Broad-spectrum anti-coronavirus lipopeptides

Abstract

The invention provides a novel antiviral polypeptide, a lipopeptide, a preparation method thereof, a pharmaceutical composition, a pharmaceutical composition and application. The lipopeptide provided by the invention is obtained by conjugating an antiviral polypeptide, an optional linking arm, an amino acid residue and a lipophilic compound. The lipopeptide provided by the invention has the effect of resisting novel coronavirus.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biological medicine, and particularly relates to polypeptides, lipopeptides for inhibiting viruses. BACKGROUND

[0002] Coronaviruses (CoVs) are a common family of viruses that widely exist in nature, can infect humans, and easily cause acute and chronic respiratory system diseases. The wide host characteristics of the virus, as well as the structural characteristics of its own genome, make it extremely easy to undergo gene recombination and variation in the evolutionary process, showing genetic diversity. Coronaviruses are divided into four genera, i.e., alpha, beta, gamma and delta, and the beta genus coronavirus can be further divided into four lineages, i.e., A, B, C and D. Alpha coronaviruses and beta coronaviruses are usually found in mammals, while gamma coronaviruses and delta coronaviruses are mainly related to the infection of birds. There are currently seven known coronaviruses that can infect humans. Among them, human coronavirus NL63 (HCoV-NL63) and human coronavirus 229E (HCoV-229E) belong to the alpha genus coronavirus; the other five are all beta genus coronaviruses. HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 have low pathogenicity, and generally only cause mild respiratory symptoms similar to common cold; while SARS-CoV, MERS-CoV and SARS-CoV-2 are highly pathogenic human coronaviruses, which pose a serious threat to human health. For all human coronavirus infections including SARS-CoV-2, there is currently no sufficient, specific and effective treatment drug for symptomatic treatment, highlighting the urgency of developing new anti-coronavirus drugs. At the same time, the rapid emergence of variants is also an important reason for the urgent development of a new generation of anti-coronavirus drugs for active defense against future new and recurring highly pathogenic human coronavirus infection epidemics.

[0003] The spike protein (S protein) of HCoV is a type I envelope protein, mainly composed of S1 and S2 subunits, and presented on the virus surface in a trimeric state. Among them, the S1 subunit is responsible for the recognition of receptors; the S2 subunit mainly mediates the fusion of virus and host cell membranes. When HCoV invades host cells, the S1 subunit first binds to the receptors on the surface of target cells; then, the S2 subunit undergoes a conformational change: the HR1 (heptad repeat 1, HR1) and HR2 (heptad repeat 2, HR2) domains of the S2 subunit interact with each other to form a six-helix bundle (6-HB), and release energy, thereby pulling the virus membrane and the host cell membrane connected at both ends of S2, and ultimately leading to fusion. Current research reports have analyzed the 6-HB crystal structure of various HCoVs. By comparison, it can be found that the three-dimensional structure of β-HCoV 6-HB is very similar; further alignment of the amino acid sequences of the corresponding regions of 6-HB can find that their primary structures are also highly conserved. Coronavirus will undergo significant variation during human-to-human transmission, for example, the continuous variation of SARS-CoV-2 has led to the prevalence of multiple variant strains such as Alpha, Beta, Gamma, Delta, and Omicron. Among these mutant strains, the receptor binding domain (RBD) of the S1 subunit is the main site of mutation. For example, among the more than 30 mutation sites of the Omicron strain, 15 mutation sites are located in the RBD region. These mutations in the RBD will bring many difficulties to the development of related antibody drugs and vaccine research. Compared with the S1 subunit, the S2 subunit is very conservative. The universal and conserved 6-HB structure in the process of HCoV entering host cells is considered a potential target for drug development.

[0004] The polypeptide fragment derived from the viral HR2 domain interferes with the formation of the virus's own 6-HB, which can effectively block the fusion of the virus with the target cell membrane. Currently, a number of universal fusion-inhibiting polypeptides for β-HCoVs have been discovered. Crystal structures show that there are very obvious differences in the lengths of the HR1 and HR2 helices in the 6-HB formed by β-HCoVs. Among them, the HR1 helix is composed of 45 amino acid residues, while the α-helix core region of the HR2 domain contains only 19 amino acids. Previous studies have shown that the polypeptide sequence directly cut from the α-helix core region of the HR2 domain cannot interact with the target HR1 due to its short length, and thus cannot exert an antiviral effect. Therefore, the currently reported broad-spectrum anti-coronavirus polypeptides all contain the sequence of the HR2 α-helix core region and the tail sequences at the N- and C-termini of the region, resulting in a long amino acid sequence, for example, the EK1 polypeptide and the IPB series polypeptides currently in the clinical trial stage each contain more than 36 amino acid residues. The length of the polypeptide is undoubtedly closely related to its preparation difficulty and cost. At the same time, another obvious defect of polypeptide drugs is their poor enzymatic stability and short in vivo half-life. These defects result in the need for frequent administration of the drug to patients in clinical practice, which seriously limits the compliance. Based on the highly conserved 6-HB structure of the HCoV spike protein, it is of great clinical significance to find a new generation of broad-spectrum anti-coronavirus polypeptide drug for active prevention of future new and recurring highly pathogenic human coronavirus infection epidemics. SUMMARY

[0005] To solve the above technical problems, the present disclosure provides a lipopeptide. The lipopeptide provided by the present disclosure not only has broad-spectrum antiviral activity, but also has a sequence length of only about 22 amino acids, and has good enzymatic stability and a very long in vivo metabolic half-life. The lipopeptide provided by the present disclosure can be used for treating or preventing viral infection.

[0006] A first aspect of the present disclosure provides a lipopeptide or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, the lipopeptide comprising: (1) an antiviral polypeptide, (2) a linker, (3) an amino acid residue, and (4) a lipophilic compound, wherein the linker (2) can be absent; the amino acid residue (3) is selected from K or C, or an amino acid residue having similar properties thereto.

