Application of GRFT and fusion protein thereof in paramyxovirus or new coronavirus infection

Abstract

The invention provides application of GRFT and fusion protein thereof in paramyxovirus or new coronavirus infection. The GRFT or the fusion protein GREK1 thereof can be used for preparing medicines for (a) preventing or treating paramyxovirus or new coronavirus infection, and / or (b) diseases caused by paramyxovirus or new coronavirus infection. The GRFT can comprise an amino acid sequence as shown in SEQ ID NO: 2, and the GREK1 can comprise an amino acid sequence as shown in SEQ ID NO: 1.

Description

Technical Field

[0001] The present invention relates to the field of protein drugs, and particularly to the application of two recombinant expression proteins, GREK1 and GRFT, in drugs for preventing and / or treating paramyxovirus or novel coronavirus infection. Background Art

[0002] Paramyxovirus is a class of single-stranded negative-strand RNA enveloped viruses with a diameter of 150–300 nm, consisting of an envelope, matrix protein, nucleocapsid, etc. The genome length is between 15 and 19 kb, encoding different structural proteins and non-structural proteins in sequence, namely N (nucleoprotein), P (phosphoprotein), M (matrix protein), F (fusion protein), G (attachment glycoprotein), and L (polymerase protein). A variety of paramyxoviruses are highly infectious and pathogenic to humans, such as Nipah Virus (NiV), Hendra Virus (HeV), Measles virus (MeV), Respiratory syncytial virus (RSV), etc., thus seriously threatening human survival and health. In addition, paramyxoviruses also cause many infectious diseases in poultry or animals. For example, Newcastle disease virus (NDV) can infect poultry such as chickens, causing Newcastle disease, a highly contagious infectious disease that greatly harms the poultry industry. Diseased poultry show symptoms such as dyspnea and neurological symptoms, with a high mortality rate. Therefore, paramyxoviruses also have a serious impact on the global economic development.

[0003] Nipah virus and Hendra virus are Biosafety Level 4 (BSL-4) viruses, highly pathogenic zoonotic viruses. The mortality rate of infected patients can be as high as 60% - 75%, often presenting serious clinical symptoms such as acute respiratory diseases and fatal encephalitis. Currently, there are no specific antiviral drugs for this type of virus in clinical practice, so the research and development of relevant antiviral drugs are urgently needed. Measles virus is also a highly infectious pathogen, and its infected patients are often children. Its clinical manifestations are symptoms such as fever, rash, and cough. In severe cases, it can cause complications such as pneumonia and encephalitis, even endangering life. In recent years, the number of measles infections globally has been continuously increasing, also seriously threatening human life and health.

[0004] On the other hand, coronaviruses exist widely in nature, and a variety of coronaviruses pose a serious threat to the survival and health of humans. Among them, the most pathogenic one is Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the mortality rate of its infected patients is as high as about 30%. Recently, a variety of newly emerging coronaviruses have been successively reported, such as MjHKU4r-CoV, NeoCoV, PDF-2180, MOW15-22, PnNL2018B, etc. The S proteins of these newly emerging coronaviruses have changed very significantly compared with traditional coronaviruses (such as SARS-CoV-2, etc.), and their patterns of recognition and binding to receptors are significantly different from those of previously reported coronaviruses (Nature. 2022, 12(7941):748-757; PLoS Pathog. 2024, 20(11):e1012695; Cell. 2025, 188(6):1693-1710.e18). And currently, neither the immune sera of coronavirus vaccines nor the convalescent sera of coronavirus-infected patients can effectively inhibit the infection of these newly emerging viruses (Nature. 2022, 12(7941):748-757; Cell. 2025, 188(6):1465-1468). Once the newly emerging coronaviruses such as MjHKU4r-CoV, NeoCoV, PDF-2180, MOW15-22, PnNL2018B and their progeny spread to humans, they may trigger another pandemic (Nature. 2022, 12(7941):748-757; Cell. 2023, 186(4):850-863.e16; Cell. 2025, 188(6):1465-1468). Therefore, it is urgent to develop highly effective and broad-spectrum antiviral drugs against such viruses. Summary of the Invention

[0005] To solve the above problems, the present invention explores and discovers new uses of GRFT and GREK1 in the infection of paramyxoviruses and newly emerging coronaviruses.

[0006] Specifically, the first aspect of the present invention provides the application of a product selected from any one of the following a) to e) in inhibiting paramyxoviruses or newly emerging coronaviruses:

[0007] a) GRFT protein;

[0008] b) GRFT fusion protein;

[0009] c) Nucleic acid encoding GRFT protein;

[0010] d) Nucleic acid encoding GRFT fusion protein;

[0011] e) A composition comprising any one of the above a)-d).

[0012] In some embodiments, the fusion protein is a fusion protein GREK1 of GRFT protein and EK1 peptide.

[0013] In some embodiments, the inhibition is in vitro inhibition. In some embodiments, the inhibition includes inhibiting virus entry into host cells, inhibiting virus genetic material replication, inhibiting virus protein synthesis and processing, inhibiting virus assembly and release, and / or inhibiting virus immune escape. Preferably, the inhibition is inhibiting virus entry into host cells.

[0014] In some embodiments, the novel coronavirus is selected from MjHKU4r-CoV, MOW15-22, PnNL2018B, NeoCoV, PDF-2180, and progeny variants.

[0015] In some embodiments, the paramyxovirus is selected from Nipah virus (NiV), Hendra virus (HeV), and Measles virus (MeV).

[0016] In some embodiments, the GRFT protein is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 2; 2) a variant amino acid sequence obtained by one or more amino acid mutations on the basis of SEQ ID NO: 2; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2.

[0017] In some embodiments, the EK1 peptide is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 3; 2) a variant amino acid sequence obtained by one or more amino acid mutations on the basis of SEQ ID NO: 3; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3.

[0018] In some embodiments, GRFT and EK1 in the fusion protein GREK1 are directly linked or linked through a linker; preferably, the linker is a flexible linker; preferably, the linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, where n is an integer of 1-20, 2-15, 3-10, or 4-8; preferably, the linker is (GGGGS)5.

[0019] In some embodiments, the fusion protein is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 1; 2) a variant amino acid sequence obtained by one or more amino acid mutations based on SEQ ID NO: 1; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1.

[0020] In some embodiments, the mutation is a substitution, insertion, deletion, and / or addition, preferably a conservative amino acid substitution.

