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

Abstract

Provided are polypeptides capable of inhibiting MERS-like coronavirus infection and applications thereof. Based on the membrane fusion invasion characteristics of the S2 subunit of the MjHKU4r-CoV coronavirus S protein, the inventors take the HR1 functional domain as a target, and invent a group of polypeptides capable of efficiently inhibiting the membrane fusion invasion process of the MjHKU4r-CoV coronavirus. These polypeptides can inhibit the formation of the 6-HB six-helix fusion core by competitively binding to the HR1 functional domain of the virus, thereby efficiently blocking the process of the MjHKU4r-CoV coronavirus invading target cells. The present application can provide efficient preventive and therapeutic candidate drugs for the prevention and treatment of the MjHKU4r-CoV coronavirus with potential high pathogenicity and cross-species transmission.

Description

[0001] This application claims the priority of Chinese Invention Patent Application No. 202410986087.8, filed on July 22, 2024, 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 cross-species transmission and infect humans, thus seriously threatening human survival and health. For example, the 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 broken out many times in recent years. Therefore, it is urgent to develop effective antiviral drugs for the prevention and treatment of high-risk coronaviruses with the potential to infect humans.

[0004] MERS-CoV 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 currently no effective drug, neutralizing antibody, and vaccine for the envelope glycoprotein (S protein) of this 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] In one aspect, provided herein are polypeptides comprising a sequence of Formula 1 below:

[0008] 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,

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

[0010] In one embodiment, the polypeptide can comprise a sequence of Formula 2 below:

[0011] 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,

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

[0013] In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 by 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.

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

[0015] In one aspect, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is a substitution, addition, deletion, or insertion of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 2, 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.

[0016] In one aspect, provided herein is a stapling peptide that 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 sidechain-annexable unnatural amino acids, the sidechain-annexable unnatural amino acids are alpha-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, to form a staple.

[0017] In one embodiment, 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.

[0018] In one aspect, provided herein is a composition comprising a polypeptide, a nucleic acid, a vector, a host cell, a polypeptide derivative, and / or a stapling peptide as described herein. In one embodiment, 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 preparation, 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 product, such as a hand washing liquid, or a shower gel.

[0019] In one aspect, provided herein is use of a polypeptide described herein or a polypeptide described in any one of SEQ ID NOs: 6-10, a polypeptide derivative, or a stapling peptide 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.

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

[0021] In one aspect, provided herein is a method of inhibiting a MERS-like coronavirus MjHKU4r-CoV in vitro, comprising the step of contacting a polypeptide described herein or a polypeptide derivative of the polypeptide described in any one of SEQ ID NOs: 6-10 or staple peptide with the MERS-like coronavirus MjHKU4r-CoV.

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

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

[0024] (2) harvesting and isolating the polypeptide.

[0025] In one aspect, provided herein is a method of screening for an active ingredient that inhibits a MERS-like coronavirus MjHKU4r-CoV, comprising:

[0026] (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 described herein;

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

[0028] (3) selecting the polypeptide having the ability to inhibit intercellular fusion or the polypeptide having the ability to inhibit MjHKU4r-CoV entry into cells.

[0029] In a first aspect, there is provided the use of a polypeptide described herein, a staple peptide formed from the polypeptide, a polypeptide derivative, a nucleic acid encoding the polypeptide, a composition comprising the polypeptide or staple peptide or polypeptide derivative or nucleic acid, for the manufacture of a medicament or a kit for the treatment or prevention of a MERS-like coronavirus MjHKU4r-CoV infection or a disease caused by a MERS-like coronavirus MjHKU4r-CoV infection, the polypeptide comprising the C-terminal heptad repeat domain 2 of a coronavirus S protein.

[0030] In a second aspect, there is provided a method for the treatment or prevention of a MERS-like coronavirus MjHKU4r-CoV infection or a disease caused by a MERS-like coronavirus MjHKU4r-CoV infection, comprising administering to a subject a polypeptide, staple formed from the polypeptide, polypeptide derivative, nucleic acid encoding a polypeptide, composition comprising said polypeptide or polypeptide derivative or nucleic acid, of the present disclosure, said polypeptide comprising C-terminal heptad repeat domain 2 of a coronavirus S protein.

[0031] In a third aspect, there is provided a polypeptide, staple formed from the polypeptide, polypeptide derivative, nucleic acid encoding a polypeptide, composition comprising said polypeptide or polypeptide derivative or nucleic acid, of the present disclosure, for use in the treatment or prevention of a MERS-like coronavirus MjHKU4r-CoV infection or a disease caused by a MERS-like coronavirus MjHKU4r-CoV infection, said polypeptide comprising C-terminal heptad repeat domain 2 of a coronavirus S protein.

[0032] In a fourth aspect, there is provided a method of inhibiting a MERS-like coronavirus MjHKU4r-CoV in vitro, comprising the step of contacting a polypeptide, staple formed from the polypeptide, polypeptide derivative, nucleic acid encoding a polypeptide, composition comprising said polypeptide or polypeptide derivative or nucleic acid, of the present disclosure, said polypeptide comprising C-terminal heptad repeat domain 2 of a coronavirus S protein, with a MERS-like coronavirus MjHKU4r-CoV.

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

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

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

[0036] Formula (I):

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

[0038] X1is a non-polar uncharged residue, preferably P, or is absent;

[0039] X2is a hydrophilic uncharged residue, preferably N, or is absent;

[0040] X3is an aromatic residue, preferably F, or is absent;

[0041] X4is an aliphatic uncharged residue, preferably A, or is absent;

[0042] X5is E, S or D; X6is I, A, L or V; X7is S, N, T, D or E; X8is K, Q or G; X9is I, F or Y; X10is N; X11is T, Q, V or A; X12is T, A or S; X13is L, F, I or V; X14is L, G or V; X15is D, A or N; X16is L or M; X17is S, Q, E, N or T; X18is D, T, K or S; X19is E, G or F; X20is M, F, L, I; X21is A, T, K, M, L, D or S; X22is I, T, K, M, V, S or R; X23is L or S; X24is Q, N, L, E or S; X25is E, L, Q or N; X26is V, A or K; X27is V, F, I, A or S; X28is K, A or S; X29is Q, K, A, N or E; X30is L or V; X31is N, Q or E; X32is D, E or Y; X33is S, A or T; X34is Y, V or L; X35is I, N or Q; X36is D, A or K; X37is L or N; X38is K, A or Q; X39is E, N or T; X40is L or A; X41is L, I or absent; X42is S, D or absent; X43is K, N or absent; X44is L, I or absent; X45is A, N or absent; X46is S or absent; X47is E, T or absent; X48is L or absent; X49is V or absent; X50is D or absent; X51is L or absent; X52is K or absent; X53is W or absent; X54is L or absent.

