Polypeptide capable of inhibiting MERS-like coronavirus infection and application thereof

Abstract

The invention provides a polypeptide capable of inhibiting MERS-like coronavirus infection and application of the polypeptide. The inventor invents a group of polypeptides on the basis of MjHKU4r-CoV coronavirus S protein S2 subunit mediated membrane fusion invasion characteristics by taking an HR1 functional domain as a target spot, and the group of polypeptides can efficiently inhibit the membrane fusion invasion process of MERS-like coronavirus MjHKU4r-CoV. The polypeptides are competitively combined with a virus HR1 functional domain to inhibit the formation of a virus 6-HB six-helix fusion core, so that the process that the coronavirus MjHKU4r-CoV invades target cells is efficiently blocked. According to the application disclosed by the invention, an efficient preventive and therapeutic candidate drug can be provided for preventing and treating the MERS-like coronavirus MjHKU4r-CoV with potential high pathogenicity and cross-species transmission.

Description

[0001] This application claims the priority benefit of a Chinese invention patent application with a filing date of July 22, 2024, application number: 202410986087.8, and invention name: “Polypeptides capable of inhibiting MERS-like coronavirus infection and their applications”. Technical Field

[0002] The present invention belongs to the field of biomedicine and relates to a group of polypeptides capable of inhibiting infection by MERS-like coronavirus MjHKU4r-CoV. Background Art

[0003] Coronaviruses have a huge natural reservoir in nature and can infect poultry, livestock and wild mammals including birds, cats, dogs, pigs, mice, bats, etc. At the same time, these coronaviruses have the potential to spread across races and infect humans, thus seriously threatening human survival and health, such as the highly pathogenic human coronaviruses that have broken out many times in recent years, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, it is urgent to develop effective antiviral drugs for the prevention and treatment of high-risk coronaviruses with the potential to infect humans.

[0004] MERS-CoV first broke out in the Middle East in 2012. Dromedary camels are its intermediate hosts, and they infect human cells through hDPP4 as a receptor. So far, there have been more than 2,500 confirmed cases of MERS worldwide, with a mortality rate of 34%, making it the human coronavirus with the highest mortality rate. Previously, two MERS-like coronaviruses, HKU4-CoV and HKU5-CoV, were found in bats, but their infectivity to human cells was very limited. Recently, a new MERS-like coronavirus, MjHKU4r-CoV, was isolated from pangolins. It can directly use hDPP4 as a functional receptor to infect human cells or tissues and organs, and has significant pathogenicity in hDPP4-transgenic mice. Therefore, the MERS-like coronavirus MjHKU4r-CoV has the potential to spread across species and infect humans. In addition, there are currently no effective drugs, neutralizing antibodies, or vaccines targeting the envelope glycoprotein (S protein) of the virus (reference: Chen J, Yang X, Si H, et al. A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cellentry. Cell. 2023;186(4):850-863.e16. doi:10.1016 / j.cell.2023.01.019). Therefore, once it breaks out in the population, it will pose a serious threat to human survival and health.

[0005] There is a need in the art for drugs against MERS-like coronavirus MjHKU4r-CoV. Summary of the invention

[0006] The purpose of the present invention is to provide a group of polypeptide entry inhibitors (SEQ ID NO: 1-19) that can effectively inhibit the infection of MERS-like coronavirus MjHKU4r-CoV. The polypeptide entry inhibitors interfere with the formation process of the virus's own six helices by targeting the HR1 region in the S2 protein of MjHKU4r-CoV, thereby inhibiting the fusion infection process of the virus. The specific sequence of the group of polypeptides is shown in Table 1. The present invention is based in part on the following findings: the uniqueness of the polypeptide antiviral mechanism (HR1 target), the high efficiency of the antiviral effect (IC50 at the nanomolar level), and the novelty against the virion (MjHKU4r-CoV, its specific antiviral drugs or protective vaccines have not been reported yet). The polypeptide length of the polypeptide entry inhibitor can be 30-60 amino acid residues, especially 30-40 amino acid residues.

[0007] In one aspect, provided herein is a polypeptide comprising the sequence of Formula 1:

[0008] EISKIN-Y1-T-Y2-L-Y3-L-Y4-Y5-Y6-Y7-Y8-Y9-L-Y10-E-Y11-Y12-K-Y13-L-Y14-Y15-S-Y16-IDLKEL Formula 1,

[0009] Wherein Y1 is T or V; Y2 is L or F; Y3 is D or N; Y4 is S, E or N; Y5 is D or T; Y6 is E or F; Y7 is M or L; Y8 is A, K or M; Y9 is I, K, M or V; Y10 is Q, E, S or L; Y11 is V or A; Y12 is V or I; Y13 is Q or K; Y14 is N or E; Y15 is S, D or E; Y16 is Y or L.

[0010] In one embodiment, the polypeptide may comprise the sequence of Formula 2:

[0011] EISKIN-Y17-TLLDLSDEMAIL-Y18-E-Y19-Y20-KQLNDSYIDLKEL Formula 2,

[0012] Y17 is T or V, Y18 is L or Q, Y19 is V or A, and Y20 is V or I.

[0013] In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or inserted into the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 1;

[0014] In one embodiment, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1, 3-5 and 11-18.

[0015] In one aspect, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or inserted into the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 2.

[0016] On the one hand, the present invention provides a stapled peptide, which is a stapled peptide of the amino acid sequence of any one of SEQ ID NOs: 1, 3-5 and 11-18, wherein two amino acids in the amino acid sequence are replaced by non-natural amino acids whose side chains can be linked, and the non-natural amino acids whose side chains can be linked are α-amino acids with alkenyl side chains, such as one or more of the following: (S)-2-(4'-pentenyl)alanine (S5), (R)-2-(4'-pentenyl)alanine (R5), (S)-2-(7'-octenyl)alanine (S8), (R)-2-(7'-octenyl)alanine (R8), (S)-2-(4'-pentenyl)glycine (Sg5) and (R)-2-(4'-pentenyl)alanine (Rg5); the positions of the replaced amino acids are 25th and 29th, respectively, to form a staple.

[0017] In one embodiment, the stapled peptide comprises the amino acid sequence of EISKINVTLLDLSDEMAILLEAIK-S5-LND-S5-YIDLKEL, wherein S5 is (S)-2-(4'-pentenyl)alanine, and a staple is formed between two S5s.

[0018] On the one hand, provided herein is a composition comprising a polypeptide, nucleic acid, vector, host cell, polypeptide derivative and / or stapled peptide as described herein. In one embodiment, the composition is a drug, preferably a tablet, capsule, drop pill, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, suppository or lyophilized powder injection, or an external preparation, preferably an external smear preparation, such as an external gel or an external infiltration preparation; the composition is a mask, a tissue, a glove, a garment, such as a protective suit, a hand washing product, such as a hand sanitizer, or a shower gel.

[0019] On the one hand, the present invention provides the use of the polypeptide described herein or the polypeptide, polypeptide derivative or stapled peptide described in any one of SEQ ID NOs: 6-10 in the preparation of a medicament or kit for treating or preventing MERS-like coronavirus MjHKU4r-CoV infection or a disease caused by MERS-like coronavirus MjHKU4r-CoV infection.

[0020] In one embodiment, the disease is a respiratory tract disease or a digestive tract disease.

[0021] On the one hand, the present invention provides a method for inhibiting MERS-like coronavirus MjHKU4r-CoV in vitro, which comprises the step of contacting a polypeptide described herein or a polypeptide derivative or stapled peptide of any one of SEQ ID NOs: 6-10 with MERS-like coronavirus MjHKU4r-CoV.

[0022] In one aspect, provided herein is a method for preparing a polypeptide, comprising the steps of:

[0023] (1) culturing the host cell described herein in a suitable culture medium; and

[0024] (2) Harvesting and isolating peptides.

[0025] On the one hand, the present invention provides a method for screening an active ingredient that inhibits MERS-like coronavirus MjHKU4r-CoV, comprising:

[0026] (1) Producing a polypeptide derived from the C-terminal heptad repeat domain 2 of the coronavirus S protein; preferably, the coronavirus is an alpha or beta coronavirus, preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV, and preferably, the polypeptide is a polypeptide described herein;

[0027] (2) determining the ability of the polypeptide to inhibit cell-cell fusion in a MjHKU4r-CoV S protein-mediated cell fusion assay, preferably inhibiting fusion between cells expressing the MjHKU4r-CoV S protein and cells expressing the human coronavirus receptor, or determining the ability of the polypeptide to inhibit the entry of MjHKU4r-CoV into cells, preferably cells expressing the human coronavirus receptor, in a cell-based assay; and

[0028] (3) Selecting peptides that have the ability to inhibit cell-cell fusion or the ability to inhibit MjHKU4r-CoV from entering cells.

[0029] In the first aspect, provided is the use of the polypeptide, the stapled peptide formed by the polypeptide, the polypeptide derivative, the nucleic acid encoding the polypeptide, and the composition comprising the polypeptide or stapled peptide or polypeptide derivative or nucleic acid in the preparation of a medicament or kit for treating or preventing MERS-like coronavirus MjHKU4r-CoV infection or a disease caused by MERS-like coronavirus MjHKU4r-CoV infection, wherein the polypeptide comprises the C-terminal heptad repeat domain 2 of the coronavirus S protein.

[0030] In a second aspect, a method for treating or preventing infection with a MERS-like coronavirus MjHKU4r-CoV or a disease caused by infection with a MERS-like coronavirus MjHKU4r-CoV is provided, which comprises administering to a subject the polypeptide described herein, a stapled peptide formed by the polypeptide, a polypeptide derivative, a nucleic acid encoding the polypeptide, a composition comprising the polypeptide or polypeptide derivative or nucleic acid, wherein the polypeptide comprises the C-terminal heptad repeat domain 2 of the coronavirus S protein.

[0031] In the third aspect, provided are the polypeptides of the present invention, stapled peptides formed by the polypeptides, polypeptide derivatives, nucleic acids encoding the polypeptides, and compositions comprising the polypeptides or polypeptide derivatives or nucleic acids for use in treating or preventing MERS-like coronavirus MjHKU4r-CoV infection or diseases caused by MERS-like coronavirus MjHKU4r-CoV infection, wherein the polypeptide comprises the C-terminal heptapeptide repeat domain 2 of the coronavirus S protein.

[0032] In a fourth aspect, a method for inhibiting MERS-like coronavirus MjHKU4r-CoV in vitro is provided, which comprises the step of contacting the polypeptide described herein, a stapled peptide formed by the polypeptide, a polypeptide derivative, a nucleic acid encoding the polypeptide, a composition comprising the polypeptide or polypeptide derivative or nucleic acid with MERS-like coronavirus MjHKU4r-CoV, wherein the polypeptide comprises the C-terminal heptad repeat domain 2 of the coronavirus S protein.

[0033] In various embodiments of the above aspects, the coronavirus is an alpha or beta coronavirus.