[0021] The second aspect of the present invention provides the use of a product selected from any one of a) to e) below in the preparation of a drug:

[0022] a) GRFT protein;

[0023] b) GRFT fusion protein;

[0024] c) nucleic acid encoding the GRFT protein;

[0025] d) nucleic acid encoding the GRFT fusion protein;

[0026] e) a composition comprising any one of a)-d) above.

[0027] In some embodiments, the drug is used for preventing or treating paramyxovirus infection.

[0028] In some embodiments, the paramyxovirus is selected from Nipah virus (NiV), Hendra virus (HeV), and Measles virus (MeV).

[0029] In some embodiments, the drug is used for preventing or treating novel coronavirus infection.

[0030] In some embodiments, the novel coronavirus is selected from MjHKU4r-CoV, MOW15-22, PnNL2018B, NeoCoV, PDF-2180, and progeny variants.

[0031] In some embodiments, the drug is used for preventing or treating diseases caused by paramyxovirus infection.

[0032] In some embodiments, the disease is selected from Nipah virus disease, Hendra virus disease, and Measles.

[0033] In some embodiments, the drug is used for preventing or treating diseases caused by novel coronavirus infection.

[0034] In some embodiments, the disease includes pneumonia caused by the novel coronavirus.

[0035] In some embodiments, the fusion protein is the fusion protein GREK1 of GRFT protein and EK1 peptide.

[0036] In some embodiments, the GRFT protein is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 2; 2) variant amino acid sequences obtained by one or more amino acid mutations based on SEQ ID NO: 2; and 3) amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2; wherein the mutations are substitutions, insertions, deletions, and / or additions, preferably conservative amino acid substitutions.

[0037] In some embodiments, the EK1 peptide is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 3; 2) variant amino acid sequences obtained by one or more amino acid mutations based on SEQ ID NO: 3; and 3) amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 3; wherein the mutations are substitutions, insertions, deletions, and / or additions, preferably conservative amino acid substitutions.

[0038] In some embodiments, the fusion protein is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 1; 2) variant amino acid sequences obtained by one or more amino acid mutations based on SEQ ID NO: 1; and 3) amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1; wherein the mutations are substitutions, insertions, deletions, and / or additions, preferably conservative amino acid substitutions.

[0039] In some embodiments, the drug inhibits virus from invading target cells.

[0040] In some embodiments, the drug is a drug for nasal administration.

[0041] In some embodiments, the drug is a prophylactic drug.

[0042] The third aspect of the present invention provides a method for inhibiting paramyxovirus or novel coronavirus, which includes contacting GRFT or its fusion protein or a composition comprising the GRFT or fusion protein with a sample containing or suspected of containing paramyxovirus or novel coronavirus.

[0043] In some embodiments, the fusion protein is the fusion protein GREK1 of GRFT protein and EK1 peptide.

[0044] In some embodiments, the GRFT is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 2; 2) a variant amino acid sequence obtained by performing one or more amino acid mutations on the basis of SEQ ID NO: 2; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 2; wherein the mutations are substitutions, insertions, deletions and / or additions, preferably conservative amino acid substitutions.

[0045] In some embodiments, the EK1 is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 3; 2) a variant amino acid sequence obtained by performing one or more amino acid mutations on the basis of SEQ ID NO: 3; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 3; wherein the mutations are substitutions, insertions, deletions and / or additions, preferably conservative amino acid substitutions.

[0046] In some embodiments, the fusion protein is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 1; 2) a variant amino acid sequence obtained by performing one or more amino acid mutations on the basis of SEQ ID NO: 1; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 1; wherein the mutations are substitutions, insertions, deletions and / or additions, preferably conservative amino acid substitutions.

[0047] In some embodiments, the paramyxovirus is selected from Nipah virus (NiV), Hendra virus (HeV) and Measles virus (MeV).

[0048] In some embodiments, the emerging coronavirus is selected from MjHKU4r-CoV, MOW15-22, PnNL2018B, NeoCoV, PDF-2180 and progeny variants.

[0049] In some embodiments, the method is carried out in vitro.

[0050] In some embodiments, the inhibition includes inhibiting virus entry into host cells, inhibiting virus genetic material replication, inhibiting virus protein synthesis and processing, inhibiting virus assembly and release, and / or inhibiting virus immune escape. Preferably, the inhibition is inhibiting virus entry into host cells.

[0051] The fourth aspect of the present invention provides a method for preventing or treating a disease caused by paramyxovirus or novel coronavirus infection, the method comprising administering to a subject in need a product selected from any one of a) to e) below:

[0052] a) GRFT protein;

[0053] b) GRFT fusion protein;

[0054] c) nucleic acid encoding GRFT protein;

[0055] d) nucleic acid encoding GRFT fusion protein;

[0056] e) a composition comprising any one of a)-d) above.

[0057] The fifth aspect of the present invention provides a product selected from any one of a) to e) below for inhibiting paramyxovirus or novel coronavirus, or for preventing or treating a disease caused by paramyxovirus or novel coronavirus infection:

[0058] a) GRFT protein;

[0059] b) GRFT fusion protein;

[0060] c) nucleic acid encoding GRFT protein;

[0061] d) nucleic acid encoding GRFT fusion protein;

[0062] e) a composition comprising any one of a)-d) above.

[0063] The advantages of the present invention include:

[0064] The present invention discovers for the first time that both GRFT and the fusion protein GREK1 can efficiently inhibit the infection of a variety of paramyxoviruses (NiV, HeV, and MeV), and unexpectedly, compared with the antiviral properties of the GRFT polypeptide itself, the component EK1 against coronavirus in the fusion protein, which has no paramyxovirus inhibitory activity itself, can significantly enhance the paramyxovirus inhibitory activity of GREK1.

[0065] The present invention also discovers that GRFT and GREK1 can inhibit newly emerging coronaviruses (such as MjHKU4r-CoV, MOW15-22, PnNL2018B, NeoCoV, PDF-2180, etc.). These newly emerging coronaviruses have great changes compared with traditional coronaviruses, especially in the sequences of their S proteins (target proteins). Moreover, GREK1 has extremely strong antiviral activity against the newly emerging coronaviruses detected (MjHKU4r-CoV, NeoCoV, PDF-2180), with IC50 values all at the low picomolar level, and no drugs and / or preparations with similar activities have been found in the research and development of drugs against these newly emerging coronaviruses. The inhibitory activities of previously reported proteins such as GRFT and GREK1 against SARS-CoV-2 are all at the nanomolar level (mBio. 2024, 15(5): e0074124). In contrast, their antiviral activities against these newly emerging coronaviruses are increased by 100 - 1000 times. Brief Description of the Drawings

[0066] Figure 1 . Schematic diagram of GREK1 and SDS-PAGE of its prokaryotic expression in PET-32a vector and TEV protease digestion. Figure 1 A, Schematic diagram of GREK1; Figure 1 B, SDS-PAGE diagram of the prokaryotic expression product of GREK1; Figure 1 C, SDS-PAGE diagram of the TEV protease digestion product of GREK1.

