Broad-spectrum Anti-β-coronavirus short polypeptide and derivative thereof

Abstract

Disclosed in the present invention are a short polypeptide having broad-spectrum inhibition effect on β human coronaviruses invading host cells, and the pharmaceutical use thereof. Reasonable drug design is carried out on the basis of the universality and conservation of a six-α-helix bundle (6-HB) structure of spike proteins of coronaviruses during the process of β human coronaviruses entering host cells; and α-helix conformation restriction is performed with respect to a polypeptide derived from the HR2 domain in the 6-HB, so as to provide the short polypeptide that has a broad-spectrum inhibitory effect on infections of two or more β human coronaviruses (such as SARS-CoV, MERS-CoV, SARS-CoV-2 and OC43). The polypeptide has important advantages such as strong antiviral activity, a short sequence and easy synthesis. The present invention will prospectively provide candidate drug reserve and technical reserve for research and development of drugs used for emergency prevention and control of emerging or re-emerging human coronavirus infection epidemics in the future.

Description

Short peptides and their derivatives with broad-spectrum anti-β coronavirus effects Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a polypeptide and its use, a pharmaceutical composition comprising the polypeptide and its use, and also specifically to a method for treating a disease using the polypeptide or the pharmaceutical composition. Background Art

[0002] Coronaviruses (CoVs) are widespread in nature, infecting humans and causing respiratory illness. The Coronaviridae family is divided into four genera: alpha, beta, gamma, and delta. Betacoronaviruses are further divided into four lineages: A, B, C, and D. Alphacoronaviruses and betacoronaviruses are commonly found in mammals, while gammacoronaviruses and deltacoronaviruses are primarily associated with infecting birds. Currently, there are seven known coronaviruses capable of infecting humans. Human coronavirus NL63 (HCoV-NL63) and human coronavirus 229E (HCoV-229E) are alphacoronaviruses. The other five are all betacoronaviruses: human coronavirus OC43 (HCoV-OC43), Hong Kong coronavirus type 1 (HCoV-HKU1), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and novel coronavirus (SARS-CoV-2). From the perspective of pathogenicity, among the betacoronaviruses discovered so far, HCoV-OC43 and HCoV-HKU1 are low-pathogenic coronaviruses, while SARS-CoV, MERS-CoV, and SARS-CoV-2 are highly pathogenic human coronaviruses. Coronaviruses are able to cross species barriers and infect humans rapidly and unpredictably. Moreover, their genetic mutation characteristics during human-to-human transmission can easily lead to the emergence of variant strains with greater infectivity and pathogenicity. Currently, there are no clinically effective specific therapeutic drugs for human coronavirus infections. Therefore, to address the threat posed by betacoronaviruses to human health and social public security, there is an urgent need to develop broad-spectrum antiviral drugs targeting common and highly conserved potential targets in the replication cycle of this type of virus to proactively defend against future outbreaks of highly pathogenic betacoronavirus (β-HCoV) infections.

[0003] When β-HCoV invades host cells, the spike glycoprotein (S protein) on the viral surface plays a crucial role. The S protein is divided into S1 and S2 subunits. Initially, the S1 subunit is responsible for binding to receptors on the host cell surface; subsequently, the S2 subunit undergoes conformational changes. During this process, the HR1 (heptad repeat 1) and HR2 (heptad repeat 2) domains of the S2 subunit interact to form a six-helix bundle (6-HB), releasing energy that draws the viral and host cell membranes connected at either end of the S2 subunit closer together, ultimately leading to fusion. Current research reports have resolved the crystal structures of the fusion core 6-HB of various β-HCoVs. Comparison reveals that the three-dimensional structures of the β-HCoV 6-HB are highly similar. Further alignment of the amino acid sequences of the corresponding 6-HB regions reveals that their primary structures are also highly conserved. The addition of a peptide fragment derived from the coronavirus HR2 domain (called C peptide) can effectively interfere with the formation of the virus's own 6-HB, thereby blocking the fusion of the virus with the target cell membrane. Currently, multiple specific β-HCoV fusion inhibitors have been discovered, such as the peptide CP-1 derived from the SARS-CoV HR2 domain, the peptide HR2P derived from the MERS-CoV HR2 domain, and the peptide 2019-nCoV-HR2P derived from the SARS-CoV-2 HR2 domain. They all exert antiviral effects by specifically inhibiting the formation of the corresponding coronavirus 6HB. Based on these works, the Jiang Shibo / Lu Lu team of Fudan University found that the peptide OC43-HR2P derived from the HR2 domain of human coronavirus OC43 (HCoV-OC43) and its derivative EK1 can block the formation of multiple β-HCoV 6-HB and exhibit a broad-spectrum inhibitory effect on β-HCoV at the micromolar level; the He Yuxian team of the Chinese Academy of Medical Sciences found that the lipopeptide IPB02 derived from the HR2 domain of SARS-CoV-2 can also exert broad-spectrum anti-β-HCoV activity at the low micromolar level.

[0004] Interestingly, most of these anti-coronavirus peptides have long amino acid sequences. For example, the specific anti-SARS-CoV peptide CP-1 has 37 amino acid residues; the specific anti-MERS-CoV peptide HR2P has 36 amino acid residues; the specific anti-SARS-CoV-2 peptide 2019-nCoV-HR2P has 36 amino acid residues; and the broad-spectrum anti-coronavirus peptides EK1 and IPB02 both contain 36 amino acid residues. Research by the Jiang Shibo / Lu Lu team showed that truncation of the anti-MERS-CoV peptide HR2P to 19 amino acids completely abolished its anti-MERS-CoV activity. Research by He Yuxian's team also demonstrated that truncating only 10 residues from the amino terminus of IPB02 significantly reduced its anti-coronavirus activity. Undoubtedly, peptide length is closely related to the difficulty and cost of its preparation. High production costs significantly limit the clinical application of peptide-based therapeutics. For example, T20 peptide (containing 36 amino acids), an AIDS treatment drug approved by the US FDA in 2003, is not used as a first-line drug because its annual treatment cost per patient is as high as US$20,000.

[0005] In summary, there is an urgent need in this field to discover short, broad-spectrum anti-β-HCoV peptides that are short in sequence length, easy to synthesize, low in cost, and have potential clinical application value.

[0006] It should be noted that the approaches described in this section are not necessarily approaches that have been previously conceived or employed. Unless otherwise indicated, it should not be assumed that any approach described in this section is prior art simply because it is included in this section. Similarly, unless otherwise indicated, the issues mentioned in this section should not be considered to have been recognized in any prior art.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to overcome the defects of the prior art and provide a polypeptide with a length of 21-22 amino acid residues, a pharmaceutically acceptable salt thereof, or a derivative thereof that can broadly inhibit infection by human β coronaviruses.