[0034] In various embodiments of the above aspects, the coronavirus is HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV.

[0035] In various embodiments of the above aspects, the polypeptide comprises the amino acid sequence of the following formula (I):

[0036] Formula (I):

[0037] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28- X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39-X40-X41-X42-X43-X44-X45-X46-X47-X48-X49-X50-X51-X52-X53-X54;

[0038] X1 is a nonpolar uncharged residue, preferably P, or is absent;

[0039] X2 is a hydrophilic uncharged residue, preferably N, or is absent;

[0040] X3 is an aromatic residue, preferably F, or is absent;

[0041] X4 is an aliphatic uncharged residue, preferably A, or is absent;

[0042] X5 is E, S or D; X6 is I, A, L or V; X7 is S, N, T, D or E; X8 is K, Q or G; X9 is I, F or Y; X10 is N; X11 is T, Q, V or A; X12 is T, A or S; X13 is L, F, I or V; X14 is L, G or V; X15 is D, A or N; X16 is L or M; X17 is S, Q, E, N or T; X1 X8 is D, T, K or S; X19 is E, G or F; X20 is M, F, L, I; X21 is A, T, K, M, L, D or S; X22 is I, T, K, M, V, S or R; X23 is L or S; X24 is Q, N, L, E or S; X25 is E, L, Q or N; X26 is V, A or K; X27 is V, F, I, A or S; X28 is K, A or S ; X29 is Q, K, A, N or E; X30 is L or V; X31 is N, Q or E; X32 is D, E or Y; X33 is S, A or T; X34 is Y, V or L; X35 is I, N or Q; X36 is D, A or K; X37 is L or N; X38 is K, A or Q; X39 is E, N or T; X40 is L or A; X41 is L, I or absent; X42 is S, D or absent; X43 is K, N or absent; X44 is L, I or absent; X45 is A, N or absent; X46 is S or absent; X47 is E, T or absent; X48 is L or absent; X49 is V or absent; X50 is D or absent; X51 is L or absent; X52 is K or absent; X53 is W or absent; X54 is L or absent.

[0043] In various embodiments of the above aspects, the polypeptide derivative is a cholesterol-modified derivative of the polypeptide.

[0044] In various embodiments of the above aspects, the polypeptide is linked to the cholesterol moiety at the C-terminus via a linker.

[0045] In various embodiments of the above aspects, the linker is PEGylated.

[0046] In various embodiments of the above aspects, the polypeptide is linked to the cholesterol-modified cysteine ​​at the C-terminus via a linker.

[0047] In various embodiments of the above aspects, the linker comprises (GSG)n or (GSGSG)n, wherein n is 1 or 2.

[0048] In various embodiments of the above aspects, the linker is -(GSG)n-DPEG4- or -(GSGSG)n-DPEG4-.

[0049] In one embodiment of the first aspect, the dosage form of the drug is a tablet, capsule, pellet, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, suppository or lyophilized powder injection.

[0050] In one embodiment of the first aspect, the drug is administered by the following methods: injection, including subcutaneous injection, intravenous injection, intramuscular injection and intraperitoneal injection, intracisternal injection or infusion, etc., cavity administration, such as rectal, vaginal and sublingual, respiratory administration, such as nasal cavity; mucosal administration, or surface administration.

[0051] In various embodiments of the above aspects, X5 is E; and / or, X6 is I; and / or, X7 is S; and / or, X8 is K; and / or, X9 is I; and / or, X10 is N; and / or, X11 is T or V; and / or, X12 is T; and / or, X13 is L or F; and / or, X14 is L; and / or, X15 is D or N; and / or, X16 is L; and / or, X17 is S, E or N; and / or, X18 is D or T; and / or, X19 is E or F; and / or, X20 is M or L; and / or, X21 is A, K or M; and / or, X22 is I, K, M or or X32 is D or E; or X33 is S; or X34 is Y or L; or X35 is I; or X36 is D; or X37 is L; or X38 is K; or X39 is E; or X40 is L; or X41-X54 is absent.

[0052] In various embodiments, the stapled peptide is a stapled peptide of the amino acid sequence of any one of SEQ ID NOs: 1, 3-5 and 11-18, wherein two amino acids in the amino acid sequence are replaced by non-natural amino acids whose side chains can be linked, and the non-natural amino acids whose side chains can be linked are α-amino acids with alkenyl side chains, such as one or more of the following: (S)-2-(4'-pentenyl)alanine (S5), (R)-2-(4'-pentenyl)alanine (R5), (S)-2-(7'-octenyl)alanine (S8), (R)-2-(7'-octenyl)alanine (R8), (S)-2-(4'-pentenyl)glycine (Sg5) and (R)-2-(4'-pentenyl)alanine (Rg5); the positions of the replaced amino acids are 25th and 29th, respectively. In one embodiment, the stapled peptide comprises an amino acid sequence of EISKINVTLLDLSDEMAILLEAIK-S5-LND-S5-YIDLKEL, wherein S5 is (S)-2-(4'-pentenyl)alanine and a staple is formed between two S5s. In various embodiments of the above aspects, the polypeptide comprises (1) an amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; (2) an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or replaced in the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; or (3) an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19.

[0053] In various embodiments of the above aspects, the polypeptide comprises an amino acid sequence of the following formula (II):

[0054] X1 -

[0055] Wherein Y1 is T or V; Y2 is L or F; Y3 is D or N; Y4 is S, E or N; Y5 is D or T; Y6 is E or F; Y7 is M or L; Y8 is A, K or M; Y9 is I, K, M or V; Y10 is Q, E, S or L; Y11 is V or A; Y12 is V or I; Y13 is Q or K; Y14 is N or E; Y15 is S, D or E; Y16 is Y or L.

[0056] In various embodiments of the above aspects, the polypeptide is the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19.

[0057] In various embodiments of the above aspects, the polypeptide derivative is SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-(GSGSG)n-PEG4-C-cholesterol, wherein n is 1 or 2.

[0058] In one embodiment, the polypeptide is derived from the C-terminal heptad repeat domain 2 of the coronavirus S protein.

[0059] In one embodiment, the coronavirus is an alpha or beta coronavirus, preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV.

[0060] In one embodiment, the polypeptide has MERS coronavirus MjHKU4r-CoV inhibitory activity.

[0061] In one embodiment, the polypeptide comprises (1) an amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; (2) an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or replaced in the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; or (3) an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19.

[0062] In a sixth aspect, a nucleic acid is provided that encodes any of the polypeptides described herein.

[0063] In a seventh aspect, a vector is provided, comprising a nucleic acid as described herein.

[0064] In an eighth aspect, a host cell is provided, comprising a nucleic acid as described herein or a vector as described herein. In one embodiment, the host cell is a eukaryotic host cell or a prokaryotic host cell. In one embodiment, the eukaryotic host cell is a yeast cell, such as a Pichia cell. In one embodiment, the prokaryotic host cell is an Escherichia coli cell.

[0065] In the ninth aspect, a composition is provided, comprising a polypeptide or stapled peptide as described herein, a nucleic acid as described herein, a vector as described herein, and / or a host cell as described herein. In one embodiment, the composition is a drug. In one embodiment, the drug is a tablet, a capsule, a drop pill, an aerosol, a pill, a powder, a solution, a suspension, an emulsion, a granule, a liposome, a transdermal agent, a suppository or a lyophilized powder injection, or an external preparation, preferably an external application preparation, such as an external gel or an external infiltration preparation; the composition is a mask, a tissue, a glove, a garment, such as a protective suit, a hand washing product, such as a hand sanitizer, or a shower gel.

[0066] In a tenth aspect, a polypeptide derivative is provided which is a cholesterol-modified derivative of a polypeptide described herein.

[0067] In one embodiment, the polypeptide is linked to the cholesterol moiety at the C-terminus via a linker.

[0068] In one embodiment, the linker is PEGylated.

[0069] In one embodiment, the polypeptide is linked to a cholesterol-modified cysteine ​​at the C-terminus via a linker.

[0070] In one embodiment, the linker comprises (GSG)n or (GSGSG)n, wherein n is 1 or 2.

[0071] In one embodiment, the linker is -(GSG)n-DPEG4- or -(GSGSG)n-DPEG4-.

[0072] In an eleventh aspect, a method for preparing a polypeptide is provided, comprising the steps of:

[0073] (1) culturing the host cell described herein in a suitable culture medium; and

[0074] (3) Harvesting and isolating peptides.

[0075] In the twelfth aspect, a method for screening an active ingredient that inhibits MERS-like coronavirus MjHKU4r-CoV is provided, comprising:

[0076] (1) Producing a polypeptide or stapled peptide derived from the C-terminal heptad repeat domain 2 of a coronavirus S protein; preferably, the coronavirus is an alpha or beta coronavirus, preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV;

[0077] (2) measuring the ability of the polypeptide or stapled peptide to inhibit cell-cell fusion in a MjHKU4r-CoV S protein-mediated cell fusion assay, or measuring the ability of the polypeptide to inhibit MjHKU4r-CoV entry into cells in a cell-based assay; and

[0078] (3) Selecting a polypeptide or stapled peptide that has the ability to inhibit cell-cell fusion or a polypeptide or stapled peptide that has the ability to inhibit MjHKU4r-CoV from entering cells.

[0079] In some embodiments, the ability of the polypeptide or stapled peptide to inhibit fusion between cells expressing the MjHKU4r-CoV S protein and cells expressing the human coronavirus receptor is determined. In some embodiments, the ability of the polypeptide or stapled peptide to inhibit the entry of MjHKU4r-CoV into cells expressing the human coronavirus receptor is determined.

[0080] In one embodiment, the coronavirus is an alpha or beta coronavirus, preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV.

[0081] In one embodiment, the polypeptide comprises the amino acid sequence of formula (I) herein.

[0082] In one embodiment, the stapled peptide comprises an amino acid sequence in which two or more of the amino acids in the amino acid sequence of formula (I) are replaced by non-natural amino acids to which the side chains can be attached; wherein the non-natural amino acids to which the side chains can be attached are α-amino acids with alkenyl side chains, such as one or more of the following: (S)-2-(4'-pentenyl)alanine (S5), (R)-2-(4'-pentenyl)alanine (R5), (S)-2-(7'-octenyl)alanine (S8), (R)-2-(7'-octenyl)alanine (R8), (S)-2-(4'-pentenyl)glycine (Sg5) and (R)-2-(4'-pentenyl)alanine (Rg5).

[0083] In one embodiment, the number of substituted amino acids is two, and the positions of the substituted amino acids are respectively the i-th and i+3-th from X1, where 1≤i≤51, or the i-th and i+4-th, where 1≤i≤50.

[0084] In one embodiment, the polypeptide comprises (1) an amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; (2) an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or replaced in the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; or (3) an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19.

