂为可注射或可推注制剂；
优选地，药物组合物为疫苗组合物；
此类药物可以是片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、栓剂或冻干粉针剂。可以通过各种施用方式应用这些药物或药物组合物，例如注射给药，包括皮下注射、静脉注射、肌肉注射和腹腔注射、脑池内注射或灌输等，腔道给药，如经直肠、阴道和舌下，呼吸道给药，如经鼻腔；粘膜给药，或者表面给药。
发明提供了预防或治疗受试者中的冠状病毒感染或由冠状病毒引起的疾病的方法，其包括施用本发明的多肽或多肽衍生物。本发明还提供了在预防或治疗受试者中的冠状病毒感染或由冠状病毒引起的疾病中使用的多肽或多肽衍生物。
本发明还涉及预防或治疗受试者中的冠状病毒感染或由冠状病毒引起的疾病的方法，其包括给受试者/患者施用本发明所述的多肽或多肽衍生物的组合物，
优选地，其中冠状病毒是原始毒株Prototype或变异毒株Alpha、Beta、Gamma、Delta、Omicron、Kappa、Lambda、Deltacron。
本发明还涉及多肽或多肽衍生物或的组合物，用于预防或治疗受试者中的冠状病毒感染或由冠状病毒引起的疾病，优选地，其中所述冠状病毒原始毒株Prototype或变异毒株Alpha、Beta、Gamma、Delta、Omicron、Kappa、Lambda、Deltacron。
本发明还涉及用于预防或治疗受试者/患者中的多肽或多肽衍生物或的组合物，药物组合物，和试剂盒，优选地，其中所述冠状病毒是原始毒株Prototype或变异毒株Alpha、Beta、Gamma、Delta、Omicron、Kappa、Lambda、Deltacron。
实施例
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
以下，对本发明进行详述。
本发明基于SARS-CoV-2S蛋白的HR2区域，设计出多肽P3，并对P3多肽进行了一系列 的改造(包括一个或多个氨基酸的增加或替换)，改造后的多肽分别命名为P3-1、P3-2、P3-3，并对多肽P3-1、P3-2、P3-3进行连接子和胆固醇修饰得到相应的多肽衍生物，通过本领域常规使用的方法直接合成得到。
实验仪器和材料：
假病毒包装骨架病毒G*VSV-delG(购自武汉枢密脑科学技术有限公司)。
真核系统蛋白pCAGGS表达载体(由苏州金唯智公司提供)。
HEK293T ACE2细胞(购自苏州金唯智公司提供)。
多肽：
发明人通过将人感染冠状病毒蛋白中HR1和HR2序列的同源性对比，设计了多肽P3，并对P3多肽进行了一系列的改造(包括一个或多个氨基酸的增加或替换)，改造后的多肽分别命名为P3-1、P3-2、P3-3，并对多肽P3-1、P3-2、P3-3进行一系列连接子和胆固醇修饰得到相应的多肽衍生物，得到表1中显示的这些多肽及多肽衍生物的具体序列和结构。由深圳翰宇药业股份有限公司进行合成及纯化，其纯度>95％。
表1.实施例中所用的氨基酸序列
Figure PCTCN2022084473-appb-000009
Figure PCTCN2022084473-appb-000010
本发明的SARS-CoV-2原始毒株及其变异毒株假病毒为发明者实验室包装得到(参见实施例2)。
实施例1
SARS-CoV-2原始毒株及变异毒株假病毒的包装
截短的S蛋白的表达质粒的制备
1)将编码SARS-CoV-2原始毒株(Prototype)及变异毒株(Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Delta(B.1.617.2)和Omicron(BA.1))的S蛋白的后18位氨基酸的核苷酸去掉，分别得到核苷酸序列SARS-CoV-2-Prototype-S-del18、B.1.1.7-S-del18、B.1.351-S-del18、P.1-S-del18、B.1.617.2-S-del18、BA.1-S-del18，并由苏州金唯智公司进行合成。
2)分别将1)中获得的各核苷酸序列克隆到pCAGGS表达载体上，得到表达质粒pCAGGS-SARS-CoV-2-Prototype-S-del18、pCAGGS-B.1.1.7-S-del18、pCAGGS-B.1.351-S-del18、pCAGGS-P.1-S-del18、pCAGGS-B.1.617.2-S-del18和pCAGGS-BA.1-S-del18。
SARS-CoV-2原始毒株及变异毒株假病毒的包装
a.细胞准备：在10cm细胞培养皿中铺HEK293T细胞，使第二天细胞汇合密度至80％左右。
b.转染：取上述步骤2)中获得的各S蛋白的表达质粒，用PEI转染30μg质粒/10cm细胞培养皿，目的质粒与PEI按1:3比例混匀后转染，4-6h换培养液(含10％FBS的DMEM培养基)，37℃培养24h。
c.加毒：将假病毒包装骨架病毒G*VSV-delG(购自武汉枢密脑科学技术有限公司)加入上述转染后的HEK293T细胞，37℃孵育2h，换培养液(含10％FBS的DMEM培养基)，并加入VSV-G抗体(表达该抗体杂交瘤细胞购自ATCC细胞库)，在培养箱中继续培养30h。