[0007] A second aspect of the present disclosure provides an antiviral polypeptide, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, the antiviral polypeptide comprising an amino acid sequence according to any one of (a)-(f):

[0008] (a) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 5, preferably the amino acid sequence shown in SEQ ID NO: 5;

[0009] (b) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 6, preferably the amino acid sequence shown in SEQ ID NO: 6;

[0010] (c) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 7, preferably the amino acid sequence shown in SEQ ID NO: 7;

[0011] (d) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 8, preferably the amino acid sequence shown in SEQ ID NO: 8.

[0012] (e) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 9, preferably the amino acid sequence shown in SEQ ID NO: 9.

[0013] (f) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 10, preferably the amino acid sequence shown in SEQ ID NO: 10.

[0014] A third aspect of this disclosure provides a pharmaceutical composition comprising a lipopeptide of the first aspect of this disclosure or a stereoisomer thereof, derivative thereof, pharmaceutically acceptable salt thereof, mixture thereof or a functionally equivalent variant thereof, or an antiviral polypeptide of the second aspect of this disclosure, or a stereoisomer thereof, derivative thereof, pharmaceutically acceptable salt thereof, mixture thereof or a functionally equivalent variant thereof, and a pharmaceutically acceptable carrier.

[0015] The fourth aspect of this disclosure provides a pharmaceutical combination comprising a lipopeptide of the first aspect of this disclosure or a stereoisomer thereof, derivative thereof, pharmaceutically acceptable salt, mixture thereof or a functionally equivalent variant thereof, an antiviral polypeptide of the second aspect of this disclosure, or a stereoisomer thereof, derivative thereof, pharmaceutically acceptable salt, mixture thereof or a functionally equivalent variant thereof, or a pharmaceutical composition of the third aspect of this disclosure, and a second therapeutic agent.

[0016] The fifth aspect of this disclosure provides the use of the lipopeptide of the first aspect of this disclosure, or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures, or functionally equivalent variants thereof; the antiviral polypeptide of the second aspect of this disclosure, or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures, or functionally equivalent variants thereof; the pharmaceutical composition of the third aspect of this disclosure; or the pharmaceutical composition of the fourth aspect of this disclosure in the preparation of a medicament for treating or preventing viral infections.

[0017] The sixth aspect of this disclosure provides a method for preparing the lipopeptide of the first aspect of this disclosure or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof, comprising (1) synthesizing the antiviral polypeptide, (2) optionally, linking the antiviral polypeptide to a linker arm, (3) loading amino acid residues, and (4) conjugating a lipophilic compound.

[0018] The seventh aspect of this disclosure provides the use of the lipopeptide of the first aspect of this disclosure, or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures, or functionally equivalent variants thereof; the antiviral polypeptide of the second aspect of this disclosure, or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures, or functionally equivalent variants thereof; the pharmaceutical composition of the third aspect of this disclosure; or the pharmaceutical composition of the fourth aspect of this disclosure for the treatment or prevention of viral infection.

[0019] The eighth aspect of this disclosure provides a method for treating or preventing viral infection, comprising administering to a subject in need an effective amount of a lipopeptide of the first aspect of this disclosure or a stereoisomer, derivative thereof, pharmaceutically acceptable salt, mixture thereof or a functionally equivalent variant thereof, an antiviral polypeptide of the second aspect of this disclosure, or a stereoisomer, derivative thereof, pharmaceutically acceptable salt, mixture thereof or a functionally equivalent variant thereof, a pharmaceutical composition of the third aspect of this disclosure, or a pharmaceutical combination of the fourth aspect of this disclosure. Attached Figure Description

[0020] Figure 1A The inhibitory activity of BC1-A on the replication of Omicron BA.5.2 was measured in Caco-2 cells. The experiment was repeated three times, and the data are expressed as mean ± standard deviation.

[0021] Figure 1B This diagram illustrates the administration of BC1-A peptide and SARS-CoV-2 infection. hACE2 transgenic C57BL / 6 mice (6-8 weeks old) in the prevention and vector control groups were intranasally treated with BC1-A peptide (16 mg / kg) or the vector control, respectively, 30 minutes before Omicron BA.5.2 infection. Mice in the treatment group received BC1-A peptide (16 mg / kg) intranasally 30 minutes after Omicron BA.5.2 challenge. Seven days after infection, the mice were sacrificed and tissues were collected.

[0022] Figure 1C The viral titer in the lungs of mice in each group was measured on day 7 post-infection. An asterisk indicates a significant difference, **p<0.01.

[0023] Figure 1D Histopathological examination of lung tissue from mice given or not given BC1-A. Lung tissue sections were stained with hematoxylin and eosin for histopathological examination. Graduation, 100 μm. Detailed Implementation

[0024] Unless otherwise stated, the terminology used herein has the common meaning understood by one of ordinary skill in the art. It may vary for those skilled in the art depending on the desired properties and effects sought through this application, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or as understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the experimental procedures in organic chemistry, medicinal chemistry, and biology described herein are well-known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Where multiple definitions exist for terms used herein, the definitions in this section shall prevail unless otherwise stated.

[0025] Unless otherwise stated, all figures used in this specification and claims to indicate content, concentration, proportion, weight, particle size, percentage, technical effect, etc., shall in any event be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. “About” or “approximately” can be understood to mean a range of plus or minus 10%, 20%, 30%, 40%, or 50% of the indicated values.

[0026] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0027] As used herein, the terms “include,” “contain,” or “comprising” mean that in addition to having the listed elements, other elements are not excluded.