[0085] In one embodiment, the polypeptide comprises the amino acid sequence of the following formula (II):

[0086] EISKIN-Y1-T-Y2-L-Y3-L-Y4-Y5-Y6-Y7-Y8-Y9-L-Y10-E-Y11-Y12-K-Y13-L-Y14-Y15-S-Y16-IDLKEL;

[0087] Wherein Y1 is T or V; Y2 is L or F; Y3 is D or N; Y4 is S, E or N; Y5 is D or T; Y6 is E or F; Y7 is M or L; Y8 is A, K or M; Y9 is I, K, M or V; Y10 is Q, E, S or L; Y11 is V or A; Y12 is V or I; Y13 is Q or K; Y14 is N or E; Y15 is S, D or E; Y16 is Y or L.

[0088] Advantages of the present invention include:

[0089] 1. The present invention provides a group of polypeptide entry inhibitors (any one of SEQ ID NOs: 1-19) that can effectively inhibit infection by MERS-like coronavirus MjHKU4r-CoV.

[0090] 2. EK1 and EK1C4 of the present invention have more effective inhibitory activity against MERS-like coronavirus MjHKU4r-CoV compared with other polypeptides (SEQ ID NOs: 1-6, any one of SEQ ID NOs: 9-19), which is unexpected. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 MjHKU4r-HR1P and MjHKU4r-HR2P polypeptide designs are shown.

[0092] Figure 2 The biophysical activities and characteristics of MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides are shown.

[0093] Figure 3 The results showed that MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides have inhibitory activity on MjHKU4r-CoV S protein-mediated membrane fusion and infection process.

[0094] Figure 4 It was shown that HR2-derived peptides and EK1 and EK1C4 of closely related coronaviruses inhibited MjHKU4r-CoV infection.

[0095] Figure 5 The inhibitory effects of optimized peptides based on MjHKU4r-HR2P peptide: MjHKU4r-HR2P2 -HR2P8 on MjHKU4r-CoV infection were shown. DETAILED DESCRIPTION

[0096] In the present invention, a cell-cell fusion system mediated by MjHKU4r-CoV S protein was established to detect the inhibitory activity of a series of polypeptides on the corresponding virus fusion process, such as Figure 2 As shown. In 2014, Lu Lu (one of the inventors of this patent) et al. constructed a cell-cell fusion system for MERS-CoV by co-expressing the viral S protein with GFP, and successfully designed and evaluated a series of MERS-CoV polypeptide entry inhibitors with good activity on this system. The results were published in Nature Communications, a subsidiary of Nature (reference: Lu, L., Q. Liu, Y. Zhu, KH Chan, L. Qin, Y. Li, Q. Wang, JF Chan, L. Du, F. Yu, C. Ma, S. Ye, KY Yuen, R. Zhang, and S. Jiang. 2014. Structure-based discovery of MiddleEast respiratory syndrome coronavirus fusion inhibitor. Nat Commun 5:3067). In the present invention, the same method was used to detect the fusion inhibition effect of the invented series of polypeptides on MjHKU4r-CoV. In the cell-cell fusion experiment of MjHKU4r-CoV, both the EK1 series peptides and the HR2-derived series peptides showed good inhibitory effects, and the cholesterol-modified EK1 peptide (EK1C4) showed the highest antiviral activity, with an IC50 at the low nanomolar level.

[0097] These HR2P peptides and EK series peptides can have a good inhibitory effect on MERS-like coronavirus MjHKU4r-CoV, which will provide a new strategy for the prevention and control of related epidemics in the future, and also provide a new theoretical basis for accelerating the development of highly effective anti-coronavirus peptide small molecule drugs. In addition, these peptides have clear antiviral targets and clear mechanisms, which can ensure the safety of their application and the clarity of the optimization pathway, facilitating further development in the future.

[0098] Herein, the polypeptide may be a polypeptide comprising any one of SEQ ID NOs: 1-19 or a variant thereof.

[0099] Table 1: Specific sequence of polypeptide:

[0100]

[0101] For example, the polypeptide may include an amino acid sequence in which one or more, preferably 2, 3, 4 or 5 amino acid residues are substituted, added, deleted or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 1-19.

[0102] Amino acid addition refers to adding amino acids to the C-terminus or N-terminus of an amino acid sequence, such as any one of SEQ ID NOs: 1-19, as long as the polypeptide has inhibitory activity against MERS-like coronavirus MjHKU4r-CoV.

[0103] Amino acid substitution refers to the replacement of an amino acid residue at a certain position in an amino acid sequence, such as any one of SEQ ID NOs: 1-19, by another amino acid residue, as long as the polypeptide has inhibitory activity against MERS-like coronavirus MjHKU4r-CoV.

[0104] Amino acid insertion refers to inserting an amino acid residue at an appropriate position in an amino acid sequence, such as any one of SEQ ID NOs: 1-19. The inserted amino acid residues may also be adjacent to each other in whole or in part, or the inserted amino acids may not be adjacent to each other, as long as the polypeptide has inhibitory activity against MERS-like coronavirus MjHKU4r-CoV.

[0105] Amino acid deletion means that 1, 2 or 3 or more amino acids can be deleted from an amino acid sequence, such as any one of SEQ ID NOs: 1-19, as long as the polypeptide has inhibitory activity against coronavirus.

[0106] In the present invention, the substitution may be a conservative amino acid substitution, which means that compared with the amino acid sequence of any one of SEQ ID NOs: 1-19, 3, preferably 2 amino acids or 1 amino acid are replaced by amino acids with similar or similar properties to form a peptide. These conservative variant peptides can be generated by amino acid substitution according to Table 2.

[0107] In this document, a plurality may refer to any integer greater than 1, such as 2-15 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15).

[0108] In the context of the present invention, conservative substitutions may be defined according to substitutions within the amino acid classes reflected in one or more of the following three tables:

[0109] Table 2: Categories of conservatively substituted amino acid residues

[0110]

[0111] Table 3: Alternative conservative amino acid residue substitution categories

[0112]

[0113] Table 4: Alternative physical and functional classifications of amino acid residues

[0114]

[0115] For purposes of the present invention, use Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48: 443-453) to measure the sequence identity between two amino acid sequences, as in EMBOSS bag (EMBOSS:The European Molecular Biology Open Software Suite, Rice etc., 2000, Trends Genet.16: 276-277), preferably implement in the Needle program more than version 5.0.0. The parameter used is gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.The output (using-nobrief option to obtain) of the Needle labeled as "longest identity" is used as percentage identity, and is calculated as follows:

[0116] (Identical residues x 100) / (length of alignment - total number of gaps in the alignment).

[0117] The polypeptide of the present invention can be synthetically generated or the polypeptide of the present invention can be cell expressed. For example, the polypeptide of the present invention can be synthesized by chemical means. Alternatively, the polypeptide of the present invention can be expressed in a recombinant cell. The type of cell is not limited, for example, the cell can be a eukaryotic cell or a prokaryotic cell. The eukaryotic cell can be a fungal cell, such as a yeast cell, or an insect cell or a mammalian cell, such as a mouse cell. The prokaryotic cell can be a bacterial cell, such as an Escherichia coli cell.

[0118] As used herein, "nucleic acid" refers to a plurality of nucleotides linked by internucleotides. Internucleotide linkages may be, for example, phosphodiester bonds. The nucleic acid herein may comprise a polynucleotide encoding a polypeptide of the present invention. In order to facilitate subsequent processing of the polypeptide, the nucleic acid of the present invention may also comprise nucleotides encoding a purification tag, such as a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag, and a nucleotide sequence encoding a leader sequence when necessary.

[0119] The nucleic acid of the polypeptide of the present invention can be codon optimized with the host cell used. The nucleic acid can be cloned into a suitable expression vector, and then the expression vector is introduced into the host cell for expression. The type of expression vector is not limited and is well known to those skilled in the art.

[0120] As used herein, the term "vector" is a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc. The vector may contain a variety of elements for controlling expression, including but not limited to promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may contain a nucleic acid of the present invention to facilitate introduction into cells for expression. The vector may contain expression control elements such as promoters, terminators and / or enhancers that are operably linked to the nucleic acid.

[0121] As used herein, the term "host cell" is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection of viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. The host cell can be a eukaryotic cell or a prokaryotic cell. For example, a eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. A prokaryotic cell can be an E. coli cell.

[0122] As used herein, "treatment" and the like refer to the administration of a drug in order to obtain an effect in a subject. "Prevention" refers to the complete or partial prevention of a disease or its symptoms, and treatment refers to the partial or complete cure of a disease and / or the symptoms caused by the disease. "Treatment" may include: (a) inhibiting a disease, i.e., preventing its development; (b) alleviating a disease or its symptoms.

[0123] As used herein, the term "coronavirus" belongs to the order Nidovirales, the family Coronaviridae, and the genus Coronavirus. It is a type of RNA virus with an envelope and a linear single-stranded positive-strand genome. It is a large class of viruses that are widely present in nature. The 5' end of the viral genome has a methylated cap structure, the 3' end has a poly (A) tail, and the total length of the genome is about 27-32kb. It is the virus with the largest genome among the currently known RNA viruses. Coronaviruses only infect vertebrates and are associated with a variety of diseases in humans and animals. They can cause respiratory, digestive and nervous system diseases in humans and animals. According to the phylogenetic tree, coronaviruses can be divided into four genera: α, β, γ, and δ, among which the β-genus coronavirus can be divided into four independent subgroups A, B, C and D.

[0124] As used herein, the term "coronavirus S protein" refers to the spike protein of the coronavirus, and may refer to a specific S protein, such as SARS-CoV-2 S protein, MERS-CoV S protein, SARS-CoV S protein or MjHKU4r-CoV S protein. For example, SARS-CoV-2-spike protein is a type I membrane glycoprotein that is assembled into a trimer of spikes or envelope protrusions constituting the surface of enveloped coronavirus particles. Spike protein has two basic functions, namely host receptor binding and membrane fusion, which is attributed to the N-terminal (S1) half and the C-terminal (S2) half of the S protein. The coronavirus S protein binds to its cognate receptor via the receptor binding domain (RBD) present in the S1 subunit.

[0125] As used herein, the term "MERS-like coronavirus MjHKU4r-CoV" is a recently discovered novel pangolin-derived MERS-like coronavirus. It is closely related to bat HKU4-CoV and is infectious in human organs and transgenic mice. MjHKU4r-CoV-1 utilizes the dipeptidyl peptidase 4 receptor for virus entry and has a broad host tropism.

[0126] As used herein, the term "coronavirus infection" or "CoV infection" refers to an infection with a coronavirus such as SARS-CoV-2, MERS-CoV, SARS-CoV, or MjHKU4r-CoV. Coronavirus infections include respiratory infections, usually in the lower respiratory tract. Symptoms may include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms such as diarrhea, organ failure (renal failure and renal insufficiency), septic shock, and even death in severe cases.

[0127] The term "subject" refers to a mammal for which treatment is being evaluated and / or is being treated. In some embodiments, the mammal is a human. The term "subject" includes, but is not limited to, an individual suffering from a disease. The subject can be a human, but also includes other mammals, particularly mammals that can be used as laboratory models of human diseases, such as rodents (e.g., mice), primates, etc.