d.收毒：收上清3000rpm离心10min,在超净工作台中经0.45μm无菌滤器过滤，去除 细胞碎片，分装，-80℃冰箱冻存。
经过上述步骤，分别得到SARS-CoV-2原始毒株(SARS-CoV-2prototype)及变异毒株(Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Delta(B.1.617.2)及Omicron(BA.1))的假病毒。
实施例2
多肽对SARS-CoV-2原始毒株及其变异毒株假病毒抑制效果评价
本实施例旨在测定表1中多肽对原始毒株(SARS-CoV-2Prototype)及变异毒株Omicron(BA.1))的假病毒的抑制效果。
实验分组：HEK293T ACE2细胞(空白对照组，细胞未感染病毒)；HEK293T ACE2细胞+原始毒株或变异毒株假病毒+DMEM培养基(阴性对照组，细胞感染了病毒，但未用多肽处理)；HEK293T ACE2细胞+原始毒株或变异毒株假病毒+多肽P3(P3处理组)；HEK293T ACE2细胞+原始毒株或变异毒株假病毒+P3-1(P3-1处理组)、HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-2(P3-2处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-3(P3-3处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-1-GS-Chol(P3-1-GS-Chol处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-2-GS-Chol(P3-2-GS-Chol处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-3-GS-Chol(P3-3-GS-Chol处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-3-GS2-Chol(P3-3-GS2-Chol处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-1-PEG-Chol(P3-1-PEG-Chol处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-2-PEG-Chol(P3-2-PEG-Chol处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-3-PEG-Chol(P3-3-PEG-Chol处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-1-GS-PEG-Chol(P3-1-GS-PEG-Chol处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-2-GS-PEG-Chol(P3-2-GS-PEG-Chol处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-3-GS-PEG-Chol(P3-3-GS-PEG-Chol处理组)。
多肽原液配制：表1中的各多肽先分别用DMSO配制成10mM母液，再用含10％FBS的DMEM培养基将母液稀释成20μM，作为进一步梯度稀释的原液。
多肽梯度稀释液的配制：用含10％FBS的DMEM培养基将上述20μM的各多肽原液以2倍的倍比稀释，共稀释9个梯度(分别为10μM、5μM、2.5μM、1.25μM、0.625μM、0.3125μM、0.156μM、0.078μM、0.039μM)，每个梯度3个复孔，每孔50μL。
假病毒用量的确定：将SARS-CoV-2原始毒株或变异毒株假病毒原液及一系列稀释液在 HEK 293T ACE2细胞上进行定量，将出现1000个FFU时的稀释度作为评价多肽抑制效果时的病毒用量(稀释倍数介于6～20倍之间)。
病毒抑制效果的测定方法为：
a.在96孔细胞培养板中铺HEK 293T ACE2细胞，培养使第二天细胞汇合密度达到80-90％。
b.根据上述实验分组，取上述各多肽梯度稀释液，加入等体积(50μL)的定量的假病毒稀释液，混匀后，在37℃孵育1h。
c.将步骤a中的96孔细胞培养板上清小心弃去，加入步骤b中的多肽-假病毒混合液(100μL/孔)，在培养箱中继续培养15h-24h。
d.利用高内涵显微镜统计被感染的细胞数，计算各多肽在不同浓度下的抑制率，再利用GraphPad计算出各多肽的EC 50(如图1、图2所示)，共进行两次平行实验，结果标记为1st和2nd，结果见表2。
表2.多肽对SARS-CoV-2原始毒株及其变异毒株假病毒抑制效果(EC 50)
EC 50(μM) Prototype 1st Omicron 1st Prototype 2nd Omicron 2nd
P3 0.3665 8.896 N.D. N.D.
P3-1 N.D. 7.899 N.D. N.D.
P3-2 0.1904 6.59 N.D. 7.289
P3-3 0.5011 22.66 N.D. N.D.
