Polypeptide capable of inhibiting MERS-like coronavirus infection, and use thereof

Abstract

Provided are a polypeptide capable of inhibiting a MERS-like coronavirus infection, and a use thereof. On the basis of a membrane fusion invasion feature mediated by an S2 subunit of a MjHKU4r-CoV coronavirus S protein, a group of polypeptides are invented by using the HR1 functional domain of the protein as a target. The polypeptides can efficiently inhibit the membrane fusion invasion process of a MERS-like coronavirus MjHKU4r-CoV. By competitively binding to a viral HR1 functional domain, these polypeptides inhibit the formation of a viral 6-helix bundle (6-HB) fusion core, thereby efficiently blocking the process of the coronavirus MjHKU4r-CoV invading a target cell. The present invention can provide an efficient preventive and therapeutic candidate drug for prevention and treatment of the MERS-like coronavirus MjHKU4r-CoV having potential high pathogenicity and cross-species transmission.

Description

Polypeptides capable of inhibiting mers-like coronavirus infection and applications thereof

[0001] This application claims priority to the following applications: Chinese Invention Patent Application No. 202410986087.8, filed on July 22, 2024, entitled “Polypeptides capable of inhibiting mers-like coronavirus infection and applications thereof”; and Chinese Invention Patent Application No. 202510146852.X, filed on February 10, 2025, entitled “Polypeptides capable of inhibiting mers-like coronavirus infection and applications thereof”. TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine, and relates to a group of polypeptides capable of inhibiting the infection of mers-like coronavirus MjHKU4r-CoV. BACKGROUND

[0003] Coronaviruses have a huge natural reservoir in nature, and can infect poultry and livestock such as birds, cats, dogs, pigs, mice, bats, and wild mammals. At the same time, these coronaviruses have the potential to spread across species and infect humans, thus seriously threatening human survival and health. For example, in recent years, highly pathogenic human coronaviruses such as Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory Syndrome coronavirus (MERS-CoV), and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have repeatedly broken out. Therefore, effective antiviral drugs are urgently needed for the prevention and treatment of high-risk coronaviruses that have the potential to infect humans.

[0004] MERS-CoV was first identified in the Middle East in 2012, and single-humped camels are the intermediate host, which infects human cells through hDPP4 as a receptor. As of now, more than 2,500 cases of MERS have been confirmed worldwide, with a mortality rate of 34%, which is the highest among human coronaviruses. Previously, two types of 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, which can directly use hDPP4 as a functional receptor to infect human cells or tissue-like organs, and has significant pathogenicity in hDPP4-transgenic mice. Therefore, MjHKU4r-CoV has the potential to cross-species transmission and infect humans. In addition, there is no effective drug, neutralizing antibody and vaccine for 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 cell entry. Cell. 2023; 186(4): 850-863.e16. doi: 10.1016 / j.cell.2023.01.019). Therefore, once it breaks out in the human population, it will pose a serious threat to human survival and health.

[0005] There is a need in the art for drugs against MjHKU4r-CoV. SUMMARY

[0006] The purpose of the present application is to provide a group of polypeptide entry inhibitors (any one of SEQ ID NO: 1-19) that can have high inhibitory function against MjHKU4r-CoV infection. The polypeptide entry inhibitor targets the HR1 region in the S2 protein of MjHKU4r-CoV, interferes with the formation process of the virus's own six-helix, and thus inhibits the fusion infection process of the virus. The specific sequences of the group of polypeptides are shown in Table 1. The present application 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 of the virus body (MjHKU4r-CoV, for which no specific antiviral drugs or protective vaccines have been reported). The polypeptide length of the polypeptide entry inhibitor can be 30-60 amino acid residues, in particular 30-40 amino acid residues.

[0007] On the one hand, this article provides a polypeptide comprising the sequence of 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,

[0008] Where 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.

[0009] In one embodiment, the polypeptide may comprise the sequence of Formula 2: EISKIN-Y17-TLLDLSDEMAIL-Y18-E-Y19-Y20-KQLNDSYIDLKEL Formula 2,

[0010] Y17 is T or V, Y18 is L or Q, Y19 is V or A, and Y20 is V or I.

[0011] 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 in 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;

[0012] In one embodiment, the polypeptide comprises the amino acid sequence of any one of SEQ ID NO:1, 3-5, and 11-18.

[0013] On the one hand, this article provides 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 in 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.

[0014] On the one hand, this article provides a staple peptide, which is a staple peptide with an amino acid sequence of any one of SEQ ID NO: 1, 3-5 and 11-18, wherein two amino acids in the amino acid sequence are replaced with non-natural amino acids that can be linked by side chains. The non-natural amino acids that can be linked by side chains 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'-octene)alanine (S8), (R)-2-(7'-octene)alanine (R8), (S)-2-(4'-pentene)glycine (Sg5) and (R)-2-(4'-pentene)alanine (Rg5); the replaced amino acids are positioned at positions 25 and 29, respectively, to form a staple.

[0015] In one embodiment, the staple peptide comprises the amino acid sequence EISKINVTLLDLSDEMAILLEAIK-S5-LND-S5-YIDLKEL, wherein S5 is (S)-2-(4'-pentenyl)alanine, and a staple is formed between the two S5s.

[0016] On one hand, this document provides compositions comprising polypeptides, nucleic acids, carriers, host cells, polypeptide derivatives, and / or stapled peptides as described herein. In one embodiment, the composition is a pharmaceutical product, preferably in the form of tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal formulations, suppositories, or lyophilized powder for injection, or a topical preparation, preferably a topical ointment, such as a topical gel or topical infiltration preparation; the composition is a mask, tissue, gloves, clothing, such as protective clothing, handwashing products, such as hand sanitizer, or shower gel.

[0017] On the one hand, this document provides the use of the polypeptides described herein or any of the polypeptides, polypeptide derivatives or stapled peptides described herein or in any of the SEQ ID NO:6-10 in the preparation of medicaments or kits for the treatment or prevention of MERS-like coronavirus MjHKU4r-CoV infection or disease caused by MERS-like coronavirus MjHKU4r-CoV infection.

[0018] In one implementation, the disease is a respiratory disease or a digestive disease.

[0019] On the one hand, this article provides a method for inhibiting MERS-like coronavirus MjHKU4r-CoV in vitro, which includes the step of contacting the polypeptide described herein or any polypeptide derivative or stapled peptide in any of SEQ ID NO:6-10 with MERS-like coronavirus MjHKU4r-CoV.

[0020] On the one hand, this article provides a method for preparing peptides, which includes the following steps:

[0021] (1) The host cells described herein are cultured in a suitable culture medium; and

[0022] (2) Harvest and separate the polypeptides.

[0023] On the one hand, this article provides a method for screening active ingredients that inhibit MERS-like coronavirus MjHKU4r-CoV, which includes:

[0024] (1) Generate a polypeptide derived from the C-terminal heptapeptide repeat domain 2 of the coronavirus S protein; preferably, the coronavirus is an α or β coronavirus, more preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV, and preferably, the polypeptide is the polypeptide described herein;

[0025] (2) In a MjHKU4r-CoV S protein-mediated cell fusion assay, the ability of the peptide to inhibit cell-cell fusion is determined, preferably inhibiting the fusion between cells expressing the MjHKU4r-CoV S protein and cells expressing the human coronavirus receptor; or in a cell assay, the ability of the peptide to inhibit MjHKU4r-CoV entry into cells is determined, preferably in cells expressing the human coronavirus receptor; and

[0026] (3) Select peptides that have the ability to inhibit intercellular fusion or peptides that have the ability to inhibit MjHKU4r-CoV from entering cells.

[0027] In a first aspect, the use of the polypeptide of this article, a staple peptide formed from the polypeptide, a polypeptide derivative, a nucleic acid encoding the polypeptide, and a composition comprising the polypeptide or staple 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 disease caused by MERS-like coronavirus MjHKU4r-CoV infection is provided, the polypeptide comprising the C-terminal heptapeptide repeat domain 2 of the coronavirus S protein.

[0028] In a second aspect, a method for treating or preventing MERS-like coronavirus MjHKU4r-CoV infection or disease caused by MERS-like coronavirus MjHKU4r-CoV infection is provided, comprising administering to a subject a polypeptide of the present invention, a stapled peptide formed from the polypeptide, a polypeptide derivative, a nucleic acid encoding the polypeptide, or a composition comprising said polypeptide or polypeptide derivative or nucleic acid, said polypeptide comprising the C-terminal heptapeptide repeat domain 2 of the coronavirus S protein.

[0029] In a third aspect, the present invention provides a polypeptide, a stapled peptide formed from the polypeptide, a polypeptide derivative, a nucleic acid encoding the polypeptide, and a composition comprising the polypeptide or a polypeptide derivative or a nucleic acid for the treatment or prevention of MERS-like coronavirus MjHKU4r-CoV infection or disease caused by MERS-like coronavirus MjHKU4r-CoV infection, wherein the polypeptide comprises a C-terminal heptapeptide repeat domain 2 of the coronavirus S protein.