[0128] The term "stapled peptide" refers to a polypeptide modified by chemical means, the main feature of which is the stabilization of the α-helical structure of the polypeptide by introducing a full carbon backbone or other chemical bonds.

[0129] The term "side chain-attachable non-natural amino acid" refers to an α-amino acid with an alkenyl side chain, for example selected from: (S)-2-(4'-pentenyl)alanine (S5), (R)-2-(4'-pentenyl)alanine (R5), (S)-2-(7'-octenyl)alanine (S8), (R)-2-(7'-octenyl)alanine (R8), (S)-2-(4'-pentenyl)glycine (Sg5), (R)-2-(4'-pentenyl)alanine (Rg5).

[0130] The present invention also provides a method for preparing a polypeptide. The method comprises introducing an expression vector into a host cell, then culturing the cell in a suitable culture medium, then harvesting or isolating the polypeptide or fusion protein in the supernatant, or collecting the host cell in the case of intracellular expression, then lysing the cell to collect the polypeptide. In the case of a polypeptide, the polypeptide may be linked to a suitable signal peptide. The signal peptide may or may not be cleaved after harvesting. These techniques are well known to those skilled in the art.

[0131] The polypeptides of the present invention can be prepared in the form of derivatives. For example, the polypeptides can be cholesterol-modified. Methods for cholesterol modification are well known. In one embodiment, cholesterol modification can be performed at the C-terminus of the polypeptide so that the polypeptide is connected to the cholesterol moiety at the C-terminus. The connection can be a direct connection or can be connected through a linker.

[0132] The type and length of the linker can vary. For example, the linker can be (GSG)n or (GSGSG)n, where n can be any integer, such as 1, 2, 3, 4, 5, 6, etc.

[0133] The joint can be PEGylated. The means of PEGylation modification are known to those skilled in the art. As used herein, the term PEGylated joint refers to a joint to which one or more PEGs are attached. In this article, a PEGylated joint can be a PEGylated (GSG)n or (GSGSG)n. N can be any integer, such as 1, 2, 3, 4, 5, 6, etc. PEG4 represents that the number of repeated ethylene glycol residues is 4.

[0134] The polypeptide or polypeptide derivative or stapled peptide of the present invention can be used alone or in combination, or in combination with other agents having MERS coronavirus MjHKU4r-CoV inhibitory activity.

[0135] Herein, the polypeptide or polypeptide derivative or stapled peptide of the present invention is prepared in the form of a composition or a kit. The composition may include a suitable carrier, such as a pharmaceutically acceptable carrier. Such compositions may be external, for example, used as external preparations, external smear preparations, such as external gels or external infiltration preparations. Such compositions may be applied to articles that need to suppress viruses, such as, but not limited to, masks, paper towels, gloves, clothing, such as protective clothing, etc. Alternatively, it may be added as an active ingredient to hand washing products, such as hand sanitizers, shower gels, etc. Such polypeptides or polypeptide derivatives or stapled peptides may be used to inhibit coronaviruses in vitro to prevent and / or reduce viral infection. Such polypeptides or polypeptide derivatives or stapled peptides may be used to prevent or treat coronavirus infection in a subject or a disease caused by a coronavirus.

[0136] The polypeptide or polypeptide derivative or stapled peptide of the present invention can also be prepared into a medicine or pharmaceutical composition. Such medicines can be tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, suppositories or freeze-dried powder injections. These medicines or pharmaceutical compositions can be applied by various administration methods, such as injection, including subcutaneous injection, intravenous injection, intramuscular injection and intraperitoneal injection, intracisternal injection or infusion, etc., cavity administration, such as rectal, vaginal and sublingual, respiratory administration, such as nasal; mucosal administration, or surface administration, etc.

[0137] The present invention provides a method for preventing or treating a MERS-like coronavirus MjHKU4r-CoV infection in a subject or a disease caused by a MERS-like coronavirus MjHKU4r-CoV, comprising administering any polypeptide or polypeptide derivative or stapled peptide of the present invention. The present invention also provides any polypeptide or polypeptide derivative or stapled peptide herein used in preventing or treating a MERS-like coronavirus MjHKU4r-CoV in a subject or a disease caused by a MERS-like coronavirus MjHKU4r-CoV.

[0138] The present invention also relates to a method for preventing or treating MERS-like coronavirus MjHKU4r-CoV infection or a disease caused by MERS-like coronavirus MjHKU4r-CoV in a subject, which comprises administering the polypeptide or derivative or stapled peptide, nucleic acid, vector, host cell or composition or kit described in the present invention to the subject / patient.

[0139] The present invention also relates to a polypeptide or derivative or stapled peptide, nucleic acid, vector, host cell, or composition or kit for preventing or treating MERS-like coronavirus MjHKU4r-CoV infection in a subject or a disease caused by MERS-like coronavirus MjHKU4r-CoV.

[0140] The present invention also relates to polypeptides or derivatives or stapled peptides, nucleic acids, vectors, host cells, compositions, pharmaceutical compositions or kits for preventing or treating MERS-like coronavirus MjHKU4r-CoV infection or diseases caused by MERS-like coronavirus MjHKU4r-CoV in subjects / patients.

[0141] Coronavirus

[0142] Coronavirus is a large class of viruses that are widely present in nature. Coronavirus includes mammalian coronavirus and avian coronavirus. Mammalian coronavirus mainly belongs to α and β coronavirus, which can infect many animals including pigs, dogs, cats, mice, cattle, horses, etc. Avian coronavirus mainly comes from γ and δ coronavirus, which can cause disease in many birds such as chickens, turkeys, sparrows, ducks, geese, pigeons, etc. The coronaviruses described herein may belong to any subgenus of the genus Alphacoronavirus, including, but not limited to, Colacovirus (e.g., bat coronavirus CDPHE15), Decacovirus (e.g., bat coronavirus HKU10, Rhinolophus ferrumequinum alphacoronavirus HuB-2013), Duvinacovirus (e.g., human coronavirus 229E), Luchacovirus (e.g., Lucheng Rn rat coronavirus), Minacovirus (e.g., mink coronavirus 1), Minunacovirus (e.g., Miniopterus bat coronavirus 1 and Miniopterus bat coronavirus HKU8), Myotacovirus (e.g., Myotis ricketti alphacoronavirus Sax-2011), Nyctacovirus (e.g., Nyctalus velutinusalphacoronavirus SC-2013 and Pipistrellus kuhlii alphacoronavirus), coronavirus) 3398), Pedacovirus (e.g., porcine epidemic diarrhea virus and Scotophilus bat coronavirus 512), Rhinacovirus (e.g., Rhinolophus bat coronavirus HKU2), Setracovirus (e.g., human coronavirus NL63 and NL63-related bat coronavirus strain BtKYNL63-9b), Soracovirus (e.g., Sorex araneus coronavirus T14), Sunacovirus (e.g., Suncus murinus coronavirus X74), and Tegacovirus (e.g., alpha coronavirus 1).

[0143] The genus β-coronavirus described herein may include Embecovirus (lineage A), Sarbecovirus (lineage B), Merbecovirus (lineage C), Nobecovirus (lineage D) and Hibecovirus. Therefore, in some embodiments, the coronavirus described herein may be any strain or species in any subgenus or pedigree of β-coronavirus. The Embecovirus subgenus may include, but is not limited to, β-coronavirus 1 (e.g., bovine coronavirus and human coronavirus OC43), Chinese mouse coronavirus HKU24, human coronavirus HKU1, mouse coronavirus (e.g., mouse hepatitis virus), and Myodes coronavirus 2JL14. Sarbecovirus subgenus may include, but are not limited to, SARS-CoV, SARS-CoV2, 16BO133, bat SARS CoV Rf1, bat coronavirus HKU3 (BtCoV HKU3), LYRa11, bat SARS-CoV / Rp3, bat SL-CoV YNLF_31C, bat SL-CoV YNLF_34C, SHC014-CoV, WIV1, WIV16, civet SARS-CoV, Rc-o319, SL-ZXC21, SL-ZC45, pangolin SARSr-COV-GX, pangolin SARSr-COV-GD, RshSTT182, RshSTT200, RacCS203, RmYN02, RpYN06, RaTG13, bat CoV BtKY72, and bat CoVBM48-31. The Merbecovirus subgenus may include, but is not limited to, Hedgehog coronavirus 1, MERS-CoV, Pipistrellus bat coronavirus HKU5, and Tylonycteris bat coronavirus HKU4. The Nobecovirus subgenus may include, but is not limited to, Eidolon bat coronavirus C704, Rousettus bat coronavirus GCCDC1, and Rousettus bat coronavirus HKU9. Coronavirus antigens may belong to any species or strain in the Hibecovirus subgenus, including, but not limited to, bat Hp-β coronavirus Zhejiang 2013.

[0144] The coronavirus described herein can be clustered in different branches in terms of phylogeny. For example, using the nucleotide sequences of the nonstructural protein genes ORF1a and ORF1b, the coronavirus of β-coronavirus (Sarbecovirus) can be clustered into branch 1, branch 2, branch 1 / 2 or branch 3 (see, for example, Hu et al., PLoS Pathog 13 (11): e1006698). Therefore, coronavirus can belong to any species or strain in any branch in these branches. Coronavirus can belong to any species or strain in branch 1, including but not limited to SARS-CoV, WIV1, LYRa11, Rs7327, Rs4231, Rs4084 and SHC014. Coronavirus can belong to any species or strain in clade 2, including but not limited to: As6526, 279-2005, Rs4237, Rs4081, Rp3, Rs4247, HKU3-8, HKU3-13, GX2013, Rf4092, ZXC21, ZC45, JL2012, HuB2013, Rf1, HeB2013 and 273-2005. Coronavirus can belong to any species or strain in clade 1 / 2, including but not limited to SARS-CoV2. Coronavirus can belong to any species or strain in clade 3, including but not limited to BM48-31. Coronavirus described herein can be, for example, the coronavirus of SARS, SARS-2, WIV1, SHC014, Rf1, RmYN02, pang17, RaTG13 and Rs4081.

[0145] In some preferred embodiments, the coronavirus described herein may be HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, and MERS-CoV.

[0146] MjHKU4r-CoV virus

[0147] The MERS-like coronavirus MjHKU4r-CoV is a recently discovered novel pangolin-derived MERS-like coronavirus. The MjHKU4r-CoV virus was found in Malayan pangolins. Whether pangolins play a role in the zoonotic transmission of bat coronaviruses is unclear. Evolutionary analysis identified MjHKU4r-CoV-1 as belonging to the bat HKU4 coronavirus species of the subgenus Merbecovirus. There are 16 key residues in the receptor binding motif (RBM) of the S protein of MERS-CoV that determine binding to hDPP4. By comparison with MERS-CoV, it was found that the RBD of MjHKU4r-CoV-1 has 8 residues consistent with MERS-CoV. The potential furin cleavage site (RQQR) in the S protein subdomain 1 (SD1) in the MjHKU4r-CoV-1 genome is similar to that in the MERS-CoV genome, suggesting that MjHKU4r-CoV-1 may also bind to hDPP4. MjHKU4r-CoV-1 uses hDPP4 as a receptor to enter cells. In vivo infection experiments showed that MjHKU4r-CoV-1 was contagious and could cause interstitial pneumonia in mice.