[0067] Figure 2 . Pseudovirus inhibitory activities of GRFT, EK1, and GREK1 against paramyxoviruses NiV-M, NiV-B, HeV, and measles virus.

[0068] Figure 3 . Inhibitory activities of GRFT, EK1, and GREK1 against cell-cell fusion and pseudovirus infection mediated by the S protein of the newly emerging coronavirus MjHKU4r-CoV.

[0069] Figure 4 . Inhibitory activities of GRFT, EK1, and GREK1 against cell-cell fusion and pseudovirus infection mediated by the S protein of the newly emerging coronavirus NeoCoV.

[0070] Figure 5 . Inhibitory activities of GRFT, EK1, and GREK1 against cell-cell fusion and pseudovirus infection mediated by the S protein of the newly emerging coronavirus PDF-2180.

[0071] Figure 6 . Inhibitory activities of GRFT, EK1, and GREK1 against the infection of the newly emerging coronaviruses MOW15-22 and PnNL2018B-VSV chimeric virus. Detailed implementation manners

[0072] To further understand the present invention, the preferred implementation manners of the present invention are described below. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred implementation manners, and those related can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Although it is believed that those of ordinary skill in the art are fully aware of the following terms, the following definitions are still stated to help illustrate the subject matter disclosed by the present invention.

[0073] As used herein, Griffithsin can be used interchangeably with the GRFT polypeptide and is a seaweed-derived lectin, which is one of the most effective viral entry inhibitors discovered so far. It is currently being developed as a microbicide and has broad-spectrum activity against several enveloped viruses. GRFT can inhibit human immunodeficiency virus (HIV) infection at picomolar concentrations, exceeding the capabilities of most anti-HIV drugs. In this article, Griffithsin can have the amino acid sequence shown in SEQ ID NO.2 or a variant amino acid sequence with one or more amino acid mutations.

[0074] As used herein, the "EK1 peptide" is an artificially designed synthetic polypeptide, mainly used as a broad-spectrum coronavirus fusion inhibitor. By targeting the HR1 (Heptad Repeat1) domain of the viral spike protein (S protein), it blocks the fusion process with the host cell membrane, thereby inhibiting virus infection. The EK1 peptide mimics the function of the viral HR2 domain, competitively binds to the HR1 region, interferes with the formation of the viral S protein six-helix bundle (6-HB), and prevents the fusion of the viral envelope with the host cell membrane. In this article, EK1 can have the amino acid sequence shown in SEQ ID NO:3 or a variant amino acid sequence with one or more amino acid mutations.

[0075] As used herein, "paramyxovirus" is a common pathogen clinically. The symptoms of its infectious diseases include fever, muscle or body pain, gastrointestinal infection symptoms, acute respiratory tract infection, encephalitis, etc. In this article, the paramyxovirus can be selected from NiV, HeV, and MeV, but is not limited to the above paramyxoviruses.

[0076] As used herein, the newly emerging coronaviruses may include MjHKU4r-CoV, MOW15-22, PnNL2018B, NeoCoV, PDF-2180, etc. and their progeny variant strains, etc., but are not limited to the above newly emerging coronaviruses.

[0077] As used herein, "Progeny Variant Strain" refers to a new generation of virus variants produced during the replication and proliferation of a virus in a host cell due to mutations in the genetic material (such as RNA or DNA). Such variant strains usually carry different gene sequences from the original virus strain and may cause changes in their biological characteristics (such as transmission efficiency, pathogenicity, immune escape ability, etc.).

[0078] As used herein, the pathogen of "Nipah virus disease" is Nipah virus, belonging to the genus Henipavirus, family Paramyxoviridae. The clinical manifestations of Nipah virus include fever, headache, myalgia, vomiting, and can progress to encephalitis (confusion, epilepsy) or severe respiratory symptoms.

[0079] As used herein, the pathogen of "Hendra virus disease" is Hendra virus. The clinical manifestations in humans are influenza-like symptoms (fever, cough), and can progress to meningitis or pneumonia, with a fatality rate of approximately 57%.

[0080] As used herein, the pathogen of "measles" is measles virus. Its typical course is high fever, cough, runny nose, conjunctivitis, and after 3-5 days, erythematous papules and Koplik spots appear. Complications include pneumonia, encephalitis, blindness; subacute sclerosing panencephalitis (SSPE) is a rare late sequela.

[0081] As used herein, "infectious diseases" include respiratory tract infections, gastrointestinal infections, nervous system infections, and infections of other tissue systems.

[0082] As used herein, a peptide or polypeptide refers to multiple amino acid residues linked by peptide bonds.

[0083] As used herein, a mutation refers to an addition, deletion, substitution, and / or insertion of an amino acid sequence. In this article, a mutation is a conservative amino acid substitution. Any amino acid sequence herein, such as any one of SEQ ID NO.1-3, can be appropriately modified while retaining the desired activity (such as anti-paramyxovirus activity and / or anti-newly emerging coronavirus activity).

[0084] As used herein, the term "one or more" refers to any number greater than 1. There is no particular limit on the upper limit of the number, and it can be determined by those skilled in the art. For example, one or more can be 1 to 50 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50).