[0030] In a fourth aspect, a method for inhibiting MERS-like coronavirus MjHKU4r-CoV in vitro is provided, comprising the steps of contacting a polypeptide of the present invention, a stapled peptide formed from the polypeptide, a polypeptide derivative, a nucleic acid encoding the polypeptide, or a composition comprising the polypeptide or a polypeptide derivative or nucleic acid with MERS-like coronavirus MjHKU4r-CoV, said polypeptide comprising the C-terminal heptapeptide repeat domain 2 of the coronavirus S protein.

[0031] In the various implementations of the above aspects, the coronavirus is an α or β coronavirus.

[0032] In the various implementations of the above aspects, the coronaviruses are HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, and MERS-CoV.

[0033] In various embodiments of the above aspects, the polypeptide comprises the amino acid sequence of formula (I):

[0034] Formula (I):

[0035] 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 -

[0036] X1 is a nonpolar, uncharged residue, preferably P, or it may not exist.

[0037] X2 is a hydrophilic, uncharged residue, preferably N, or it may not be present.

[0038] X3 is an aromatic residue, preferably F, or it may not be present.

[0039] X4 is an aliphatic, uncharged residue, preferably A, or it may not exist.

[0040] 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 8 is D, T, K, or S; X19 is E, G, or F; X20 is M, F, L, or 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 does not exist; X42 is S, D, or does not exist; X43 is K, N, or does not exist; X44 is L, I, or does not exist; X45 is A, N, or does not exist; X46 is S or does not exist; X47 is E, T, or does not exist; X48 is L or does not exist; X49 is V or does not exist; X50 is D or does not exist; X51 is L or does not exist; X52 is K or does not exist; X53 is W or does not exist; X54 is L or does not exist.

[0041] In various embodiments of the above aspects, the polypeptide derivative is a cholesterol-modified derivative of the polypeptide.

[0042] In various embodiments of the above aspects, the peptide is linked to the cholesterol portion at its C-terminus via a linker.

[0043] In various implementations of the above aspects, the connector is PEGylated.

[0044] In various embodiments of the above aspects, the peptide is linked to a cholesterol-modified cysteine ​​residue at its C-terminus via a linker.

[0045] In various embodiments of the above aspects, the connector comprises (GSG)n or (GSGSG)n, where n is 1 or 2.

[0046] In various embodiments of the above aspects, the connector is -(GSG)n-DPEG4- or -(GSGSG)n-DPEG4-.

[0047] In one embodiment of the first aspect, the dosage form of the drug is a tablet, capsule, drop, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal preparation, suppository, or lyophilized powder for injection.

[0048] In one embodiment of the first aspect, the drug is administered by means of: injection, including subcutaneous injection, intravenous injection, intramuscular injection and intraperitoneal injection, intracerebrospinal injection or infusion, cavity administration, such as rectal, vaginal and sublingual administration, respiratory administration, such as nasal administration; mucosal administration, or surface administration.

[0049] In the 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 V; and / or, X23 is L; and / or, X24 is Q, L, or E; and / or, X25 is E; X26 is V or A; and / or, X27 is V or I; and / or, X28 is K; and / or, X29 is Q or K; and / or, X30 is L; and / or, X31 is N or E; and / or, X32 is D or E; and / or, X33 is S; and / or, X34 is Y or L; and / or, X35 is I; and / or, X36 is D; and / or, X37 is L; and / or, X38 is K; and / or, X39 is E; and / or, X40 is L; and / or, X41-X54 do not exist.

[0050] In various embodiments, the staple peptide is a staple peptide with an amino acid sequence of any one of SEQ ID NO: 1, 3-5, and 11-18, wherein two amino acids in the amino acid sequence are replaced with non-natural amino acids that can be linked by side chains. The non-natural amino acids that can be linked by side chains are α-amino acids having 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'-octene)alanine (S8), (R)-2-(7'-octene)alanine (R8), (S)-2-(4'-pentene)glycine (Sg5), and (R)-2-(4'-pentene)alanine (Rg5); the positions of the replaced amino acids are at positions 25 and 29, respectively. In one embodiment, the staple peptide comprises the amino acid sequence EISKINVTLLDLSDEMAILLEAIK-S5-LND-S5-YIDLKEL, wherein S5 is (S)-2-(4'-pentenyl)alanine, and the two S5s form a staple. In various embodiments of the foregoing aspects, the polypeptide comprises (1) the amino acid sequence of any one of SEQ ID NO: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 NO: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%, or at least 99% identity with the amino acid sequence of any one of SEQ ID NO:1-7 and 9-19.

[0051] In various embodiments of the above aspects, the polypeptide comprises the amino acid sequence of formula (II):

[0052] X1 -

[0053] Where 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.

[0054] In various embodiments of the above aspects, the polypeptide is an amino acid sequence of any one of SEQ ID NO:1-7 and 9-19.

[0055] In various embodiments of the above aspects, the polypeptide derivative is SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-(GSGSG)n-PEG4-C-cholesterol, where n is 1 or 2.

[0056] In one embodiment, the peptide is derived from the C-terminal heptapeptide repeat domain 2 of the coronavirus S protein.

[0057] In one implementation, the coronavirus is an α or β coronavirus, preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, and MERS-CoV.

[0058] In one embodiment, the peptide has MERS coronavirus MjHKU4r-CoV-like inhibitory activity.

[0059] In one embodiment, the polypeptide comprises (1) the amino acid sequence of any one of SEQ ID NO:1-7 and 9-19; (2) the amino acid sequence of any one of SEQ ID NO:1-7 and 9-19 in which one or more amino acid residues are substituted, added, deleted or replaced; or (3) the 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 any one of SEQ ID NO:1-7 and 9-19.

[0060] In the sixth aspect, nucleic acids are provided that encode any of the polypeptides described herein.

[0061] In the seventh aspect, a vector containing the nucleic acid described herein is provided.

[0062] In an eighth aspect, a host cell is provided, which contains the nucleic acid or the vector 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 Pichia pastoris cells. In one embodiment, the prokaryotic host cell is an Escherichia coli cell.

[0063] In a ninth aspect, a composition is provided comprising the polypeptide or stapled peptide described herein, the nucleic acid described herein, the carrier described herein, and / or the host cell described herein. In one embodiment, the composition is a pharmaceutical product. In one embodiment, the pharmaceutical product is a tablet, capsule, drop, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal preparation, suppository, or lyophilized powder for injection, or a topical preparation, preferably a topical ointment, such as a topical gel or topical infiltration preparation; the composition is a mask, tissue, gloves, clothing, such as protective clothing, handwashing products, such as hand sanitizer, or shower gel.

[0064] In the tenth aspect, a polypeptide derivative is provided, which is a cholesterol-modified derivative of the polypeptide described herein.

[0065] In one implementation, the peptide is linked to the cholesterol moiety at its C-terminus via a linker.

[0066] In one implementation, the connector is PEGylated.

[0067] In one embodiment, the peptide is linked to a cholesterol-modified cysteine ​​residue at its C-terminus via a linker.

[0068] In one embodiment, the connector comprises (GSG)n or (GSGSG)n, where n is 1 or 2.

[0069] In one implementation, the connector is -(GSG)n-DPEG4- or -(GSGSG)n-DPEG4-.

[0070] In the eleventh aspect, a method for preparing a polypeptide is provided, comprising the following steps:

[0071] (1) The host cells described herein are cultured in a suitable culture medium; and

[0072] (3) Harvest and separate the polypeptides.

[0073] In the twelfth aspect, a method is provided for screening active ingredients that inhibit MERS-like coronaviruses MjHKU4r-CoV, comprising:

[0074] (1) Generate a polypeptide or staple peptide derived from the C-terminal heptapeptide repeat domain 2 of the coronavirus S protein; preferably, the coronavirus is an α or β coronavirus, more preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV;

[0075] (2) To determine the ability of the peptide or stapling peptide to inhibit intercellular fusion in a MjHKU4r-CoV S protein-mediated cell fusion assay, or to determine the ability of the peptide to inhibit MjHKU4r-CoV entry into cells in a cell assay; and

[0076] (3) Select peptides or staple peptides that have the ability to inhibit intercellular fusion or that have the ability to inhibit MjHKU4r-CoV from entering cells.

[0077] In some embodiments, the ability of the peptide or stapler peptide to inhibit the 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 peptide or stapler peptide to inhibit the entry of MjHKU4r-CoV into cells expressing the human coronavirus receptor is determined.

[0078] In one implementation, the coronavirus is an α or β coronavirus, preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, and MERS-CoV.

[0079] In one embodiment, the polypeptide comprises the amino acid sequence of formula (I) herein.

[0080] In one embodiment, the staple peptide comprises two or more amino acids in the amino acid sequence of formula (I) replaced with an amino acid sequence of non-natural amino acids that can be linked by side chains; wherein the non-natural amino acids that can be linked by side chains are α-amino acids having 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'-octene)alanine (S8), (R)-2-(7'-octene)alanine (R8), (S)-2-(4'-pentene)glycine (Sg5) and (R)-2-(4'-pentene)alanine (Rg5).