[0028] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "identity" between two sequences, expressed as a percentage, is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum possible identity. Such comparisons can be performed using, for example, the method readily available through computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. Alternatively, the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0) can be used to determine the percentage identity between two amino acid sequences using a PAM120 weighted residue table, a 12-bit gap length penalty, and a 4-bit gap penalty. In addition, the Needleman and Wunsch (J MoI Biol.48:444-453(1970)) algorithm, which is integrated into the GAP program (available at www.gcg.com), can be used to determine the identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0029] As defined herein, the terms “polypeptide,” “protein,” “peptide,” and “amino acid sequence” are used interchangeably herein and refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, may contain modified amino acids or amino acid analogs, and may be broken down by non-amino acid chemical motifs. The term also includes amino acid polymers that have been naturally or artificially modified (e.g., disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a label or bioactive component). The term “peptide” includes two or more naturally occurring or synthetic amino acids linked by covalent bonds (e.g., amide bonds).

[0030] As used herein, unless otherwise indicated, site numbering of polypeptide or amino acid sequences, as is customary in the art, is in order from the N-terminus to the C-terminus. For example, the first amino acid starting from the N-terminus of the polypeptide or amino acid sequence is the first amino acid, the second amino acid starting from the N-terminus of the polypeptide or amino acid sequence is the second amino acid, the third amino acid starting from the N-terminus of the polypeptide or amino acid sequence is the third amino acid, and so on. As used herein, unless otherwise indicated, the left-hand end of the polypeptide or amino acid sequence is the N-terminus, i.e., the amino terminus, and the right-hand end is the C-terminus, i.e., the carboxyl terminus.

[0031] In this article, when referring to peptides or polypeptides, "amino acid" and "amino acid residue" have the same meaning, referring to the amino acid residue that remains after some groups are lost due to participation in the formation of the linking bond when amino acids are linked by chemical bonds. The twenty common amino acids mentioned in this article are listed according to conventional usage. See, for example, Immunology-A Synthesis (2 nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this document, amino acids are generally represented by single-letter and three-letter abbreviations well-known in the art. For example, alanine may be represented as A or Ala.

[0032] The term “amino acid” as used in this article includes natural amino acids, as well as other “non-protein” α-amino acids / non-natural amino acids commonly used in the field of peptide chemistry to prepare analogs of natural peptides.

[0033] Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, ornithine, and lysine. Examples of “non-protein” α-amino acids / non-natural amino acids include ortholeucine, orthovaline, alloleucine, homoarginine, thioproline, dehydroproline, hydroxyproline (Hyp), homoserine, cyclohexylglycine (Chg), α-amino-butyric acid (Aba), cyclohexylalanine (Cha), aminophenylbutyric acid (Pba), phenylalanine with the phenyl moiety replaced by alkyl, alkoxy, halogen, or nitro groups, O-alkylated derivatives of serine, threonine, and tyrosine, S-alkylated cysteine, O-sulfate esters of tyrosine, and D-isomers of natural amino acids. In some embodiments of this document, non-natural amino acids include amino acids with olefin side chains, such as (S)-2-(4'-pentenyl)alanine (i.e., S5), (R)-2-(4'-pentenyl)alanine (i.e., R5), (S)-2-(7'-octenyl)alanine (i.e., S8), (R)-2-(7'-octenyl)alanine (i.e., R8), and 2-amino-2-(4'-pentenyl)-6-heptenoic acid (i.e., B5).

[0034] As used herein, the term "stereoisomer" refers to an isomer formed by having one or more stereoisomeric centers, each of which may be in an R or S configuration or a combination thereof. Similarly, the polypeptides described herein may have one or more double bonds, each of which may be in an E (trans) or Z (cis) configuration or a combination thereof. A particular stereoisomer, regioisomer, diastereomer, enantiomer, or epiisomer should be understood to include all possible stereoisomers, regioisomers, diastereomers, enantiomers, or epiisomers and mixtures thereof. Therefore, the polypeptides described herein include all configurationally different stereoisomers, regioisomers, diastereomers, enantiomers, or epiisomers and their corresponding mixtures. Preferred structurally pure isomers of the polypeptides of the present invention are enantiomers or diastereomers. Techniques for converting or maintaining a specific stereoisomer, as well as techniques for separating mixtures of stereoisomers, are well known in the art, and those skilled in the art can select the appropriate method for the specific circumstances.

[0035] As used herein, a stapled peptide is a polypeptide in which two amino acid side chains form a covalent bond through an orthogonal reaction. A double-bridged stapled peptide is a polypeptide in which two independent covalent bonds are formed between the amino acid side chains. Peptide stapleting can be used to physically bind a peptide to form or maintain a specific conformation (e.g., physically binding a peptide to maintain its original α-helical state). Stapleting can enhance the pharmaceutical properties of peptides by helping to maintain the original structure required for interaction with target molecules, increasing cell penetration, and / or protecting peptides from protein degradation.

[0036] As used herein, the term "functionally equivalent variant" or "functional variant" refers to a polypeptide derived from the polypeptide sequence of the present invention through modification by substitution, deletion, or addition of amino acids, or a derivative polypeptide having a certain sequence identity with the polypeptide of the present invention, provided that the mentioned derivative polypeptide maintains at least 50%, at least 80%, or at least 100% of the function based on the corresponding unmodified polypeptide. This functionally equivalent variant also includes polypeptides with increased functional activity compared to the unmodified polypeptide.

[0037] antiviral peptides

[0038] This application provides an antiviral polypeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof, the antiviral polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NO: 1-10 or a functional variant thereof.

[0039] This application provides an antiviral polypeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof, the antiviral polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NO: 5-10 or a functional variant thereof.

[0040] This application provides an antiviral polypeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures, or functionally equivalent variants thereof, the antiviral polypeptide comprising the amino acid sequence of any one of the following (a)-(f):

[0041] (a) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 5, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO: 5.

[0042] (b) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 6, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO: 6;

[0043] (c) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 7, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO: 7;

[0044] (d) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 8, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO: 8.

[0045] (e) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 9, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO: 9.

[0046] (f) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 10, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO: 10.

[0047] In some embodiments, the polypeptide derivatives provided in this disclosure are selected from derivatives modified with maleimide, derivatives obtained by attaching an amino-terminal protecting group to the amino terminus and / or a carboxyl-terminal protecting group to the carboxyl terminus, derivatives modified with protein or polyethylene glycol, derivatives obtained by attaching an oligopeptide or lipophilic group or cholesterol to the amino terminus and / or carboxyl terminus, or derivatives obtained by replacing amino acids with a D-type conformation, artificially modified amino acids, and rare amino acids found in nature.