[0085] As used herein, "linker" refers to one or more amino acid residues that connect multiple functional polypeptide moieties. For example, the linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, where n is an integer from 1 - 20, 2 - 15, 3 - 10, or 4 - 8. For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0086] As used herein, the degree of relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity". For the purposes of the present invention, the Needleman - Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443 - 453) implemented by the Needle program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276 - 277) (preferably version 5.0.0 or later) is used to determine the sequence identity between two amino acid sequences. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the non - simplified option) is used as the percentage identity and is calculated as follows:

[0087] (Identical residues × 100) / (Alignment length - Total number of gaps in the alignment)

[0088] For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) implemented by the needle program as in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 5.0.0 or later) is used to determine the sequence identity between two deoxynucleotide sequences. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of needle labeled "longest identity" (obtained using the non-simplified option) is used as the percentage identity and is calculated as follows:

[0089] (Number of identical deoxyribonucleotides x 100) / (Alignment length - total number of gaps in the alignment)

[0090] In the context of the present invention, a conservative amino acid substitution or a conservative substitution can be defined by a substitution within an amino acid class as reflected in one or more of the following tables:

[0091] Table 1: Amino acid residues of conservative classes:

[0092] Acidic residue D and E Basic residue K, R, and H Hydrophilic uncharged residue S, T, N, and Q Aliphatic uncharged residue G, A, V, L, and I Nonpolar uncharged residue C, M, and P Aromatic residue F, Y, and W

[0093] Table 2: Physical and functional classification of alternative amino acid residues:

[0094] Alcohol group-containing residue S and T Aliphatic residue I, L, V, and M Cycloalkenyl-related residue F, H, W, and Y Hydrophobic residue A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residue D and E Polar residue C, D, E, H, K, N, Q, R, S, and T Positively charged residue H, K, and R Small residue A, C, D, G, N, P, S, T, and V Minimal residue A, G, and S Residue involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residue Q, T, K, S, G, P, D, E, and R

[0095] Pharmaceutical uses of the GRFT polypeptide and the GRFT-EK1 fusion protein (GREK1)

[0096] The GRFT polypeptide described herein may comprise SEQ ID NO.2 or a variant sequence thereof, provided that the variant sequence retains the antiviral activity of the GRFT polypeptide, particularly the activity against paramyxoviruses (NiV, etc.). The EK1 polypeptide described herein may comprise SEQ ID NO.3 or a variant sequence thereof, provided that the variant sequence retains the antiviral activity of the EK1 polypeptide, particularly the activity against novel coronaviruses. The GREK1 described herein may comprise the amino acid sequence of SEQ ID NO:1 or a variant thereof, provided that the variant sequence retains the antiviral activity of GREK1, particularly the activity against paramyxoviruses or novel coronaviruses.

[0097] The present invention provides the use of GRFT (SEQ ID NO: 2) and / or its fusion protein GREK1 (SEQ ID NO: 1) described herein in the preparation of a medicament. The medicament can be used for preventing / or treating a) preventing or treating paramyxovirus or novel coronavirus infection, and / or b) diseases caused by paramyxovirus or novel coronavirus infection. In some embodiments, the infection includes respiratory tract infection, gastrointestinal tract infection, nervous system infection, and other tissue system infections. In some embodiments, the diseases include Nipah virus disease, Hendra virus disease, measles, or pneumonia caused by novel coronavirus.

[0098] The paramyxoviruses herein include NiV, HeV, and MeV, but are not limited to these paramyxoviruses; the novel coronaviruses herein include MjHKU4r-CoV, MOW15-22, PnNL2018B, NeoCoV, PDF-2180, and their progeny variants, but are not limited to these novel coronaviruses. Method for inhibiting paramyxovirus and / or novel coronavirus infection

[0099] The present invention provides a method for inhibiting paramyxovirus and novel coronavirus infection, which includes contacting virus particles with GRFT (SEQ ID NO: 2) and / or its fusion protein GREK1 (SEQ ID NO: 1) described herein or contacting with a composition containing GRFT (SEQ ID NO: 2) or GREK1 (SEQ ID NO: 1) to protect cells from infection by these pathogens.

[0100] In this context, the paramyxoviruses include NiV, HeV, and MeV but are not limited to these paramyxoviruses, and the novel coronaviruses include MjHKU4r-CoV, MOW15-22, PnNL2018B, NeoCoV, PDF-2180, and their progeny variants, but are not limited to these novel coronaviruses.

[0101] In this context, the inhibition is in vivo inhibition or in vitro inhibition.

[0102] In this context, the contact is in vivo contact or in vitro contact.

[0103] In this text, the composition may further comprise additional paramyxovirus drugs and / or coronavirus drugs in addition to GRFT or GREK1 to enhance the therapeutic effect. For example, the coronavirus drugs may be selected from molnupiravir, nirmatrelvir, ritonavir, azvudine, etc. The composition in this text may be in the form of tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, or lyophilized powders. Preferably, the fusion polypeptide GREK1, GRFT protein or its composition of the present invention is prepared into a dosage form for nasal administration. The method of the present invention is particularly suitable for preventing viral infections or related diseases. Prevention does not necessarily mean before infection.

[0104] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0105] Examples

[0106] The following examples are provided to illustrate the present invention. Those skilled in the art should understand that the examples are merely illustrative and not restrictive. The present invention is solely defined by the scope of the appended claims.

[0107] Example 1 Recombinant Proteins and Their Synthesis

[0108] EK1 used in the following examples of the present invention has the amino acid sequence shown in SEQ ID NO.3 and was synthesized by Shanghai Jiepeptide Co., Ltd. GRFT has the amino acid sequence shown in SEQ ID NO.2, and the GREK1 fusion protein has the amino acid sequence shown in SEQ ID NO.1, which were synthesized internally by the applicant. The sequence information of EK1, GRFT and GREK1 is shown in Table 3 below. The synthesis methods of GRFT and GREK1 are shown in 1.1 and 1.2 below.

[0109] Table 3: Polypeptides of the Present Invention

[0110]

[0111] 1.1 Expression, Purification and Identification of GREK1 Recombinant Fusion Protein

[0112] The method comprises the following steps:

[0113] Step 1: After codon-optimizing the gene sequence of GREK1 protein (the optimized sequence is shown in Table 4), it was constructed onto the Escherichia coli expression vector pET32a(+) by double digestion with KpnI and XhoⅠ to obtain a recombinant plasmid. Subsequently, the recombinant plasmid was transformed into Escherichia coli BL21(DE3) and then spread on an LB plate containing 50 μg / mL kanamycin.

[0114] Table 4

[0115]

[0116] Step 2: A single positive clone was selected and inoculated into 5 mL of LB culture medium (containing 50 μg / mL kanamycin) and pre-cultured overnight at 37 °C and 220 rpm. Subsequently, the pre-culture solution was inoculated into 1 L of LB culture medium (containing 50 μg / mL kanamycin) at a ratio of 1:200 and cultured at 37 °C and 220 rpm until the OD600 value of the bacterial solution reached about 0.5. The bacterial solution was placed at 4 °C for half an hour, 1 mM IPTG was added, and the culture was continued at 16 °C and 180 rpm for 20 hours.