[0081] In one embodiment, the number of amino acids replaced is two, and the positions of the replaced amino acids are the i-th position and the (i+3)-th position starting from X1, where 1 ≤ i ≤ 51, or the i-th position and the (i+4)-th position, where 1 ≤ i ≤ 50.

[0082] In one embodiment, the polypeptide comprises (1) the amino acid sequence of any one of SEQ ID NO:1-7 and 9-19; (2) the amino acid sequence of any one of SEQ ID NO:1-7 and 9-19 in which one or more amino acid residues are substituted, added, deleted or replaced; or (3) the 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 any one of SEQ ID NO:1-7 and 9-19.

[0083] In one embodiment, the polypeptide comprises the amino acid sequence of formula (II):

[0084] 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;

[0085] Where 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.

[0086] The advantages of this invention include:

[0087] 1. This invention provides a group of polypeptide entry inhibitors (any one of SEQ ID NO: 1-19) that can effectively inhibit MERS-like coronavirus MjHKU4r-CoV infection.

[0088] 2. The EK1 and EK1C4 of the present invention have unexpectedly more effective inhibitory activity against MERS-like coronavirus MjHKU4r-CoV compared to other peptides (any one of SEQ ID NO:1-6, SEQ ID NO:9-19). Attached Figure Description

[0089] Figure 1 shows the design of MjHKU4r-HR1P and MjHKU4r-HR2P peptides.

[0090] Figure 2 shows the biophysical activities and characteristics of the MjHKU4r-HR1P and MjHKU4r-HR2P peptides.

[0091] Figure 3 shows the inhibitory activity of MjHKU4r-HR1P and MjHKU4r-HR2P peptides on the MjHKU4r-CoV S protein-mediated membrane fusion and infection process.

[0092] Figure 4 shows the inhibition of MjHKU4r-CoV infection by HR2-derived peptides and EK1 and EK1C4 of closely related coronaviruses.

[0093] Figure 5 shows the inhibitory effect of the optimized peptide based on MjHKU4r-HR2P, MjHKU4r-HR2P2-HR2P8, on MjHKU4r-CoV infection. Detailed Implementation

[0094] In this invention, a cell-cell fusion system mediated by the MjHKU4r-CoV S protein was established to detect the inhibitory activity of a series of peptides on the corresponding viral fusion process, as shown in Figure 2. 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. On this system, they successfully designed and evaluated a series of MERS-CoV peptide entry inhibitors with good activity. This work was published in Nature Communications (Reference: Lu, L., Q. Liu, Y. Zhu, KHChan, L. Qin, Y. Li, Q. Wang, JFChan, L. Du, F. Yu, C. Ma, S. Ye, KY Yuen, R. Zhang, and S. Jiang. 2014. Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor. Nat Commun 5:3067). In this invention, the same method was used to detect the inhibitory effects of the invented series of peptides on the fusion of 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 a low nanomolar level.

[0095] These HR2P and EK series peptides exhibit excellent inhibitory effects against the MERS-like coronavirus MjHKU4r-CoV, providing a new strategy for future epidemic control and offering new theoretical support for accelerating the development of highly effective anti-coronavirus peptide small molecule drugs. Furthermore, the antiviral targets and mechanisms of these peptides are clearly defined, ensuring their safety and clarifying optimization pathways, facilitating further development.

[0096] In this document, a polypeptide can be a polypeptide or a variant thereof that includes any one of SEQ ID NO:1-19.

[0097] Table 1: Specific sequences of polypeptides:

[0098] For example, a polypeptide may include an amino acid sequence in which one or more amino acid residues, preferably two, three, four or five, are substituted, added, deleted or inserted in any of the amino acid sequences shown in SEQ ID NO:1-19.

[0099] Amino acid addition refers to the addition of an amino acid to the C-terminus or N-terminus of an amino acid sequence, such as any one of SEQ ID NO:1-19, provided that the polypeptide has inhibitory activity against the MERS-like coronavirus MjHKU4r-CoV.

[0100] Amino acid substitution refers to the substitution of an amino acid residue at a certain position in an amino acid sequence, such as any of the sequences in SEQ ID NO:1-19, by another amino acid residue, as long as the polypeptide has inhibitory activity against the MERS-like coronavirus MjHKU4r-CoV.

[0101] Amino acid insertion refers to the insertion of an amino acid residue at an appropriate position in an amino acid sequence, such as any one of the sequences in SEQ ID NO:1-19. The inserted amino acid residues may be all or partly adjacent to each other, or the inserted amino acids may not be adjacent to each other, as long as the polypeptide has inhibitory activity against the MERS-like coronavirus MjHKU4r-CoV.

[0102] Amino acid deletion refers to the deletion of one, two, or three or more amino acids from an amino acid sequence, such as any one of SEQ ID NO:1-19, as long as the polypeptide has inhibitory activity against coronaviruses.

[0103] In this invention, substitution can be a conserved amino acid substitution, meaning that compared to the amino acid sequence of any one of SEQ ID NO:1-19, three, more preferably two, or one amino acid are replaced by amino acids with similar or related properties to form a peptide. These conserved variant peptides can be generated by amino acid substitution according to Table 2.

[0104] In this article, "multiple" can refer to any integer more than one, such as 2-15 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15).

[0105] In the context of this invention, conservative substitution can be defined according to substitutions within the amino acid classes reflected in one or more of the following three tables:

[0106] Table 2: Categories of conserved substituted amino acid residues

[0107] Table 3: Categories of alternative conserved amino acid residue substitutions

[0108] Table 4: Alternative physical and functional classifications of amino acid residues

[0109] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) was used to determine sequence identity between two amino acid sequences, as in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), preferably implemented in Needle program version 5.0.0 or higher. The parameters used were a nick opening penalty of 10, a nick extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "Longest Identity" (obtained using the -nobrief option) was used as the percentage identity and calculated as follows:

[0110] (identical residues x 100) / (alignment length - total number of gaps in alignment).

[0111] The polypeptides of the present invention can be synthesized or expressed in cells. For example, the polypeptides of the present invention can be synthesized by chemical means. Alternatively, the polypeptides of the present invention can be expressed in recombinant cells. The type of cell is not limited; for example, the cells can be eukaryotic cells or prokaryotic cells. Eukaryotic cells can be fungal cells, such as yeast cells, or insect cells or mammalian cells, such as mouse cells. Prokaryotic cells can be bacterial cells, such as Escherichia coli cells.

[0112] As used herein, "nucleic acid" refers to a plurality of nucleotides linked together by nucleotides. The nucleotide linkages can be, for example, phosphodiester bonds. The nucleic acids described herein may comprise polynucleotides encoding the polypeptides of the present invention. To facilitate subsequent processing of the polypeptides, the nucleic acids of the present invention may also comprise nucleotides encoding purification tags, such as His tags, GST tags, MBP tags, SUMO tags, or NusA tags, and, when necessary, nucleotide sequences encoding a leader sequence.

[0113] The nucleic acid of the polypeptide of the present invention can be codon-optimized according to the host cell used. This nucleic acid can be cloned into a suitable expression vector, and then the expression vector is introduced into a host cell for expression. The type of expression vector is not limited and is well known to those skilled in the art.

[0114] As used herein, the term "vector" is a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage, and animal viruses. Vectors may contain various elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may contain the nucleic acids of this invention for introduction into cells for expression. Vectors may contain expression control elements operatively linked to the nucleic acids, such as promoters, terminators, and / or enhancers.

[0115] As used herein, the term "host cell" refers to a cell into which nucleic acid molecules have been introduced using molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA via electroporation, lipid transfection, and particle gun techniques. Host cells can be eukaryotic or prokaryotic cells. For example, eukaryotic cells include yeast cells, animal cells, and / or insect cells. Prokaryotic cells can be E. coli cells.

[0116] As used herein, “treatment” refers to the administration of a drug to achieve 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 its symptoms. “Treatment” may include: (a) suppressing a disease, i.e., preventing its progression; and (b) alleviating a disease or its symptoms.

[0117] As used in this article, the term "coronavirus" belongs to the order Nidovirales, family Coronaviridae, and genus Coronavirus. It is a large family of enveloped, linear, single-stranded, positive-sense RNA viruses, widely distributed in nature. The viral genome has a methylated cap-like structure at the 5' end and a poly(A) tail at the 3' end. The total genome length is approximately 27-32 kb, making it the largest known RNA virus genome. Coronaviruses infect only vertebrates and are associated with various diseases in humans and animals, causing respiratory, digestive, and nervous system disorders. According to the phylogenetic tree, coronaviruses can be divided into four genera: α, β, γ, and δ. The β-genus coronaviruses can be further divided into four independent subgroups: A, B, C, and D.

[0118] As used herein, the term "coronavirus S protein" refers to the spike protein of coronaviruses and can refer to specific S proteins such as the SARS-CoV-2 S protein, MERS-CoV S protein, SARS-CoV S protein, or MjHKU4r-CoV S protein. For example, the SARS-CoV-2 spike protein is a type I membrane glycoprotein that assembles into a trimer that forms spikes or enveloped protrusions on the surface of enveloped coronavirus particles. The spike protein has two basic functions: host receptor binding and membrane fusion, which are attributed to the N-terminal (S1) and C-terminal (S2) halves of the S protein. The coronavirus S protein binds to its homologous receptor via a receptor-binding domain (RBD) located in the S1 subunit.