[0048] In some embodiments, the amino-terminal protecting group provided by the present invention is selected from: H, a polymer derived from polyethylene glycol, acyclic substituted or unsubstituted aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted heteroarylalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted aralkyl groups, and R5-CO-, wherein R5 is selected from: H, acyclic substituted or unsubstituted aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted aralkyl groups, substituted or unsubstituted heterocyclic groups, and substituted or unsubstituted heteroarylalkyl groups. Preferably, the amino-terminal protecting group is selected from: H, acetyl (Ac), lauroyl, myristyl, and palmitoyl.

[0049] In some embodiments, the carboxyl-terminal protecting group provided by the present invention is selected from: -NR3R4, -OR3 and -SR3, wherein R3 and R4 are independently selected from: H, a polymer derived from polyethylene glycol, a non-cyclic substituted or unsubstituted aliphatic group, substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heteroarylalkyl group, substituted or unsubstituted aryl group, and substituted or unsubstituted aralkyl group. Preferably, the carboxyl-terminal protecting group is selected from: -NR3R4 and -OR3, wherein R3 and R4 are independently selected from H, methyl, ethyl, hexyl, dodecyl and hexadecyl.

[0050] Lipopeptides

[0051] This application provides a lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof, the lipopeptide comprising: (1) an antiviral polypeptide, (2) a linker arm, (3) an amino acid residue, and (4) a lipophilic compound, wherein (2) the linker arm may be absent; and (3) the amino acid residue is selected from K or C, or amino acid residues having similar properties to them.

[0052] In some embodiments, (1) the antiviral peptide is selected from binding peptides and non-binding peptides. In some preferred embodiments, (1) the antiviral peptide is selected from dual-bridged binding peptides.

[0053] In some preferred embodiments, the lipopeptide provided in this application comprises, from the N-terminus to the C-terminus, the following components in sequence: (1) an antiviral polypeptide, (2) a linker arm, (3) an amino acid residue, and (4) a lipophilic compound, wherein (1) the antiviral polypeptide is a binding peptide, preferably a double-bridged binding peptide; (2) the linker arm may be absent; and (3) the amino acid residue is selected from K or C, or amino acid residues with similar properties to them.

[0054] In some embodiments, the lipopeptide provided in this application comprises the following amino acid sequence:

[0055] (a) The amino acid sequence shown in Formula I,

[0056] L-X1-LQ-X2-X3-X4-X5-X6-LQ-X7-X8-I-X9-X 10 -LNX 11 -X 12 -YX 13 -KX 14 Formula I,

[0057] Where X1 represents E or K, or an amino acid residue with similar properties;

[0058] X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n Furthermore, the side chains of X2 and X6, as well as X... 10 and X 12 The side chains are all connected by covalent bonds, or

[0059] X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n And X2, X6, X 10 X 12 They do not form covalent bonds with each other, or

[0060] X2, X6, X 10 X 12 Each is independently 2-aminoisobutyric acid (Aib);

[0061] X3 represents K or E, or an amino acid residue with similar properties;

[0062] X4 represents L-ortholeucine (Nle);

[0063] X5 represents N, K, or E, or an amino acid residue with similar properties;

[0064] X7 represents E or K, or an amino acid residue with similar properties;

[0065] X8 represents 2-aminoisobutyric acid Aib;

[0066] X9 represents E or K, or an amino acid residue with similar properties;

[0067] X 11 It represents Q, E, or K, or amino acid residues with similar properties;

[0068] X 13 This indicates a connecting arm, or that it does not exist.

[0069] X 14 Indicates a lipophilic compound;

[0070] The symbol "-" indicates a covalent bond between residues;

[0071] (b) An amino acid sequence having 1-5 conserved substitutions as described in Formula I; or

[0072] (c) The sequence shown in Formula I is obtained by deleting 1-2 amino acids at the N-terminus or C-terminus.

[0073] In some preferred embodiments, the lipopeptide provided in this application comprises the following amino acid sequence from the N-terminus to the C-terminus:

[0074] (a) The amino acid sequence shown in Formula I,

[0075] L-X1-LQ-X2-X3-X4-X5-X6-LQ-X7-X8-I-X9-X 10 -LNX 11 -X 12 -YX 13 -KX 14 Formula I,

[0076] Where X1 represents E or K, or an amino acid residue with similar properties;

[0077] X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n Furthermore, the side chains of X2 and X6, as well as X... 10 and X 12 The side chains are all connected by covalent bonds, or

[0078] X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n And X2, X6, X 10 X 12 They do not form covalent bonds with each other, or

[0079] X2, X6, X 10 X 12 Each is independently 2-aminoisobutyric acid (Aib);

[0080] X3 represents K or E, or an amino acid residue with similar properties;

[0081] X4 represents L-ortholeucine (Nle);

[0082] X5 represents N, K, or E, or an amino acid residue with similar properties;

[0083] X7 represents E or K, or an amino acid residue with similar properties;

[0084] X8 represents 2-aminoisobutyric acid Aib;

[0085] X9 represents E or K, or an amino acid residue with similar properties;

[0086] X 11 It represents Q, E, or K, or amino acid residues with similar properties;

[0087] X 13 This indicates a connecting arm, or that it does not exist.

[0088] X 14 Indicates a lipophilic compound;

[0089] The symbol "-" indicates a covalent bond between residues;

[0090] (b) An amino acid sequence having 1-5 conserved substitutions as described in Formula I; or

[0091] (c) The sequence shown in Formula I is obtained by deleting 1-2 amino acids at the N-terminus or C-terminus.