[0148] S protein of coronavirus

[0149] The S protein of coronavirus can be divided into two important functional subunits, namely the N-terminal S1 subunit and the C-terminal S2 region. The N-terminal S1 subunit forms the globular head of the S protein. The C-terminal S2 region forms the stem of the protein, which is directly embedded in the viral envelope. When interacting with potential host cells, the S1 subunit will recognize and bind to receptors on the host cell, especially the angiotensin-converting enzyme 2 (ACE2) receptor, while the S2 subunit is the most conserved component of the S protein and will be responsible for fusing the viral envelope with the host cell membrane. See, for example, Shang et al., PLoS Pathog. 2020 Mar; 16(3):e1008392.). Each monomer of the trimer of the trimer of the trimeric S protein contains two subunits, S1 and S2, which mediate attachment and membrane fusion, respectively. As part of the in vivo infection process, these two subunits are separated from each other by an enzymatic cleavage process. The S protein is first cleaved at the S1 / S2 site of the infected cell by furin-mediated cleavage. In vivo, a subsequent serine protease-mediated cleavage event occurs at the S2' site within S1.

[0150] The term "S1 subunit" (e.g., S1 subunit antigen) refers to the N-terminal subunit of the spike protein, starting with the N-terminus of the S protein and ending with the S1 / S2 cleavage site, while the term "S2 subunit" (e.g., S2 subunit antigen) refers to the C-terminal subunit of the spike protein, starting with the S1 / S2 cleavage site of the spike protein and ending with the C-terminus. Within the S1 subunit, the domains include the N-terminal domain (NTD) and the receptor binding domain (RBD), and the RBD domain also includes a receptor binding motif (RBM). Within the S2 subunit, the domains include a fusion peptide (FP), a heptad repeat sequence 1 (HR1), a heptad repeat sequence 2 (HR2), a transmembrane domain (TM), and a cytoplasmic domain (also known as a cytoplasmic tail (CT)). For SARS-CoV-2, the HR1 and HR2 domains can be referred to as the "fusion core region". The S1 subunit includes the N-terminal domain (NTD), a linker region, a receptor binding domain (RBD), a first subdomain (SD1), and a second subdomain (SD2). The S2 subunit may include a first heptad repeat sequence (HR1), a second heptad repeat sequence (HR2), a transmembrane domain (TM), and a cytoplasmic tail.

[0151] In some embodiments herein, the polypeptide described herein comprises the amino acid sequence of the C-terminal heptad repeat domain 2 of the coronavirus S protein. In some embodiments, the coronavirus is an alpha or beta coronavirus, preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV. The polypeptide described herein can interact with the MjHKU4r-HR2P polypeptide of the MjHKU4r-CoV virus. In some embodiments, the polypeptide described herein can inhibit membrane fusion and infection mediated by the MjHKU4r-CoV S protein. In some embodiments, the polypeptide described herein can inhibit MjHKU4r-CoV infection.

[0152] Peptides

[0153] The polypeptide described herein is a polypeptide derived from the C-terminal heptad repeat domain 2 of the coronavirus S protein. The polypeptide may comprise the full length or a portion of the C-terminal heptad repeat domain 2 of the coronavirus S protein, provided that the polypeptide can interact with the MjHKU4r-HR2P polypeptide of the MjHKU4r-CoV virus (e.g., form a six-helix complex), or the polypeptide can inhibit membrane fusion and infection mediated by the MjHKU4r-CoV S protein, or inhibit MjHKU4r-CoV infection. The polypeptide may comprise the general structure of X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39-X40-X41-X42-X43-X44-X45-X46-X47-X48-X49-X50-X51-X52-X53-X54, wherein X1-X54 are as defined above. In some embodiments, the polypeptide may comprise the general structure of X1-X2-X3-X4-EISKIN-Y1-T-Y2-L-Y3-L-Y4-Y5-Y6-Y7-Y8-Y9-L-Y10-E-Y11-Y12-K-Y13-L-Y14-Y15-S-Y16-IDLKEL, wherein X1-X4 and Y1-Y16 are as defined above.

[0154] The polypeptides described herein may comprise any one of the amino acid sequences (SEQ ID NOs. 1-19) shown in Table 1. In some embodiments, the polypeptides described herein may comprise a variant of any one of the amino acid sequences (SEQ ID NOs. 1-19) shown in Table 1. The variant may have an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NOs. 1-19. The variant may be a variant obtained by performing one or more mutations (e.g., 2-30, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) in any one of SEQ ID NO. 1-19. The mutation may be one or more of substitution, addition, insertion, and deletion. The variants described herein may interact with the MjHKU4r-HR2P polypeptide of the MjHKU4r-CoV virus. In some embodiments, the variants described herein may inhibit membrane fusion and infection mediated by the MjHKU4r-CoV S protein. In some embodiments, the variants described herein may inhibit MjHKU4r-CoV infection. In some embodiments, the polypeptide or variant inhibits fusion between cells expressing the MjHKU4r-CoV S protein and cells expressing the human coronavirus receptor. In some embodiments, the polypeptide or variant inhibits the entry of MjHKU4r-CoV into cells expressing the human coronavirus receptor.

[0155] Stapling peptide

[0156] Also provided herein are stapled peptides. Stapled peptides can be stapled peptides of the polypeptides shown in Table 1. Methods for preparing stapled peptides (e.g., by using olefin metathesis reactions) are known to those skilled in the art. For example, two or more non-natural amino acids to which side chains can be connected can be replaced in the amino acid sequence of a polypeptide, and the side chains of the non-natural amino acids can be connected. The non-natural amino acids to which side chains can be connected are α-amino acids with alkenyl side chains, for example, selected from: (S)-2-(4'-pentene)alanine (S5), (R)-2-(4'-pentene)alanine (R5), (S)-2-(7'-octene)alanine (S8), (R)-2-(7'-octene)alanine (R8), (S)-2-(4'-pentene)glycine (Sg5), (R)-2-(4'-pentene)alanine (Rg5). The side chains of the non-natural amino acids can form 1 ring or 2 rings or more rings after connection, preferably 1 or 2 rings.

[0157] The stapled peptide may comprise an amino acid sequence of any one of SEQ ID NOs: 2-7 and 9-19, wherein relative to SEQ ID NO: 18, the amino acid residues at positions 25 and 29 are replaced with non-natural amino acids to which side chains can be connected, preferably (S)-2-(4'-pentene)alanine (S5) or (R)-2-(4'-pentene)alanine (R5). Non-natural amino acids can form staplers through side chains. In particular, the stapled peptide comprises an amino acid sequence of EISKINVTLLDLSDEMAILLEAIK-S5-LND-S5-YIDLKEL, wherein S5 is (S)-2-(4'-pentene)alanine, and a covalent connection is formed between two S5s.

[0158] Peptide derivatives

[0159] The polypeptides described herein can be modified to produce polypeptide derivatives. In some embodiments, the polypeptides can be modified by conjugation with PEG. The PEG modification sites of the polypeptide are at the N-terminus, C-terminus, Lys side chain and Cys thiol of the polypeptide. The molecular weight range of the PEG single molecule used for modification is between PEG2 and PEG24. PEG modification can be used to improve proteolytic stability, biodistribution and peptide solubility. The introduction of PEG chains on polypeptides can improve their pharmacological properties and inhibit the hydrolysis of polypeptides by proteolytic enzymes. Since the effective half-life of PEG polypeptides in the body is extended, normal therapeutic levels can be maintained by using lower doses and lower frequencies of polypeptide drugs. In some embodiments, the polypeptides described herein can be cholesterol modified. Cholesterol-modified polypeptide drugs can improve the drug activity and in vivo / in vitro half-life of polypeptides, have weaker toxic side effects, and can also cross the blood-brain barrier. In some embodiments, the polypeptide derivative is a cholesterol-modified derivative of the polypeptide. In some embodiments, the polypeptide derivative is a PEG-modified derivative of the polypeptide. In some embodiments, the polypeptide derivative is a cholesterol-modified and PEG-modified derivative of the polypeptide. In some embodiments, the polypeptide is connected to the cholesterol moiety at the C-terminus via a linker. In some embodiments, the linker is PEGylated. In some embodiments, the polypeptide is connected to a cholesterol-modified cysteine ​​at the C-terminus via a linker. In some embodiments, the linker comprises (GSG)n or (GSGSG)n, wherein n is 1 or 2. In some embodiments, the linker is -(GSG)n-DPEG4- or -(GSGSG)n-DPEG4-, wherein n is 1 or 2.

[0160] Pharmaceutical compositions and kits

[0161] In some aspects, the invention relates to a pharmaceutical composition or kit comprising a polypeptide described herein, a nucleic acid encoding a polypeptide, a vector comprising a nucleic acid, a cell comprising a nucleic acid or a vector.

[0162] The pharmaceutical composition may also include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any and all solvents, cosolvents, compounding agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, etc., that have no adverse effects biologically or otherwise. The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agents are incompatible with the active ingredient, it is considered to be used in the pharmaceutical composition. Other active ingredients may also be incorporated into the pharmaceutical composition. In addition, various excipients such as excipients commonly used in the art may be included. These and other such compounds are described in the literature, for example, in Merck Index, Merck & Company, Rahway, NJ. The considerations for incorporating various components into the pharmaceutical composition are described in, for example, Gilman et al. (ed.) (2010); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 12th edition, The McGraw-Hill Companies. In some embodiments, the pharmaceutical composition is a tablet, capsule, drop pill, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, suppository or freeze-dried powder injection. In some embodiments, the pharmaceutical composition is administered by the following ways: injection, including subcutaneous injection, intravenous injection, intramuscular injection and intraperitoneal injection, intracisternal injection or infusion, etc., cavity administration, such as rectal, vaginal and sublingual, respiratory tract administration, such as nasal cavity; mucosal administration, or surface administration.

[0163] In some aspects, the present invention relates to a kit comprising a polypeptide described herein, a nucleic acid encoding a polypeptide, a vector comprising a nucleic acid, a cell comprising a nucleic acid or a vector. In some embodiments, the present invention provides a kit for producing a single-dose administration unit. In some aspects, the kit of the present invention can accommodate a first container containing a polypeptide described herein, a nucleic acid encoding a polypeptide, a vector comprising a nucleic acid, a cell comprising a nucleic acid or a vector and a second container containing an aqueous formulation. In some embodiments, the kit contains solutions such as diluents, buffers, etc. for diluting or reconstructing powders. In some embodiments, the kit contains instructions for use of a polypeptide described herein, a nucleic acid encoding a polypeptide, a vector comprising a nucleic acid, a cell comprising a nucleic acid or a vector.