[0119] As used in this article, 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 uses the dipeptidyl peptidase 4 receptor to enter the virus and exhibits broad host tropism.

[0120] As used in this article, the term "coronavirus infection" or "CoV infection" refers to infection with coronaviruses such as SARS-CoV-2, MERS-CoV, SARS-CoV, or MjHKU4r-CoV. Coronavirus infections include respiratory infections, typically in the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms such as diarrhea, organ failure (kidney failure and renal insufficiency), septic shock, and in severe cases, even death.

[0121] The term "subject" refers to a mammal that is being evaluated for treatment and / or is receiving treatment. In some implementations, "mammal" refers to a human. The term "subject" includes, but is not limited to, individuals suffering from a disease. Subjects can be humans, but also include other mammals, particularly those that can be used as laboratory models of human diseases, such as rodents (e.g., mice), primates, etc.

[0122] The term "sizing peptide" refers to a polypeptide that has been chemically modified, characterized by the introduction of a full-carbon backbone or other chemical bonds to stabilize the α-helix structure of the polypeptide.

[0123] The term “non-natural amino acid with a side chain that can be linked” refers to an α-amino acid having an alkenyl side chain, such as (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), and (R)-2-(4'-pentene)alanine (Rg5).

[0124] This invention also provides a method for preparing peptides. The method includes introducing an expression vector into host cells, culturing them in a suitable culture medium, and then harvesting or separating the peptide or fusion protein from the supernatant. Alternatively, in the case of intracellular expression, host cells can be collected, followed by cell lysis to collect the peptides. In the case of peptides, the peptides can 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.

[0125] The polypeptides of the present invention can be prepared in derivative form. For example, the polypeptides can be modified with cholesterol. Methods for cholesterol modification are well known. In one embodiment, cholesterol modification can be performed at the C-terminus of the polypeptide, such that the polypeptide is linked to a cholesterol moiety at the C-terminus. This linking can be a direct linking or a linking via a adapter.

[0126] The type and length of the connector can vary. For example, the connector can be (GSG)n or (GSGSG)n, where n can be any integer, such as 1, 2, 3, 4, 5, 6, etc.

[0127] The linker can be PEGylated. Methods of PEGylation modification are known to those skilled in the art. As used herein, the term PEGylated linker refers to a linker with one or more PEG residues attached. In this document, a PEGylated linker can be PEGylated (GSG)n or (GSGSG)n. n can be any integer, such as 1, 2, 3, 4, 5, 6, etc. PEG4 indicates a number of repeating ethylene glycol residues of 4.

[0128] The polypeptides or polypeptide derivatives or staple peptides of the present invention can be used alone or in combination, or in combination with other agents having MERS coronavirus MjHKU4r-CoV inhibitory activity.

[0129] In this document, the polypeptides or polypeptide derivatives or staple peptides of the present invention are prepared in the form of compositions or kits. The compositions may contain a suitable carrier, such as a pharmaceutically acceptable carrier. Such compositions may be for external use, for example, as topical formulations, topical application formulations, such as topical gels or topical infiltration formulations. Such compositions may be applied to items requiring viral inhibition, such as, but not limited to, masks, tissues, gloves, clothing, such as protective clothing, etc. Alternatively, they may be added as active ingredients to handwashing products, such as hand sanitizers, shower gels, etc. Such polypeptides or polypeptide derivatives or staple peptides can be used to inhibit coronaviruses in vitro to prevent and / or reduce viral infection. Such polypeptides or polypeptide derivatives or staple peptides can be used to prevent or treat coronavirus infection or disease caused by coronaviruses in subjects.

[0130] The polypeptides or polypeptide derivatives or stapled peptides of the present invention can also be prepared into pharmaceuticals or pharmaceutical compositions. Such pharmaceuticals can be tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, suppositories, or lyophilized powder injections. These pharmaceuticals or pharmaceutical compositions can be applied by various routes of administration, such as injection (including subcutaneous, intravenous, intramuscular, and intraperitoneal injections, intracerebrospinal injections or infusions), cavity administration (such as rectal, vaginal, and sublingual), respiratory administration (such as nasal administration), mucosal administration, or topical administration, etc.

[0131] This invention provides a method for preventing or treating MERS-like coronavirus MjHKU4r-CoV infection or disease caused by MERS-like coronavirus MjHKU4r-CoV in subjects, comprising administering any polypeptide or polypeptide derivative or staple peptide of the present invention. The invention also provides any polypeptide or polypeptide derivative or staple peptide of the present invention for use in preventing or treating MERS-like coronavirus MjHKU4r-CoV infection or disease caused by MERS-like coronavirus MjHKU4r-CoV in subjects.

[0132] The present invention also relates to a method for preventing or treating MERS-like coronavirus MjHKU4r-CoV infection or disease caused by MERS-like coronavirus MjHKU4r-CoV in a subject, comprising administering to a subject / patient the polypeptide or derivative or staple peptide, nucleic acid, vector, host cell or composition or kit described in the present invention.

[0133] This invention also relates to polypeptides or derivatives or stapled peptides, nucleic acids, vectors, host cells, or compositions or kits for the prevention or treatment of MERS-like coronavirus MjHKU4r-CoV infection or disease caused by MERS-like coronavirus MjHKU4r-CoV in subjects.

[0134] The present invention also relates to polypeptides or derivatives or stapled peptides, nucleic acids, vectors, host cells, compositions, pharmaceutical compositions or kits for the prevention or treatment of MERS-like coronavirus MjHKU4r-CoV infection or disease caused by MERS-like coronavirus MjHKU4r-CoV in subjects / patients.

[0135] coronavirus

[0136] Coronaviruses are a large family of viruses that are widespread in nature. Coronaviruses include mammalian coronaviruses and avian coronaviruses. Mammalian coronaviruses are mainly alpha and beta coronaviruses, which can infect a variety of animals, including pigs, dogs, cats, rats, cattle, and horses. Avian coronaviruses are mainly derived from gamma and delta coronaviruses, which can cause illness in various birds such as chickens, turkeys, sparrows, ducks, geese, and pigeons. The coronaviruses described herein can belong to any subgenus of the α-coronavirus genus, 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 coronavirus HKU8), Myotacovirus (e.g., Myotis ricketti alphacoronavirus Sax-2011), Nyctacovirus (e.g., Nyctalus velutinus alphacoronavirus SC-2013 and Pipistrellus kuhlii coronavirus). Coronaviruses such as 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., A coronavirus 1).

[0137] The genus *Embecovirus* described herein may include *Embecovirus* (lineage A), *Sarbecovirus* (lineage B), *Merbecovirus* (lineage C), *Nobecovirus* (lineage D), and *Hibecovirus*. Therefore, in some embodiments, the coronaviruses described herein may be any strain or species of any subgenus or lineage of *Embecovirus*. Subgenuses of *Embecovirus* may include, but are not limited to: *Embecovirus* 1 (e.g., bovine coronavirus and human coronavirus OC43), *Chinese mouse coronavirus* HKU24, human coronavirus HKU1, mouse coronaviruses (e.g., mouse hepatitis virus), and *Myodes coronavirus* 2JL14. The subgenus Sarbecovirus may include, but is 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 CoV BM48-31. The subgenus *Merbecovirus* can include, but is not limited to: Hedgehog coronavirus 1, MERS-CoV, Pipistrellus bat coronavirus HKU5, and Tylonycteris bat coronavirus HKU4. The subgenus *Nobecovirus* can include, but is not limited to: Eidolon bat coronavirus C704, Rousettus bat coronavirus GCCDC1, and Rousettus bat coronavirus HKU9. Coronavirus antigens can belong to any species or strain within the subgenus *Hibecovirus*, including, but not limited to: bat *Hp-β* coronavirus (Zhejiang 2013).

[0138] The coronaviruses described in this article can phylogenetically cluster into functionally distinct clades. For example, using the nucleotide sequences of the non-structural protein genes ORF1a and ORF1b, coronaviruses of β-coronavirus (Sarbecovirus) can be clustered into clades 1, 2, 1 / 2, or 3 (see, for example, Hu et al., PLoS Pathog 13(11):e1006698). Therefore, coronaviruses can belong to any species or strain within any of these clades. Coronaviruses can belong to any species or strain within clade 1, including but not limited to SARS-CoV, WIV1, LYRa11, Rs7327, Rs4231, Rs4084, and SHC014. Coronaviruses 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. Coronaviruses can belong to any species or strain in clade 1 / 2, including but not limited to SARS-CoV2. Coronaviruses can belong to any species or strain in clade 3, including but not limited to BM48-31. The coronaviruses described herein can be, for example, coronaviruses of SARS, SARS-2, WIV1, SHC014, Rf1, RmYN02, pang17, RaTG13, and Rs4081.

[0139] In some preferred embodiments, the coronaviruses described herein may be HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, and MERS-CoV.