[0009] According to one embodiment of the present application, a conformationally restricted polypeptide of the 19-peptide shown in SEQ ID NO: 1 can be provided, characterized in that 2-3 natural amino acids are added to the N-terminus of the 19-peptide shown in SEQ ID NO: 1, or 1-2 natural amino acids are added to the N-terminus and the C-terminus respectively to form a conformationally restricted polypeptide backbone; the conformationally restricted polypeptide backbone is sequentially substituted with a first, second, third, and fourth non-natural amino acid (4) from the N-terminus to the C-terminus. n ), the first and second unnatural amino acids (S n ) are separated by 3 amino acids and the first and second unnatural amino acids (S n) form a covalent bond between the third and fourth unnatural amino acids (S n ) are separated by 3 amino acids and between the third and fourth unnatural amino acids (S n ) are covalently linked to form a stable α-helical structure; the conformationally restricted polypeptide backbone corresponds to SEQ ID NO: 1, wherein the first amino acid at the N-terminus is L, the fifth amino acid is M or Nle, the eighth amino acid is L, the 12th amino acid is I, the 15th amino acid is L, and the 19th amino acid is Y; the first, second, third, and fourth unnatural amino acids (S n ) is selected from positions other than position 1, position 5, position 8, position 12, position 15, and position 19 at the N-terminus of SEQ ID NO: 1.

[0010] In some embodiments, the polypeptide comprises an amino acid sequence as shown in Formula I: (X1)L(X2)LQ(X3)E(X4)(X5)(X6)LQ(X7)(X8)IK(X9)LN(X 10 )(X 11 ) Y Formula I

[0011] Wherein, (X1) is I or absent; (X2) is D or K; (X4) is M or Nle; (X5) is N or K; (X7) is (S n ) or E; (X8) is A or Aib; (X 10 ) is Q or E; (X 11 ) is (S n ) or S; and (X3), (X6), (X9) are independently unnatural amino acids;

[0012] The polypeptide contains an unnatural amino acid (S) selected from the group consisting of n ) are covalently linked so that the polypeptide forms an α-helical structure:

[0013] (i) (X3) and (X6), (X9) and (X 11 );or

[0014] (ii) (X3) and (X6), (X7) and (X9).

[0015] According to one embodiment of the present application, a pharmaceutical composition may be provided, characterized in that the pharmaceutical composition comprises the polypeptide described in the present application or a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex thereof, and a pharmaceutically, physiologically or nutritionally acceptable excipient.

[0016] According to one embodiment of the present application, the use of the polypeptide described in the present application and the pharmaceutical composition described in the present application in the preparation of a drug for detecting, preventing or treating a disease can be provided. Preferably, the disease is a coronavirus infection disease.

[0017] According to one embodiment of the present application, the use of the polypeptide described in the present application and the pharmaceutical composition described in the present application in inhibiting membrane fusion between coronavirus and host cells can be provided.

[0018] According to one embodiment of the present application, a method for detecting, preventing or treating a disease may be provided, comprising administering the polypeptide described herein or the pharmaceutical composition described herein to a subject in need thereof, preferably, the disease is a coronavirus infection disease.

[0019] The short peptides described in this invention exhibit low-micromolar antiviral activity against more than two human β-coronaviruses, with activity comparable to or higher than that of the broad-spectrum anti-coronavirus peptide EK1, currently in clinical trials. Furthermore, their peptide sequence is nearly half the length of EK1, significantly reducing the difficulty and cost of synthesis.

[0020] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings illustrate exemplary embodiments and constitute a part of the specification. Together with the description of the specification, they serve to explain exemplary implementation of the embodiments. The illustrated embodiments are for illustrative purposes only and do not limit the scope of the claims. Throughout the drawings, the same reference numerals designate similar, but not necessarily identical, elements.

[0022] FIG1 shows the HPLC chart of polypeptide C21DS6 in Example 1.

[0023] FIG2 shows the HPLC chart of polypeptide C21DS6-1 in Example 1.

[0024] FIG3 shows the HPLC chart of polypeptide C21DS6-3 in Example 1.

[0025] FIG4 shows the HPLC chart of polypeptide C21DS6-4 in Example 1.

[0026] FIG5 shows the HPLC chart of polypeptide C21DS6-5 in Example 1.

[0027] FIG6 shows the HPLC chart of polypeptide C21DS6-6 in Example 1.

[0028] FIG7 shows the HPLC chart of polypeptide C21DS6-8 in Example 1.

[0029] FIG8 shows the HPLC chart of polypeptide C21DS6-10 in Example 1.

[0030] FIG9 shows the HPLC chart of polypeptide C21DS6-11 in Example 1.

[0031] FIG10 shows the HPLC chart of polypeptide C21DS4 in Example 1.

[0032] FIG11 shows the HPLC chart of polypeptide EK1 in Comparative Example 1.

[0033] FIG12 shows the mass spectrum of polypeptide C21DS4 in Example 1.

[0034] FIG13 shows the mass spectrum of polypeptide C21DS6 in Example 1.

[0035] FIG14 shows the mass spectrum of polypeptide C21DS6-1 in Example 1.

[0036] FIG15 shows the mass spectrum of polypeptide C21DS6-3 in Example 1.

[0037] FIG16 shows the mass spectrum of polypeptide C21DS6-4 in Example 1.

[0038] FIG17 shows the mass spectrum of polypeptide C21DS6-5 in Example 1.

[0039] FIG18 shows the mass spectrum of polypeptide C21DS6-6 in Example 1.

[0040] FIG19 shows the mass spectrum of polypeptide C21DS6-8 in Example 1.

[0041] FIG20 shows the mass spectrum of polypeptide C21DS6-10 in Example 1.

[0042] FIG21 shows the mass spectrum of polypeptide C21DS6-11 in Example 1.

[0043] FIG22 shows the mass spectrum of polypeptide EK1 in Comparative Example 1. DETAILED DESCRIPTION

[0044] Unless otherwise indicated, the terms used herein have the usual meanings understood by those skilled in the art. Unless otherwise indicated, all numbers used in this specification and claims to represent content, concentration, ratio, weight, particle size, percentage, technical effect, etc. should be understood as being modified by the term "about" or "approximately" under any circumstances. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained through this application, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or in a manner understood by those skilled in the art.

[0045] Although the numerical ranges and parameters setting forth the broad scope of this application are approximations, the numerical values ​​set forth in the specific examples are provided as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in its respective testing measurements. Every numerical range given herein will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were expressly written herein.