[0164] Peptide drug screening method

[0165] The present invention also provides a method for screening active ingredients that inhibit MERS-like coronavirus MjHKU4r-CoV. The method may include generating a polypeptide library, which may include polypeptides derived from the C-terminal heptad repeat domain 2 of the coronavirus S protein. The coronavirus may be an alpha or beta coronavirus, preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV. The polypeptide library may comprise the amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39-X40-X41-X42-X43-X44-X45-X46-X47-X48-X49-X50-X51-X52-X53-X54 of formula I. The polypeptide library may comprise Formula II: X1-X2-X3-X4-EISKIN-Y1-T-Y2-L-Y3-L-Y4-Y5-Y6-Y7-Y8-Y9-L-Y10-E-Y11-Y12-K-Y13-L-Y14-Y15-S-Y16-IDLKEL. The variables X1-X54 and Y1-Y16 are defined as described elsewhere herein. Methods for generating polypeptide libraries are known to those skilled in the art, for example by chemical synthesis or by biological synthesis.

[0166] Alternatively, the method may comprise generating a stapled peptide library of polypeptides, which stapled peptide library may comprise stapled peptides derived from the amino acid sequence of Formula I or Formula II.

[0167] In some embodiments, the stapled peptide library comprises an amino acid sequence in which two or more amino acids in the amino acid sequence of formula (I) are replaced with non-natural amino acids to which the side chains can be connected; wherein the non-natural amino acids to which the side chains can be connected are α-amino acids with alkenyl side chains, such as one or more of the following: (S)-2-(4'-pentene)alanine (S5), (R)-2-(4'-pentene)alanine (R5), (S)-2-(7'-octenyl)alanine (S8), (R)-2-(7'-octenyl)alanine (R8), (S)-2-(4'-pentene)glycine (Sg5) and (R)-2-(4'-pentene)alanine (Rg5). In some embodiments, the number of replaced amino acids is two, and the positions of the replaced amino acids are respectively the i-th and i+3-th positions from X1, wherein 1≤i≤51, or the i-th and i+4-th positions, wherein 1≤i≤50.

[0168] In some embodiments, the stapled peptide comprises an amino acid sequence in which two or more amino acids in the amino acid sequence of formula (II) are replaced with non-natural amino acids to which the side chains can be connected; wherein the non-natural amino acids to which the side chains can be connected are α-amino acids with alkenyl side chains, such as one or more of the following: (S)-2-(4'-pentene)alanine (S5), (R)-2-(4'-pentene)alanine (R5), (S)-2-(7'-octenyl)alanine (S8), (R)-2-(7'-octenyl)alanine (R8), (S)-2-(4'-pentene)glycine (Sg5) and (R)-2-(4'-pentene)alanine (Rg5). In some embodiments, the number of replaced amino acids is two, and the positions of the replaced amino acids are respectively the i-th position and the i+4th position from X1, wherein 1≤i≤50.

[0169] In some embodiments, after generating a polypeptide or stapled peptide library described herein, the polypeptide or stapled peptide library can be contacted with the MjHKU4r-HR1P polypeptide. If a member of the polypeptide or stapled peptide library forms a six-helix complex with the MjHKU4r-HR1P polypeptide, the member is selected as a candidate.

[0170] In some embodiments, the ability of a member of a polypeptide library or a stapled peptide library to inhibit cell-to-cell fusion can be determined in a MjHKU4r-CoV S protein-mediated cell fusion assay. If a member of a polypeptide library or a stapled peptide library inhibits cell-to-cell fusion, the member is selected as a candidate. In some embodiments, the ability of a member to inhibit fusion between a cell expressing the MjHKU4r-CoV S protein and a cell expressing a human coronavirus receptor is determined.

[0171] In some embodiments, members of the polypeptide library or stapled peptide library can be contacted with MjHKU4r-CoV pseudovirus and target cells expressing human coronavirus receptors (e.g., Caco2). If a member of the polypeptide library or stapled peptide library inhibits the invasion of the target cell by the MjHKU4r-CoV pseudovirus, the member is selected as a candidate. In some embodiments, the ability of the member to inhibit the entry of the MjHKU4r-CoV pseudovirus into cells expressing human coronavirus receptors is determined.

[0172] In some embodiments, members of the polypeptide library or stapled peptide library can be contacted with MjHKU4r-CoV and a target cell expressing a human coronavirus receptor (e.g., Caco2). If a member of the polypeptide library or stapled peptide library inhibits the invasion of the target cell by MjHKU4r-CoV, the member is selected as a candidate. In some embodiments, the ability of the member to inhibit the entry of MjHKU4r-CoV into a cell expressing a human coronavirus receptor is determined.

[0173] In some embodiments, the polypeptide library comprises (1) an amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; (2) an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or replaced in the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; or (3) an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19.

[0174] In some embodiments, the candidate can be an amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto.

[0175] In some embodiments, the stapled peptide library comprises the amino acid sequence of any one of SEQ ID NOs: 2-7 and 9-19 or variant amino acid sequences thereof as defined above by identity. In some embodiments, relative to SEQ ID NO: 18, the amino acid residues are replaced with non-natural amino acids to which the side chain can be attached, preferably (S)-2-(4'-pentene)alanine (S5) or (R)-2-(4'-pentene)alanine (R5) at positions i and i+3 or i and i+4. In some embodiments, the stapled peptide library comprises the amino acid sequence of SEQ ID NO: 18, the amino acid residues are replaced with non-natural amino acids to which the side chain can be attached, preferably (S)-2-(4'-pentene)alanine (S5) or (R)-2-(4'-pentene)alanine (R5) at positions i and i+3 or i and i+4.

[0176] Example

[0177] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims. In combination with this specification and common knowledge in the art, a person of ordinary skill in the art can clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, a person of ordinary skill in the art can make any modification or change to the technical solution of the present invention, and such modification and change are also included in the scope of the present invention.

[0178] General methods of PCR, cloning, ligation, transfection, etc. of nucleic acids are well known to those skilled in the art and can be found, for example, in the following literature: "Molecular cloning: A laboratory manual", Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, New York; Ausubel, FM et al. (eds.); "Current protocols in Molecular Biology", John Wiley and Sons (1995); Harwood, CR and Cutting, SM (eds.); "DNA Cloning: A Practical Approach, Volumes I and II", DN Glover, ed. (1985); "Oligonucleotide Synthesis", MJ Gait, ed. (1984); "Nucleic Acid Hybridization", BD Hames & SJ Higgins, eds. (1985); "A Practical Guide to DNA Cloning", 1996; To Molecular Cloning[A Practical Guide]", B. Perbal (1984).

[0179] Example 1: MjHKU4r-HR1P and MjHKU4r-HR2P polypeptide design

[0180] like Figure 1 As shown, through homology comparison with MERS-CoV S2 protein, the specific positioning of HR1 and HR2 functional domains in MjHKU4r-CoV S2 protein and the distribution of key amino acid sites were clarified, thereby designing MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides. Figure 1 Sequence alignments between MjHKU4r-CoV HR1 and MERS-CoV HR1, and between MjHKU4r-CoVHR2 and MERS-CoV HR2, were also shown, revealing high homology between MjHKU4r-CoV HR1 and MERS-CoV HR1, and between MjHKU4r-CoV HR2 and MERS-CoV HR2.

[0181] Example 2: Biophysical characteristics of MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides

[0182] Experimental materials: MjHKU4r-HR1P and MjHKU4r-HR2P peptides were synthesized and purified by Shanghai Jiepeptide Biotechnology Co., Ltd., with a purity of >95%. All peptides in Table 1 were synthesized and purified by Shanghai Jiepeptide Biotechnology Co., Ltd., with a purity of >95%.

[0183] Experimental methods:

[0184] 1. Non-denaturing polyacrylamide gel electrophoresis (N-PAGE). The peptides of gradient concentrations of MjHKU4r-HR1P and MjHKU4r-HR2P were mixed, where the final concentrations of MjHKU4r-HR1 peptide were 0, 100, 300, and 600 µM, and the final concentration of MjHKU4r-HR2 peptide was 100 µM, and incubated at 37°C for 30 minutes. Take 5x high pH loading buffer (supplier: TIANDZ, product number 81212A-30) and mix it with 4 times the volume of the peptide mixture. Then spot (20 µl per well) in 12% non-denaturing gel and electrophoresed at 125V constant voltage at room temperature for 4 hours. Coomassie brilliant blue staining was performed. The results showed that after the MjHKU4r-HR2P and MjHKU4r-HR1P polypeptides were mixed, a new band (i.e., 6-HB) was produced, which may be a six-helix complex ( Figure 2 (a in the figure).

[0185] 2. Determination of peptide secondary structure by circular dichroism. Free peptide (MjHKU4r-HR1P or MjHKU4r-HR2P) or peptide mixture (MjHKU4r-HR1P and MjHKU4r-HR2P) was diluted to a final concentration of 50 µmol / L with 300 mmol / L PBS, pH 7.2. Circular dichroism spectra were measured using a spectropolarimeter (J-815, Jasco Inc, Japan). The detection temperature was 4°C, the bandwidth was 5.0 nm, the resolution was 0.1 nm, the optical path was 0.1 cm, the reaction time was 4.0 s, and the scanning speed was 50 nm / min. The thermal denaturation of the peptide was monitored at 222 nm with a temperature gradient of 5°C / min. The blank control of the buffer solution was subtracted to correct the spectrum. The melting curve was smoothed, and the midpoint temperature of the thermal dissociation transition, i.e., the Tm value, was calculated using Jasco.

[0186] Results and analysis:

[0187] 1. If Figure 2As shown in Figure a, after mixing different concentrations of MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides, a new band that may be a six-helix complex was generated, indicating that MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides can interact with each other;

[0188] 2. If Figure 2 As shown in the bc figure, the single polypeptide is a low-helical structure. When MjHKU4r-HR2P and MjHKU4r-HR1P are mixed, a highly helical structure appears, indicating that the MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides interact with each other in the form of a six-helix formation, and the Tm value of the mixture of MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides is relatively high (=84.8℃), indicating that the six-helix structure is stable.

[0189] Example 3: MjHKU4r-HR2P inhibits MjHKU4r-CoV S protein-mediated membrane fusion and infection process

[0190] Experimental method 1 (evaluation of membrane fusion inhibitory activity, Figure 3 ab diagram in ):

[0191] 1. Transfect 293T cells with a plasmid encoding the coronavirus S protein and culture them for 36-48 hours after transfection as effector cells. Specifically, the MjHKU4r-S protein encoding gene was connected to the pAAV-IRES-GFP vector through the two restriction sites of BamhI and Xhol to obtain the plasmid pAAV-IRES-GFP-MjHKU4r-S, which can encode the MjHKU4r-CoV S protein. 2 μL of Vigofect transfection reagent (Vigos Biotech (Beijing) Co., Ltd., used with DMEM medium at a ratio of 1:1000) was added to another 1 mL of 0.9% NaCl solution and allowed to stand at room temperature for 5 minutes; the Vigofect dilution was added dropwise to the plasmid (pAAV-IRES-GFP-MjHKU4r-S, 10 μg) mixture, allowed to stand at room temperature for 15 minutes, and then added to 293T cells. The transfected cells were called 293T / MjHKU4r / EGFP cells. 293T cells were transfected with the empty vector plasmid pAAV-IRES-GFP, and the resulting cells were called 293T / EGFP cells, which served as negative control cells.