[0140] MjHKU4r-CoV virus

[0141] MjHKU4r-CoV, a recently discovered novel pangolin-derived MERS-like coronavirus, was found in the Malayan pangolin. Whether pangolins play a role in the zoonotic transmission of bat coronaviruses remains unclear. Evolutionary analysis identified MjHKU4r-CoV-1 as belonging to the subgenus *Merbecovirus*, specifically the species *HKU4*. The receptor-binding motif (RBM) of the MERS-CoV spike protein contains 16 key residues that determine its binding to hDPP4. Comparison with MERS-CoV revealed that 8 residues in the RBD of MjHKU4r-CoV-1 are identical to those in MERS-CoV. The potential furin cleavage site (RQQR) in the S protein subdomain 1 (SD1) of 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 is infectious and can cause interstitial pneumonia in mice.

[0142] The S protein of coronavirus

[0143] The S protein of coronaviruses can be divided into two important functional subunits: 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 recognizes and binds to receptors on the host cell, particularly the angiotensin-converting enzyme 2 (ACE2) receptor, while the S2 subunit, the most conserved component of the S protein, is 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 trimeric S protein trimer 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, the subsequent serine protease-mediated cleavage event occurs at the S2' site within S1.

[0144] The term "S1 subunit" (e.g., S1 subunit antigen) refers to the N-terminal subunit of the spike protein, beginning at the N-terminus of the S protein and ending at 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, beginning at the S1 / S2 cleavage site and ending at the C-terminus. Within the S1 subunit, the domains include an N-terminal domain (NTD) and a receptor-binding domain (RBD), the RBD domain further including a receptor-binding motif (RBM). Within the S2 subunit, the domains include a fusion peptide (FP), heptapeptide repeat 1 (HR1), heptapeptide repeat 2 (HR2), a transmembrane domain (TM), and a cytoplasmic domain (also known as the cytoplasmic tail (CT)). For SARS-CoV-2, the HR1 and HR2 domains may 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 heptacapeptide repeat (HR1), a second heptacapeptide repeat (HR2), a transmembrane domain (TM), and a cytoplasmic tail.

[0145] In some embodiments herein, the polypeptide described herein comprises the amino acid sequence of the C-terminal heptapeptide repeat domain 2 of the coronavirus S protein. In some embodiments, the coronavirus is an α or β 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 MjHKU4r-CoV S protein-mediated membrane fusion and infection. In some embodiments, the polypeptide described herein can inhibit MjHKU4r-CoV infection.

[0146] polypeptide

[0147] The polypeptide described herein is a polypeptide derived from the C-terminal heptapeptide repeat domain 2 of the coronavirus S protein. This polypeptide may contain the full length or a portion of the C-terminal heptapeptide 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., forming a six-hexahelix complex), or that the polypeptide can inhibit MjHKU4r-CoV S protein-mediated membrane fusion and infection, or inhibit MjHKU4r-CoV infection. A polypeptide can contain the general formula 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, where X1-X54 are as defined above. In some embodiments, the polypeptide may comprise the general formula 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.

[0148] The polypeptides described herein may comprise any of the amino acid sequences (SEQ ID NO. 1-19) shown in Table 1. In some embodiments, the polypeptides described herein may comprise variants of any of the amino acid sequences (SEQ ID NO. 1-19) shown in Table 1. Variants may have amino acid sequences 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 any of the sequences in SEQ ID NO. 1-19. Variants can be those resulting from one or more mutations (e.g., 2-30, such as 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 of SEQ ID NOs 1-19. Mutations can be one or more of substitution, addition, insertion, and deletion. The variants described herein can interact with the MjHKU4r-HR2P peptide of the MjHKU4r-CoV virus. In some embodiments, the variants described herein can inhibit MjHKU4r-CoV S protein-mediated membrane fusion and infection. In some embodiments, the variants described herein can inhibit MjHKU4r-CoV infection. In some embodiments, the peptide or variant inhibits the fusion between cells expressing the MjHKU4r-CoV S protein and cells expressing the human coronavirus receptor. In some implementations, the peptide or its variants inhibit MjHKU4r-CoV from entering cells expressing human coronavirus receptors.

[0149] Bookbinding peptide

[0150] This document also provides staple peptides. Staple peptides can be staple peptides of the polypeptides shown in Table 1. Methods for preparing staple peptides (e.g., by using an olefin metathesis reaction) are known to those skilled in the art. For example, two or more non-natural amino acids with linkable side chains can be substituted in the amino acid sequence of the polypeptide, and the side chains of the non-natural amino acids are linked. The non-natural amino acids with linkable side chains are α-amino acids having 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 linking of the non-natural amino acid side chains can form one ring, two rings, or more rings, preferably one or two rings.

[0151] The staple peptide may comprise the amino acid sequence of any one of SEQ ID NO: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 that can be linked by side chains, preferably (S)-2-(4'-pentenyl)alanine (S5) or (R)-2-(4'-pentenyl)alanine (R5). The non-natural amino acids can form staples via side chains. Specifically, the staple peptide comprises the amino acid sequence EISKINVTLLDLSDEMAILLEAIK-S5-LND-S5-YIDLKEL, wherein S5 is (S)-2-(4'-pentenyl)alanine, and the two S5s are covalently linked.

[0152] polypeptide derivatives

[0153] The peptides described herein can be modified to produce peptide derivatives. In some embodiments, the peptides can be modified by conjugation with PEG. The PEG modification sites of the peptide are at the N-terminus, C-terminus, Lys side chain, and Cys thiol group. The molecular weight range of the PEG single molecules used for modification is between PEG2 and PEG24. PEG modification can be used to improve protein hydrolytic stability, biodistribution, and peptide solubility. Introducing a PEG chain onto the peptide can improve its pharmacological properties and inhibit the hydrolysis of the peptide by proteolytic enzymes. Because PEG peptides have a prolonged effective half-life in vivo, normal therapeutic levels can be maintained by using peptide drugs at lower doses and lower frequencies. In some embodiments, the peptides described herein can be cholesterol-modified. Cholesterol-modified peptide drugs can improve the drug activity and in vivo / in vitro half-life of the peptide, have weaker toxic side effects, and can cross the blood-brain barrier. In some embodiments, the peptide derivative is a cholesterol-modified derivative of the peptide. In some embodiments, the peptide derivative is a PEG-modified derivative of the peptide. In some embodiments, the peptide derivative is a cholesterol-modified and PEG-modified derivative of the peptide. In some embodiments, the peptide is linked to a cholesterol moiety at its C-terminus via a linker. In some embodiments, the linker is PEGylated. In some embodiments, the peptide is linked to a cholesterol-modified cysteine ​​residue at its C-terminus via a linker. In some embodiments, the linker comprises (GSG)n or (GSGSG)n, where n is 1 or 2. In some embodiments, the linker is -(GSG)n-DPEG4- or -(GSGSG)n-DPEG4-, where n is 1 or 2.

[0154] Pharmaceutical Compositions and Kits

[0155] In some aspects, the present invention relates to pharmaceutical compositions or kits comprising polypeptides described herein, nucleic acids encoding polypeptides, carriers containing nucleic acids, or cells containing nucleic acids or carriers.

[0156] Pharmaceutical compositions may also contain pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" includes any and all solvents, co-solvents, complexes, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc., which are biologically or otherwise unadverse. The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in a pharmaceutical composition is considered. Additional active ingredients may also be incorporated into the pharmaceutical composition. Additionally, various excipients may be included, such as those commonly used in the art. These and other such compounds are described in the literature, for example, in the Merck Index, Merck & Company, Rahway, NJ. Considerations for incorporating various components into a pharmaceutical composition are described, for example, in Gilman et al. (eds.) (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, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal preparation, suppository, or lyophilized powder for injection. In some embodiments, the pharmaceutical composition is administered via: injection, including subcutaneous, intravenous, intramuscular, and intraperitoneal injection, intracerebrospinal injection or infusion, cavity administration, such as rectal, vaginal, and sublingual administration, respiratory administration, such as nasal administration; mucosal administration, or topical administration.

[0157] In some aspects, the present invention relates to a kit comprising a polypeptide described herein, a nucleic acid encoding the polypeptide, a carrier containing the nucleic acid, or cells containing the nucleic acid or the carrier. In some embodiments, the present invention provides a kit for generating a single-dose administration unit. In some aspects, the kit of the present invention can contain a first container containing a polypeptide described herein, a nucleic acid encoding the polypeptide, a carrier containing the nucleic acid, or cells containing the nucleic acid or the carrier, and a second container containing an aqueous formulation. In some embodiments, the kit contains a diluent, buffer, or other solution for diluting or reconstituted powder. In some embodiments, the kit includes instructions for use of the polypeptide described herein, the nucleic acid encoding the polypeptide, the carrier containing the nucleic acid, or cells containing the nucleic acid or the carrier.

[0158] Peptide drug screening methods

[0159] The present invention also provides a method for screening active ingredients that inhibit MERS-like coronavirus MjHKU4r-CoV. The method may include generating a peptide library, which may contain peptides derived from the C-terminal heptapeptide repeat domain 2 of the coronavirus S protein. The coronavirus may be an α or β coronavirus, preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, and MERS-CoV. A polypeptide library can contain the amino acid sequence of formula I: 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. The peptide library may contain 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 elsewhere herein. Methods for generating the peptide library are known to those skilled in the art, for example, by chemical synthesis or by biosynthesis.