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

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

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

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

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

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

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

[0050] In one embodiment of the first aspect, the drug 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.

[0051] In various embodiments of the above aspect, 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 is absent.

[0052] In various embodiments, the staple peptide is a staple peptide of the amino acid sequence of any one of SEQ ID NOs: 1, 3-5, and 11-18, in which two amino acid substitutions are to side-chain connectable unnatural amino acids, the side-chain connectable unnatural amino acids are alpha-amino acids with alkenyl side chains, such as one or more of the following: (S)-2-(4’-pentenyl)alanine (S5), (R)-2-(4’-pentenyl)alanine (R5), (S)-2-(7’-octenyl)alanine (S8), (R)-2-(7’-octenyl)alanine (R8), (S)-2-(4’-pentenyl)glycine (Sg5), and (R)-2-(4’-pentenyl)alanine (Rg5); the positions of the substituted amino acids are positions 25 and 29, respectively. In one embodiment, the staple 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. In various embodiments of the above aspect, the polypeptide comprises (1) the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; (2) an amino acid sequence that is a substitution, addition, deletion, or substitution of one or more amino acid residues in the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; or (3) an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19.

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

[0054] 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;

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

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

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

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

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

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

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

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

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

[0064] In an eighth aspect, provided is a host cell comprising the nucleic acid described herein 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, for example, a Pichia pastoris cell. In one embodiment, the prokaryotic host cell is an Escherichia coli cell.

[0065] In a ninth aspect, a composition comprising a polypeptide or staple described herein, a nucleic acid described herein, a vector described herein, and / or a host cell described herein is provided. In one embodiment, the composition is a medicament. In one embodiment, 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, or a topical preparation, preferably a topical smearing preparation, such as a topical gel or a topical infiltrating preparation; the composition is a face 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 body wash.

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

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

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

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

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

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

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

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

[0074] (3) harvesting and isolating the polypeptide.

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

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

[0077] (2) determining the ability of the polypeptide or staple to inhibit intercellular fusion in an MjHKU4r-CoV S protein-mediated cell fusion assay, or the ability of the polypeptide to inhibit MjHKU4r-CoV entry into a cell in a cell assay; and

[0078] (3) selecting the polypeptide or staple having the ability to inhibit intercellular fusion or the polypeptide or staple having the ability to inhibit MjHKU4r-CoV entry into a cell.

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

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

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

[0082] In one embodiment, the staple comprises an amino acid sequence of two or more of the amino acid substitutions in the amino acid sequence of Formula (I) to a side chain- connectable unnatural amino acid; wherein the side chain- connectable unnatural amino acid is an alpha-amino acid having an alkenyl side chain, such as one or more of the following: (S)-2-(4'- pentenyl)alanine (S5), (R)-2-(4'-pentenyl)alanine (R5), (S)-2-(7'-octenyl)alanine (S8), (R)-2-(7'-octenyl)alanine (R8), (S)-2-(4'-pentenyl)glycine (Sg5) and (R)-2-(4'-pentenyl)alanine (Rg5).

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

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

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

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

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

[0088] Advantages of the present application include:

[0089] 1. The present application provides a group of polypeptide class MERS coronavirus MjHKU4r-CoV infection with high efficiency of inhibition function of the entry inhibitor (any one of SEQ ID NO: 1-19).

[0090] 2. EK1 and EK1C4 of the present application have more effective class MERS coronavirus MjHKU4r-CoV inhibitory activity compared with other polypeptides (any one of SEQ ID NO: 1-6, SEQ ID NO: 9-19), which is unexpected. BRIEF DESCRIPTION OF DRAWINGS

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

[0092] Figure 2 Biophysical activity and characteristics of MjHKU4r-HR1P, MjHKU4r-HR2P polypeptides are shown.

[0093] Figure 3 The inhibitory activity of MjHKU4r-HR1P, MjHKU4r-HR2P polypeptides on MjHKU4r-CoV S protein-mediated membrane fusion and infection process is shown.

[0094] Figure 4 HR2-derived peptides and EK1, EK1C4 of the closely related coronaviruses are shown to inhibit MjHKU4r-CoV infection.

[0095] Figure 5 The inhibitory effect of the optimized polypeptides based on MjHKU4r-HR2P polypeptides: MjHKU4r-HR2P2 - HR2P8 on MjHKU4r-CoV infection is shown. DETAILED DESCRIPTION

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

[0097] These HR2P polypeptides and EK series polypeptides have good inhibitory effect on MERS-like coronavirus MjHKU4r-CoV, which will provide a new strategy for the prevention and control of future related epidemic, and also provide a new theoretical basis for accelerating the research and development of efficient anti-coronavirus polypeptide small molecule drugs. Moreover, the antiviral target of these polypeptides is clear, and the mechanism is clear, which can ensure the safety of its application and the clearness of the optimization path, and is convenient for further development in the future.

[0098] In this document, the polypeptide can be a polypeptide comprising any one of SEQ ID NOs: 1-19 or a variant thereof.

[0099] Table 1: Specific sequences of polypeptides:

[0100]

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

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

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

[0104] Amino acid insertion refers to the insertion of an amino acid residue at an appropriate position in the sequence of an amino acid sequence, for example, any one of SEQ ID NOs: 1-19, and the inserted amino acid residues can be all or part adjacent to each other, or none of the inserted amino acids are adjacent to each other, as long as the polypeptide has inhibitory activity against MERS-like coronavirus MjHKU4r-CoV.

[0105] Amino acid deletion refers to the deletion of 1, 2, or more than 3 amino acids from an amino acid sequence, for example, any one of SEQ ID NOs: 1-19, as long as the polypeptide has inhibitory activity against coronavirus.

[0106] In the present application, substitution can be a conservative amino acid substitution, meaning that a peptide is formed in which 3, more preferably 2 or 1 amino acid is replaced by an amino acid of similar or comparable properties compared to the amino acid sequence of any one of SEQ ID NOs: 1-19. These conservatively varied peptides can be generated by amino acid substitution according to Table 2.