[0046] As used herein, "polypeptide" and "peptide" are used interchangeably to refer to amino acid polymers of any length. Thus, polypeptides, oligopeptides, proteins, antibodies, and enzymes are all included within the definition of polypeptide.

[0047] The "conformationally constrained peptide" described in this application refers to a peptide that has been chemically modified to obtain a stable secondary structure (e.g., α-helix, β-sheet, turn). For the types and modification methods of common conformationally constrained peptides, see Ram Dharanipragada, Future Med. Chem., 2013, 5(7), 831–849. Conformationally constrained methods include, but are not limited to, hydrocarbon-stapled side chain to side chain cyclization, azide-alkyne cyclization, hydrogen bond replacement stapled peptides, main chain cyclization, peptide grafting strategy, and lasso peptides. In some embodiments, the conformationally constrained method includes olefin metathesis reaction and 1,3-dipolar cycloaddition reaction.

[0048] As used herein, "amino acid" refers to natural amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that function in a manner similar to the natural amino acids.

[0049] As used herein, "natural amino acids" refer to proteinogenic amino acids known to those skilled in the art, including amino acids encoded by the genetic code and their post-translationally modified variants. The amino acids encoded by the genetic code include the 20 common amino acids: A (alanine), V (valine), L (leucine), I (isoleucine), F (phenylalanine), W (tryptophan), M (methionine), P (proline), G (glycine), T (threonine), S (serine), C (cysteine), N (asparagine), Q (glutamine), Y (tyrosine), K (lysine), R (arginine), H (histidine), D (aspartic acid), E (glutamic acid), B (aspartic acid or asparagine), Z (glutamine or glutamic acid), J (leucine or isoleucine), as well as the less common pyrrolysine and selenocysteine. Natural amino acids also include post-translationally modified variants of the amino acids encoded by the genetic code, non-limiting examples of which include prenylated amino acids, prenylated amino acids, myristylated amino acids, palmitoylated amino acids, N-linked glycosylated amino acids, O-linked glycosylated amino acids, phosphorylated amino acids, and acylated amino acids.

[0050] "Non-natural amino acid" as used herein refers to synthetic amino acids, amino acid analogs, and amino acid mimetics other than natural amino acids. In some embodiments, the non-natural amino acid (S n ) include 2-amino-2-methyl-6-heptenoic acid, 2-amino-2-methyl-9-decenoic acid, 2-(4'-pentenyl)alanine, 2-(7'-octenyl)alanine and 2-amino-2-(4-pentenyl)-6-heptenoic acid. Other non-limiting examples of unnatural amino acids include, but are not limited to, Nle (norleucine), Aib (2-aminoisobutyric acid, α-Aminoisobutanoic acid or 2-Aminoisobutyric acid).

[0051] As used herein, "amino acid analogs" refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bonded to a hydrogen, carboxyl, amino, and R groups, for example, homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium; amino acid analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids.

[0052] As used herein, "amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0053] As used herein, "amino acid side chain" refers to the moiety attached to the α-carbon of an amino acid. For example, the amino acid side chain of alanine is a methyl group, the amino acid side chain of phenylalanine is a phenylmethyl group, the amino acid side chain of cysteine ​​is a thiomethyl group, the amino acid side chain of aspartic acid is a carboxymethyl group, the amino acid side chain of tyrosine is a 4-hydroxybenzyl group, and so on. Other non-naturally occurring amino acid side chains are also included, such as those occurring naturally (e.g., amino acid metabolites) or those produced synthetically (e.g., α,α-disubstituted amino acids).

[0054] " olefin " and its synonymous variant (such as alkene or alkenyl) described in this application represent the monovalent group that is derived from the straight chain or branched hydrocarbon part with at least one carbon-carbon double bond by removing single hydrogen atom.Alkenyl moiety contains the carbon atom of specified number.For example, C2-C10 represents that this group may have 2 to 10 carbon atoms.This means that it has C2, C3, C4, C5, C6, C7, C8, C9 or C10 alkenyl.In the case of any numerical designation, " alkenyl " refers to the alkyl with one or more carbon-carbon double bonds, and example alkenyl includes but is not limited to vinyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl etc.

[0055] As used herein, "treat" and synonyms thereof refer to the improvement of a disease, disorder, and / or condition. "Treatment" can be an improvement in at least one measurable physical parameter, which is not necessarily discernible to the patient. "Treatment" can also be the inhibition of the progression of a disease, disorder, and / or condition, either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both. "Treatment" can also be the slowing down of the progression of a disease, disorder, and / or condition or its reversal.

[0056] As used herein, "prevent," "prevent," and its synonyms refer to delaying the onset of a particular disease, disorder, and / or condition, or symptoms associated with such disease, disorder, and / or condition, or reducing the risk of acquiring such disease, disorder, and / or condition.

[0057] As used herein, a "pharmaceutically acceptable excipient" refers to a carrier, diluent, or adjuvant used in the formulation or administration of a drug, which is not itself an essential active ingredient and is not unduly toxic upon administration. Suitable pharmaceutically acceptable excipients are well known to those of ordinary skill in the art and include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and surfactants, including, for example, polysorbate 20.

[0058] The "physiologically acceptable excipient" and "nutritionally acceptable excipient" mentioned in the present application refer to carriers, diluents or adjuvants that do not cause significant irritation to an organism and do not eliminate the biological activity and properties of the administered polypeptide.

[0059] Conformationally restricted peptides

[0060] According to one embodiment of the present application, a conformationally restricted polypeptide of the 19-peptide shown in SEQ ID NO: 1 can be provided, characterized in that 2-3 natural amino acids are added to the N-terminus of the 19-peptide shown in SEQ ID NO: 1, or 1-2 natural amino acids are added to the N-terminus and the C-terminus respectively to form a conformationally restricted polypeptide backbone; the conformationally restricted polypeptide backbone is sequentially substituted with a first, second, third, and fourth non-natural amino acid (4) from the N-terminus to the C-terminus. n ), the first and second unnatural amino acids (S n ) are separated by 3 amino acids and the first and second unnatural amino acids (S n ) form a covalent bond between the third and fourth unnatural amino acids (S n ) are separated by 3 amino acids and between the third and fourth unnatural amino acids (S n ) are covalently linked to form a stable α-helical structure; the conformationally restricted polypeptide backbone corresponds to SEQ ID NO: 1, wherein the first amino acid at the N-terminus is L, the fifth amino acid is M or Nle, the eighth amino acid is L, the 12th amino acid is I, the 15th amino acid is L, and the 19th amino acid is Y; the first, second, third, and fourth unnatural amino acids (S n ) is selected from positions other than position 1, position 5, position 8, position 12, position 15, and position 19 at the N-terminus of SEQ ID NO: 1.