P3-1-GS-Chol 0.03368 8.614 3.067 0.6463
P3-2-GS-Chol 0.0929 0.02208 0.05333 0.01469
P3-3-GS2-Chol 0.1991 0.6057 8.955 1.694
P3-3-GS-Chol 0.1615 N.D. 15.67 1.729
P3-1-PEG-Chol 0.1903 N.D. 24.02 0.959
P3-2-PEG-Chol 0.2113 0.004168 0.1955 0.01631
P3-3-PEG-Chol 0.1946 N.D. 1.916 0.5712
P3-1-GS-PEG-Chol 0.668 0.2818 0.6805 0.25
P3-2-GS-PEG-Chol 0.1535 0.007021 0.0454 0.007624
P3-3-GS-PEG-Chol 0.2596 0.1397 0.1758 0.0198
注：N.D.表示本次实验中该多肽在设计的浓度范围内未能测得抑制该毒株的半数起效浓度(EC 50)。
结果与讨论：
从表2中可看出，发明所有的多肽及其衍生物都显示出具有冠状病毒SARS-CoV-2抑制效果。其中，P3-2对于原始毒株Prototype和变异毒株Omicron的抑制效果优于原始P3，但针对原始毒株的抑制结果重复性较差；
P3-1-GS-Chol、P3-2-GS-Chol、P3-3-GS2-Chol对于原始毒株Prototype和变异毒株Omicron的抑制效果显著优于与各自对应的未进行修饰的P3-1、P3-2、P3-3，其中P3-2-GS-Chol的抑制效果提升幅度较大，其针对原始毒株Prototype的两次实验EC50分别为0.0929μM和0.05333μM，其针对变异毒株Omicron的两次实验EC50分别为0.02208μM和0.01469μM；
P3-1-PEG-Chol、P3-2-PEG-Chol、P3-3-PEG-Chol对于原始毒株Prototype和变异毒株Omicron的抑制效果显著优于与各自对应的未进行修饰的P3-1、P3-2、P3-3，其中P3-2-PEG-Chol的抑制效果提升幅度较大，其针对原始毒株Prototype的两次实验EC50分别为0.2113μM和0.1955μM，其针对变异毒株Omicron的两次实验EC50分别为0.004168μM和0.01631μM；
P3-1-GS-PEG-Chol、P3-2-GS-PEG-Chol、P3-3-GS-PEG-Chol对于原始毒株Prototype和变异毒株Omicron优于与各自对应的未进行修饰的P3-1、P3-2、P3-3，其中P3-2-GS-PEG-Chol的抑制效果提升幅度较大，其针对原始毒株Prototype的两次实验EC50分别为0.1535μM和0.0454μM，其针对变异毒株Omicron的两次实验EC50分别为0.007021μM和0.007624μM；
且，P3-2-GS-Chol、P3-2-PEG-Chol、P3-2-GS-PEG-Chol对原始毒株Prototype和变异毒株Omicron的抑制效果最好，特别是针对变异毒株Omicron的抑制效果显著，且P3-2衍生物两次实验结果重复性好，表明其对病毒的抑制能力最稳定。
实施例3
多肽P3-2及其衍生物对SARS-CoV-2原始毒株及其变异毒株假病毒抑制效果评价
本实施例旨在测定多肽P3-2，P3-2-GS-Chol、P3-2-PEG-Chol、P3-2-GS-PEG-Chol、P3-1-GS-PEG-Chol对原始毒株(SARS-CoV-2Prototype)及变异毒株(Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Delta(B.1.617.2)及Omicron(BA.1))的假病毒的抑制效果。
实验分组：HEK293T ACE2细胞(空白对照组，细胞未感染病毒)；HEK293T ACE2细胞+原始毒株或变异毒株假病毒+DMEM培养基(阴性对照组，细胞感染了病毒，但未用多肽处理)；HEK293T ACE2细胞+原始毒株或变异毒株假病毒+多肽P3-2(P3-2处理组)；HEK293T ACE2细胞+原始毒株或变异毒株假病毒+P3-2-GS-Chol(P3-2-GS-Chol处理组)；HEK293T ACE2细胞+原始毒株或变异毒株假病毒+P3-2-PEG-Chol(P3-2-PEG-Chol处理组)；HEK293T ACE2细胞+原始毒株或变异毒株假病毒+P3-2-GS-PEG-Chol(P3-2-GS-PEG-Chol处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-1-GS-PEG-Chol (P3-1-GS-PEG-Chol处理组)。
多肽原液配制：分别表1中的各多肽先用DMSO配制成10mM母液，再用含10％FBS的DMEM培养基将母液稀释成20μM，作为进一步梯度稀释的原液。
多肽梯度稀释液的配制：用含10％FBS的DMEM培养基将上述20μM的各多肽原液以2倍的倍比稀释，共稀释9个梯度(分别为10μM、5μM、2.5μM、1.25μM、0.625μM、0.3125μM、0.156μM、0.078μM、0.039μM)，每个梯度3个复孔，每孔50μL。
假病毒用量的确定：将SARS-CoV-2原始毒株或变异毒株假病毒原液及一系列稀释液在HEK 293T ACE2细胞上进行定量，将出现1000个FFU时的稀释度作为评价多肽抑制效果时的病毒用量(稀释倍数介于6～20倍之间)。
病毒抑制效果的测定方法为：