[0117] Step 3: The culture solution after the end of the culture was centrifuged at 8000 rpm for 3 minutes to collect the bacterial solution, which was then resuspended with 100 mL of PBS. The resuspended bacterial solution was sonicated and then centrifuged at 10000 rpm and 4 °C for 30 minutes. The supernatant was filtered through a 0.45 μm filter membrane. 1 mL of nickel column material pre-equilibrated with binding buffer (10 mM imidazole, 20 mM Na2HPO4, 0.5 M NaCl, pH 7.4) was added to the culture supernatant and bound at room temperature for 2 hours.

[0118] Step 4: The liquid in Step 3 was passed through a purification column and washed with 50 volumes of washing buffer (20 mM imidazole, 20 mM Na2HPO4, 0.5 M NaCl, pH 7.4) to remove impurities. Finally, it was eluted with 5 volumes of elution buffer (500 mM imidazole, 20 mM Na2HPO4, 0.5 M NaCl, pH 7.4). The eluted protein was ultrafiltered 5 - 6 times to replace the buffer with PBS, obtaining a purified fusion protein.

[0119] Step 5: The purified protein was subjected to SDS-PAGE protein electrophoresis. After the electrophoresis was completed, Coomassie Brilliant Blue staining was performed. The results are as Figure 1 shown, where Figure 1 A is the schematic diagram of the structure of GREK1, Figure 1 B is the SDS-PAGE identification diagram of the recombinant expressed GREK1 protein, Figure 1C is GREK1 after being digested by TeV enzyme (protein: enzyme = 10:1, digested overnight at 4°C, and after digestion, Ni column was added, and the flow-through was collected after binding for 3 - 4 h and verified by gel electrophoresis) and removing the purification tag. As Figure 1 shown in C, the digested GREK1 protein migrated to the position corresponding to the expected band size (GREK1: 21 kDa).

[0120] 1.2 Expression, purification and identification of GRFT recombinant protein

[0121] The method includes the following steps:

[0122] Step 1: After codon optimization of the GRFT gene sequence, it was digested with KpnI and XhoⅠ double enzymes and constructed into the Escherichia coli expression vector PET28a(+), obtaining a recombinant plasmid. Subsequently, after transforming the recombinant plasmid into Escherichia coli BL21(DE3), it was spread on an LB plate containing 50 μg / mL kanamycin.

[0123] Table 4 Nucleic acid sequence of GRFT fusion protein

[0124]

[0125] Step 2: Place the competent BL21 on ice for 3 - 5 minutes, add 100 ng of pET-28a-GL25E plasmid to the competent cells, mix well, and then place the mixture on ice for 30 - 40 minutes. After placing it at 42°C for 90 seconds, place the competent cells on ice for 3 - 5 minutes again. Add 1 mL of medium and continue to culture at 220 rpm for 45 - 60 minutes, and then centrifuge at 3000 rpm for 2 minutes. Discard the supernatant, spread the precipitate on a Kan+LB plate, and culture at 37°C for 12 hours.

[0126] Step 3: Pick a single clone and immediately inoculate it into an LB culture medium (containing kanamycin), and culture it at 220 rpm and 37°C for 12 hours. Inoculate the bacteria from the small-scale culture into an LB culture medium (1000 mL) containing kanamycin at a ratio of 1:200, and continue to culture at 220 rpm and 37°C until the OD 600 value of the bacteria reaches 0.5 - 1.0. At this time, add IPTG (stock concentration is 1 M) to the bacteria culture medium at a ratio of 1:1000, and continue to culture at 180 rpm and 16°C for 20 hours.

[0127] Step 4: Centrifuge the bacteria at 8000 rpm to collect them, remove the supernatant as much as possible, and resuspend the bacteria in 100 mL of PBS. Ultrasonically disrupt the resuspended bacteria until they become clear. After centrifuging at 10000 rpm to remove the precipitate, filter the supernatant through a 0.45-μm filter membrane. Add 1 mL of nickel column material that has been equilibrated with binding buffer (10 mM imidazole, 20 mM Na2HPO4, 0.5 M NaCl, pH 7.4) to the culture supernatant and bind at room temperature for 2 hours.

[0128] Step 5: Pass the liquid from Step 4 through a purification column and wash away impurities with 50 volumes of washing buffer (20 mM imidazole, 20 mM Na2HPO4, 0.5 M NaCl, pH 7.4). Finally, elute with 5 volumes of elution buffer (500 mM imidazole, 20 mM Na2HPO4, 0.5 M NaCl, pH 7.4). Ultrafilter the eluted protein 5 - 6 times to replace the buffer with PBS, obtaining the purified GRFT protein.

[0129] Example 2: Preparation of paramyxovirus pseudovirus and novel coronavirus pseudovirus

[0130] Prepare pseudoviruses using methods known in the art. For example, refer to a previous report (L. Lu, Q. Liu, Y. Zhu, K.-H. Chan, L. Qin, Y. Li, Q. Wang, J.F.-W. Chan, L. Du, F. Yu, C. Ma, S. Ye, K.-Y. Yuen, R. Zhang, S. Jiang, Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor. Nat. Commun. 5, 3067 (2014)).

[0131] The envelope plasmids used for packaging paramyxovirus pseudoviruses are: pcDNA3.1-NiV-M-G / F, pcDNA3.1-NiV-B-G / F, pCAGGS-HeV-G / pcDNA3.1-HEV-F, pCAGGS-MeV-H / F, and the VSV backbone (VSV-del-G-rLuc) vector containing the luciferase reporter gene.

[0132] The envelope plasmids used for packaging novel coronaviruses are: pcDNA3.1-MjHKU4r-CoV-S, pcDNA3.1-MOW15-22-S, pcDNA3.1-PnNL2018B-S, pcDNA3.1-PDF-2180-S, pcDNA3.1-NeoCoV-S, and the HIV backbone plasmid (pNL4-3.Luc.R-.E-).

[0133] The information of the above envelope plasmids and the complete genomic sequences of each virus are shown in Table 5 below.

[0134] The envelope plasmids of the above paramyxovirus pseudoviruses and novel coronaviruses were either prepared in our laboratory or can be directly purchased. The used HIV backbone plasmid (pNL4-3.Luc.R-.E-) and VSV virus backbone vector (VSV-del-G-rLuc) were both stocked in our laboratory (Signal Transduct Target Ther. 2021, 6(1): 288).

[0135] Table 5 Information of envelope plasmids and virus genomes

[0136]

[0137] Specifically, the preparation process of the paramyxovirus pseudovirus is as follows:

[0138] 1. Culture HEK293T cells in DMEM medium containing 10% fetal bovine serum in a 10 cm culture dish, and place it in an incubator at 37 °C and 5% CO2. Wait until the cell confluence rate reaches 50% - 70% for transfection.