[0107] In the present context, multiple can mean any integer more than 1, such as 2-15 (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15)

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

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

[0110]

[0111] Table 3: Alternative classes of conservatively substituted amino acid residues

[0112]

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

[0114]

[0115] For the purposes of the present application, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle is also modified as follows to give the percentage sequence identity: the "highest scoring matching" segments of the "full" alignment (obtained using the -nobrief option) are identified and then used to calculate the percentage sequence identity.

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

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

[0118] As used herein, "nucleic acid" refers to a plurality of nucleotides linked by internucleotide linkages. The internucleotide linkages can be, for example, phosphodiester bonds. The nucleic acid herein can comprise a polynucleotide that encodes a polypeptide of the present application. The nucleic acid of the present application can further comprise nucleotides that encode a purification tag, for example, a His tag, a GST tag, a MBP tag, a SUMO tag, or a NusA tag, and, if necessary, a nucleotide sequence that encodes a leader sequence, for facilitating subsequent processing of the polypeptide.

[0119] The nucleic acid of the polypeptide of the present application can be codon-optimized for the host cell used. The nucleic acid can be cloned into a suitable expression vector, which is then 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.

[0120] As used herein, the term "vector" is a nucleic acid vehicle into which a polynucleotide can be inserted. The vector is referred to as an expression vector when the vector is capable of causing expression of the protein encoded by the inserted polynucleotide. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector are expressed in the host cell. The vector is well known to those skilled in the art, including but not limited to: a plasmid; a phagemid; a cosmid; an artificial chromosome, such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1 -derived artificial chromosome (PAC); a bacteriophage, such as a lambda phage or a M13 phage; and an animal virus, etc. The vector can contain various elements for controlling expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector can contain a replication initiation site. The vector can comprise the nucleic acid of the present application so as to be introduced into a cell for expression. The vector can comprise expression control elements, such as a promoter, a terminator, and / or an enhancer, operably linked to the nucleic acid.

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

[0122] As used herein, “treatment” and the like, refer to the administration of a drug to a subject for the purpose of obtaining an effect. “Prevention” refers to complete or partial prevention of a disease or symptoms thereof, and treatment refers to achieving partial or complete cure of a disease and / or symptoms caused by the disease. “Treatment” can include: (a) inhibiting the disease, i.e., arresting its development; (b) relieving the disease or symptoms thereof.

[0123] As used herein, the term “coronavirus” belongs to the order Nidovirales, family Coronaviridae, genus Coronavirus, is a class of viruses with envelope, linear single-stranded positive strand RNA genome, is a large class of viruses widely existing in nature. The 5' end of the viral genome has a methylated cap-like structure, and the 3' end has a poly(A) tail, and the full length of the genome is about 27-32 kb, which is the largest known RNA virus genome. Coronaviruses only infect vertebrates and are associated with a variety of diseases in humans and animals, and can cause respiratory, digestive and nervous system diseases in humans and animals. According to the phylogenetic tree, coronaviruses can be divided into four genera: alpha, beta, gamma, and delta, and the beta genus coronavirus can be divided into four independent subgroups: A, B, C, and D groups.

[0124] As used herein, the term “coronavirus S protein” refers to the spike protein of a coronavirus and can refer to a specific S protein, such as a SARS-CoV-2 S protein, a MERS-CoV S protein, a SARS-CoV S protein, or a MjHKU4r-CoV S protein. For example, the SARS-CoV-2 spike protein is a type I membrane glycoprotein that assembles into trimers that constitute the spikes or membrane 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 cognate receptor via a receptor-binding domain (RBD) present in the S1 subunit.

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

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

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

[0128] The term "stapled peptide" is a polypeptide engineered by chemical means, the main feature of which is the stabilization of the alpha-helical structure of the polypeptide by the introduction of all-carbon backbone or other chemical bonds.

[0129] The term "side chain linkable unnatural amino acid" is an alpha-amino acid with an alkenyl side chain, for example selected from the group consisting 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), (R)-2-(4'-pentenyl)alanine (Rg5).

[0130] The present application also provides a method of producing the polypeptides. The method comprises introducing the expression vector into a host cell, followed by culturing in a suitable medium, and then harvesting or isolating the polypeptides or fusion proteins in the supernatant, or in the case of intracellular expression, the host cells can be collected, and then the cells are lysed, thereby collecting the polypeptides. In the case of polypeptides, the polypeptides can be linked to a suitable signal peptide. The signal peptide can be cleaved or not cleaved after harvesting. These techniques are well known to those skilled in the art.

[0131] The polypeptides of the present application can be prepared in a derivative form. For example, the polypeptides can be cholesterol-modified. Methods of cholesterol modification are well known. In one embodiment, the 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. The linkage can be a direct linkage or can be through a linker.

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

[0133] The linker can be PEGylated. Means of PEGylation modification are known to those skilled in the art. As used herein, the term PEGylated linker refers to a linker appended with one or more PEGs. In this context, the PEGylated linker can be PEGylated (GSG)n or (GSGSG)n. n can be any integer, for example 1, 2, 3, 4, 5, 6, and so on. PEG4 indicates the number of repeating ethylene glycol residues is 4.

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

[0135] In this context, the polypeptide or polypeptide derivative or staple peptide of the present application is prepared in the form of a composition or kit. The composition can comprise a suitable carrier, for example a pharmaceutically acceptable carrier. Such a composition can be for external use, for example for use as an external preparation, an external application preparation, for example an external gel or an external infiltrating preparation. Such a composition can be coated on an article in need of viral inhibition, for example but not limited to a mask, a paper towel, a glove, a clothing, for example a protective clothing, and the like. Alternatively, it can be added as an active ingredient to a hand washing article, for example a hand sanitizer, a body wash, and the like. Such a polypeptide or polypeptide derivative or staple peptide can be used to inhibit a coronavirus in vitro to prevent and / or reduce viral infection. Such a polypeptide or polypeptide derivative or staple peptide can be used to prevent or treat a coronavirus infection or a disease caused by a coronavirus in a subject.

[0136] The polypeptide or polypeptide derivative or staple peptide of the present application can also be prepared as a medicament or a pharmaceutical composition. Such a medicament can be 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 agent, a suppository, or a lyophilized powder injection. These medicaments or pharmaceutical compositions can be applied by various administration methods, for example injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, and intraperitoneal injection, intracisternal injection or infusion, and the like, cavity administration, such as rectal, vaginal, and sublingual, respiratory tract administration, such as nasal cavity; mucosal administration, or surface administration, and the like.