a.在96孔细胞培养板中铺HEK 293T ACE2细胞，培养使第二天细胞汇合密度达到80-90％。
b.根据上述实验分组，取上述各多肽梯度稀释液，加入等体积(50μL)的定量的假病毒稀释液，混匀后，在37℃孵育1h。
c.将步骤a中的96孔细胞培养板上清小心弃去，加入步骤b中的多肽-假病毒混合液(100μL/孔)，在培养箱中继续培养15h-24h。
d.利用高内涵显微镜统计被感染的细胞数，计算各多肽在不同浓度下的抑制率，再利用GraphPad计算出各多肽的EC 50(如图3-7所示)结果见表3。
表3.多肽P3-2及其衍生物对SARS-CoV-2原始毒株及其变异毒株假病毒抑制效果(EC 50)
EC 50(μM) Prototype Alpha Beta Gamma Delta Omicron
P3-2 N.D. 327.9 N.D. 1304 280.7 7.289
P3-2-GS-Chol 0.05333 0.1618 0.3385 0.09824 0.0586 0.01469
P3-2-PEG-Chol 0.1955 0.1443 0.5096 1.257 0.2623 0.01631
P3-2-GS-PEG-Chol 0.0454 0.1605 0.1359 0.05655 0.05991 0.007624
P3-1-GS-PEG-Chol 0.6805 4.093 1.373 1.285 0.9383 0.25
注：N.D.表示本次实验中该多肽在设计的浓度范围内未能测得抑制该毒株的半数起效浓度(EC 50)。
结果与讨论
从表3看出，多肽P3-2的修饰衍生物P3-2-GS-Chol、P3-2-PEG-Chol、P3-2-GS-PEG-Chol不仅对新冠病毒的原始毒株Prototype、变异毒株Omicron有良好的抑制效果，同时对其他变异毒株Alpha、Beta、Gamma、Delta亦拥有良好的数据效果，而且对原始毒株和变异毒株Omicron 的抑制实验结果重复性好；
相比于多肽P3-1的衍生物P3-1-GS-PEG-Chol，多肽P3-2的衍生物P3-2-GS-Chol、P3-2-PEG-Chol、P3-2-GS-PEG-Chol对原始毒株Prototype、变异毒株Alpha、Beta、Gamma、Delta、Omicron的抑制效果更好；
相比于多肽P3-2，P3-2-GS-Chol、P3-2-PEG-Chol、P3-2-GS-PEG-Chol对原始毒株Prototype、变异毒株Alpha、Beta、Gamma、Delta、Omicron的抑制效果更好，其中P3-2-GS-PEG-Chol的抑制效果要优于P3-2-GS-Chol、P3-2-PEG-Chol；
针对各种变异毒株Alpha、Beta、Gamma、Delta、Omicron，P3-2-GS-Chol、P3-2-PEG-Chol、P3-2-GS-PEG-Chol对毒株Omicron的抑制效果最好。
实施例4
多肽P3-2-GS-PEG-Chol截短多肽及P3-2-GS-Chol截短多肽对SARS-CoV-2原始毒株及其变异毒株假病毒抑制效果评价
本实施例旨在评价多肽P3-2-GS-Chol和P3-2-GS-PEG-Chol从N端逐个敲除氨基酸后的截短多肽对原始毒株(SARS-CoV-2Prototype)及变异毒株(Delta(B.1.617.2)和Omicron(BA.1))的假病毒的抑制效果。
其中，P3-2-GS-Chol和P3-2-GS-PEG-Chol从N端逐个敲除氨基酸后的截短多肽的氨基酸序列如表4、表5所示。
表4.P3-2-GS-Chol从N端逐个敲除氨基酸的截短多肽的氨基酸序列
Figure PCTCN2022084473-appb-000011
Figure PCTCN2022084473-appb-000012
表5.P3-2-GS-PEG-Chol从N端逐个敲除氨基酸的截短多肽的氨基酸序列
Figure PCTCN2022084473-appb-000013
其中，P3-2-GS-Chol-1和P3-2-GS-PEG-Chol-1在合成制备的纯化过程中性质极为不稳定，因此不继续参与对SARS-CoV-2原始毒株及其变异毒株假病毒抑制效果的评价实验。
实验分组：HEK 293T ACE2细胞(空白对照组，细胞未感染病毒)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+DMEM培养基(阴性对照组，细胞感染了病毒，但未用多肽处理)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-2-GS-PEG-Chol(P3-2-GS-PEG-Chol处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+多肽P3-2-GS-PEG-Chol-2(P3-2-GS-PEG-Chol-2处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-2-GS-PEG-Chol-3(P3-2-GS-PEG-Chol-3处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-2-GS-Chol-2(P3-2-GS-Chol-2处理组)；HEK 293T ACE2细胞+原始毒株或变异毒株假病毒+P3-2-GS-Chol-3(P3-2-GS-Chol-3处理组)。
多肽原液配制：分别表4、表5中的各多肽先用DMSO配置成10mM母液，再用含10％FBS的DMEM培养基将母液稀释成20μM，作为进一步梯度稀释的原液。