[0139] 2. The G / F expression plasmids used for pseudovirus packaging are 20 μg and 20 μg respectively. Mix them together in 1 mL of 0.9% NaCl solution and let it stand at room temperature for 5 minutes.

[0140] 3. Add 10 μL of Vigofect transfection reagent (Vigorous Biotechnology (Beijing) Co., Ltd., used in a ratio of 1:1000 with the medium) to another 1 mL of 0.9% NaCl solution and let it stand at room temperature for 5 minutes.

[0141] 4. Slowly add the Vigofect dilution drop by drop into the plasmid mixture. After standing at room temperature for 15 minutes, add it drop by drop into the HEK 293T cells cultured in a T75 culture flask.

[0142] 5. Replace the medium with fresh DMEM medium containing 10% fetal bovine serum 6 hours after transfection.

[0143] 6. Add VSV backbone (VSV-del-G-rLuc) particles. Wash twice and replace with fresh medium 2 hours later.

[0144] 7. 48 hours after changing the cell medium, collect the supernatant of the cell culture medium. Centrifuge at 3000 rpm for 20 minutes, then filter the supernatant through a 0.22 μm filter membrane, and aliquot and store it at -80 °C.

[0145] The preparation method of the pseudovirus of the novel coronavirus is as follows:

[0146] The backbone plasmid (pNL4-3.Luc.R-E-) and the envelope plasmid corresponding to the pseudovirus to be packaged are used for pseudovirus packaging in a ratio of 20 μg of the backbone plasmid to 20 μg of the envelope plasmid, that is, a 1:1 ratio. Specifically, the preparation process of the pseudovirus of the novel coronavirus is as follows:

[0147] 1. Culture HEK 293T cells in DMEM culture medium containing 10% fetal bovine serum in a 10 cm culture dish and place it in an incubator at 37 °C and 5% CO2. Transfect when the cell confluence rate reaches 50% - 70%;

[0148] 2. Mix 20 μg of the pcDNA3.1-S plasmid and 20 μg of the pNL4-3.Luc.R-E- backbone plasmid used for pseudovirus packaging in 1 mL of 0.9% NaCl solution and let it stand at room temperature for 5 minutes;

[0149] 3. Add 10 μL of Vigofect transfection reagent (Vigorous Biotechnology (Beijing) Co., Ltd., used in a ratio of 1:1000 with the culture medium) to another 1 mL of 0.9% NaCl solution and let it stand at room temperature for 5 minutes;

[0150] 4. Dropwise add the Vigofect dilution to the plasmid mixture. After standing at room temperature for 15 minutes, dropwise add it to the HEK 293T cells cultured in a T75 culture flask;

[0151] 5. Replace with fresh DMEM medium containing 10% fetal bovine serum 12 hours after transfection;

[0152] 6. 48 hours after changing the cell medium, collect the supernatant of the cell culture medium. After centrifuging at 3000 rpm for 20 minutes, filter the supernatant through a 0.22 μm filter membrane and store it in aliquots at -80 °C.

[0153] Example 3 GRFT and GREK1 inhibit paramyxovirus pseudovirus infection

[0154] Using the NiV-M / NiV-B / HeV / MeV pseudovirus system obtained in Example 2 as a tool, detect the activities of GRFT, GREK1, and EK1 in inhibiting pseudovirus infection on U87 / Vero target cells. The specific steps are as follows:

[0155] 1. Gradiently dilute GRFT, GREK1, and EK1 in a 96-well round-bottom plate using serum-free DMEM medium. The drug addition volume per well is 100 μL, and three replicates are set for each gradient (1000, 250, 62.5, 15.625, 3.906, 0.977 nanomoles).

[0156] 2. Add 100 μL of pseudovirus solution (RLU: 20000 - 100000) to the 96-well plate in step 1 of this example (the wells with drugs and virus are denoted as drug wells). At the same time, set up a positive control group for virus infection (with virus and without drug, i.e., virus wells) and a negative control group (without virus and without drug, i.e., drug-free wells). Incubate the virus and drugs at 37 °C for 1 hour.

[0157] 3. Add 200 μL of the mixture from step 2 to a 96-well plate seeded with target cells (U87 / Vero / Caco2, all from the Cell Bank of the Chinese Academy of Sciences' Type Culture Collection Committee). Culture at 37 °C and 5% CO2 for 12 hours, and then change the medium to DMEM medium containing 10% FBS.

[0158] 4. After continuing to culture for 48 hours, discard the culture supernatant, wash the cells once with PBS, add 50 μL of cell lysate (purchased from Promega, catalog number 0000389797), and lyse at room temperature for 1 hour. Pipette 40 μL of the lysate supernatant into a 96-well detection plate, add 40 μL of Luciferase Assay System substrate (purchased from Promega, catalog number 0000369690), and immediately measure the fluorescence value.

[0159] 5. Calculate the inhibition rate of pseudovirus infection. The calculation formula is: Inhibition rate = (virus well - drug well) / (virus well - drug-free well) × 100%.

[0160] Figure 2 Shows the inhibition effect diagrams of two proteins, GRFT and GREK1, provided by the present invention against 4 paramyxoviruses. As Figure 2 shown, GRFT and GREK1 can efficiently inhibit the infection of NiV-M, NiV-B, MeV, and HeV pseudoviruses. In particular, the half-maximal inhibitory concentration (IC 50 ) values of GREK1 are 16.73 nM (NiV-M), 11.66 nM (NiV-B), 2.03 nM (MeV), and 9.44 nM (HeV) respectively, which are all significantly higher than the activity of GRFT by 3 - 4 times. In addition, the inventors unexpectedly found that in the case where EK1 has no inhibitory activity against paramyxoviruses, GREK1 has an improved IC50 compared to GRFT. Figure 2) Detection of the inhibitory effect of GRFT and GREK1 against pseudoviruses of novel coronaviruses (MjHKU4r-CoV, NeoCoV, PDF-2180)

[0161] Construct a pseudovirus system of novel coronaviruses (MjHKU4r-CoV, NeoCoV, PDF-2180) according to the method of Example 2, and detect the inhibitory effects of GRFT, GREK1, and EK1 against these pseudoviruses respectively. The specific steps are as follows:

[0162] 1. Gradient dilute GRFT, GREK1, and EK1 in a 96-well round-bottom plate using serum-free DMEM medium. The drug addition volume per well is 60 μL, and 3 replicates are set for each gradient (5000, 1250, 312.5, 78.125, 19.531, 4.883, 1.221, 0.305, 0.076, or 0.019 nanomoles);

[0163] 2. Add 60 μL of pseudovirus solution (RLU: 10000 - 100000) to the 96-well round-bottom plate (the wells with drugs and viruses are recorded as drug wells), and simultaneously set a positive control group for virus infection (with virus and without drug, i.e., virus wells) and a negative control group (without virus and without drug, i.e., drug-free wells). Incubate the virus and drugs at 37°C for 1 hour;

[0164] 3. Add 100 μL of the mixture in step (2) of this example to a 96-well plate seeded with Caco2 cells. Culture at 37°C and 5% CO2 for 12 hours, and then change the medium to DMEM medium containing 10% FBS;

[0165] 4. After continuing to culture for 48 hours, discard the culture supernatant, wash the cells once with PBS, add 50 μL of cell lysate (purchased from Promega, catalog number 0000389797), and lyse at room temperature for 1 hour. Pipette 40 μL of the lysate supernatant into a 96-well detection plate, add 40 μL of the substrate (purchased from Promega, catalog number 0000369690), and immediately detect the fluorescence value;

[0166] 5. Calculate the inhibition rate of pseudovirus infection. The calculation formula is: Inhibition rate = (virus well - drug well) / (virus well - drug-free well) × 100%.

[0167] The results are as Figure 3 、 Figure 4 、 Figure 5As shown in Table 6, the effect of GREK1 in inhibiting the infection of newly emerging coronaviruses (MjHKU4r-CoV, NeoCoV, PDF-2180) is significantly better than that of EK1 and GRFT. This indicates that GRFT can improve the anti-viral activity of EK1 against these three newly emerging coronaviruses. The ability of GREK1 to counter these newly emerging coronaviruses (MjHKU4r-CoV, NeoCoV, PDF-2180) is increased by 392.5 to over 10,545 times compared to EK1, and by 14.8 to 24 times compared to GRFT. Among them, GREK1 has extremely strong anti-viral activity against the newly emerging coronaviruses (MjHKU4r-CoV, NeoCoV, PDF-2180) detected, with IC50 values all at the low picomolar level, and no drugs or formulations with similar activities have been found in the research and development of drugs against these newly emerging coronaviruses.

[0168] Table 6 Inhibitory effects of GRFT and GREK1 against pseudoviruses of newly emerging coronaviruses (MjHKU4r-CoV, NeoCoV, PDF-2180, MOW15-22, PnNL2018B)

[0169]

[0170] Example 5 Membrane fusion and infection processes mediated by the S proteins of GRFT and GREK1 against newly emerging coronaviruses (MjHKU4r-CoV, NeoCoV, PDF-2180)

[0171] The S protein expression genes of newly emerging coronaviruses (MjHKU4r-CoV, NeoCoV, PDF-2180) (gene sequences are from the viral genomes shown in Table 5 above) were cloned into the pAAV-IRES-GFP plasmid vector to obtain the pAAV-S-IRES-GFP recombinant plasmid.

[0172] After 293T cells were transfected with the plasmid encoding the coronavirus S protein and cultured for 36 - 48 h, they were used as effector cells. For example, 293T cells transfected with the plasmid encoding the S protein - pAAV-MjHKU4r / NeoCoV / PDF-2180-S-IRES-GFP are called 293T / S / EGFP cells. 293T cells transfected with the empty vector plasmid - pAAV-IRES-GFP are 293T / EGFP cells, which are used as negative control cells.

[0173] The above cells were blown and aspirated, centrifuged, resuspended with fresh DMEM medium, and the cell concentration was adjusted to 2×10 5 cells / mL. 50 μL was taken out and added to the gradient-diluted drug (50 μL), and incubated at 37 °C for 30 min.

[0174] Add 100 μL of the cell / drug mixture to the target cells (Caco2) that have been plated on a 96-well plate. Incubate at 37 °C with 5% CO2 for 2 - 4 hours, and observe and record the cell fusion under the green fluorescence channel of a fluorescence microscope.

[0175] Count the number of fused cells in each well. The formula for the fusion inhibition rate is: Inhibition rate = (positive fusion well - drug well) / (positive fusion well - negative control well) × 100%.

[0176] The results are as Figure 3 、 Figure 4 and Figure 5 shown. GREK1 significantly outperforms EK1 and GRFT in inhibiting S-mediated membrane fusion of novel coronaviruses (MjHKU4r-CoV, NeoCoV, PDF-2180), which is consistent with the results of the pseudovirus inhibition experiment in Example 4.

[0177] Example 6. Inhibition of GRFT and GREK1 against novel coronavirus (MOW15-22, PnNL2018B)-VSV chimeric virus infection

[0178] Use novel coronavirus (MOW15-22, PnNL2018B)-VSV chimeric virus, and detect the inhibitory effects of GRFT and GREK1 on these virus particles respectively. The specific steps are as follows:

[0179] ① The novel coronavirus MOW15-22-VSV, PnNL2018B-VSV chimeric virus and the target cells Caco2-P.nat-ACE2 cells were kindly provided by the team of Professor Huan Yan from the Wuhan Institute of Virology, Chinese Academy of Sciences (Cell, 4: S0092-8674(24)01474-0).

[0180] ② The process of the inhibition experiment of novel coronavirus-VSV chimeric virus infection:

[0181] (1) Gradient dilute GRFT, GREK1 and EK1 in a 96-well round bottom plate using serum-free DMEM medium. The drug volume added to each well is 100 μL. Set 3 replicates for each gradient (5000, 1250, 312.5, 78.125, 19.531, 4.883, 1.221, 0.305, 0.076 or 0.019 nM). Use GRFT or EK1 as the positive control.

[0182] (2) Add 100 μL of the chimeric virus solution (RLU: 20000 - 100000) to the 96-well round bottom plate (the wells with drugs and virus are recorded as drug wells). At the same time, set a positive control group for virus infection (with virus and without drug, i.e., virus wells) and a negative control group (without virus and without drug, i.e., no drug wells). Incubate the virus and drugs at 37 °C for 1 hour.

[0183] (3) Add 200 μL of the mixed solution obtained in step (2) to a 96-well plate seeded with target cells (Caco2-P.nat-ACE2). Incubate at 37 °C and 5% CO2 for 12 hours, and then change the medium to DMEM medium containing 10% FBS.