[0160] Alternatively, the method may include generating a staple peptide library containing staple peptides derived from an amino acid sequence of Formula I or Formula II.

[0161] In some embodiments, the stapled peptide library includes an amino acid sequence in which two or more amino acids of formula (I) are replaced with an amino acid sequence of non-natural amino acids that can be linked by side chains; wherein the non-natural amino acids that can be linked by side chains are α-amino acids having 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'-octene)alanine (S8), (R)-2-(7'-octene)alanine (R8), (S)-2-(4'-pentene)glycine (Sg5), and (R)-2-(4'-pentene)alanine (Rg5). In some embodiments, the number of substituted amino acids is two, and the positions of the substituted amino acids are the i-th and i+3-th positions from X1, where 1 ≤ i ≤ 51, or the i-th and i+4-th positions, where 1 ≤ i ≤ 50.

[0162] In some embodiments, the staple peptide comprises two or more amino acids in the amino acid sequence of formula (II) replaced with an amino acid sequence of non-natural amino acids that can be linked by side chains; wherein the non-natural amino acids that can be linked by side chains are α-amino acids having 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'-octene)alanine (S8), (R)-2-(7'-octene)alanine (R8), (S)-2-(4'-pentene)glycine (Sg5), and (R)-2-(4'-pentene)alanine (Rg5). In some embodiments, the number of substituted amino acids is two, and the positions of the substituted amino acids are the i-th and i+4-th positions from X1, respectively, where 1 ≤ i ≤ 50.

[0163] In some embodiments, after generating the polypeptide or stapler peptide library described herein, the polypeptide or stapler peptide library can be contacted with the MjHKU4r-HR1P polypeptide. If a member of the polypeptide or stapler peptide library forms a six-helix complex with the MjHKU4r-HR1P polypeptide, that member is selected as a candidate.

[0164] In some embodiments, the ability of members of a peptide library or stapled peptide library to inhibit intercellular fusion can be determined in an MjHKU4r-CoV S protein-mediated cell fusion assay. If a member of the peptide library or stapled peptide library inhibits intercellular fusion, that member is selected as a candidate. In some embodiments, the ability of a member to inhibit fusion between cells expressing the MjHKU4r-CoV S protein and cells expressing the human coronavirus receptor is determined.

[0165] In some embodiments, members of a peptide library or stapled peptide library may be exposed to MjHKU4r-CoV pseudovirus and target cells expressing human coronavirus receptors (e.g., Caco2). If a member of the peptide library or stapled peptide library inhibits the invasion of MjHKU4r-CoV pseudovirus into target cells, that member is selected as a candidate. In some embodiments, the ability of the member to inhibit the entry of MjHKU4r-CoV pseudovirus into cells expressing human coronavirus receptors is determined.

[0166] In some embodiments, members of a peptide library or stapled peptide library may be exposed to MjHKU4r-CoV and target cells expressing human coronavirus receptors (e.g., Caco2). If a member of the peptide library or stapled peptide library inhibits the invasion of MjHKU4r-CoV into the target cells, that member is selected as a candidate. In some embodiments, the ability of a member to inhibit the entry of MjHKU4r-CoV into cells expressing human coronavirus receptors is determined.

[0167] In some embodiments, the polypeptide library comprises (1) an amino acid sequence of any one of SEQ ID NO: 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 any one of SEQ ID NO: 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%, or at least 99% identity with an amino acid sequence of any one of SEQ ID NO: 1-7 and 9-19.

[0168] In some embodiments, the candidate may be an amino acid sequence of any one of SEQ ID NO: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%, or at least 99% identity with it.

[0169] In some embodiments, the stapled peptide library comprises the amino acid sequence of any one of SEQ ID NO:2-7 and 9-19 or a variant amino acid sequence thereof defined above by identity. In some embodiments, relative to SEQ ID NO:18, at positions i and i+3 or i and i+4, the amino acid residues are replaced with non-natural amino acids that can be linked by side chains, preferably (S)-2-(4'-pentene)alanine (S5) or (R)-2-(4'-pentene)alanine (R5). In some embodiments, the stapled peptide library comprises the amino acid sequence of SEQ ID NO:18, at positions i and i+3 or i and i+4, the amino acid residues are replaced with non-natural amino acids that can be linked by side chains, preferably (S)-2-(4'-pentene)alanine (S5) or (R)-2-(4'-pentene)alanine (R5).

[0170] Example

[0171] The present invention is further illustrated by the following embodiments, but any embodiment 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, and those skilled in the art will clearly understand the scope defined by the claims in conjunction with this specification and common knowledge in the art. Without departing from the spirit and scope of the present invention, those skilled in the art can make any modifications or changes to the technical solutions of the present invention, and such modifications and changes are also included within the scope of the present invention.

[0172] The general methods of PCR, cloning, ligation, and transfection of nucleotides 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, NY; 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," DNGlover (ed.) (1985); "Oligonucleotide Synthesis," MJ Gait (ed.) (1984); "Nucleic Acid Hybridization," BD Hames & S.J. Higgins (eds.) (1985); "A Practical Guide to..." "Molecular Cloning: A Practical Guide" by B. Perbal (1984).

[0173] Example 1: Design of MjHKU4r-HR1P and MjHKU4r-HR2P peptides

[0174] As shown in Figure 1, by comparing the homology with the MERS-CoV S2 protein, the specific locations of the HR1 and HR2 functional domains in the MjHKU4r-CoV S2 protein, as well as the distribution of key amino acid sites, were clarified, thus enabling the design of the MjHKU4r-HR1P and MjHKU4r-HR2P peptides. Figure 1 also shows the sequence alignment between MjHKU4r-CoV HR1 and MERS-CoV HR1, and between MjHKU4r-CoV HR2 and MERS-CoV HR2, revealing high homology between them.

[0175] Example 2: Biophysical characteristics of MjHKU4r-HR1P and MjHKU4r-HR2P peptides

[0176] Experimental materials: MjHKU4r-HR1P and MjHKU4r-HR2P peptides were synthesized and purified by Shanghai Jietai Biotechnology Co., Ltd., with a purity >95%. All peptides in Table 1 were synthesized and purified by Shanghai Jietai Biotechnology Co., Ltd., with a purity >95%.

[0177] Experimental methods:

[0178] 1. Non-denaturing polyacrylamide gel electrophoresis (N-PAGE). A gradient concentration of MjHKU4r-HR1P and MjHKU4r-HR2P peptides was mixed, with final concentrations of MjHKU4r-HR1 peptide at 0, 100, 300, and 600 μM, and a final concentration of MjHKU4r-HR2 peptide at 100 μM. The mixture was incubated at 37°C for 30 minutes. 5x high-pH loading buffer (supplier: TIANDZ, product number 81212A-30) was mixed with 4 times the volume of the peptide mixture. The mixture was then loaded (20 μL per well) onto a 12% non-denaturing gel and electrophoresed at 125 V for 4 hours at room temperature. Coomassie brilliant blue staining was performed. The results showed that both MjHKU4r-HR2P and MjHKU4r-HR1P peptides produced a new band (6-HB), which is likely a six-helix complex (Figure 2a).

[0179] 2. Circular dichroism spectroscopy for determining the secondary structure of peptides. Free peptides (MjHKU4r-HR1P or MjHKU4r-HR2P) or a mixture of peptides (MjHKU4r-HR1P and MjHKU4r-HR2P) were diluted to a final concentration of 50 μmol / L with 300 mmol / L, pH 7.2 PBS. Circular dichroism spectroscopy was performed using a spectrophotometer (J-815, Jasco Inc, Japan). The detection temperature was 4℃, bandwidth was 5.0 nm, resolution was 0.1 nm, optical path was 0.1 cm, reaction time was 4.0 s, and scan rate was 50 nm / min. Thermal denaturation of the peptides was monitored by a temperature gradient of 5℃ / min at 222 nm. The spectral values ​​were corrected by subtracting a buffer solution blank control. The melting curves were smoothed, and the midpoint temperature of the thermal dissociation transition, Tm, was calculated using Jasco.

[0180] Results and Analysis:

[0181] 1. As shown in Figure 2a, different concentrations of MjHKU4r-HR1P and MjHKU4r-HR2P peptides can produce a new band that may be a six-helix complex after mixing, indicating that MjHKU4r-HR1P and MjHKU4r-HR2P peptides can interact.

[0182] 2. As shown in Figure 2bc, the individual peptides have a low-helix structure. When MjHKU4r-HR2P and MjHKU4r-HR1P are mixed, a high-helix structure appears, indicating that the peptides MjHKU4r-HR1P and MjHKU4r-HR2P interact in a hexagonal form. The Tm value of the mixture of MjHKU4r-HR1P and MjHKU4r-HR2P peptides is high (=84.8℃), indicating that the hexagonal structure is stable.