多肽梯度稀释液的配制：用含10％FBS的DMEM培养基将上述20μM的各多肽原液以3倍的倍比稀释，共稀释9个梯度(分别为6.67、2.22、0.74、0.25、0.082、0.027、0.009、0.003、0.001μM)，每个梯度3个复孔，每孔50μL。
假病毒用量的确定：将SARS-CoV-2原始毒株或变异毒株假病毒原液及一系列稀释液在HEK 293T ACE2细胞上进行定量，将出现1000个FFU时的稀释度作为评价多肽抑制效果时的病毒用量(稀释倍数介于6～20倍之间)。
病毒抑制效果的测定方法为：
a.在96孔细胞培养板中铺HEK 293T ACE2细胞，培养使第二天细胞汇合密度达到80-90％。
b.根据上述实验分组，取上述各多肽梯度稀释液，加入等体积(50μL)的定量的假病毒稀释液，混匀后，在37℃孵育1h。
c.将步骤a中的96孔细胞培养板上清小心弃去，加入步骤b中的多肽-假病毒混合液(100μL/孔)，在培养箱中继续培养15h-24h。
d.利用高内涵显微镜统计被感染的细胞数，计算各多肽在不同浓度下的抑制率，再利用GraphPad计算出各多肽的EC 50结果见表6。
表6.多肽P3-2-GS-PEG-Chol截短多肽及P3-2-GS-Chol截短多肽对SARS-CoV-2原始毒株及其变异毒株假病毒抑制效果(EC50)
EC 50(μM) Prototype Delta Omicron
P3-2-GS-PEG-Chol 0.03110 0.01589 0.01085
P3-2-GS-PEG-Chol-2 0.2100 0.02260 0.01156
P3-2-GS-PEG-Chol-3 1.585 0.04737 0.1563
P3-2-GS-Chol-2 0.8645 1.248 0.1928
P3-2-GS-Chol-3 0.7789 0.6295 0.06472
结果与讨论
本实施例中，通过固相合成了P3-2-GS-Chol以及P3-2-GS-PEG-Chol的截短多肽P3-2-GS-Chol-1和P3-2-GS-PEG-Chol-1，但这两条多肽的稳定性差，通过纯化无法得到高纯度样品，无法用于假病毒中和试验评估对病毒抑制效果。
从表6和图8-10看出，多肽P3-2-GS-PEG-Chol的截短多肽P3-2-GS-PEG-Chol-2、P3-2-GS-PEG-Chol-3对新冠病毒的原始毒株Prototype、变异毒株Delta、Omicron也显示了较好的抑制效果，但抑制效果均弱于P3-2-GS-PEG-Chol，且这种抑制效果并不是随着肽序的逐渐截短而逐渐减弱的；多肽P3-2-GS-Chol的截短多肽P3-2-GS-Chol-2、P3-2-GS-Chol-3、P3-2-GS-Chol-4、P3-2-GS-Chol-5对原始毒株Prototype和变异毒株Delta、Omicron同样具有一定的抑制效果，但抑制效果均弱于P-2-GS-Chol。
对比截短多肽和原始长度多肽的假病毒中和试验数据，可以发现P3-2-GS-Chol和P3-2-GS-PEG-Chol截短后抗病毒效果均会有不同程度的下降。
实施例5
多肽P3-2的衍生物在Vero细胞上对SARS-CoV-2原始毒株及其变异毒株活病毒抑制效果 评价
本实施例旨在测定表1中多肽P3-2-GS-Chol、P3-2-PEG-Chol、P3-2-GS-PEG-Chol在Vero细胞上对原始毒株(SARS-CoV-2WH01)及变异毒株Delta(B.1.617.2)及Omicron(BA.1)的活病毒的抑制效果。
实验分组：Vero细胞(空白对照组，细胞未感染病毒)；Vero细胞+原始毒株或变异毒株活病毒+DMEM培养基(阴性对照组，细胞感染了病毒，但未用多肽处理)；Vero细胞+原始毒株或变异毒株活病毒+P3-2-GS-Chol(P3-2-GS-Chol处理组)；Vero细胞+原始毒株或变异毒株活病毒+P3-2-PEG-Chol(P3-2-PEG-Chol处理组)；Vero细胞+原始毒株或变异毒株活病毒+P3-2-GS-PEG-Chol(P3-2-GS-PEG-Chol处理组)。
多肽原液配制：表1中的测定表1中多肽P3-2、P3-2-GS-Chol、P3-2-PEG-Chol、P3-2-GS-PEG-Chol先用DMSO配置成5mM母液，再用含2％FBS的DMEM培养基将母液稀释成5μM，作为进一步梯度稀释的原液。多肽P3-2-GS-Chol、P3-2-PEG-Chol对三种毒株均从5μM开始3倍倍比稀释，共11个梯度；多肽P3-2-GS-PEG-Chol对SARS-CoV-2Prototype和Delta毒株从5μM开始3倍倍比稀释，对Omicron从5μM开始5倍倍比稀释，共11个梯度；每个梯度4个重复孔，每孔50μl。
活病毒用量的确定：按TCID50计算，分别稀释SARS-CoV-2WH01、Delta和Omicron的病毒液至2000TCID50/mL，每种病毒液共需要9.6mL(按12mL准备)。稀释时使用DMEM(含2％FBS)。
病毒抑制效果的测定方法为：
a.根据上述实验分组，取上述各多肽梯度稀释液，加入等体积(50μL)的定量的活病毒稀释液，混匀后，在37℃孵育1h。
b.将悬浮的Vero细胞加入混合液中，在培养箱中继续培养48h-120h。
c.观察细胞病变情况，分别记录48h、72h和120h时细胞病变孔数，计算各多肽在不同浓度下的抑制率，再利用GraphPad计算出各多肽的EC50(如图11-13所示)结果见表7。