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

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

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

[0140] The present application also relates to a polypeptide or derivative or staple peptide, a nucleic acid, a vector, a host cell, a composition, a pharmaceutical composition or a kit for use in preventing or treating a MERS-like coronavirus MjHKU4r-CoV infection or a disease caused by a MERS-like coronavirus MjHKU4r-CoV in a subject / patient.

[0141] Coronavirus

[0142] Coronaviruses are a large family of viruses that circulate widely in nature. Coronaviruses include mammalian coronaviruses and avian coronaviruses. Mammalian coronaviruses are primarily of the alpha, beta genera of coronaviruses and can infect a variety of animals including pigs, dogs, cats, mice, cattle, horses, and the like. Avian coronaviruses are primarily derived from the gamma, delta genera of coronaviruses and can cause disease in a variety of avian species such as chickens, turkeys, sparrows, ducks, geese, pigeons, and the like. The coronavirus described herein can belong to any subgenus of the alpha-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 bat coronavirus HKU8), Myotacovirus (e.g., Myotis ricketti alphacoronavirus Sax-2011), Nyctacovirus (e.g., Nyctalus velutinus alphacoronavirus SC-2013 and Pipistrellus kuhlii coronavirus 3398), Pedacovirus (e.g., porcine epidemic diarrhea virus and Scotophilus bat coronavirus 512), Rhinacovirus (e.g., Rhinolophus bat coronavirus HKU2), Setracovirus (e.g., human coronavirus NL63 and NL63-related bat coronavirus strain BtKYNL63-9b), Soracovirus (e.g., Sorex araneus coronavirus T14), Sunacovirus (e.g., Suncus murinus coronavirus X74), and Tegacovirus (e.g., coronavirus 1A).

[0143] The betacoronavirus genera described herein can include Embecovirus (lineage A), Sarbecovirus (lineage B), Merbecovirus (lineage C), Nobecovirus (lineage D), and Hibecovirus. Thus, in some embodiments, the coronavirus described herein can be any strain or species in any subgenus or lineage of betacoronavirus. The Embecovirus subgenus can include, but is not limited to: betacoronavirus 1 (e.g., bovine coronavirus and human coronavirus OC43), China rat coronavirus HKU24, human coronavirus HKU1, murine coronavirus (e.g., mouse hepatitis virus), and Myodes coronavirus 2JL14. The Sarbecovirus subgenus can include, but is not limited to: SARS-CoV, SARS-CoV2, 16B0133, bat SARS CoV Rfl, 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 Merbecovirus subgenus can include, but is not limited to: Hedgehog coronavirus 1, MERS-CoV, Pipistrellus bat coronavirus HKU5, and Tylonycteris bat coronavirus HKU4. The Nobecovirus subgenus can include, but is not limited to: Eidolon bat coronavirus C704, Rousettus bat coronavirus GCCDC1, and Rousettus bat coronavirus HKU9. The coronavirus antigen can belong to any species or strain in the Hibecovirus subgenus, including, but not limited to: bat Hp-beta coronavirus Zhejiang2013.

[0144] The coronavirus described herein can be phylogenetically clustered into functionally distinct clades. For example, using the nucleotide sequences of the non-structural protein genes ORFla and ORFlb, the coronavirus of the genus Betacoronavirus (Sarbecovirus) can be clustered into clade 1, clade 2, clade 1 / 2, or clade 3 (see, e.g., Hu et al., PLoS Pathog 13(11):e1006698). Thus, the coronavirus can be of any species or strain in any of these clades. The coronavirus can be of any species or strain in clade 1, including but not limited to SARS-CoV, WIV1, LYRa11, Rs7327, Rs4231, Rs4084, and SHC014. The coronavirus can be of 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, Rfl, HeB2013, and 273-2005. The coronavirus can be of any species or strain in clade 1 / 2, including but not limited to SARS-CoV2. The coronavirus can be of any species or strain in clade 3, including but not limited to BM48-31. The coronavirus described herein can be, for example, a coronavirus of SARS, SARS-2, WIV1, SHC014, Rfl, RmYN02, pangl7, RaTG13, and Rs4081.

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

[0146] MjHKU4 r-CoV virus

[0147] MERS-like coronavirus MjHKU4r-CoV is a newly discovered pangolin- derived MERS-like coronavirus. MjHKU4r-CoV virus was found in Malayan pangolin. It is unclear whether pangolins play a role in the transmission of bat coronaviruses to humans. Evolutionary analysis identified MjHKU4r-CoV-1 as a bat HKU4 coronavirus species belonging to the Merbecovirus subgenus. There are 16 key residues in the receptor binding motif (RBM) of the S protein of MERS-CoV that determine binding to hDPP4. By comparison with MERS-CoV, it was found that the RBD of MjHKU4r-CoV-1 has 8 residues identical to MERS-CoV. The potential furin cleavage site (RQQR) in the S protein subdomain 1 (SD1) in the genome of MjHKU4r-CoV-1 is similar to that in the genome of MERS-CoV, suggesting that MjHKU4r-CoV-1 can 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.

[0148] S protein of a coronavirus

[0149] The S protein of a coronavirus can be divided into two important functional subunits, the N-terminal SI subunit and the C-terminal S2 region. The N-terminal SI 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. In interaction with the potential host cell, the SI subunit will recognize and bind to a receptor on the host cell, in particular the angiotensin-converting enzyme 2 (ACE2) receptor, while the S2 subunit is the most conserved component in the S protein, which will be responsible for fusing the envelope of the virus with the host cell membrane. See, e.g., Shang et al., PLoS Pathog. 2020 Mar; 16(3):e1008392.). Each monomer of the trimeric S protein trimer contains two subunits SI 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 SI / S2 site of the infected cell by a furin-mediated cleavage. In vivo, a subsequent serine protease-mediated cleavage event occurs at the S2’ site within SI.

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

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

[0152] Polypeptides

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

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

[0155] Stapled peptides

[0156] Also provided herein are stapled peptides. The stapled peptides can be stapled peptides of the polypeptides shown in Table 1. Methods of making stapled peptides (e.g., by using olefin metathesis reactions) are known to those of skill in the art. For example, two or more side chain-attachable unnatural amino acids can be substituted in the amino acid sequence of the polypeptide, and the side chains of the unnatural amino acids are attached. The side chain-attachable unnatural amino acids are alpha-amino acids having alkenyl side chains, e.g., selected from the group consisting 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), (R)-2-(4’-pentenyl)alanine (Rg5). The side chains of the unnatural amino acids can form 1 or 2 or more rings, preferably 1 or 2 rings, upon attachment.