[0092] In some embodiments, the non-natural amino acid S provided in this application n The amino acids are selected independently from those with olefin side chains. In some preferred embodiments, S n The amino acid is selected from 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 more preferred embodiments, S n It is 2-amino-2-methyl-6-heptenoic acid.

[0093] In some embodiments, the non-natural amino acid S provided in this application n The covalent bonds between the side chains are selected from disulfide bonds, hydrocarbon chains, intramolecular amide bonds, thioether bonds, and covalent bonds formed by cycloaddition reactions of azide-alkyne hydrocarbons.

[0094] In some embodiments, the non-natural amino acid S provided in this application n The covalent bonds between the side chains are formed through olefin metathesis reactions, and in some preferred embodiments, they are formed through all-hydrocarbon chain linking bridges.

[0095] In some implementations, when X2, X6, X... 10 X 12 Each is an independent non-natural amino acid S n Furthermore, the side chains of X2 and X6, as well as X... 10 and X 12 When the side chains of S are all covalently connected, n It is 2-amino-2-methyl-6-heptenic acid; when X2, X6, X... 10 X 12 Each is an independent non-natural amino acid S n And X2, X6, X 10 X 12 When there are no covalent bonds between them, S n It is 2-amino-2-methyl-6-heptenoic acid.

[0096] In some embodiments, the connecting arm provided in this application comprises a flexible or rigid connecting arm.

[0097] In some embodiments, the linker provided in this application comprises at least one of small molecule polyethylene glycol linkers, chain alkane linkers, and oligopeptide linkers. In some preferred embodiments, the linker is selected from EAAAK, PEG4, PEG8, and PEG12.

[0098] In some embodiments, the lipophilic compounds provided in this application include fatty acids, cholesterol derivatives, hydroxycholesterol derivatives, dihydrosphingosine, and vitamin E.

[0099] In some embodiments, the lipophilic compound provided in this application is selected from cholesterol monosuccinate, 2-cholesterolacetic acid, 2-cholesterolpropionic acid, 3-cholesterolpropionic acid, 2-cholesterolbutyric acid, 2-cholesterol isobutyric acid, 3-cholesterolbutyric acid, 3-cholesterol isobutyric acid, 4-cholesterolbutyric acid, 2-cholesterol valerate, 2-cholesterol isovaleric acid, 3-cholesterol valerate, 5-cholesterol valerate, 2-cholesterol hexanoic acid, 6-cholesterol hexanoic acid, 2-cholesterol heptanoic acid, 7-cholesterol heptanoic acid, 2-cholesterol octanoic acid, or 8-cholesterol octanoic acid.

[0100] In some embodiments, the lipophilic compound provided in this application comprises CH3(CH2). nCOOH, where n is an integer from 1 to 30. In some preferred embodiments, the lipophilic compound provided in this application is palmitic acid.

[0101] In some embodiments, the lipopeptide provided in this application comprises the sequence shown in any one of (a)-(m), a sequence having 1-5 conserved substitutions with the sequence shown in any one of (a)-(m) below, or the sequence shown in any one of (a)-(m) with 1-2 amino acids deleted from the N-terminus or C-terminus:

[0102] (a)LKLQ-X2-EZN-X6-LQEBIKX 10 -LNQX 12 -YKC 16 ;

[0103] (b)LKLQ-X2-EZN-X6-LQEBIKX 10 -LNQX 12 -YEAAAKKC 16 ;

[0104] (c)LKLQ-X2-EZN-X6-LQEBIKX 10 -LNQX 12 -Y-PEG4-KC 16 ;

[0105] (d)LKLQ-X2-EZN-X6-LQEBIKX 10 -LNQX 12 -Y-PEG8-KC 16 ;

[0106] (e)LKLQ-X2-EZN-X6-LQEBIKX 10 -LNQX 12 -Y-PEG12-KC 16 ;

[0107] (f)LKLQ-X2-EZK-X6-LQEBIKX 10 -LNQX 12 -YKC 16 ;

[0108] (g)LKLQ-X2-EZN-X6-LQEBIKX 10 -LNEX 12 -YKC16 ;

[0109] (h)LKLQ-X2-EZK-X6-LQEBIKX 10 -LNEX 12 -YKC 16 ;

[0110] (i)LELQ-X2-KZN-X6-LQEBIKX 10 -LNQX 12 -YKC 16 ;

[0111] (j)LELQ-X2-KZE-X6-LQKBIKX 10 -LNQX 12 -YKC 16 ;

[0112] (k)LELQ-X2-KZN-X6-LQEBIEX 10 -LNKX 12 -YKC 16 ;

[0113] (l)LELQ-X2-KZE-X6-LQKBIEX 10 -LNKX 12 -YKC 16 ;

[0114] (m)LKLQBEZKBLQEBIKBLNQBY-KC 16 ;

[0115] Among them, X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n Furthermore, the side chains of X2 and X6, as well as X... 10 and X 12 The side chains are all covalently connected, or X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n And X2, X6, X 10 X 12 They do not form covalent bonds with each other. Preferably, when X2, X6, X 10 X 12 Each is an independent non-natural amino acid S nFurthermore, the side chains of X2 and X6, as well as X... 10 and X 12 When the side chains of S are all covalently connected, n It is 2-amino-2-methyl-6-heptenic acid; when X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n And X2, X6, X 10 X 12 When there are no covalent bonds between them, S n It is 2-amino-2-methyl-6-heptenoic acid;

[0116] “Z” represents the special amino acid L-ortholeucine (Nle).

[0117] “B” represents the special amino acid 2-aminoisobutyric acid Aib;

[0118] “PEG4” represents polyethylene glycol, “PEG8” represents polyethylene glycol octadecyl glycol, and “PEG12” represents polyethylene glycol dodecyl glycol.

[0119] “C 16 "Represents palmitic acid;

[0120] The symbol "-" indicates a covalent bond between residues;

[0121] Preferably, C 16 It is connected to K via a side chain (a)-(m)(a)-(m)(a)-(m).