表7.多肽P3-2及其衍生物对SARS-CoV-2原始毒株及其变异毒株活病毒抑制效(EC50)
Figure PCTCN2022084473-appb-000014
Figure PCTCN2022084473-appb-000015
结果与讨论
从表7可以看出，P3-2-GS-Chol、P3-2-PEG-Chol和P3-2-GS-PEG-Chol在Vero细胞上具有优异的新冠病毒活病毒抑制效果。
P3-2-GS-Chol的活病毒中和试验中，对于新冠病毒原型毒株(WH01)，在48小时观察CPE(细胞病变效应)，计算得到EC50为0.5280μM。对于新冠病毒Delta变异株，在48小时的实验组中未观察到CPE，此时多肽对病毒抑制率为100％，延长观察时间至72小时，可观察到CPE，计算得到EC50为0.0206μM。对于新冠病毒Omicron变异株，在48和72小时的实验组中均未观察到CPE，此时多肽对病毒抑制率为100％，延长观察时间至120小时，可观察到CPE，计算得到EC 50为0.0362μM。
P3-2-PEG-Chol的活病毒中和试验中，对于新冠病毒原型毒株(WH01)，在48小时观察CPE，计算得到EC 50为0.5292μM。对于新冠病毒Delta变异株，在48小时的实验组中未观察到CPE，此时多肽对病毒抑制率为100％，延长观察时间至72小时，可观察到CPE，计算得到EC 50为0.0206μM。对于新冠病毒Omicron变异株，在48和72小时的实验组中均未观察到CPE，此时多肽对病毒抑制率为100％，延长观察时间至120小时，可观察到CPE，计算得到EC50为0.1852μM。
P3-2-GS-PEG-Chol的活病毒中和试验中，对于新冠病毒原型毒株(WH01)，在48小时观察CPE，计算得到EC 50为0.0587μM。对于新冠病毒Delta变异株，在48小时的实验组中未观察到CPE，此时多肽对病毒抑制率为100％，延长观察时间至72小时，可观察到CPE，计算得到EC 50为0.0406μM。对于新冠病毒Omicron变异株，在48和72小时的实验组中均未观察到CPE，此时多肽对病毒抑制率为100％，延长观察时间至120小时，可观察到CPE，计算得到EC 50为0.0216μM。
综合观察时间和EC50分析，P3-2-GS-Chol、P3-2-PEG-Chol和P3-2-GS-PEG-Chol在Vero细胞上对新冠病毒活病毒有效，尤其针对Omicron的抑制效果最好。
实施例6
多肽P3-2的衍生物在Vero E6细胞上对SARS-CoV-2原始毒株及其变异毒株活病毒抑制效果评价
本实施例旨在测定表1中多肽P3-2-GS-Chol、P3-2-PEG-Chol、P3-2-GS-PEG-Chol在Vero E6细胞上对原始毒株(SARS-CoV-2WH01)及变异毒株Delta(B.1.617.2)及Omicron(BA.1)的 活病毒的抑制效果。
实验分组：Vero E6细胞(空白对照组，细胞未感染病毒)；Vero E6细胞+原始毒株或变异毒株活病毒+DMEM培养基(阴性对照组，细胞感染了病毒，但未用多肽处理)；Vero E6细胞+原始毒株或变异毒株活病毒+P3-2-GS-Chol(P3-2-GS-Chol处理组)；Vero E6细胞+原始毒株或变异毒株活病毒+P3-2-PEG-Chol(P3-2-PEG-Chol处理组)；Vero E6细胞+原始毒株或变异毒株活病毒+P3-2-GS-PEG-Chol(P3-2-GS-PEG-Chol处理组)。
多肽原液配制：表1中的测定表1中多肽P3-2、P3-2-GS-Chol、P3-2-PEG-Chol、P3-2-GS-PEG-Chol先用DMSO配置成5mM母液，再用含2％FBS的DMEM培养基将母液稀释成5μM，作为进一步梯度稀释的原液。多肽P3-2-GS-Chol、P3-2-PEG-Chol对三种毒株均从5μM开始3倍倍比稀释，共11个梯度；多肽P3-2-GS-PEG-Chol对SARS-CoV-2Prototype和Delta毒株从5μM开始3倍倍比稀释，对Omicron从5μM开始5倍倍比稀释，共11个梯度；每个梯度4个重复孔，每孔50μl。
活病毒用量的确定：按TCID50计算，分别稀释SARS-CoV-2WH01、Delta和Omicron的病毒液至2000TCID50/mL，每种病毒液共需要9.6mL(按12mL准备)。稀释时使用DMEM(含2％FBS)。
病毒抑制效果的测定方法为：
a.根据上述实验分组，取上述各多肽梯度稀释液，加入等体积(50μL)的定量的活病毒稀释液，混匀后，在37℃孵育1h。
b.将悬浮的Vero E6细胞加入混合液中，在培养箱中继续培养48-72h。
c.观察细胞病变情况，记录48h和72h时细胞病变孔数，计算各多肽在不同浓度下的抑制率，再利用GraphPad计算出各多肽的EC50(如图14-16所示)结果见表8。
表8. 48h和72h多肽P3-2及其衍生物在Vero E6细胞上对SARS-CoV-2原始毒株及其变异毒株活病毒抑制效(EC50)
Figure PCTCN2022084473-appb-000016
注：N.D.表示本次实验中该多肽在设计的浓度范围内未能测得抑制该毒株的半数起效浓度 (EC 50)。
结果与讨论
从表8可以看出，P3-2-GS-Chol、P3-2-PEG-Chol和P3-2-GS-PEG-Chol在Vero E6细胞上具有优异的新冠病毒活病毒抑制效果。由于48h后即能够观察到各多肽的EC 50，主要对比48h的数据。