[0157] The staple peptide can comprise the amino acid sequence of any one of SEQ ID NOs: 2-7 and 9-19, wherein the amino acid residues at positions 25 and 29 are replaced with a side chain-attachable unnatural amino acid, preferably (S)-2-(4'-pentenyl)alanine (S5) or (R)-2-(4'-pentenyl)alanine (R5), relative to SEQ ID NO: 18. The unnatural amino acid can form a covalent attachment through the side chain. In particular, the staple peptide comprises the amino acid sequence of EISKINVTLLDLSDEMAILLEAIK-S5-LND-S5-YIDLKEL, wherein S5 is (S)-2-(4'-pentenyl)alanine, and a covalent attachment is formed between the two S5s.

[0158] Polypeptide derivatives

[0159] The polypeptides described herein can be modified to produce polypeptide derivatives. In some embodiments, the polypeptides can be modified by conjugation with PEG. The PEG modification sites of the polypeptides are at the N-terminus, C-terminus, Lys side chain, and Cys thiol group. The PEG used for modification has a monomolecular weight ranging between PEG2-PEG24. PEG modification can be used to improve proteolytic stability, biodistribution, and peptide solubility. The introduction of PEG chains on the polypeptides can improve their pharmacological properties and also inhibit the hydrolysis of the polypeptides by proteolytic enzymes. Because of the prolonged effective half-life of PEG polypeptides in vivo, normal therapeutic levels can be maintained by using lower doses and less frequent polypeptide drugs. In some embodiments, the polypeptides described herein can be cholesterol-modified. Cholesterol-modified polypeptide drugs can improve the drug activity and in vivo / ex vivo half-life of the polypeptides, have weaker toxic side effects, and also cross the blood-brain barrier. In some embodiments, the polypeptide derivative is a cholesterol-modified derivative of the polypeptide. In some embodiments, the polypeptide derivative is a PEG-modified derivative of the polypeptide. In some embodiments, the polypeptide derivative is a cholesterol-modified and PEG-modified derivative of the polypeptide. In some embodiments, the polypeptide is linked to a cholesterol moiety at the C-terminus via a linker. In some embodiments, the linker is PEGylated. In some embodiments, the polypeptide is linked to a cholesterol-modified cysteine at the 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.

[0160] Pharmaceutical compositions and kits

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

[0162] The pharmaceutical composition can also include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any and all solvents, co-solvents, vehicles, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are biologically compatible and otherwise not deleterious in use. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the pharmaceutical composition is contemplated. Additional active ingredients can also be incorporated into the pharmaceutical composition. In addition, various excipients can 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 the inclusion of 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, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal, suppository, or lyophilized powder injection. In some embodiments, the pharmaceutical composition 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.

[0163] In some aspects, the present application relates to a kit comprising a polypeptide described herein, a nucleic acid encoding a polypeptide, a vector comprising a nucleic acid, a cell comprising a nucleic acid or a vector. In some embodiments, the present application provides a kit for producing a single dose administration unit. In some aspects, the kit of the present application can contain a first container containing a polypeptide described herein, a nucleic acid encoding a polypeptide, a vector comprising a nucleic acid, a cell comprising a nucleic acid or a vector and a second container containing an aqueous formulation. In some embodiments, the kit contains a diluent, a buffer, etc. solution for diluting or reconstituting a powder. In some embodiments, the kit comprises instructions for use of a polypeptide described herein, a nucleic acid encoding a polypeptide, a vector comprising a nucleic acid, a cell comprising a nucleic acid or a vector.

[0164] Polypeptide drug screening method

[0165] The present application also provides a method of screening for an active ingredient that inhibits a MERS-like coronavirus MjHKU4r-CoV. The method can comprise producing a library of polypeptides, which can comprise polypeptides derived from the C-terminal heptad repeat domain 2 of the S protein of a coronavirus. The coronavirus can be an alpha or beta genus coronavirus, preferably HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV. The library of polypeptides can comprise an 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 library of polypeptides can comprise Formula II: X1-X2-X3-X4-EISKIN-Y1-T-Y2-L-Y3-L-Y4-Y5-Y6-Y7-Y8-Y9-L-Y10-E-Y11-Y12-K-Y13-L-Y14-Y15-S-Y16-IDLKEL. The variables X1-X54 and Y1-Y16 are defined elsewhere herein. Methods of producing a library of polypeptides are known to those skilled in the art, for example can be by chemical synthesis or by biological synthesis.

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

[0167] In some embodiments, the stapled peptide library comprises an amino acid sequence of Formula (I) with two or more amino acids replaced by a side chain-attachable unnatural amino acid; wherein the side chain-attachable unnatural amino acid is an alpha-amino acid with an alkenyl side chain, for example 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). In some embodiments, the number of replaced amino acids is two, and the positions of the replaced amino acids are the i-th and i+3-th positions from X1, respectively, where 1≤i≤51, or the i-th and i+4-th positions, where 1≤i≤50.

[0168] In some embodiments, the staple peptide comprises an amino acid sequence of Formula (II) with two or more amino acid replacements to side chain attachable unnatural amino acids; wherein the side chain attachable 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). In some embodiments, the number of replaced amino acids is two, and the positions of the replaced amino acids are the i-th and i+4-th positions from X1, respectively, where 1≤i≤50.

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

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

[0171] In some embodiments, a member of a polypeptide library or staple peptide library can be contacted with MjHKU4r-CoV pseudovirus and target cells (e.g., Caco2) expressing a human coronavirus receptor. If a member of the polypeptide library or staple peptide library inhibits MjHKU4r-CoV pseudovirus entry into the target cells, then the member is selected as a candidate. In some embodiments, the member is assayed for the ability to inhibit MjHKU4r-CoV pseudovirus entry into cells expressing a human coronavirus receptor.

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

[0173] In some embodiments, the polypeptide library comprises (1) an amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; (2) an amino acid sequence that is a substitution, addition, deletion, or substitution of one or more amino acid residues in an amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19; or (3) an amino acid sequence that is at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence of any one of SEQ ID NOs: 1-7 and 9-19.