[0192] Amino acid sequence of coronavirus MjHKU4r S protein:

[0193]

[0194] 2. The above-mentioned 293T / MjHKU4r / EGFP cells and 293T / EGFP cells were blown off and centrifuged, and the cells were resuspended in fresh DMEM medium and the cell concentration was adjusted to 2x10 5 / mL, take out 50 µL and add it to the gradient diluted peptide drug (50 µL), incubate at 37°C for 30 min.

[0195] 3. Take 100 μL of the cell / peptide drug mixture and add it to the target cells Caco2 (ATCCHTB-37) that have been plated on a 96-well plate. Incubate at 5% CO2 and 37°C for 2-4 hours, and observe and record the cell fusion using the green fluorescence channel of a fluorescence microscope. For the membrane fusion and infection process mediated by the coronavirus S protein, reference can also be made to the applicant's previous patent applications, such as CN117777247A and CN116440285A. Figure 3 Panel a shows fluorescent photographs of membrane fusion under different concentrations of MjHKU4r-HR1P and MjHKU4r-HR2P. Figure 3 Panel b shows that the IC50 of MjHKU4r-HR1P and MjHKU4r-HR2P are 0.037 μM and greater than 10 μM, respectively.

[0196] Experimental method 2 (evaluation of virus invasion inhibition activity, Figure 3 (c in Figure ):

[0197] 1. Dissolve the peptides MjHKU4r-HR1P and MjHKU4r-HR2P in DMSO and measure the peptide concentration.

[0198] 2. MjHKU4r-CoV pseudovirus packaging (see L. Lu, Q. Liu, Y. Zhu, K.-H. Chan, L.Qin, Y. Li, Q. Wang, JF-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)): PC-MjHKU4r-S plasmid was obtained by ligating the MjHKU4r-S coding gene into the PC-DNA3.1(+) vector at the two restriction sites of BamHI and XhoI. Then, the MjHKU4r-CoV pseudovirus was packaged using the PC-MjHKU4r-S plasmid and HIV-1 backbone plasmid (pNL4-3.Luc.R-.E-). Specifically, 293T cells were digested 24 h before transfection and plated in 10 cm tissue culture dishes (2 × 10 6 / culture dish). 2 h before transfection, replace the cells with pre-warmed fresh DMEM medium (containing 10% FBS). Use two 1.5 mL EP tubes for transfection. Add 500 μL 0.9% NaCl solution to EP tube 1, which contains 20 μg of PC-MjHKU4r-S plasmid and pNL4-3.Luc.R-.E- plasmid. Add 500 μL 0.9% NaCl to EP tube 2, and then add 2 μL of transfection reagent vigofect, and let stand for 5 minutes. Add 500 μL of solution in EP tube 2 dropwise to EP tube 1, mix with a pipette tip while adding, and let it stand at room temperature for 15 min; add the above 1 mL of mixed liquid dropwise and evenly to the previously laid 293T cell culture dish. 8-10 h after transfection, replace with 10 mL of fresh DMEM medium containing 10% FBS; 48 h later, collect the supernatant containing pseudovirus, centrifuge at 4000 rpm for 4 min to remove cell debris, filter with a 0.45 μm sterile filter, aliquot and store at -80°C for later use, and aliquot and store at -80°C.

[0199] 3. Prepare a suspension of Caco2 (ATCC HTB-37) cells by trypsin digestion. After adjusting the cell concentration, add 10 4 cells.

[0200] 4. Use DMEM culture medium to dilute the peptide drug 4 times in a 96-well plate, 50 µL per well. Add 50 µL / well of MjHKU4r-CoV pseudovirus to the peptide drug dilution plate. Incubate at 37℃ for 30 min to allow the peptide drug and virus to fully interact. Add 100 µL of the peptide drug and virus mixture to each well and add it to the target cells after removing the supernatant. After culturing at 37℃ for 12 h, replace with fresh DMEM medium containing 10% FBS.

[0201] 6. After 72 hours, discard the original culture medium in the 96-well cell plate, add 40 μl of cell lysis solution (Promega, catalog number E1531) to each well, and shake on a decolorizing shaker for 45 minutes. Take 30 μl of cell lysis solution from each well to the 96-well ELISA plate, and then add 30 μl of firefly luciferase substrate (purchased from Promega, catalog number E1501) to each well. Put the above ELISA plate into an ELISA reader (purchased from PerkinElmer) to read the fluorescence value of each well.

[0202] Calculate the inhibition rate curve and calculate the half effective dose (IC50) of the drug. Specifically, the virus inhibition rate of each well in the drug-treated wells was calculated by the following formula: Inhibition rate = (fluorescence value of virus well - fluorescence value of drug-treated well) / (fluorescence value of virus well - fluorescence value of cell well) x 100%, virus wells do not include peptide treatment, drug-treated wells include virus treatment and peptide treatment, and cell wells do not include virus treatment. Import the virus inhibition rate calculated in each well into Graphpad Prism software, select Variable Slope in Dose-Response-Inhibition mode, and generate the inhibition rate curve and IC50 value.

[0203] Results and Discussion:

[0204] 1. As Figure 3 As shown in Figure b and Table 5, MjHKU4r-HR2P polypeptide and MjHKU4r-HR2 PEK polypeptide have a good inhibitory effect on the cell-cell fusion mediated by the S protein of MjHKU4r-CoV, thereby preliminarily showing the antiviral activity of MjHKU4r-CoV HR2-derived polypeptide in inhibiting MjHKU4r-CoV.

[0205] 2. If Figure 3 As shown in Figure c and Table 5, MjHKU4r-HR2P polypeptide and MjHKU4r-HR2PEK polypeptide have a good inhibitory effect on the pseudovirus infection process mediated by the S protein of MjHKU4r-CoV, further proving that the MjHKU4r-CoV HR2-derived polypeptide has antiviral activity in inhibiting MjHKU4r-CoV infection.

[0206] Table 5: Inhibitory activity of peptides on cell-cell fusion and pseudovirus infection mediated by the S protein of MjHKU4r-CoV coronavirus:

[0207]

[0208] Example 4: Broad-spectrum anti-β-HCoV activity of MjHKU4r-HR2P9 and MjHKU4r-HR2P10

[0209] Experimental method (evaluation of viral entry inhibition activity):

[0210] 1. Caco2 cells (purchased from ATCC, product number HTB-37 Caco2) were used as target cells. 4 Cells were plated per well and cultured at 37°C for 24 hours.

[0211] 2. Use DMEM culture medium to dilute the peptide drugs MjHKU4r-HR2P9 and MjHKU4r-HR2P10 in a 96-well plate in a 4-fold gradient (5 µM initial concentration and 5 4-fold dilution gradients), 50 µL per well.

[0212] 3. Add 50 µL / well of MERS-CoV or SARS-CoV-2 variant pseudovirus (SARS-CoV-2-Delta, SARS-CoV-2-BA.2.75, SARS-CoV-2-XBB.1.16 and SARS-CoV-2-KP.2) (reference: Xia S, LiuM, Wang C, et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infectionby a highly potent pan-coronavirus fusion inhibitor targeting its spikeprotein that harbors a high capacity to mediate membrane fusion. Cell Res.2020;30(4):343-355. doi:10.1038 / s41422-020-0305-x) to the plate wells containing peptide drug dilutions and incubate at 37°C for 30 min to allow the peptide drug to fully interact with the virus. 100 μL of the peptide drug and virus mixture was added to each well and added to the target cells after removing the supernatant. After incubation at 37°C for 12 h, the medium was replaced with fresh DMEM containing 10% FBS.

[0213] 4. After 72 hours, add 40 μl of cell lysis buffer (Promega, catalog number E1531) to each well, shake and lyse on a decolorizing shaker for 45 minutes, take out 30 μl of cell lysis buffer from each well to a 96-well ELISA plate, and then add 30 μl of firefly luciferase substrate (purchased from Promega, catalog number E1501) to each well. Place the above ELISA plate in an ELISA reader (purchased from PerkinElmer) to read the fluorescence value of each well. Calculate the inhibition rate curve and calculate the half-maximal inhibitory concentration (IC50) of the peptide drug.

[0214] Inhibition rate = (fluorescence value of virus wells - fluorescence value of drug-treated wells) / (fluorescence value of virus wells - fluorescence value of cell wells) x 100%; virus wells do not include peptide treatment, drug-treated wells include virus treatment and peptide treatment, and cell wells do not include virus treatment.

[0215] Results and Discussion:

[0216] As shown in Table 6, the MjHKU4r-HR2P9 peptide and the MjHKU4r-HR2P10 stapled peptide have high-efficiency and broad-spectrum antiviral activity, and can significantly inhibit the activity of pseudoviruses such as MERS-CoV, SARS-CoV-2-Delta, SARS-CoV-2-XBB.1.16, and SARS-CoV-2-KP.2. Compared with MjHKU4r-HR2P9, the antiviral activity of the stapled peptide MjHKU4r-HR2P10 is significantly improved, further suggesting that the HR2-derived peptide can significantly improve the broad-spectrum antiviral activity of the peptide by introducing a pair of unnatural amino acids (S5) and forming an intrapeptide linker arm, which is unexpected.

[0217] Table 6: Broad-spectrum and highly effective antiviral activity of MjHKU4r-HR2P9 peptide and MjHKU4r-HR2P10 stapled peptide:

[0218]

[0219] Example 5: HR2-derived peptides of closely related coronaviruses inhibit MjHKU4r-CoV infection

[0220] Experimental methods (evaluation of viral entry inhibition activity, Figure 4 ):

[0221] 1. Through sequence alignment, we designed and synthesized peptides such as MERS-HR2P, HKU4-HR2P, HKU5-HR2P, SARS-HR2P, 229E-HR2P, EK1, EK1C4, etc. All peptides were synthesized and purified by Shanghai Jiepeptide Biotechnology Co., Ltd. with a purity of >95%. Figure 4 Figure a shows the closeness between the coronaviruses. Figure 4Figure b shows the HR2 sequence of each coronavirus and the amino acid sequence alignment of EK1 and EK1C4.

[0222] 2. Using Caco2 cells (ATCC HTB-37Caco2) as target cells, 10 4 Cells / well were plated.

[0223] 3. Use DMEM culture medium to dilute the peptide drug in a 4-fold gradient in a 96-well plate, 50 µL per well.