[0122] In some embodiments, the lipopeptides provided in this disclosure comprise the sequence shown in any one of SEQ ID NOs: 14-27, the sequence having 1-5 conserved substitutions with the sequence shown in any one of SEQ ID NOs: 14-27, the sequence of any one of SEQ ID NOs: 14-27 with 1-2 amino acids deleted at the N-terminus or C-terminus, or the functional variants described above.

[0123] As used herein, “derivatives” of polypeptides refer to products obtained by modifying amino acids, substituting conserved amino acids, and / or substituting hydrogen in amino acid residues based on the polypeptides mentioned herein.

[0124] When used herein, the term "amino acid modification" includes, but is not limited to, N-terminal modification, C-terminal modification, and side-chain modification. The manner of "amino acid modification" includes, but is not limited to, hydroxylation, carboxylation, alkylation, acylation, phosphorylation, sulfonation, amidation, aldehydeation, alcoholylation, mercaptoethylamineation, esterification, and glycosylation.

[0125] As used herein, "conservative amino acid substitution" can generally be described as the substitution of one amino acid residue by another amino acid having a similar chemical structure and / or similar chemical properties, with little effect on the function, activity, or other biological properties of the peptide. Such conserved amino acid substitutions are well known in the art. Such conserved substitutions can be, for example, the substitution of one amino acid from the following groups (a)-(e) by another amino acid from the same group: (a) small aliphatic, nonpolar, or weakly polar amino acid residues: Ala, Ser, Thr, Pro, and Gly; (b) negatively charged polar amino acid residues and their amides: Asp, Asn, Glu, and Gln; (c) positively charged polar amino acid residues: His, Arg, and Lys; (d) large aliphatic, nonpolar amino acid residues: Met, Leu, Ile, Val, and Cys; (e) aromatic amino acid residues: Phe, Tyr, and Trp. Conservative amino acid substitution can also be the substitution of an amino acid residue by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphiphilic properties of the residues involved.

[0126] As used in this paper, substitutions between amino acid residues with similar properties have little effect on the function, activity, or other biological properties of the peptide.

[0127] When used herein, the substitution of hydrogen in an amino acid residue can be the substitution of hydrogen in the amino acid residue by any conventional substituent known in the art, including but not limited to alkyl, alkenyl, alkynyl, aromatic, alkoxy, carboxyl, aldehyde, carbonyl, hydroxyl, halogen, cyano, acyl, sulfonic acid, amino, mercapto, or nitro groups.

[0128] In some embodiments, the lipopeptide derivatives provided in this application comprise amino acid-modified derivatives.

[0129] In some embodiments, the amino acid modifications provided in this application include replacing one or more L-type amino acids with D-type amino acids, artificially modified amino acids, or rare amino acids found in nature, as well as C-terminal and N-terminal modifications of amino acids.

[0130] As illustrated herein, the term "lipopeptide" refers to a molecule comprising at least one lipophilic compound and a peptide fragment. The lipophilic compound in a lipopeptide can be a straight-chain, branched, or cyclic fatty acid or a derivative thereof, typically linked to the polypeptide moiety via an amide bond or ester bond. The peptide fragment can contain L-, D-, α-, or β-amino acid residues. The amino acids described herein can be of natural or non-natural origin.

[0131] As used herein, the term "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts. Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include, but are not limited to: acetates, adipates, aspartates, benzoates, benzenesulfonates, bicarbonates / carbonates, hydrogen sulfates / sulfates, borates, camphor sulfonates, citrates, cyclohexylamine sulfonates, ethanedisulfonates, formates, fumarates, gluconate, glucuronates, glucuronates, hexafluorophosphates, 2-(4-hydroxybenzyl)benzoates, hydrochlorides / chlorides, hydrobromines / bromines, hydroiodides / iodides, 2-hydroxyethanesulfonates, lactates, malates, maleates, malonates, methanesulfonates, methyl sulfates, naphthalates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, oxalates, hexadecates, phosphates / hydrogen phosphates / dihydrogen phosphates, pyroglutamates, gluconate, stearates, salicylates, tannins, tartrates, toluenesulfonates, and trifluoroacetates. Suitable base addition salts are formed by bases that form non-toxic salts. Examples include, but are not limited to: aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. They can also form hemisalts of acids and bases, such as hemisulfates and hemicalcium salts. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahland Wermuth (Wiley-VCH, 2002).

[0132] Preparation method

[0133] This disclosure provides a method for preparing the lipopeptide or stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof, comprising (1) synthesizing the antiviral polypeptide provided herein, (2) optionally, linking the antiviral polypeptide to a linker arm, (3) loading amino acid residues, and (4) conjugating a lipophilic compound.

[0134] In some embodiments, the peptides are synthesized using methods conventional in the art, such as solid-phase synthesis, liquid-phase synthesis, biosynthesis, and enzyme-catalyzed synthesis.

[0135] In some embodiments, the conjugation of the lipophilic compound is achieved by a condensation method. In some embodiments, the conjugation of the lipophilic compound onto the peptide is achieved by exposing a side-chain amino acid, such as lysine, and then covalently attaching the lipophilic compound to the side-chain amino acid.

[0136] Drug composition, drug combination

[0137] This disclosure provides pharmaceutical compositions comprising the lipopeptide provided herein, or a stereoisomer, derivative thereof, pharmaceutically acceptable salt, mixture thereof, or a functionally equivalent variant thereof, or an antiviral polypeptide, or a stereoisomer, derivative thereof, pharmaceutically acceptable salt, mixture thereof, or a functionally equivalent variant thereof, and a pharmaceutically acceptable carrier.

[0138] This disclosure provides pharmaceutical combinations comprising the lipopeptide provided herein or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof, the antiviral polypeptide provided herein or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof, or the pharmaceutical composition provided herein, and a second therapeutic agent.

[0139] In some embodiments, the second therapeutic agent is selected from antiviral drugs, including but not limited to protease inhibitors, reverse transcriptase inhibitors, coronavirus neutralizing antibodies, intravenous COVID-19 human immunoglobulin, and convalescent plasma.