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

[0175] In some embodiments, the staple peptide library comprises an amino acid sequence of any one of SEQ ID NOs: 2-7 and 9-19 or a variant amino acid sequence thereof as defined above in terms of identity. In some embodiments, the amino acid residue is replaced with a side chain connectable unnatural amino acid, preferably (S)-2-(4’-pentenyl)alanine (S5) or (R)-2-(4’-pentenyl)alanine (R5), at position i and i+3 or i and i+4 relative to SEQ ID NO: 18. In some embodiments, the staple peptide library comprises an amino acid sequence of SEQ ID NO: 18, wherein the amino acid residue is replaced with a side chain connectable unnatural amino acid, preferably (S)-2-(4’-pentenyl)alanine (S5) or (R)-2-(4’-pentenyl)alanine (R5), at position i and i+3 or i and i+4.

[0176] Examples

[0177] The present application is further illustrated by the following examples, but any example or combination thereof should not be interpreted as limiting the scope or implementation of the present application. The scope of the present application is defined by the appended claims, and those skilled in the art can clearly understand the scope defined by the claims in combination with the present specification and general common knowledge in the art. Any modification or change to the technical solutions of the present application made by those skilled in the art without departing from the spirit and scope of the present application shall also be included in the scope of the present application.

[0178] General methods for PCR, cloning, ligation, transfection of nucleotides, etc. are well known to those skilled in the art and can be found, for example, in "Molecular cloning: A laboratory manual", Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, New York; Ausubel, F. M. et al. (eds); "Current protocols in Molecular Biology", John Wiley and Sons (1995); Harwood, C. R. and Cutting, S. M. (eds); "DNA Cloning: A Practical Approach, Volumes I and II", D. N. Glover ed. (1985); "Oligonucleotide Synthesis", M. J. Gait ed. (1984); "Nucleic Acid Hybridization", B. D. Hames & S. J. Higgins eds. (1985); "A Practical Guide To Molecular Cloning", B. Perbal (1984).

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

[0180] As shown in Figure 1, by homology alignment with MERS-CoV S2 protein, the specific location of HR1 and HR2 functional domains in MjHKU4r-CoV S2 protein and the distribution of key amino acid sites were identified, thus MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides were designed. Figure 1 As shown in Figure 1, by homology alignment with MERS-CoV S2 protein, the specific location of HR1 and HR2 functional domains in MjHKU4r-CoV S2 protein and the distribution of key amino acid sites were identified, thus MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides were designed. Figure 1 As shown in Figure 1, by homology alignment with MERS-CoV S2 protein, the specific location of HR1 and HR2 functional domains in MjHKU4r-CoV S2 protein and the distribution of key amino acid sites were identified, thus MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides were designed.

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

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

[0183] Experimental methods:

[0184] 1. Native polyacrylamide gel electrophoresis (N-PAGE). Gradient concentrations of MjHKU4r-HR1P and MjHKU4r-HR2P polypeptide mixtures were prepared, with MjHKU4r-HR1 polypeptide final concentrations of 0, 100, 300, and 600 µM, and MjHKU4r-HR2 polypeptide final concentration of 100 µM, incubated at 37°C for 30 minutes. 5x high pH loading buffer (Supplier: Tian'enze TIANDZ, Product No. 81212A-30) was mixed with 4x volume of the peptide mixture. Then, 20 µl of each sample was loaded onto a 12% native gel and electrophoresed at 125V constant voltage for 4 hours at room temperature. Coomassie brilliant blue staining was performed. The results showed that, after mixing MjHKU4r-HR2P and MjHKU4r-HR1P polypeptides, a new band possibly a six-helix complex (i.e., 6-HB) was produced (Figure Figure 2 a in the figure).

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

[0186] Results and analysis:

[0187] 1. As shown in Figure 2As shown in Figure 1a, after mixing MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides at different concentrations, a new band possibly a six-helix complex was produced, indicating that MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides can interact with each other.

[0188] 2. As Figure 2 As shown in Figure 1b-c, the polypeptide alone is a low helix structure, and when MjHKU4r-HR2P and MjHKU4r-HR1P are mixed, a high helix structure appears, indicating that MjHKU4r-HR1P and MjHKU4r-HR2P polypeptides interact in the form of a six-helix. The Tm value of the MjHKU4r-HR1P and MjHKU4r-HR2P polypeptide mixture is higher (= 84.8°C), indicating that the six-helix structure is stable.

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

[0190] Experimental method one (membrane fusion inhibition activity evaluation, Figure 3 As shown in Figure 1a-b):

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

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

[0193]

[0194] 2. Blow and suck the 293T / MjHKU4r / EGFP cells and 293T / EGFP cells mentioned above, centrifuge, resuspend the cells with fresh DMEM medium, and adjust the cell concentration to 2x10 5 6 μL of the polypeptide drug solution was added to 50 μL of the gradient diluted polypeptide drug (50 μL) at 37°C, and incubated for 30 min.

[0195] 3. Take 100 μL of the cell / polypeptide drug mixture and add it to the target cells Caco2 (ATCC HTB-37) that have been plated in a 96-well plate. Incubate at 37°C in 5% CO2 for 2-4 hours, and observe and record the fusion of the cells using the green fluorescence channel of the fluorescence microscope. For the process of coronavirus S protein-mediated membrane fusion and infection, please refer to the patent applications previously applied by the applicant, such as CN117777247A and CN116440285A. Figure 3 Figure 1a shows the fluorescence photos of membrane fusion under different concentrations of MjHKU4r-HR1P and MjHKU4r-HR2P. Figure 3 Figure 1b shows that the IC50 of MjHKU4r-HR1P and MjHKU4r-HR2P is 0.037 μM and greater than 10 μM, respectively.

[0196] Experimental method two (evaluation of virus invasion inhibition activity, Figure 3 Figure 1c):

[0197] 1. Dissolve the polypeptides MjHKU4r-HR1P and MjHKU4r-HR2P with DMSO and determine the polypeptide concentration.