[0184] (4) After continuing the incubation for 48 hours, count the number of fluorescent plaques. The formula for calculating the inhibition rate of infection is: Inhibition rate = (virus well - drug well) / (virus well - drug-free well) × 100%.

[0185] Table 7 Inhibitory effects of GRFT and GREK1 against newly emerging coronavirus (MOW15-22, PnNL2018B) chimeric viruses

[0186]

[0187] The results are as Figure 6 shown in and Table 7. The inhibitory effect of GREK1 against newly emerging coronavirus (MOW15-22, PnNL2018B)-VSV chimeric viruses is significantly better than that of EK1 and GRFT. This indicates that GRFT can improve the anti-coronavirus activity of EK1. The activity of GREK1 against these newly emerging coronavirus (MOW15-22, PnNL2018B)-VSV chimeric viruses is increased by 5580 to 45100 times compared with EK1 and by 118 to 127 times compared with GRFT. In summary, the IC50 of GREK1 against all detected newly emerging coronaviruses is at the low picomolar level, and its antiviral activity is extremely strong. No drugs or preparations with similar activity have been found in the drug research and development against these newly emerging coronaviruses.

Claims

1. Use of a product selected from any one of a) to e) below in inhibiting paramyxovirus or novel coronavirus: a) GRFT protein; b) GRFT fusion protein; c) nucleic acid encoding GRFT protein; d) nucleic acid encoding GRFT fusion protein; e) a composition comprising any one of a)-d) above; preferably, the fusion protein is the fusion protein GREK1 of GRFT protein and EK1 peptide; preferably, the inhibition is in vitro inhibition; preferably, the novel coronavirus is selected from MjHKU4r-CoV, MOW15-22, PnNL2018B, NeoCoV, PDF-2180 and progeny variants.

2. The use according to claim 1, wherein the paramyxovirus is selected from Nipah virus (NiV), Hendra virus (HeV) and Measles virus (MeV).

3. The use according to claim 1 or 2, wherein the GRFT protein is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 2; 2) a variant amino acid sequence obtained by one or more amino acid mutations on the basis of SEQ ID NO: 2; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:

2.

4. The use according to any one of claims 1-3, wherein the EK1 peptide is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 3; 2) a variant amino acid sequence obtained by one or more amino acid mutations on the basis of SEQ ID NO: 3; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:

3.

5. The use according to any one of claims 1-4, wherein GRFT and EK1 in the fusion protein GREK1 are directly linked or linked through a linker; preferably, the linker is a flexible linker; preferably, the linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, where n is an integer of 1-20, 2-15, 3-10 or 4-8; preferably, the linker is (GGGGS)5.

6. The use according to claim 5, wherein the fusion protein is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 1; 2) a variant amino acid sequence obtained by one or more amino acid mutations on the basis of SEQ ID NO: 1; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:

1.

7. The use according to any one of claims 3-6, wherein the mutation is substitution, insertion, deletion and / or addition, preferably conservative amino acid substitution.

8. Use of a product selected from any of the following a) to e) in the preparation of a drug: a) GRFT protein; b) GRFT fusion protein; c) nucleic acid encoding GRFT protein; d) nucleic acid encoding GRFT fusion protein; e) composition comprising any of a) - d) above, wherein, The drug is used for preventing or treating paramyxovirus or novel coronavirus infection, and / or diseases caused by paramyxovirus or novel coronavirus infection; preferably, the fusion protein is the fusion protein GREK1 of GRFT protein and EK1 peptide; the novel coronavirus is selected from MjHKU4r-CoV, MOW15-22, PnNL2018B, NeoCoV, PDF-2180 and progeny variant strains.

9. The application according to claim 8, wherein the paramyxovirus is selected from Nipah virus (NiV), Hendra virus (HeV) and Measles virus (MeV).

10. The application according to claim 8 or 9, wherein the diseases include one or more of Nipah virus disease, Hendra virus disease, measles and pneumonia caused by novel coronavirus.

11. The application according to any one of claims 8-10, wherein the GRFT is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 2; 2) a variant amino acid sequence obtained by one or more amino acid mutations on the basis of SEQ ID NO: 2; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2; wherein the mutations are substitutions, insertions, deletions and / or additions, preferably conservative amino acid substitutions.

12. The application according to any one of claims 8-11, wherein the EK1 is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 3; 2) a variant amino acid sequence obtained by one or more amino acid mutations on the basis of SEQ ID NO: 3; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 3; wherein the mutations are substitutions, insertions, deletions and / or additions, preferably conservative amino acid substitutions.

13. The application according to any one of claims 8-12, wherein the fusion protein is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 1; 2) a variant amino acid sequence obtained by one or more amino acid mutations on the basis of SEQ ID NO: 1; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1; wherein the mutations are substitutions, insertions, deletions and / or additions, preferably conservative amino acid substitutions.

14. The application according to any one of claims 8-13, wherein the drug inhibits virus invasion of target cells; preferably, the drug is a drug administered by nasal cavity; preferably, the drug is a prophylactic drug.

15. A method for inhibiting paramyxovirus or novel coronavirus, which comprises contacting GRFT or its fusion protein or a composition comprising the GRFT or fusion protein with a sample containing or suspected of containing paramyxovirus or novel coronavirus. Preferably, the fusion protein is GREK1, a fusion protein of GRFT protein and EK1 peptide; Preferably, the GRFT is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 2; 2) a variant amino acid sequence obtained by one or more amino acid mutations on the basis of SEQ ID NO: 2; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 2; wherein the mutations are substitution, insertion, deletion and / or addition, preferably conservative amino acid substitution; Preferably, the EK1 is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 3; 2) a variant amino acid sequence obtained by one or more amino acid mutations on the basis of SEQ ID NO: 3; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 3; wherein the mutations are substitution, insertion, deletion and / or addition, preferably conservative amino acid substitution; Preferably, the fusion protein is selected from: 1) comprising the amino acid sequence shown in SEQ ID NO: 1; 2) a variant amino acid sequence obtained by one or more amino acid mutations on the basis of SEQ ID NO: 1; and 3) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 1; wherein the mutations are substitution, insertion, deletion and / or addition, preferably conservative amino acid substitution; Preferably, the paramyxovirus is selected from Nipah virus (NiV), Hendra virus (HeV) and Measles virus (MeV); and / or the novel coronavirus is selected from MjHKU4r-CoV, MOW15-22, PnNL2018B, NeoCoV, PDF-2180 and progeny variants; Preferably, the method is carried out in vitro.

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