[0140] The pharmaceutical compositions and combinations disclosed herein can be formulated in any manner known in the art, including but not limited to dosage forms such as tablets, capsules, small capsules, suspensions, powders, lyophilized preparations, suppositories, eye drops, skin patches, oral soluble preparations, sprays, aerosols, and other solid, semi-solid, or liquid systems.

[0141] The pharmaceutical compositions disclosed herein can be immediate-release and / or modified-release formulations, including delayed-release, sustained-release, pulsatile-release, controlled-release, targeted-release, and programmed-release formulations.

[0142] In this document, "pharmaceutically acceptable carrier" includes pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, relating to carrying or delivering the peptides of this disclosure within or to a subject, enabling them to perform their intended function. Each salt or carrier must be "acceptable" in the sense of compatibility with other components of the formulation and not harmful to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate. Esters and ethyl lauryl ester; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free raw water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solution; diluents; granulators; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavoring agents; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardeners; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic, compatible substances used in pharmaceutical preparations, or any combination thereof.

[0143] use

[0144] This disclosure provides for the use of the lipopeptides provided herein, stereoisomers thereof, derivatives thereof, pharmaceutically acceptable salts, mixtures thereof, or functionally equivalent variants thereof, antiviral polypeptides, stereoisomers thereof, derivatives thereof, pharmaceutically acceptable salts thereof, mixtures thereof, or functionally equivalent variants thereof, pharmaceutical compositions, or pharmaceutical combinations in the preparation of medicaments for the treatment or prevention of viral infections.

[0145] This disclosure provides methods for treating or preventing viral infections, including administering to a subject in need an effective amount of the lipopeptide provided herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, an antiviral polypeptide, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, a pharmaceutical composition, or a combination of pharmaceuticals.

[0146] This disclosure provides for the use of lipopeptides or stereoisomers thereof, derivatives thereof, pharmaceutically acceptable salts, mixtures thereof, or functionally equivalent variants thereof, antiviral polypeptides or stereoisomers thereof, derivatives thereof, pharmaceutically acceptable salts thereof, mixtures thereof, or functionally equivalent variants thereof, pharmaceutical compositions, or pharmaceutical combinations thereof for the treatment or prevention of viral infections.

[0147] In some embodiments, the virus provided in this application includes coronaviruses. In some preferred embodiments, the virus is selected from SARS-CoV, MERS-CoV, and SARS-CoV-2.

[0148] When used in this document, the term “treatment” means to alleviate or improve a disease or disorder (i.e., to slow or stop the development of the disease or at least one clinical symptom); or to alleviate or improve at least one physical parameter or biomarker associated with the disease or disorder.

[0149] As used herein, the term “prevention” refers to methods implemented to block, reduce, suppress, prevent and / or delay the occurrence of a disease or condition or symptom (e.g., respiratory diseases and symptoms, infections or autoimmune diseases) in a subject, or methods to reduce the incidence of infectious diseases in said subject.

[0150] In some embodiments, the lipopeptides or stereoisomers thereof, derivatives, pharmaceutically acceptable salts, mixtures thereof, or functionally equivalent variants thereof provided herein, antiviral peptides or stereoisomers thereof, derivatives thereof, pharmaceutically acceptable salts, mixtures thereof, or functionally equivalent variants thereof, pharmaceutical compositions, or pharmaceutical combinations are administered by means of: injection (including but not limited to subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracerebrospinal injection, etc.), cavity administration (including but not limited to gastrointestinal administration, genital tract administration, such as oral, gastrointestinal, rectal, vaginal, sublingual, etc.), respiratory administration (including but not limited to nasal, pulmonary), mucosal administration, or topical administration, preferably injection.

[0151] When used in this document, the term “subject” includes animals such as vertebrates, preferably mammals such as dogs, cats, pigs, cattle, sheep, horses, rodents (e.g., mice, rats, or guinea pigs) or primates (e.g., gorillas, chimpanzees, and humans).

[0152] As used herein, the term "effective amount" means an amount that, compared to a corresponding subject who did not receive that amount, would result in a benefit, prevention, or treatment of disease, but is sufficiently low within the range of reasonable medical judgment to avoid serious adverse effects. Effective amounts will vary depending on factors such as the choice of polypeptide, lipopeptide, pharmaceutical composition, drug combination; route of administration; severity of the disease being treated or prevented; patient's age, body type, weight, and physical condition; patient's medical history; duration of treatment; nature of concurrent treatments; and desired therapeutic effect, but can still be determined by those skilled in the art in a conventional manner.

[0153] Example

[0154] The following description, in conjunction with the accompanying drawings, illustrates exemplary embodiments of this application, including various details of these embodiments to aid understanding. It should be understood that these are merely exemplary and are in no way intended to limit the scope of protection of this application. The scope of protection of this application is defined only by the claims. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0155] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0156] The Chinese explanations of the abbreviations or full English names used in this application are shown in Table 1 below:

[0157] Table 1. Comparison of Chinese explanations for abbreviations or full English names used.

[0158]

[0159] Example 1: Synthesis of Polypeptides

[0160] The peptides were synthesized using the standard Fmoc solid-phase synthesis (SPPS) method. All reagents used in the synthesis process, such as DMF, MeOH, DCM, and piperidine, were dried before use. Rink amide resin was used as the solid support, with a resin loading of 0.53 mmol / g.

[0161] 1.1 Swelling of Resin

[0162] Weigh 0.19 g of Rink amide resin into a 50 mL solid-phase reactor, add 5 mL of DCM and 5 mL of DMF, and allow it to swell for 15 min.