[0198] 2. MjHKU4r-CoV pseudovirus packaging (see L. Lu, Q. Liu, Y. Zhu, K.-H. Chan, L. Qin, Y. Li, Q. Wang, J. F.-W. Chan, L. Du, F. Yu, C. Ma, S. Ye, K.-Y. Yuen, R. Zhang, S. Jiang, Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor. Nat. Commun. 5, 3067 (2014)): PC-MjHKU4r-S plasmid is obtained by connecting MjHKU4r-S encoding gene into PC-DNA3.1(+) vector at two enzyme cutting sites of BamHI and Xhol. Then MjHKU4r-CoV pseudovirus is packaged by PC-MjHKU4r-S plasmid and HIV-1 backbone plasmid (pNL4-3.Luc.R-.E-). Specifically, 293T cells are digested 24 h before transfection, plated in 10 cm tissue culture dishes (2 x 10 6 / dish). The cells are replaced with pre-warmed fresh DMEM medium (containing 10% FBS) 2 h before transfection. Two 1.5 mL EP tubes are used for transfection. In EP tube 1, 500 μL of 0.9% NaCl solution is added, which contains 20 μg of PC-MjHKU4r-S plasmid and pNL4-3.Luc.R-.E- plasmid. In EP tube 2, 500 μL of 0.9% NaCl is also added, and 2 μL of transfection reagent vigofect is added, which is allowed to stand for 5 minutes. The 500 μL solution in EP tube 2 is added dropwise to EP tube 1, and the mixture is mixed with a gun head during the addition. The mixture is allowed to stand at room temperature for 15 min; the above 1 mL mixture is added dropwise and uniformly to the previously plated 293T cell culture dish. After 8~10 h of transfection, 10 mL of fresh DMEM medium containing 10% FBS is added; after 48 h, the supernatant containing the pseudovirus is collected at 4000 rpm for 4 min to remove cell debris, filtered with a 0.45 μm sterile filter, aliquoted and stored at -80°C for later use, and aliquoted and stored at -80°C.

[0199] 3. Caco2 (ATCC HTB-37) cells are prepared into a suspension after trypsin digestion, and 10 4 cells are added to each well after adjusting the cell concentration.

[0200] 4. The peptide drug was serially diluted 4-fold with DMEM medium in a 96-well plate, 50 µL per well. MjHKU4r-CoV pseudovirus was added to the peptide drug dilution plate at 50 µL / well. The plate was incubated at 37°C for 30 min to allow sufficient interaction between the peptide drug and the virus. 100 µL of the peptide drug-virus mixture was then added to each well of the target cells after supernatant removal. The plate was incubated at 37°C for 12 h, then replaced with fresh DMEM medium containing 10% FBS.

[0201] After 6.72 h, 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.

[0202] 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.

[0203] Results and Discussion:

[0204] 1. For example Figure 3 As shown in Figure b and Table 5, the MjHKU4r-HR2P peptide and the MjHKU4r-HR2 PEK 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.

[0205] 2. For example Figure 3 As shown in Figure c 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.

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

[0207]

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

[0209] Experimental method (evaluation of virus invasion inhibitory activity):

[0210] 1. Caco2 cells (purchased from ATCC, product number HTB-37 Caco2) were used as target cells, plated at 10 4 cells / well, and incubated at 37°C for 24 hours.

[0211] 2. Polypeptide drugs MjHKU4r-HR2P9 and MjHKU4r-HR2P10 were diluted in DMEM culture medium in a 96-well plate using a 4-fold gradient (5 µM initial concentration and 5 gradient gradients of 4-fold dilution), 50 µL per well.

[0212] 3. 50 µL per well of MERS-CoV or SARS-CoV-2 variant pseudovirus (SARS-CoV-2-Delta, SARS-CoV-2-BA.2.75, SARS-CoV-2-XBB.1.16, and SARS-CoV-2-KP.2) (Reference: Xia S, 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) was added to the plate wells containing polypeptide drug dilutions, and the polypeptide drugs were allowed to fully interact with the virus at 37°C for 30 min. 100 µL of polypeptide drug-virus mixture was added to the target cells in each well after removing the supernatant, and the cells were incubated at 37°C for 12 h, after which fresh DMEM culture medium containing 10% FBS was replaced.

[0213] 4. After 72 h, 40 μΐ of cell lysis solution (Promega, Cat# E1531) was added to each well, and the plate was shaken on a shaker for 45 min. Then, 30 μΐ of cell lysis solution was removed from each well to a 96-well plate, and 30 μΐ of luciferase substrate (Promega, Cat# E1501) was added to each well. The plate was read in a microplate reader (PerkinElmer) to obtain the fluorescence value of each well. The inhibition rate curve was calculated, and the half maximal inhibitory concentration (IC50) of the polypeptide drug was calculated.

[0214] Inhibition rate = (virus well fluorescence value - drug treatment well fluorescence value) / (virus well fluorescence value - cell well fluorescence value) x 100%; the virus well does not include polypeptide treatment, the drug treatment well includes virus treatment and polypeptide treatment, and the cell well does not include virus treatment.

[0215] Results and discussion:

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

[0217] Table 6: Broad-spectrum and high-efficiency antiviral activity of MjHKU4r-HR2P9 polypeptide and MjHKU4r-HR2P10 stapled peptide:

[0218]

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

[0220] Experimental method (evaluation of virus invasion inhibition activity, Figure 4 ):

[0221] 1. By sequence alignment, MERS-HR2P, HKU4-HR2P, HKU5-HR2P, SARS-HR2P, 229E-HR2P, EK1, EK1C4 and other polypeptides were designed and synthesized. All polypeptides were synthesized and purified by Shanghai Jiepeibio Technology Co., Ltd., and the purity was > 95%. Figure 4 FIG. 1A shows the relatedness between various coronaviruses. Figure 4Figure b shows the HR2 sequence of each coronavirus and the amino acid sequence alignment of EK1 and EK1C4.

[0222] 2. Using Caco2 cells (ATCC HTB-37Caco2) as target cells, with 10 4 Cell / well plating.

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

[0224] 4. Add 50 µL of MjHKU4r-CoV pseudovirus per well to a plate containing a peptide drug dilution. 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.

[0225] 5. After 72 h, cells were lysed, and firefly luciferase substrate (Promega, catalog number E1501) was added. Fluorescence values ​​were read from each well 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.

[0226] Results and Discussion:

[0227] 1. For example Figure 4 Figure c in the middle Figure 5 As shown in 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 effective antiviral activity against MjHKU4r-CoV, and their antiviral activity is significantly positively correlated with the phylogenetic relationship between the viruses.

[0228] 2. For example Figure 4As shown in Figure 6C and Table 5, EK1 and EK1C4 have high inhibitory effect on S protein-mediated pseudovirus infection of MjHKU4r-CoV, and the antiviral activity of EK1C4 is significantly improved compared with EK1, further suggesting that the modification of the C-terminal cholesterol molecule of the HR2-derived peptide can significantly improve the antiviral activity of the polypeptide. Although EK1 has sequence differences from MERS-HR2P, it has a more efficient inhibitory effect than MERS-HR2P, which is unexpected.