P3-2-GS-Chol的活病毒中和试验中，在Vero E6细胞上对于新冠病毒原型毒株(WH01)，48h和72h均未能观察到对活病毒的抑制效果。对于新冠病毒Delta变异株，在48小时的实验组中可观察到CPE，计算得到EC50为0.3485μM。对于新冠病毒Omicron变异株，在48的实验组中可观察到CPE，计算得到EC 50为0.0022μM。
P3-2-PEG-Chol的活病毒中和试验中，在Vero E6细胞上对于新冠病毒原型毒株(WH01)，48h和72h均未能观察到对活病毒的抑制效果。对于新冠病毒Delta变异株，在48小时的实验组中可观察到CPE，计算得到EC50为0.5292μM。对于新冠病毒Omicron变异株，在48的实验组中可观察到CPE，计算得到EC 50为0.0015μM。
P3-2-GS-PEG-Chol的活病毒中和试验中，在Vero E6细胞上对于新冠病毒Delta变异株，在48小时的实验组中可观察到CPE，计算得到EC50为0.1208μM。对于新冠病毒Delta变异株，在48小时的实验组中可观察到CPE，计算得到EC50为0.0403μM。对于新冠病毒Omicron变异株，在48的实验组中可观察到CPE，计算得到EC 50为0.000028μM。
综合观察时间和EC50分析，P3-2-GS-Chol和P3-2-PEG-Chol在Vero E6上对新冠病毒活病毒变异株有效，尤其针对Omicron的抑制效果最好；P3-2-GS-PEG-Chol对新冠病毒活病毒原始毒株和变异株均有效，尤其针对Omicron的抑制效果最好。
综上，在SARS-CoV-2入侵宿主细胞的过程中，本发明的多肽及其衍生物通过抑制S蛋白介导的膜融合过程抑制病毒入侵，适用于冠状病毒，尤其是SARS-CoV-2原始毒株及变异毒株引起的疾病的预防或治疗。本发明的多肽及其衍生物对SARS-CoV-2原始毒株和变异毒株Alpha、Beta、Gamma、Delta、Omicron，尤其是对变异毒株Omicron的抑制效果显著，具有潜在的广泛应用价值。
上述实施例1-6中涉及的缩写及英文的含义如下表9所示。
表9缩写及英文的含义
缩写及英文 含义
FBS 胎牛血清
CPE 细胞病变效应
胆固醇,Chol 胆固醇
PEG 聚乙二醇
A 甘氨酸
R 精氨酸
N 天门冬酰胺
D 天门冬氨酸
C 半胱氨酸
Q 谷氨酰胺
E 谷氨酸
G 甘氨酸
H 组氨酸
I 异亮氨酸
L 亮氨酸
K 赖氨酸
M 甲硫氨酸(蛋氨酸)
F 苯丙氨酸
P 脯氨酸
S 丝氨酸
T 苏氨酸
W 色氨酸
Y 酪氨酸
V 缬氨酸
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (14)

  1. 一种用于抗冠状病毒的多肽，其为以下(1)-(8)任一所述序列的多肽，其特征在于：
    (1)多肽，其包含以SEQ ID NO.1所示的氨基酸序列；
    (2)多肽，其包含以SEQ ID NO.2所示的氨基酸序列；
    (3)多肽，其包含以SEQ ID NO.3所示的氨基酸序列；
    (4)多肽，其包含以SEQ ID NO.12所示的氨基酸序列；
    (5)多肽，其包含以SEQ ID NO.13所示的氨基酸序列；
    (6)多肽，其包含以SEQ ID NO.14所示的氨基酸序列；
    (7)多肽，其包含在所述多肽(1)-(6)任一的氨基酸序列中取代、缺失、添加或插入1个或多个氨基酸残基的氨基酸序列，且该多肽具有针对冠状病毒的抑制活性；或
    (8)多肽，其包含与所述多肽(1)-(6)任一的氨基酸序列具有至少60％、65％、70％、75％、80％、85％、90％、93％、94％或95％同一性的氨基酸序列，且该多肽具有针对冠状病毒的抑制活性；
    优选地，所述多肽的序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.12、SEQ ID NO.13或SEQ ID NO.14。
  2. 一种用于抗冠状病毒的多肽衍生物，其特征在于，所述多肽衍生物为权利要求1所述的抗冠状病毒的多肽修饰有胆固醇的衍生物；
    优选地，所述胆固醇的衍生物的修饰位点在多肽的C端、N端或多肽氨基酸碱基侧链上；更优选地，所述胆固醇修饰的修饰位点在所述多肽的C端；
    优选地，所述胆固醇的衍生物通过连接子连接或者直接连接在所述多肽的C端；
    优选地，所述胆固醇的衍生物为胆固醇经过半胱氨酸的衍生物；
    或
    所述胆固醇的衍生物为胆固醇经过半胱氨酸和PEG修饰的衍生物。
  3. 根据权利要求2所述的多肽衍生物，其特征在于，
    所述胆固醇衍生物选自
    Figure PCTCN2022084473-appb-100001
    Figure PCTCN2022084473-appb-100002
    所述R 2为OH或者NH 2；
    所述n为1-20的任意整数，优选为2-6的任意整数；
    所述x为1-6的任意整数。
  4. 根据权利要求2或3所述的多肽衍生物，其特征在于，所述连接子为-NH-PEGn-(CH 2)x-CO-、(GSG)m、(GSGSG)m、(GSG)m-CONH-PEGn-(CH 2)x-CO-或(GSGSG)m-CONH-PEGn-(CH 2)x-CO-；；
    其中m为0-6的任意整数；
    所述n为1-20的任意整数，优选为2-6的任意整数；
    所述x为1-6的任意整数。
  5. 根据权利要求2所述的多肽衍生物，其特征在于，所述多肽衍生物具有如下结构I、结构II及结构III所示的结构式：
    Figure PCTCN2022084473-appb-100003