[0061] Polypeptides with a sequence length of less than 21 amino acids are difficult to assemble into a stable α-helical structure. In some embodiments, any appropriate number of amino acids are added to the N-terminus or C-terminus of the 19-peptide shown in SEQ ID NO: 1 to increase the helical stability of the short peptide. In some preferred embodiments, 2-3 natural amino acids are added to the N-terminus of the 19-peptide shown in SEQ ID NO: 1, or 1-2 natural amino acids are added to the N-terminus and C-terminus respectively to form a conformationally restricted polypeptide backbone. In some more preferred embodiments, 2 natural amino acids are added to the N-terminus of the 19-peptide shown in SEQ ID NO: 1 to form a conformationally restricted polypeptide backbone. In some more preferred embodiments, 3 natural amino acids are added to the N-terminus of the 19-peptide shown in SEQ ID NO: 1 to form a conformationally restricted polypeptide backbone. Polypeptides with a sequence length of 21 or 22 amino acids can form a complete α-helical structure, which greatly increases the helical stability of the short peptide.

[0062] Any suitable number of non-natural amino acids can be introduced into the conformationally restricted polypeptide backbone, for example, 1 pair, 2 pairs, 3 pairs, 4 pairs or 5 pairs. In some embodiments, the non-natural amino acids introduced are spaced 3 amino acids apart, and a covalent bond is formed between the non-natural amino acids spaced 3 amino acids apart, so that the polypeptide forms a stable α-helical structure. In some embodiments, the non-natural amino acids introduced are spaced 4 amino acids apart, and a covalent bond is formed between the non-natural amino acids spaced 4 amino acids apart, so that the polypeptide forms a stable α-helical structure. In some embodiments, the non-natural amino acids introduced are spaced 7 amino acids apart, and a covalent bond is formed between the non-natural amino acids spaced 7 amino acids apart, so that the polypeptide forms a stable α-helical structure. In some more preferred embodiments, the N-terminus to the C-terminus of the conformationally restricted polypeptide backbone is sequentially substituted with a first, second, third, and fourth non-natural amino acid (4 in total). n ), the first and second unnatural amino acids (S n ) are separated by 3 amino acids and the first and second unnatural amino acids (S n ) form a covalent bond between the third and fourth unnatural amino acids (S n ) are separated by 3 amino acids and between the third and fourth unnatural amino acids (S n ) form a covalent bond between the polypeptide and the peptide, resulting in a stable α-helical structure. This stable secondary structure increases the polypeptide's resistance to proteolytic cleavage and heat, and can increase target binding affinity, hydrophobicity, and cell permeability. Therefore, the covalently linked polypeptides described herein have improved biological and pharmacological activities compared to corresponding non-covalently linked polypeptides.

[0063] The amino acid residues at positions 1, 5, 8, 12, 15, and 19 of the N-terminal 19-peptide corresponding to SEQ ID NO: 1 directly interact with the two adjacent HR1 residues and are crucial for the assembly and formation of 6-HB. In some embodiments, the conformationally restricted polypeptide backbone corresponds to the amino acid at position 1 of the N-terminus of SEQ ID NO: 1, M or Nle, L, I, L, and Y. In some embodiments, other natural amino acids other than the amino acids at positions 1, 5, 8, 12, 15, and 19 of the N-terminus of the 19-peptide corresponding to SEQ ID NO: 1 can be mutated to any natural amino acid or substituted with any non-natural amino acid. In some embodiments, the first, second, third, and fourth non-natural amino acids (S n) is selected from positions other than the N-terminal 1, 5, 8, 12, 15, and 19 of SEQ ID NO: 1. In some preferred embodiments, the first and second unnatural amino acids (S n ) are selected from the N-terminal 3rd and 7th positions corresponding to SEQ ID NO: 1, respectively, and the third and fourth non-natural amino acids (S n ) are selected from the 14th and 18th positions, or the 10th and 14th positions corresponding to the N-terminus of SEQ ID NO: 1, respectively.

[0064] In some embodiments, the polypeptide comprises an amino acid sequence as shown in Formula I: (X1)L(X2)LQ(X3)E(X4)(X5)(X6)LQ(X7)(X8)IK(X9)LN(X 10 )(X 11 ) Y Formula I

[0065] Wherein, (X1) is I or absent; (X2) is D or K; (X4) is M or Nle; (X5) is N or K; (X7) is an unnatural amino acid (S n ) or E; (X8) is A or Aib; (X 10 ) is Q or E; (X 11 ) is an unnatural amino acid (S n ) or S; and (X3), (X6), (X9) are independently unnatural amino acids (S n );

[0066] The polypeptide contains an unnatural amino acid (S) selected from the group consisting of n ) are covalently linked so that the polypeptide forms an α-helical structure:

[0067] (i) (X3) and (X6), (X9) and (X 11 );or

[0068] (ii) (X3) and (X6), (X7) and (X9).

[0069] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of (i) to (ii):

[0070] (i) In the formula I, (X1) is I or absent; (X2) is D or K; (X4) is M or Nle; (X5) is N or K; (X7) is E; (X8) is A or Aib; (X 10 ) is Q or E; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );or

[0071] (ii) In the formula I, (X1) is absent; (X2) is D; (X4) is M; (X5) is N; (X8) is A; (X 10 ) is Q; (X 11 ) is S; and (X3), (X6), (X7), (X9) are independently unnatural amino acids (S n ).

[0072] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of (i) to (iv):

[0073] (i) In the formula I, (X1) is I or absent; (X2) is D; (X4) is M; (X5) is N; (X7) is E; (X8) is A or Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );

[0074] (ii) In the formula I, (X1) is I; (X2) is D or K; (X4) is Nle; (X5) is N; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );

[0075] (iii) In the formula I, (X1) is I; (X2) is D or K; (X4) is Nle; (X5) is K; (X7) is E; (X8) is Aib; (X 10 ) is Q or E; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );or

[0076] (iv) In the formula I, (X1) is absent; (X2) is D; (X4) is M; (X5) is N; (X8) is A; (X 10 ) is Q; (X 11 ) is S; and (X3), (X6), (X7), (X9) are independently unnatural amino acids (S n ).