[0224] 4. Add 50 µL / well of MjHKU4r-CoV pseudovirus to the plate containing peptide drug dilutions. Incubate at 37°C for 30 min to allow the drug and virus to fully interact. Add 100 µL of the drug and virus mixture to each well and add it to the target cells after removing the supernatant. After incubation at 37°C for 12 h, replace with fresh DMEM medium containing 10% FBS.

[0225] 5. After 72 h, cells were lysed, firefly luciferase substrate (purchased from Promega, catalog number E1501) was added, and the fluorescence value of each well was read in a microplate reader (purchased from PerkinElmer). Inhibition rate = (fluorescence value of virus well - fluorescence value of drug-treated well) / (fluorescence value of virus well - fluorescence value of cell well) * 100%, and inhibition rate curve was calculated and the half inhibitory concentration (IC50) of the drug was calculated. Virus wells do not include peptide treatment, drug-treated wells include virus treatment and peptide treatment, and cell wells do not include virus treatment.

[0226] Results and Discussion:

[0227] 1. As Figure 4 Figure c in Figure 5 As shown in Table 5, polypeptides such as MERS-HR2P, HKU4-HR2P, HKU5-HR2P, MjHKU4r-HR2P2, MjHKU4r-HR2P3, MjHKU4r-HR2P4, MjHKU4r-HR2P5, MjHKU4r-HR2P6, MjHKU4r-HR2P7 or MjHKU4r-HR2P8 have high antiviral activity against MjHKU4r-CoV, and their antiviral activity is significantly positively correlated with the relationship between viruses.

[0228] 2. If Figure 4As shown in Figure c and Table 5, EK1 and EK1C4 have a highly effective inhibitory effect on pseudovirus infection mediated by the S protein of MjHKU4r-CoV, and the antiviral activity of EK1C4 is significantly improved compared with EK1, further suggesting that the HR2-derived peptide can significantly improve the antiviral activity of the peptide by modifying the C-terminal cholesterol molecule. Although EK1 has sequence differences with MERS-HR2P, it is unexpected that it has a more efficient inhibitory effect than MERS-HR2P.

[0229] The coronavirus S protein plays an important role in mediating the virus infection process. Its S protein can be divided into S1 subunit and S2 subunit. The S1 subunit is responsible for recognizing the surface receptors of the target cell, and the S2 subunit is responsible for mediating the membrane fusion process between the virus and the host cell. The S1 subunit contains two important functional domains: the N-terminal domain (NTD) and the C-terminal domain (CTD), which can bind to the glycosyl receptor and protein receptor respectively. With the assistance of the host protease, the fusion peptide at the N-terminus of the S2 subunit is exposed, enabling it to insert into the target cell membrane, while the N-terminal heptad repeat domain 1 (HR1) forms a core trimer and interacts with its C-terminal heptad repeat domain 2 (HR2) to form a "six-helix bundle" (6HB). This "fusion core structure" can bring the viral membrane and the target cell membrane closer to each other, thereby causing membrane fusion. The viral genetic material enters the target cell through the fusion pore to replicate and produce new viral particles.

[0230] The S protein S2 subunit has become an important target for the development of antiviral entry inhibitors. Peptide drugs are the main carriers of this type of inhibitors. We found that the derivative peptides (length of 30-60 amino acid residues, especially 30-40 amino acid residues) derived from the amino acid sequence of the S2 heptad repeat region 2 (HR2) of the virus itself can competitively bind to the viral heptad repeat region 1 (HR1) trimer to form a stable heterologous 6HB, preventing the viral HR2 from binding to the viral HR1 trimer, so that they cannot form homologous 6HB, thereby inhibiting the fusion of the viral membrane and the target cell membrane, and thus inhibiting the viral gene from entering the target cell for replication. Peptide entry inhibitors with the same mechanism of action have played an important role in the treatment of human immunodeficiency virus (HIV). For example, Enfuvirtide (Fuzeon, also known as T20) is the first peptide HIV entry inhibitor approved by the US FDA. It is currently used in the clinical treatment of AIDS and has a good effect on patients who have developed resistance to HIV reverse transcriptase inhibitors. One of the inventors of this patent, Jiang Shibo, is the inventor of the T20 drug prototype peptide (peptide 637-666) (U.S. Patent No.: 5,444,044).

[0231] Therefore, we have creatively designed a group of peptides that can effectively inhibit the infection of MERS-like coronavirus MjHKU4r-CoV (Manisjavanica HKU4-related coronavirus). This invention will be of great significance for the future prevention, control and treatment of this coronavirus, and will directly provide effective preventive and therapeutic drug candidates.

[0232] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A polypeptide comprising the sequence of the following formula 1: EISKIN-Y1-T-Y2-L-Y3-L-Y4-Y5-Y6-Y7-Y8-Y9-L-Y10-E-Y11-Y12-K-Y13-L-Y14-Y15-S-Y16-IDLKEL Formula 1, wherein Y1 is T or V; Y2 is L or F; Y3 is D or N; Y4 is S, E or N; Y5 is D or T; Y6 is E or F; Y7 is M or L; Y8 is A, K or M; Y9 is I, K, M or V; Y10 is Q, E, S or L; Y11 is V or A; Y12 is V or I; Y13 is Q or K; Y14 is N or E; Y15 is S, D or E; Y16 is Y or L; Preferably, the polypeptide comprises the sequence of Formula 2: EISKIN-Y17-TLLDLSDEMAIL-Y18-E-Y19-Y20-KQLNDSYIDLKEL Formula 2, Y17 is T or V, Y18 is L or Q, Y19 is V or A, and Y20 is V or I.

2. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or inserted into the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 1; Preferably, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1, 3-5 and 11-18.

3. A polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or inserted into the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:

2.

4. A nucleic acid encoding a polypeptide according to any one of claims 1 to 3.

5. A vector comprising the nucleic acid according to claim 4.

6. A host cell comprising the nucleic acid according to claim 4 or the vector according to claim 5, preferably a eukaryotic host cell or a prokaryotic host cell; preferably, the eukaryotic host cell is a yeast cell, such as a Pichia pastoris cell; preferably, the prokaryotic host cell is an Escherichia coli cell.

7. A stapled peptide, which is a stapled peptide of the amino acid sequence of any one of SEQ ID NOs: 1, 3-5 and 11-18, wherein two amino acids in the amino acid sequence are replaced by non-natural amino acids whose side chains can be linked, and the non-natural amino acids whose side chains can be linked are α-amino acids with alkenyl side chains, such as one or more of the following: (S)-2-(4'-pentenyl)alanine (S5), (R)-2-(4'-pentenyl)alanine (R5), (S)-2-(7'-octenyl)alanine (S8), (R)-2-(7'-octenyl)alanine (R8), (S)-2-(4'-pentenyl)glycine (Sg5) and (R)-2-(4'-pentenyl)alanine (Rg5); the positions of the replaced amino acids are 25th and 29th, respectively; Preferably, the stapled peptide comprises the amino acid sequence of EISKINVTLLDLSDEMAILLEAIK-S5-LND-S5-YIDLKEL, wherein S5 is (S)-2-(4'-pentenyl)alanine, and a staple is formed between two S5s.

8. A composition comprising a polypeptide according to any one of claims 1 to 3, a nucleic acid according to claim 4, a vector according to claim 5, a host cell according to claim 6, and a stapled peptide according to claim 7; preferably, the composition is a drug, preferably a tablet, a capsule, a pellet, an aerosol, a pill, a powder, a solution, a suspension, an emulsion, a granule, a liposome, a transdermal agent, a suppository or a lyophilized powder injection, or an external preparation, preferably an external smear preparation, such as an external gel or an external infiltration preparation; the composition is a mask, a paper towel, a glove, a garment, such as a protective suit, a hand washing product, such as a hand sanitizer, or a shower gel.

9. Use of a polypeptide according to any one of claims 1 to 3 or a polypeptide or polypeptide derivative according to any one of SEQ ID NOs: 6 to 10 or a stapled peptide according to claim 7 in the preparation of a medicament or kit for treating or preventing MERS-like coronavirus MjHKU4r-CoV infection or a disease caused by MERS-like coronavirus MjHKU4r-CoV infection; Preferably, the dosage form of the drug is tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, suppositories or lyophilized powder injections; Preferably, the drug is administered by the following methods: injection, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, etc., cavity administration, such as rectal, vaginal and sublingual, respiratory administration, such as nasal cavity; mucosal administration, or surface administration; Preferably, the disease is a respiratory disease or a digestive tract disease; The polypeptide derivative is a cholesterol-modified derivative of the polypeptide; Preferably, the polypeptide is linked to the cholesterol moiety at the C-terminus via a linker; Preferably, wherein the linker is PEGylated; Preferably, the polypeptide is linked to a cholesterol-modified cysteine ​​at the C-terminus via a linker; Preferably, the linker comprises (GSG)n or (GSGSG)n, wherein n is 1 or 2, Preferably, the linker is -(GSG)n-DPEG4- or -(GSGSG)n-DPEG4-.

10. A method for inhibiting a MERS-like coronavirus MjHKU4r-CoV in vitro, comprising the step of contacting a polypeptide according to any one of claims 1 to 3 or a polypeptide or polypeptide derivative according to any one of SEQ ID NOs: 6 to 10 or a stapled peptide according to claim 7 with a MERS-like coronavirus MjHKU4r-CoV, wherein the polypeptide derivative is a cholesterol-modified derivative of the polypeptide; Preferably, the polypeptide is linked to the cholesterol moiety at the C-terminus via a linker; Preferably, wherein the linker is PEGylated; Preferably, the polypeptide is linked to a cholesterol-modified cysteine ​​at the C-terminus via a linker; Preferably, the linker comprises (GSG)n or (GSGSG)n, wherein n is 1 or 2, Preferably, the linker is -(GSG)n-DPEG4- or -(GSGSG)n-DPEG4-.

11. A method for preparing a polypeptide, comprising the steps of: (1) culturing the host cell according to claim 6 in a suitable culture medium; and (2) Harvesting and isolating peptides.

12. A method for screening active ingredients that inhibit MERS-like coronavirus MjHKU4r-CoV, comprising: (1) producing a polypeptide derived from the C-terminal heptad repeat domain 2 of the coronavirus S protein; preferably, the coronavirus is an alpha or beta coronavirus, preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV, and preferably, the polypeptide is a polypeptide according to any one of claims 1 to 3; (2) determining the ability of the polypeptide to inhibit cell-cell fusion in a MjHKU4r-CoV S protein-mediated cell fusion assay, preferably inhibiting fusion between cells expressing the MjHKU4r-CoV S protein and cells expressing the human coronavirus receptor, or determining the ability of the polypeptide to inhibit the entry of MjHKU4r-CoV into cells, preferably cells expressing the human coronavirus receptor, in a cell-based assay; and (3) Selecting peptides that have the ability to inhibit cell-cell fusion or the ability to inhibit MjHKU4r-CoV from entering cells.

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