    Figure PCTCN2022084473-appb-100004
    其中，所述R 1为(GSG)m或(GSGSG)m，m选自0-6的任意整数；优选地m为0、1或2；
    所述R 2为OH或者NH 2；优选地R 2为NH 2；
    所述多肽序列选自权利要求1中(1)-(8)任一所述多肽序列；
    优选地，所述多肽序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.12、SEQ ID NO.13或SEQ ID NO.14。
  6. 根据权利要求2所述的多肽衍生物，其特征在于，所述多肽衍生物选自：
    Figure PCTCN2022084473-appb-100005
    Figure PCTCN2022084473-appb-100006
    Figure PCTCN2022084473-appb-100007
    Figure PCTCN2022084473-appb-100008
    Figure PCTCN2022084473-appb-100009
    Figure PCTCN2022084473-appb-100010
  7. 一种多核苷酸序列，其特征在于，所述多核苷酸序列用于编码权利要求1所述的多肽。
  8. 一种载体，其特征在于，所述载体中包含权利要求7所述的多核苷酸序列。
  9. 一种细胞，其特征在于，所述细胞中包含权利要求8所述的载体。
  10. 一种融合蛋白或缀合物，其特征在于，包含权利要求1所述的多肽，和或权利要求2-5中任意一项所述的多肽衍生物。
  11. 一种药用组合物，其特征在于，其包含权利要求1所述的多肽，和或权利要求2-5中任意一项所述的多肽衍生物或其混合物、和/或权利要求10的融合蛋白或缀合物，以及药学上可接受的载体和/或赋形剂；
    优选地，所述药物组合物为吸入剂、鼻腔用制剂或口服制剂或胃肠外制剂的形式；
    优选地，所述口服制剂选自片剂、胶囊剂、颗粒剂、混悬剂、丸剂和溶液剂；
    优选地，所述胃肠外制剂为可注射或可推注制剂；
    优选地，所述药物组合物为疫苗组合物。
  12. 权利要求1所述的多肽，和或权利要求2-5中任意一项所述的多肽衍生物或其混合物、和/或权利要求10的融合蛋白或缀合物在制备药物中的用途，所述药物用于抑制冠状病毒或治疗和/或预防冠状病毒引起的疾病，
    优选地，其中所述冠状病毒选自SARS-CoV、MERS-CoV、SARS-CoV-2、 HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1的原始毒株或其变异毒株；
    优选地，所述冠状病毒选自SARS-CoV-2原始毒株、SARS-CoV-2变异毒株；
    更优选地，所述SARS-CoV-2变异毒株选自Alpha、Beta、Gamma、Delta、Omicron、Kappa、Lambda、Deltacron；
    优选地，冠状病毒引起的疾病选自新型冠状病毒肺炎(Corona Virus Disease 2019，COVID-19)、严重急性呼吸道综合症、中东呼吸综合症；
    优选地，其中所述药物的剂型是片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、栓剂或冻干粉针剂；
    优选地，其中所述药物优选通过以下方式给药：注射给药，包括皮下注射、静脉注射、肌肉注射和腹腔注射、脑池内注射或灌输等，腔道给药，如经直肠、阴道和舌下，呼吸道给药，如经鼻腔；粘膜给药，或者表面给药。
  13. 在体外抑制冠状病毒的方法，其包括施用权利要求1所述的多肽，和或权利要求2-5中任意一项所述的多肽衍生物或其混合物、权利要求10的融合蛋白或缀合物和/或权利要求11所述的药物组合物；
    优选地，其中所述冠状病毒选自SARS-CoV、MERS-CoV、SARS-CoV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1的原始毒株或其变异毒株；
    优选地，所述冠状病毒选自SARS-CoV-2原始毒株、SARS-CoV-2变异毒株；
    更优选地，所述SARS-CoV-2变异毒株选自Alpha、Beta、Gamma、Delta、Omicron、Kappa、Lambda、Deltacron。
  14. 预防或治疗受试者中的冠状病毒感染或由冠状病毒引起的疾病的方法，其包括施用权利要求1所述的多肽，和或权利要求2-5中任意一项所述的多肽衍生物或其混合物、权利要求10的融合蛋白或缀合物和/或权利要求11所述的药物组合物；
    优选地，其中所述冠状病毒选自SARS-CoV、MERS-CoV、SARS-CoV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1的原始毒株或其变异毒株。
    优选地，所述冠状病毒选自SARS-CoV-2原始毒株、SARS-CoV-2变异毒株；
    更优选地，所述SARS-CoV-2变异毒株选自Alpha、Beta、Gamma、Delta、Omicron、Kappa、Lambda、Deltacron；
    优选地，冠状病毒引起的疾病选自新型冠状病毒肺炎(Corona Virus Disease 2019，COVID-19)、严重急性呼吸道综合症、中东呼吸综合症。
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