[0077] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of (i) to (x):

[0078] (i) In the formula I, (X1) does not exist; (X2) is D; (X4) is M; (X5) is N; (X7) is E; (X8) is A; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );

[0079] (ii) In the formula I, (X1) is I; (X2) is D; (X4) is M; (X5) is N; (X7) is E; (X8) is A; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );

[0080] (iii) In the formula I, (X1) is I; (X2) is D; (X4) is M; (X5) is N; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );

[0081] (iv) In the formula I, (X1) is I; (X2) is D; (X4) is Nle; (X5) is N; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );

[0082] (v) In the formula I, (X1) is I; (X2) is K; (X4) is Nle; (X5) is N; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );

[0083] (vi) In the formula I, (X1) is I; (X2) is D; (X4) is Nle; (X5) is K; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );

[0084] (vii) In the formula I, (X1) is I; (X2) is K; (X4) is Nle; (X5) is K; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );

[0085] (viii) In the formula I, (X1) is I; (X2) is D; (X4) is Nle; (X5) is K; (X7) is E; (X8) is Aib; (X 10 ) is E; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );

[0086] (ix) In the formula I, (X1) is I; (X2) is K; (X4) is Nle; (X5) is K; (X7) is E; (X8) is Aib; (X 10 ) is E; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );or

[0087] (x) In the formula I, (X1) does not exist; (X2) is D; (X4) is M; (X5) is N; (X8) is A; (X 10 ) is Q; (X 11 ) is S; and (X3), (X6), (X7), (X9) are independently unnatural amino acids (S n ).

[0088] In some embodiments, the Nle is L-Nle.

[0089] In some embodiments, the polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-11.

[0090] In some embodiments, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 2-11.

[0091] Any suitable non-natural amino acid can be introduced into the polypeptide backbone to form a covalent bond between the introduced non-natural amino acids. In some embodiments, the non-natural amino acid (S n ) is an α,α-disubstituted non-natural amino acid having an olefin side chain. In some embodiments, the non-natural amino acid (S n) is selected from any one of 2-amino-2-methyl-6-heptenoic acid, 2-amino-2-methyl-9-decenoic acid, 2-(4'-pentenyl)alanine, 2-(7'-octenyl)alanine and 2-amino-2-(4-pentenyl)-6-heptenoic acid. In some preferred embodiments, the non-natural amino acid (S n ) is 2-amino-2-methyl-6-heptenoic acid.

[0092] Any suitable linking means can be used to form a covalent bond between the introduced non-natural amino acids. In some embodiments, the non-natural amino acid (S n ) are covalently linked via an olefin side chain. In some preferred embodiments, the non-natural amino acid (S n ) are covalently linked by olefin metathesis. In some more preferred embodiments, the two non-natural amino acids (S) comprising olefin side chains present in the polypeptide backbone n ) are covalently linked using a ring-closing metathesis reaction to form a cross-linked ring (see Blackwell et al., J. Org. Chem., 66:5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994).

[0093] Any covalent linkage known in the art can also be used, as long as it can link the introduced non-natural amino acids. In some embodiments, covalent linkage includes, but is not limited to, cross-linking comprising a disulfide bond, a hydrocarbon linkage, an intramolecular bond, a thioether bond, or a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction (see YH Lau et al., Peptide stapling techniques based on different macrocyclisation chemistries, Cheminform, 2015, 46(15); White CJ, Yudin AK, Contemporary strategies for peptide macrocyclization, Nature chemistry, 2011, 3(7): 509).

[0094] Other connection modes can also be used in the structurally stable polypeptide described herein. For example, the naturally occurring amino acids in the polypeptide backbone can be used to form a connection, rather than forming a connection by introducing two non-natural amino acid couplings. The amino acid side chains used to connect can include one or more of an ether, a thioether, an ester, an amine, or an amide or a triazole moiety. In some cases, the side chains of natural amino acids in the polypeptide backbone can be used to form a covalent bond, such as the hydroxyl in serine, the mercaptan in cysteine, the primary amine in lysine, the acid in aspartic acid or glutamic acid, or the amide in asparagine or glutamine. In addition, it is also possible to use the naturally occurring amino acids in the polypeptide backbone to form a covalent bond with the non-natural amino acids introduced.

[0095] Methods for performing different types of covalent attachment are well known in the art (see Shepherd et al., Journal of the American Chemical Society, 127:2974-2983 (2005); Madden et al., Bioorg. Med. Chem Lett., 21:1472-1475 (2011); Jackson et al., American Chemical Society, 113:9391-9392 (1991); Haney et al., Chem. Commun., 47:10915-10917 (2011); Brunel and Dawson, Chem. Commun., 552-2554 (2005); JR Kumita et al., Proc. Natl. Acad. Sci. USA, 20:109-110). =(Chem. Am. Am. Chem. USA, 97:3803-3808 (2000); Lau et al., Chem. Sci., 5:1804-1809 (2014); J.C. Phelan et al., Journal of the American Chemical Society, 119:455-460 (1997); A.M. Spokoyny et al., Journal of the American Chemical Society, 135:5946-5949 (2013).

[0096] Pharmaceutical composition

[0097] According to one embodiment of the present application, a pharmaceutical composition may be provided, characterized in that the pharmaceutical composition comprises the polypeptide described in the present application or a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex thereof, and a pharmaceutically, physiologically or nutritionally acceptable excipient.

[0098] Suitable adjuvants are well known to those of ordinary skill in the art, such as liquids, gels or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobials, isotonic agents, buffers, antioxidants, suspending agents / dispersants, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art or various combinations thereof. In some embodiments, the adjuvants include diluents, fillers, adhesives, wetting agents, absorption enhancers, surfactants, lubricants, and / or stabilizers.

[0099] use

[0100] According to one embodiment of the present application, the use of the polypeptide described in the present application and the pharmaceutical composition described in the present application in the preparation of a drug for detecting, preventing or treating a disease can be provided.

[0101] In some embodiments, the disease is a coronavirus infection. In some embodiments, the coronavirus is a beta coronavirus. In some embodiments, the coronavirus includes SARS-CoV, SARS-CoV-2, SARSr-CoVs, MERS-CoV, BtCoV-HKU4, BtCoV-HKU5, BtCoV-PML2011, BtCoV-UKR, BtCoV-HKU9, HCoV-OC43, or HCoV-HKU1. In some preferred embodiments, the coronavirus includes SARS-CoV, SARS-CoV-2, MERS-CoV, or HCoV-OC43.

[0102] According to one embodiment of the present application, the use of the polypeptide described in the present application and the pharmaceutical composition described in the present application in inhibiting membrane fusion between coronavirus and host cells can be provided.

[0103] In some embodiments, the coronavirus is a beta coronavirus. In some embodiments, the coronavirus includes SARS-CoV, SARS-CoV-2, SARSr-CoVs, MERS-CoV, BtCoV-HKU4, BtCoV-HKU5, BtCoV-PML2011, BtCoV-UKR, BtCoV-HKU9, HCoV-OC43, or HCoV-HKU1. In some preferred embodiments, the coronavirus includes SARS-CoV, SARS-CoV-2, MERS-CoV, or HCoV-OC43.