[0183] Example 3: MjHKU4r-HR2P inhibits MjHKU4r-CoV S protein-mediated membrane fusion and infection process

[0184] Experimental Method 1 (Assessment of Membrane Fusion Inhibition Activity, Figure 3, diagram ab):

[0185] 1. Transfect 293T cells with a plasmid encoding the coronavirus S protein and culture them for 36-48 hours to obtain effector cells. Specifically, the MjHKU4r-S protein encoding gene was ligated into the pAAV-IRES-GFP vector via two restriction enzyme sites, BamhI and Xhol, to obtain the plasmid pAAV-IRES-GFP-MjHKU4r-S, which encodes the MjHKU4r-CoV S protein. 2 μL of Vigofect transfection reagent (Vigofect Biotechnology (Beijing) Co., Ltd., used at a 1:1000 ratio with DMEM medium) was added to another 1 mL vial of 0.9% NaCl solution and incubated at room temperature for 5 minutes. Vigofect dilution was then added dropwise to the plasmid mixture (pAAV-IRES-GFP-MjHKU4r-S, 10 μg), and after incubation at room temperature for 15 minutes, the mixture was added to 293T cells. The transfected cells were designated as 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.

[0186] The amino acid sequence of the coronavirus MjHKU4r S protein:

[0187] 2. After detaching the 293T / MjHKU4r / EGFP and 293T / EGFP cells by pipetting and centrifugation, resuspend the cells in fresh DMEM medium and adjust the cell concentration to 2 x 10⁻⁶. 5 Take 50 μL of the sample per mL and add it to a serially diluted 50 μL of the peptide drug. Incubate at 37 °C for 30 min.

[0188] 3. Add 100 μL of the cell / peptide drug mixture to the target cells Caco2 (ATCC HTB-37) already plated in a 96-well plate. Incubate at 37°C with 5% CO2 for 2-4 hours, and observe and record the cell fusion using the green fluorescence channel of a fluorescence microscope. For information on the membrane fusion and infection process mediated by the coronavirus S protein, please refer to the applicant's previous patent applications, such as CN117777247A and CN116440285A. Figure 3a shows fluorescence photographs of membrane fusion under different concentrations of MjHKU4r-HR1P and MjHKU4r-HR2P. Figure 3b shows the IC50 values ​​of MjHKU4r-HR1P and MjHKU4r-HR2P as 0.037 μM and greater than 10 μM, respectively.

[0189] Experimental Method 2 (Assessment of Viral Invasion Inhibition Activity, Figure 3c):

[0190] 1. Dissolve peptides MjHKU4r-HR1P and MjHKU4r-HR2P in DMSO and determine the peptide concentration.

[0191] 2. Packaging of MjHKU4r-CoV pseudovirus (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)): The PC-MjHKU4r-S plasmid was obtained by ligating the MjHKU4r-S encoding gene into the PC-DNA3.1(+) vector at the BamHI and XhoI restriction sites. Then, the MjHKU4r-CoV pseudovirus was packaged using the PC-MjHKU4r-S plasmid and the HIV-1 backbone plasmid (pNL4-3.Luc.R-.E-). Specifically, 293T cells were digested 24 hours before transfection and plated in 10cm tissue culture dishes (2×10⁻⁶ cells / cm²). 6 / culture dish). Replace the pre-warmed fresh DMEM medium (containing 10% FBS) with fresh medium 2 hours before transfection. Use two 1.5 mL EP tubes for transfection. Add 500 μL of 0.9% NaCl solution to EP tube 1, containing 20 μg each of PC-MjHKU4r-S plasmid and pNL4-3.Luc.R-.E- plasmid. Add 500 μL of 0.9% NaCl to EP tube 2, then add 2 μL of Vigofect transfection reagent, and let stand for 5 minutes. Add the 500 μL solution from EP tube 2 dropwise to EP tube 1, mixing constantly with a pipette tip, and let stand at room temperature for 15 minutes. Add 1 mL of the mixture dropwise and evenly to the previously plated 293T cell culture dish. 8–10 h after transfection, replace with 10 mL of fresh DMEM culture 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℃ for later use, and store at -80℃.

[0192] 3. Caco2 (ATCC HTB-37) cells were digested with trypsin to prepare a suspension. After adjusting the cell concentration, 10 μL of the suspension was added to each well. 4 Each cell.

[0193] 4. Dilute the peptide drug 4-fold in DMEM medium to 50 μL per well in a 96-well plate. Add 50 μL of MjHKU4r-CoV pseudovirus to each well of the peptide drug dilution plate. Incubate at 37°C for 30 min to allow sufficient interaction between the peptide drug and the virus. Add 100 μL of the peptide drug and virus mixture to each well of the target cells after removing the supernatant. Incubate at 37°C for 12 h, then replace with fresh DMEM medium containing 10% FBS.

[0194] 6. After 72 hours, discard the original culture medium in the 96-well cell culture plate, add 40 μL of cell lysis buffer (Promega, catalog number E1531) to each well, and lyse the cells on a decolorizing shaker for 45 minutes. Transfer 30 μL of cell lysis buffer from each well to a 96-well microplate, and then add 30 μL of firefly luciferase substrate (Promega, catalog number E1501) to each well. Place the microplate in a microplate reader (PerkinElmer) and read the fluorescence value of each well.

[0195] Inhibition rate curves were calculated, and the half-maximal effective dose (IC50) of the drug was determined. Specifically, the viral inhibition rate of each well in the drug-treated wells was calculated using the following formula: 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 did not include peptide treatment, drug-treated wells included both virus and peptide treatment, and cell wells did not include virus treatment. The calculated viral inhibition rates for each well were imported into Graphpad Prism software, and the Variable Slope mode in Dose-Response-Inhibition was selected to generate inhibition rate curves and IC50 values.

[0196] Results and Discussion:

[0197] 1. As shown in Figure 3b and Table 5, the MjHKU4r-HR2P peptide and the MjHKU4r-HR2PEK peptide have a good inhibitory effect on cell-cell fusion mediated by the S protein of MjHKU4r-CoV, thus preliminarily demonstrating the antiviral activity of MjHKU4r-CoV HR2-derived peptides in inhibiting MjHKU4r-CoV.

[0198] 2. As shown in Figure 3c and Table 5, the MjHKU4r-HR2P peptide and the MjHKU4r-HR2PEK peptide have a good inhibitory effect on the pseudovirus infection process mediated by the S protein of MjHKU4r-CoV, which further proves that the MjHKU4r-CoV HR2-derived peptide has antiviral activity to inhibit MjHKU4r-CoV infection.

[0199] Table 5: Inhibitory activity of peptides against cell-cell fusion and pseudovirus infection mediated by the S protein of MjHKU4r-CoV coronavirus:

[0200] Example 4: Broad-spectrum anti-β-HCoV activity of MjHKU4r-HR2P9 and MjHKU4r-HR2P10

[0201] Experimental methods (assessment of viral invasion inhibitory activity):

[0202] 1. Using Caco2 cells (purchased from ATCC, product number HTB-37Caco2) as target cells, at 10 4 Cells were plated in wells and incubated at 37°C for 24 hours.

[0203] 2. In 96-well plates, use DMEM culture medium to serially dilute the peptide drugs MjHKU4r-HR2P9 and MjHKU4r-HR2P10 fourfold (5 μM initial concentration and 5 fourfold dilutions), 50 μL per well.

[0204] 3. Add 50 μL / well of MERS-CoV or SARS-CoV-2 variant pseudoviruses (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, Liu M, Wang C, et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein 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 wells containing the diluted peptide drug and incubate at 37°C for 30 min to allow the peptide drug to fully interact with the virus. Add 100 μL of the peptide drug and virus mixture to each well and add it to the target cells after removing the supernatant. Incubate at 37°C for 12 h and then replace with fresh DMEM medium containing 10% FBS.

[0205] 4. After 72 hours, add 40 μL of cell lysis buffer (Promega, catalog number E1531) to each well and lyse on a decolorizing shaker for 45 minutes. Transfer 30 μL of cell lysis buffer from each well to a 96-well microplate, and then add 30 μL of firefly luciferase substrate (Promega, catalog number E1501) to each well. Place the microplate in a microplate reader (PerkinElmer) and read the fluorescence value of each well. Calculate the inhibition rate curve and the half-maximal inhibitory concentration (IC50) of the peptide drug.

[0206] 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 both virus treatment and peptide treatment, and cell wells do not include virus treatment.

[0207] Results and Discussion:

[0208] As shown in Table 6, the MjHKU4r-HR2P9 peptide and the MjHKU4r-HR2P10 stapler peptide exhibit highly efficient and broad-spectrum antiviral activity, significantly inhibiting the activity of pseudoviruses such as MERS-CoV, SARS-CoV-2-Delta, SARS-CoV-2-XBB.1.16, and SARS-CoV-2-KP.2. Furthermore, compared to MjHKU4r-HR2P9, the stapler peptide MjHKU4r-HR2P10 showed significantly enhanced antiviral activity, further suggesting that HR2-derived peptides can unexpectedly enhance broad-spectrum antiviral activity by introducing a pair of non-natural amino acids (S5) and forming an intrapeptide linker arm.