[0229] The coronavirus S protein plays an important role in mediating the virus infection process. Its S protein can be divided into S1 and S2 subunits, S1 subunit is responsible for recognizing target cell surface receptors, and S2 subunit is responsible for mediating the fusion of virus and host cell membranes. S1 subunit contains two important functional domains: N-terminal domain (NTD) and 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, while the N-terminal heptad repeat 1 (HR1) forms a core trimer and interacts with its C-terminal heptad repeat 2 (HR2) to form a "six-helix bundle" (6HB). This "fusion core structure" allows the virus membrane and target cell membrane to be pulled close together, resulting in membrane fusion, and the genetic material of the virus enters the target cell to replicate and produce new virus particles.

[0230] The S2 subunit of the S protein has become an important target for the development of antiviral entry inhibitors. Polypeptide drugs are the main carriers of such inhibitors. We found that the derived polypeptide (30-60 amino acid residues in length, especially 30-40 amino acid residues) derived from the S2 heptad repeat region 2 (HR2) amino acid sequence of the virus itself can competitively bind to the virus heptad repeat region 1 (HR1) trimer, forming a stable heterogenous 6HB, preventing the virus HR2 from binding to the virus HR1 trimer, so that they cannot form a homogenous 6HB, thereby inhibiting the fusion of the virus membrane and the target cell membrane, and thus inhibiting the entry of the virus' s genes into the target cell for replication. Polypeptide 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 polypeptide HIV entry inhibitor approved by the FDA, and it is currently used in the clinical treatment of AIDS and has good efficacy for patients who have developed resistance to HIV reverse transcriptase inhibitors. One of the inventors of this patent, Jiang Shibob, is the inventor of the T20 prototype polypeptide (polypeptide 637-666) (U.S. Patent No. 5,444,044).

[0231] Therefore, we here creatively design a group of polypeptides which can have high efficient inhibitory effect on the infection of MERS coronavirus MjHKU4r-CoV (Manis javanica HKU4-related coronavirus), and the invention will have great significance for the future prevention and treatment of the coronavirus, and will directly provide effective prophylactic and therapeutic drug candidates.

[0232] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A polypeptide having the amino acid sequence SEQ ID NO: 1 or SEQ ID NO:

18.

2. A nucleic acid encoding the polypeptide according to claim 1.

3. A vector comprising the nucleic acid according to claim 2.

4. A host cell comprising the nucleic acid according to claim 2 or the vector according to claim 3.

5. The host cell according to claim 4, wherein it is a eukaryotic host cell or a prokaryotic host cell.

6. The host cell according to claim 5, wherein the eukaryotic host cell is a yeast cell.

7. The host cell according to claim 6, wherein the yeast cell is a Pichia pastoris cell.

8. The host cell according to claim 5, wherein the prokaryotic host cell is an Escherichia coli cell.

9. A staple peptide having the amino acid sequence EISKINVTLLDLSDEMAILLEAIK-S5-LND-S5-YIDLKEL shown in SEQ ID NO: 19, wherein S5 is (S)-2-(4'-pentene)alanine and the two S5s form a staple.

10. A composition comprising the polypeptide of claim 1, or the nucleic acid of claim 2, or the vector of claim 3, or the host cell of any one of claims 4-8, or the staple peptide of claim 9.

11. The composition according to claim 10, wherein the composition is a pharmaceutical product.

12. The composition according to claim 11, wherein the drug is a tablet, capsule, drop, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, suppository, or lyophilized powder for injection.

13. The composition according to claim 10, wherein it is a topical preparation.

14. The composition of claim 13, wherein the topical formulation is a topical application formulation.

15. The composition of claim 13, wherein the topical formulation is a topical gel or a topical soaking formulation.

16. The composition according to claim 10, wherein the composition is a mask, tissue, gloves, or clothing.

17. The composition of claim 16, wherein the clothing is protective clothing.

18. The composition according to claim 10, which is a handwashing product.

19. The composition of claim 18, wherein the handwashing product is a hand sanitizer.

20. The composition according to claim 10, wherein it is a shower gel.

21. Use of the polypeptide of claim 1 or a derivative thereof or the stapled peptide of claim 9 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, wherein the derivative thereof is a cholesterol-modified derivative of the polypeptide.

22. The use according to claim 21, wherein the dosage form of the drug is a tablet, capsule, drop, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, suppository, or lyophilized powder for injection.

23. The use according to claim 21, wherein the drug is administered by injection, cavity administration, mucosal administration, or surface administration.

24. The use according to claim 23, wherein the injection is administered subcutaneously, intravenously, intramuscularly, intraperitoneally, intracerebralally, or via infusion.

25. The use according to claim 23, wherein the drug is administered sublingually.

26. The use according to claim 21, wherein the drug is administered via the respiratory tract.

27. The use according to claim 26, wherein the respiratory administration is administered via the nasal cavity.

28. The use according to claim 21, wherein the disease is a respiratory disease or a digestive disease.

29. The use according to claim 21, wherein the polypeptide is linked to the cholesterol moiety at the C-terminus via a linker.

30. The use according to claim 29, wherein the connector is PEGylated.

31. The use according to claim 30, wherein the polypeptide is linked to a cholesterol-modified cysteine ​​residue at its C-terminus via a linker.

32. The use according to claim 31, wherein the connector comprises (GSG)n or (GSGSG)n, wherein n is 1 or 2.

33. The use according to claim 32, wherein the connector is -(GSG)n-DPEG4- or -(GSGSG)n-DPEG4-.

34. A method for inhibiting MERS-like coronavirus MjHKU4r-CoV in vitro, comprising the step of contacting a polypeptide according to claim 1 or a derivative thereof or a stapled peptide according to claim 9 with MERS-like coronavirus MjHKU4r-CoV, wherein the polypeptide derivative is a cholesterol-modified derivative of the polypeptide.

35. The method of claim 34, wherein the polypeptide is linked to the cholesterol moiety at its C-terminus via a linker.

36. The method of claim 35, wherein the connector is PEGylated.

37. The method of claim 36, wherein the polypeptide is linked to a cholesterol-modified cysteine ​​residue at its C-terminus via a linker.

38. The method of claim 37, wherein the connector comprises (GSG)n or (GSGSG)n, wherein n is 1 or 2.

39. The method of claim 38, wherein the connector is -(GSG)n-DPEG4- or -(GSGSG)n-DPEG4-.

40. A method for preparing polypeptides, comprising the following steps: (1) Culture the host cell according to any one of claims 4-8 in a suitable culture medium; and (2) Harvest and separate the polypeptides.

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