[0104] In some embodiments, the host cell is a mammalian cell. In some preferred embodiments, the host cell is a human cell, such as a Caco-2 cell or a HCT-8 cell.

[0105] Disease treatment methods

[0106] According to one embodiment of the present application, a method for detecting, preventing or treating a disease may be provided, comprising administering the polypeptide described herein or the pharmaceutical composition described herein to a subject in need thereof.

[0107] In some embodiments, the disease is a coronavirus infection. In some embodiments, the coronavirus is a beta coronavirus. In some embodiments, the coronavirus includes SARS-CoV, SARS-CoV-2, SARSr-CoVs, MERS-CoV, BtCoV-HKU4, BtCoV-HKU5, BtCoV-PML2011, BtCoV-UKR, BtCoV-HKU9, HCoV-OC43, or HCoV-HKU1. In some preferred embodiments, the coronavirus includes SARS-CoV, SARS-CoV-2, MERS-CoV, or HCoV-OC43.

[0108] The subject can be selected for treatment based on, for example, a determination that the subject is infected with a coronavirus (e.g., a beta coronavirus, such as SARS-CoV-2). The polypeptides herein can also be used to determine whether a subject is infected with a coronavirus.

[0109] In general, the method comprises selecting a subject and administering to the subject an effective amount of one or more polypeptides of the present application, and optionally repeating the administration as needed for the prevention or treatment of coronavirus infection or coronavirus disease, and can be administered orally, intranasally, intravenously, subcutaneously, intramuscularly or topically, including skin administration, nasal administration, sinus administration, respiratory tree administration and lung administration. In some instances, administration is performed by topical respiratory application, which includes application to the nasal mucosa, sinus mucosa or respiratory tree, including the lungs. In some instances, topical application includes application to the skin.

[0110] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination, severity and course of the disease, condition or symptom, the patient's predisposition to the disease, condition or symptom, and the judgment of the treating physician.

[0111] An effective amount can be administered in one or more administrations, applications, or dosages. The polypeptide or composition can be administered from one or more times daily to one or more times weekly, including once every other day. Those skilled in the art will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatments, the subject's overall health and / or age, and the presence of other diseases. Furthermore, treating a subject with a therapeutically effective amount of a therapeutic compound described herein can include a single treatment or a series of treatments. For example, an effective amount can be administered at least once.

[0112] The various embodiments and preferences for the present application described above can be combined with each other (as long as they are not inherently contradictory to each other and are applicable to the purposes of the present application), and the various embodiments formed by such combination are considered to be part of the present application.

[0113] Example

[0114] The following describes exemplary embodiments of the present application in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered merely exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined solely by the claims. Therefore, it should be appreciated by those skilled in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0115] Unless otherwise stated, all reagents and instruments used in the following examples are commercially available conventional products. Unless otherwise stated, experiments were performed under conventional conditions or conditions recommended by the manufacturer.

[0116] Example 1: Synthesis of polypeptide

[0117] The peptides were synthesized using standard Fmoc solid-phase peptide synthesis (SPPS) methods. Reagents used during synthesis, such as DMF, MeOH, DCM, and piperidine, were dried prior to use. Rink amide resin was used as the solid support, with a resin loading of 0.53 mmol / g.

[0118] 1.1 Resin swelling

[0119] 0.19 g of Rink amide resin was weighed into a 50 mL solid phase reactor, and 5 mL of DCM and 5 mL of DMF were added, and the mixture was swelled for 15 min.

[0120] 1.2 Removal of Fmoc protecting group on resin

[0121] Add a deprotection reagent (20% piperidine / DMF solution (v / v)) to the swollen resin. Remove the Fmoc residue in two steps: first, add 5 mL of the deprotection reagent and stir continuously for 5 minutes, then drain. Then, add another 5 mL of the deprotection reagent and stir continuously for 25 minutes, then drain. Wash the resin twice with 5 mL of DMF, 5 mL of MeOH, and 5 mL of DCM, respectively. Then, take a small amount of the deprotected resin for testing at 110°C for 3 minutes. Remove the test tube from the heater and observe the reaction. If the resin turns blue, deprotection is complete and the next step, amino acid condensation, can be performed. Otherwise, adjust the procedure and repeat step 1.2 until the resin turns blue.

[0122] 1.3 Condensation reaction of amino acids

[0123] Add the deprotected resin to a microwave peptide synthesizer (purchased from CEM), along with 3 mL of amino acid solution (0.1 M), 5 mL of condensation reagent (0.3 M HBTU / 0.3 M HOBt / DMF), and 5 mL of activated base solution (0.6 M DIEA / NMP) and stir at room temperature. After the reaction is complete, wash the peptide resin twice with 5 mL of DMF, 5 mL of methanol, and 5 mL of dichloromethane, respectively. Then, take a small amount of the peptide resin for testing at 110°C for 3 minutes. Remove the test tube from the heater and observe the reaction. If the resin turns blue, it indicates incomplete amino acid condensation. Repeat step 1.3 until the reaction is complete and the resin turns yellow. If the resin turns yellow, repeat steps 1.2 and 1.3 to condense the next amino acid until the peptide is fully synthesized.

[0124] 1.4 Acetylation Capping

[0125] After peptide synthesis, remove the Fmoc protecting group, rinse, and add 2 mL of DIEA and 2 mL of acetic anhydride, along with 4 mL of DMF, for 30 minutes. After the reaction is complete, rinse the peptide resin and sample a small amount for testing at 110°C for 3 minutes. Remove the test tube from the heater and observe. If the resin turns yellow, end-capping is complete. Rinse and drain.

[0126] 1.5 Solid-phase olefin metathesis

[0127] The special amino acid (S n) to covalently link the side chain olefin groups of the peptide to synthesize the stapled peptide. Transfer the peptide resin (0.1 mmol) to a light-protected eggplant-shaped flask and purge with nitrogen for 5 minutes. Weigh 50 mg of the second-generation Grubbs catalyst, protecting the weighing process from light. Dissolve the weighed catalyst in 5 mL of anhydrous dichloroethane and add it to the reaction system. Stir under nitrogen for 6 hours, and repeat once after the reaction is complete. After the reaction is complete, transfer the peptide resin to a solid-phase reactor and wash twice with DMF, MeOH, and DCM for 2 minutes each. Drain the resin and set aside.