[0209] Table 6: Broad-spectrum and highly effective antiviral activity of MjHKU4r-HR2P9 peptide and MjHKU4r-HR2P10 staple peptide:

[0210] Example 5: HR2-derived peptides of closely related coronaviruses inhibit MjHKU4r-CoV infection

[0211] Experimental methods (assessment of viral invasion inhibitory activity, Figure 4):

[0212] 1. Through sequence alignment, peptides such as MERS-HR2P, HKU4-HR2P, HKU5-HR2P, SARS-HR2P, 229E-HR2P, EK1, and EK1C4 were designed and synthesized. All peptides were synthesized and purified by Shanghai Jietai Biotechnology Co., Ltd., with a purity >95%. Figure 4a shows the close relationship between the various coronaviruses. Figure 4b shows the HR2 sequence of each coronavirus and the amino acid sequence alignment of EK1 and EK1C4.

[0213] 2. Using Caco2 cells (ATCC HTB-37Caco2) as target cells, with 10 4 Cell / well plating.

[0214] 3. Dilute the peptide drug 4-fold in DMEM culture medium in a 96-well plate, 50 μL per well.

[0215] 4. Add 50 μL of MjHKU4r-CoV pseudovirus per well to a plate containing a diluted peptide drug. Incubate at 37°C for 30 min to allow the drug and virus to react fully. Add 100 μL of the drug-virus mixture to each well and then to target cells after removing the supernatant. Incubate at 37°C for 12 h, then replace with fresh DMEM medium containing 10% FBS.

[0216] After 5.72 hours, cells were lysed, and firefly luciferase substrate (Promega, catalog number E1501) was added. Fluorescence values ​​for each well were read using a microplate reader (PerkinElmer). Inhibition rate = (fluorescence value of virus wells - fluorescence value of drug-treated wells) / (fluorescence value of virus wells - fluorescence value of cell wells) * 100%. An inhibition rate curve was calculated, and the half-maximal inhibitory concentration (IC50) of the drug was also calculated. Virus wells did not include peptide treatment, drug-treated wells included both virus and peptide treatments, and cell wells did not include virus treatment.

[0217] Results and Discussion:

[0218] 1. As shown in Figure 4c, Figure 5, and Table 5, peptides such as MERS-HR2P, HKU4-HR2P, HKU5-HR2P, MjHKU4r-HR2P2, MjHKU4r-HR2P3, MjHKU4r-HR2P4, MjHKU4r-HR2P5, MjHKU4r-HR2P6, MjHKU4r-HR2P7, or MjHKU4r-HR2P8 exhibit highly efficient antiviral activity against MjHKU4r-CoV, and their antiviral activity is significantly positively correlated with the phylogenetic relationship between the viruses.

[0219] 2. As shown in Figure 4c and Table 5, EK1 and EK1C4 exhibit highly efficient inhibitory effects against S protein-mediated pseudoviral infection of MjHKU4r-CoV. Furthermore, EK1C4 shows significantly enhanced antiviral activity compared to EK1, further suggesting that C-terminal cholesterol molecule modification of HR2-derived peptides can significantly enhance their antiviral activity. Although EK1 differs from MERS-HR2P in sequence, its more efficient inhibitory effect compared to MERS-HR2P is unexpected.

[0220] The coronavirus S protein plays a crucial role in mediating viral infection. Its S protein can be divided into S1 and S2 subunits. The S1 subunit is responsible for recognizing receptors on the target cell surface, while the S2 subunit mediates 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 glycosyl receptors and protein receptors, respectively. With the assistance of host proteases, the fusion peptide at the N-terminus of the S2 subunit is exposed, allowing it to insert into the target cell membrane. The N-terminal heptapeptide repeat domain 1 (HR1) forms a core trimer, which interacts with its C-terminal heptapeptide repeat domain 2 (HR2) to form a "six-helix bundle" (6HB). This "fusion core structure" brings the viral membrane and the target cell membrane closer together, resulting in membrane fusion. The viral genetic material enters the target cell through the fusion pore and replicates to produce new viral particles.

[0221] The S2 subunit of the S protein has become an important target for the development of antiviral entry inhibitors. Peptide drugs are the main carriers of these inhibitors. We have discovered that a derivative peptide (30-60 amino acid residues in length, especially 30-40 amino acid residues) derived from the S2 heptapeptide repeat region 2 (HR2) amino acid sequence of the virus itself can competitively bind to the viral heptapeptide repeat region 1 (HR1) trimer, forming a stable hetero 6HB. This prevents the viral HR2 from binding to the viral HR1 trimer, thus preventing them from forming a homo 6HB, thereby inhibiting the fusion of the viral membrane and the target cell membrane, and thus inhibiting the entry of viral genes into the target cell for replication. Peptide entry inhibitors with the same mechanism of action have played an important role in the treatment of HIV. For example, enfuvirtide (Fuzeon, also known as T20) is the first peptide-based HIV entry inhibitor approved by the US FDA and is currently used in the clinical treatment of AIDS, showing good efficacy in 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 polypeptide (polypeptide 637-666) (US Patent No.: 5,444,044).

[0222] Therefore, we have creatively designed a group of peptides that can effectively inhibit infection of MERS-like coronavirus MjHKU4r-CoV (Manis javanica HKU4-related coronavirus). This invention will be of great significance for the future prevention and treatment of this coronavirus and will directly provide effective candidates for preventive and therapeutic drugs.

[0223] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polypeptide comprising a sequence of Formula 1 below: E-I-S-K-I-N-Y1-T-Y2-L-Y3-L-Y4-Y5-Y6-Y7-Y8-Y9-L-Y10-E-Y11-Y12-K-Y13-L-Y14-Y15-S-Y16-I-D-L-K-E-L 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 a sequence of Formula 2 below: E-I-S-K-I-N-Y17-T-L-L-D-L-S-D-E-M-A-I-L-Y18-E-Y19-Y20-K-Q-L-N-D-S-Y -I-D-L-K-E-L Formula 2, Y17 is T or V, Y18 is L or Q, Y19 is V or A, 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 that differs from the amino acid sequence of SEQ ID NO: 1 by the substitution, addition, deletion, or insertion of one or more amino acid residues, or 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%, or at least 99% identical to 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 that differs from the amino acid sequence of SEQ ID NO: 2 by the substitution, addition, deletion, or insertion of one or more amino acid residues, or 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%, or at least 99% identical to the amino acid sequence of SEQ ID NO:

2.

4. A nucleic acid encoding the polypeptide of any one of claims 1-3.

5. A vector comprising the nucleic acid of claim 4.

6. A host cell comprising the nucleic acid of claim 4 or the vector of claim 5, preferably a eukaryotic host cell or a prokaryotic host cell; preferably, the eukaryotic host cell is a yeast cell, e.g., a Pichia pastoris cell; preferably, the prokaryotic host cell is an E. coli cell. ​ ​ ​ ​ ​ ​ 7. A stapling peptide which is a stapling peptide of the amino acid sequence of any one of SEQ ID NOs: 1, 3-5 and 11-18, wherein two amino acids are replaced with side chain- connectable unnatural amino acids, the side chain-connectable unnatural amino acids are a- amino acids with alkenyl side chains, such as one or more of (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 positions 25 and 29, respectively; Preferably the stapling 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 the two S5s.

8. A composition comprising the polypeptide of any one of claims 1-3, the nucleic acid of claim 4, the vector of claim 5, the host cell of claim 6, the stapling peptide of claim 7; preferably the composition is a medicament, preferably a tablet, a capsule, a dripping pill, an aerosol, a pill, a powder, a solution, a suspension, an emulsion, a granule, a liposome, a transdermal, a suppository or a lyophilized powder injection, or a topical preparation, preferably a topical smearing preparation, such as a topical gel or a topical infiltrating preparation; the composition is a mask, a paper towel, a glove, a clothing, such as a protective clothing, a hand washing article, such as a hand washing liquid or a shower gel.

9. Use of the polypeptide of any one of claims 1-3 or the polypeptide of any one of SEQ ID NOs: 6-10 or a polypeptide derivative or the stapling peptide of claim 7 in the preparation of a medicament or a kit for treating or preventing a MERS-like coronavirus MjHKU4r-CoV infection or a disease caused by a MERS-like coronavirus MjHKU4r-CoV infection; Preferably, wherein the dosage form of the medicament is a tablet, a capsule, a dripping pill, an aerosol, a pill, a powder, a solution, a suspension, an emulsion, a granule, a liposome, a transdermal, a suppository or a lyophilized powder injection; Preferably, wherein the medicament is administered by 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; Preferably, the disease is a respiratory disease or a digestive disease; The polypeptide derivative is a cholesteryl-modified derivative of the polypeptide; Preferably, wherein the polypeptide is linked to the cholesteryl moiety via a linker at the C-terminus; Preferably, wherein the linker is PEGylated; Preferably, wherein the polypeptide is linked to the cholesteryl-modified cysteine via a linker at the C-terminus; Preferably, wherein the linker comprises (GSG)n or (GSGSG)n, wherein n is 1 or 2, Preferably, wherein the linker is -(GSG)n-DPEG4- or -(GSGSG)n-DPEG4-.

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

11. A method of producing a polypeptide comprising the steps of: (1) culturing the host cell according to claim 6 in a suitable medium; and (2) harvesting and isolating the polypeptide.

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

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