[0128] 1.6 Cracking

[0129] First, prepare a lysis buffer consisting of trifluoroacetic acid: anisole: ethanedithiol: m-cresol: water = 82.5:5:2.5:5:5 (volume percentage). It must be cooled in an ice bath for 30 minutes or stored in a refrigerator before use. Place the polypeptide resin in a 250mL eggplant-shaped flask, place it in an ice bath, add 10mL of lysis buffer, and stir electromagnetically until the resin turns orange-red. Reaction is continued in an ice bath for 30 minutes. Then, remove the ice bath and continue stirring at room temperature for another 90 minutes. Next, add 200mL of cold ether under vigorous stirring to precipitate a white precipitate. Stirring is continued for 30 minutes. Filter the precipitate using a G4 sand core suction funnel, wash it repeatedly with cold ether three times, and air dry. Subsequently, add 50mL of double-distilled water and 5mL of acetonitrile to fully dissolve the solid, filter, and freeze-dry the filtrate to obtain the crude compound.

[0130] 1.7 Purification

[0131] The crude compound was purified by high-pressure chromatography using a C8 column and acetonitrile, water, and a small amount of trifluoroacetic acid as the eluent. The specific steps were as follows: 1.00 g of the crude compound was weighed, 20 mL of water and 5 mL of acetonitrile were added to dissolve the solid, centrifuged for 10 min (3000 rpm), and the supernatant was loaded onto the sample. The column was pre-equilibrated with 200 mL of a 15% acetonitrile / water / 0.1% trifluoroacetic acid solution. After loading the sample, the column was rinsed with 200 mL of a 15% acetonitrile / water / 0.1% trifluoroacetic acid solution, and the eluate was analyzed by HPLC. Based on the results of the HPLC analysis, the acetonitrile content was gradually increased until the main peak of the purified compound was eluted. The eluates were combined, rotary evaporated to remove most of the solvent, and lyophilized to obtain the purified compound, with a purity greater than 95.0% as determined by HPLC. The molecular weight of the compound was determined by MALDI-TOF-MS. The elution gradient used for peptide purity analysis is shown in Table 1.

[0132] Table 1 Elution gradient

[0133] The HPLC spectra of the polypeptides in Example 1 are shown in Figures 1-11, and their mass spectra are shown in Figures 12-22. The sequences of the polypeptides synthesized in Example 1 are shown in SEQ ID NOs: 2-11 (Table 2). Their amino acid length is shortened by nearly half compared to EK1, greatly reducing the difficulty and cost of synthesis.

[0134] Table 2 Peptide sequences

[0135] Description: (S n ) represents (2R)-2-amino-2-methyl-6-heptenoic acid, (Z1) represents L-norleucine, and (B1) represents 2-aminoisobutyric acid.

[0136] Comparative Example 1: Synthesis of polypeptide EK1

[0137] The preparation method of Comparative Example 1 is substantially the same as that of Example 1, except that the preparation method does not include the step in "1.5 Solid-Phase Olefin Metathesis Reaction." The sequence of the polypeptide EK1 synthesized in Comparative Example 1 is shown in SEQ ID NO: 12, and its amino acid length is 36 aa.

[0138] Example 2: Determination of antiviral activity of polypeptides

[0139] To detect the inhibitory activity of peptides against coronavirus PsV infection, target cells (Caco-2 cells, American Type Culture Collection) were plated at 10 μg / well one day before infection. 4 Cells were plated in 96-well plates at a density of 10 cells / well. PsV was mixed with an equal volume of the peptide and serially diluted with PBS at 37°C for 30 minutes. This mixture was then transferred to Huh-7 cells. The medium was replaced after 12 hours, and the cells were cultured for an additional 48 hours. The cells were then washed with PBS, lysed using a lysis reagent (Promega), and relative light units (RLU) were measured using a luciferase assay kit.

[0140] The results are shown in Table 3. The results demonstrate that the novel membrane fusion inhibitors of the present invention exhibit low-micromolar antiviral activity against three highly pathogenic coronaviruses: SARS-CoV-2, SARS-CoV, and MERS-CoV. Furthermore, compared to the EK1 polypeptide currently under clinical investigation, the peptides of the present invention exhibit comparable or higher antiviral activity.

[0141] Table 3 Anti-pseudovirus activity of polypeptides

[0142] We further evaluated the inhibitory activity of the peptide against HCoV-OC43 replication in HCT-8 cells.50 OC43 was mixed with the test peptide at a gradient concentration and incubated at 37°C for 30 minutes. The mixture was then plated in triplicate on HCT-8 cell monolayers grown in 96-well microtiter plates. Virus titers in the culture medium were measured on day 5 post-infection, and TCID was calculated based on the cytopathic effect (CPE). 50 .

[0143] The assay results are shown in Table 4. We found that the novel membrane fusion inhibitor of the present invention can effectively inhibit the infection and replication of HCoV-OC43 at the cellular level, with activity comparable to that of EK1.

[0144] Table 4 Inhibitory activity of polypeptides against HCoV-OC43 replication

[0145] It should be noted that the above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Although specific implementation methods have been described, for the applicant or other skilled in the art, there may be or are currently no alternatives, modifications, changes, improvements and substantial equivalents of the above implementation methods. Therefore, the attached claims submitted and the claims that may be amended are intended to cover all such alternatives, modifications, changes, improvements and substantial equivalents. It is important to note that as technology evolves, many of the elements described herein may be replaced by equivalent elements that appear after the present application.

Claims

1. A conformationally restricted polypeptide of 19 peptides as shown in SEQ ID NO: 1, characterized in that: Adding 2-3 natural amino acids to the N-terminus of the 19-peptide shown in SEQ ID NO: 1, or adding 1-2 natural amino acids to the N-terminus and the C-terminus, respectively, to form a conformationally restricted polypeptide backbone; The conformationally restricted polypeptide backbone is substituted with a first, second, third, and fourth non-natural amino acid (S n ), the first and second unnatural amino acids (S n ) are separated by 3 amino acids and between the first and second unnatural amino acids (S n ) form a covalent bond between the third and fourth unnatural amino acids (S n ) are separated by 3 amino acids and between the third and fourth unnatural amino acids (S n ) form a covalent bond between them, so that the polypeptide forms a stable α-helical structure; The conformationally restricted polypeptide backbone corresponds to SEQ ID NO: 1, wherein the first amino acid at the N-terminus is L, the fifth amino acid is M or Nle, the eighth amino acid is L, the 12th amino acid is I, the 15th amino acid is L, and the 19th amino acid is Y; The first, second, third and fourth unnatural amino acids (S n ) is selected from other positions other than the 1st, 5th, 8th, 12th, 15th and 19th positions at the N-terminus of SEQ ID NO:

1.

2. The polypeptide according to claim 1, wherein the first and second unnatural amino acids (S n ) are selected from the 3rd and 7th positions of the N-terminus of SEQ ID NO: 1, respectively, and the third and fourth unnatural amino acids (S n ) are selected from the 14th and 18th, or the 10th and 14th N-terminal positions corresponding to SEQ ID NO: 1, respectively.

3. The polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence as shown in Formula I: (X1)L(X2)LQ(X3)E(X4)(X5)(X6)LQ(X7)(X8)IK(X9)LN(X 10 )(X 11 )Y Formula I in, (X1) is I or absent; (X2) is D or K; (X4) is M or Nle; (X5) is N or K; (X7) is an unnatural amino acid (S n ) or E; (X8) is A or Aib; (X 10 ) is Q or E; (X 11 ) is an unnatural amino acid (S n ) or S; and (X3), (X6), (X9) are independently unnatural amino acids (S n ); The polypeptide contains an unnatural amino acid (S) selected from the group consisting of n ) are covalently linked so that the polypeptide forms an alpha helical structure: (i) (X3) and (X6), (X9) and (X 11 );or (ii) (X3) and (X6), (X7) and (X9).

4. The polypeptide according to claim 3, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of (i)-(ii): (i) In Formula I, (X1) is I or absent; (X2) is D or K; (X4) is M or Nle; (X5) is N or K; (X7) is E; (X8) is A or Aib; (X 10 ) is Q or E; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );or (ii) In Formula I, (X1) is absent; (X2) is D; (X4) is M; (X5) is N; (X8) is A; (X 10 ) is Q; (X 11 ) is S; and (X3), (X6), (X7), (X9) are independently unnatural amino acids (S n ).

5. The polypeptide according to claim 3, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of (i) to (iv): (i) In Formula I, (X1) is I or absent; (X2) is D; (X4) is M; (X5) is N; (X7) is E; (X8) is A or Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n ); (ii) In Formula I, (X1) is I; (X2) is D or K; (X4) is Nle; (X5) is N; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n ); (iii) In Formula I, (X1) is I; (X2) is D or K; (X4) is Nle; (X5) is K; (X7) is E; (X8) is Aib; (X 10 ) is Q or E; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );or (iv) In Formula I, (X1) is absent; (X2) is D; (X4) is M; (X5) is N; (X8) is A; (X 10 ) is Q; (X 11 ) is S; and (X3), (X6), (X7), (X9) are independently unnatural amino acids (S n ).

6. The polypeptide according to claim 3, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of (i)-(x): (i) In Formula I, (X1) is absent; (X2) is D; (X4) is M; (X5) is N; (X7) is E; (X8) is A; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n ); (ii) In Formula I, (X1) is I; (X2) is D; (X4) is M; (X5) is N; (X7) is E; (X8) is A; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n ); (iii) In Formula I, (X1) is I; (X2) is D; (X4) is M; (X5) is N; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n ); (iv) In Formula I, (X1) is I; (X2) is D; (X4) is Nle; (X5) is N; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n ); (v) In Formula I, (X1) is I; (X2) is K; (X4) is Nle; (X5) is N; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n ); (vi) In Formula I, (X1) is I; (X2) is D; (X4) is Nle; (X5) is K; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n ); (vii) In Formula I, (X1) is I; (X2) is K; (X4) is Nle; (X5) is K; (X7) is E; (X8) is Aib; (X 10 ) is Q; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n ); (viii) In Formula I, (X1) is I; (X2) is D; (X4) is Nle; (X5) is K; (X7) is E; (X8) is Aib; (X 10 ) is E; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n ); (ix) In Formula I, (X1) is I; (X2) is K; (X4) is Nle; (X5) is K; (X7) is E; (X8) is Aib; (X 10 ) is E; and (X3), (X6), (X9), (X 11 ) are independently unnatural amino acids (S n );or (x) In Formula I, (X1) is absent; (X2) is D; (X4) is M; (X5) is N; (X8) is A; (X 10 ) is Q; (X 11 ) is S; and (X3), (X6), (X7), (X9) are independently unnatural amino acids (S n ).

7. The polypeptide according to any one of claims 1 to 6, wherein the non-natural amino acid (S n ) is an α,α-disubstituted non-natural amino acid having an olefin side chain.

8. The polypeptide according to claim 7, wherein the non-natural amino acid (S n ) is selected from any one of 2-amino-2-methyl-6-heptenoic acid, 2-amino-2-methyl-9-decenoic acid, 2-(4'-pentenyl)alanine, 2-(7'-octenyl)alanine and 2-amino-2-(4-pentenyl)-6-heptenoic acid, preferably, the unnatural amino acid (S n ) is 2-amino-2-methyl-6-heptenoic acid.

9. The polypeptide according to any one of claims 1 to 8, wherein the polypeptide is an amino acid sequence shown in any one of SEQ ID NOs: 2 to 11.

10. The polypeptide according to any one of claims 1 to 9, wherein the non-natural amino acid (S n ) is covalently linked via an olefin side chain, preferably, the non-natural amino acid (S n ) are covalently linked via olefin metathesis.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide according to any one of claims 1 to 10 or a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex thereof, and a pharmaceutically, physiologically or nutritionally acceptable auxiliary material.

12. Use of the polypeptide according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for detecting, preventing or treating a disease, preferably, the disease is a coronavirus infection disease.

13. Use of the polypeptide according to any one of claims 1 to 10 and the pharmaceutical composition according to claim 11 in inhibiting membrane fusion between coronavirus and host cells.

14. The use according to claim 12 or 13, wherein the coronavirus is a beta coronavirus.

15. The use according to claim 14, wherein the coronavirus comprises SARS-CoV, SARS-CoV-2, SARSr-CoVs, MERS-CoV, BtCoV-HKU4, BtCoV-HKU5, BtCoV-PML2011, BtCoV-UKR, BtCoV-HKU9, HCoV-OC43, or HCoV-HKU1, preferably, the coronavirus comprises SARS-CoV, SARS-CoV-2, MERS-CoV, or HCoV-OC43.

16. A method for detecting, preventing or treating a disease, the method comprising administering the polypeptide of any one of claims 1 to 10 or the pharmaceutical composition of claim 11 to a subject in need thereof, preferably, the disease is a coronavirus infection disease.

17. The method of claim 16, wherein the coronavirus is a beta coronavirus.

18. The method according to claim 17, wherein the coronavirus comprises SARS-CoV, SARS-CoV-2, SARSr-CoVs, MERS-CoV, BtCoV-HKU4, BtCoV-HKU5, BtCoV-PML2011, BtCoV-UKR, BtCoV-HKU9, HCoV-OC43, or HCoV-HKU1, preferably, the coronavirus comprises SARS-CoV, SARS-CoV-2, MERS-CoV, or HCoV-OC43.

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