[0163] 1.2 Removal of Fmoc protecting groups from resin

[0164] Add a deprotecting agent (20% piperidine / DMF solution, v / v) to the swollen resin. Remove Fmoc in two steps: first, add 5 mL of the deprotecting agent and stir continuously for 5 min, then dry under vacuum; second, add another 5 mL of the deprotecting agent and stir continuously for 25 min, then dry under vacuum. Then wash twice with 5 mL DMF, 5 mL MeOH, and 5 mL DCM, respectively. Next, take a small amount of the deprotected resin for testing at 110°C for 3 min. Remove the test tube from the heater and observe the phenomenon. If the resin turns blue, deprotection is complete, and the next step, amino acid condensation, can proceed. Otherwise, adjust the program and repeat steps 1 and 2 until the resin turns blue.

[0165] 1.3 Condensation reaction of amino acids

[0166] Deprotected resin was added 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 activating alkali solution (0.6 M DIEA / NMP). The mixture was stirred at room temperature. After the reaction was complete, the peptide resin was washed twice, sequentially with 5 mL of DMF, 5 mL of methanol, and 5 mL of dichloromethane. A small amount of the peptide resin was then tested at 110 °C for 3 min. The test tube was removed from the heater, and the phenomenon was observed. If the resin turned blue, it indicated that the amino acid condensation was incomplete. Step 1.3 was repeated until the reaction was complete, and the resin turned yellow. If the resin turned yellow, steps 1.2 and 1.3 were repeated to continue condensing the next amino acid until the peptide synthesis was complete. Lysine residues with Dde protected side chains were used for covalent conjugation. After the peptide synthesis was complete, 5 mL of 2% hydrazine hydrate / DMF solution was added, and the mixture was stirred at room temperature for 3 min, then dried. This process was repeated five times. The resin was washed five times each with DMF, DCM, and methanol. At this point, the Dde groups of the lysine residues protecting the Dde side chains in the peptide resin were removed, exposing the amino groups on the side chains. Then, the same condensation method C was used. 16 It is covalently attached to the amino side chain of lysine.

[0167] 1.5 Acetylated end capping

[0168] After peptide synthesis, the Fmoc protecting group was removed, and the mixture was washed. Then, 2 mL of DIEA and 2 mL of acetic anhydride were added, along with 4 mL of DMF. The reaction was allowed to proceed for 30 min. After the reaction was complete, the peptide resin was washed, and a small sample was taken for testing at 110 °C for 3 min. The test tube was removed from the heater, and the observed phenomena were noted. The resin turned yellow, indicating that end-capping was complete. The mixture was then washed and dried.

[0169] 1.6 Solid-phase olefin metathesis reaction

[0170] Special amino acids (S) nThe side-chain olefin groups of the peptide were covalently linked to synthesize the bound peptide. The peptide resin (0.1 mmol) was transferred to a light-protected oval flask and purged with nitrogen for 5 min. 50 mg of second-generation Grubbs catalyst was weighed, with the weighing process conducted in the dark. The weighed catalyst was dissolved in 5 mL of anhydrous dichloroethane and added to the reaction system. The mixture was stirred under nitrogen for 6 h, and the reaction was repeated once after completion. After the reaction was complete, the peptide resin was transferred to a solid-phase reactor and washed twice each for 2 min with DMF, MeOH, and DCM. The resin was then dried under vacuum for later use. If the peptide used does not undergo amino acid cyclization, step 1.6 is skipped, and the cleavage operation in step 1.7 is performed directly.

[0171] 1.7 Pyrolysis

[0172] First, prepare the lysis buffer with the following composition: trifluoroacetic acid: anisole: ethylenedithiol: m-cresol: water = 82.5:5:2.5:5:5 (volume percentage). The buffer should be pre-cooled in an ice bath for 30 minutes or stored in a refrigerator before use. Place the peptide resin in a 250 mL round-bottom flask, place it in an ice bath, add 10 mL of the lysis buffer, and stir magnetically. The resin turns orange-red. React for 30 minutes in an ice bath, then remove the ice bath and continue stirring at room temperature for another 90 minutes. Next, add 200 mL of cold diethyl ether under vigorous stirring, causing a white precipitate to form. Continue stirring for 30 minutes. Filter the precipitate using a G4 sintered glass funnel, wash three times repeatedly with cold diethyl ether, and air dry. Then, add 50 mL of double-distilled water and 5 mL of acetonitrile to fully dissolve the solid, filter, and freeze-dry the filtrate to obtain the crude compound.

[0173] 1.8 Purification

[0174] The crude compound was purified by high-performance liquid chromatography (HPLC) using a C8 column and acetonitrile, water, and a small amount of trifluoroacetic acid as eluent. The specific steps were as follows: 1.00 g of the crude compound was weighed, dissolved in 20 mL of water and 5 mL of acetonitrile, and centrifuged for 10 min (3000 rpm). The supernatant was then loaded onto the column. The column was pre-equilibrated with 200 mL of a 15% acetonitrile / water / 0.1% trifluoroacetic acid solution. After loading, the column was washed with 200 mL of the same solution, and the eluent components were analyzed by HPLC. The acetonitrile content was gradually increased based on the HPLC results until the main peak of the purified compound was eluted. The eluents were combined, most of the solvent was removed by rotary evaporation, and the compounds were lyophilized to obtain the purified compounds. The HPLC purity was greater than 95.0%. The molecular weight of the compounds was determined using MALDI-TOF-MS. The elution gradient for peptide purity identification is shown in Table 2.

[0175] Table 2 Elution gradient

[0176] Time (min) Solvent A (%) Solvent B (%) 5 50 50 10 30 70 15 10 90 20 0 100 23 0 100 25 90 10

[0177] Solvent A consists of 100% water and 0.1% trifluoroacetic acid, and solvent B consists of 70% acetonitrile, 30% water, and 0.1% trifluoroacetic acid.

[0178] Example 2: Anti-SARS-CoV-2 pseudovirus infection experiment

[0179] The polypeptides prepared in Example 1 are shown in Table 3.

[0180] Table 3 shows the prepared peptides.

[0181] Polypeptide name Structure SEQ ID NO 1 LKLQ*E ZN*LQEB IK*LNQ*Y 1 2 LKLQ*E ZK*LQEB IK*LNQ*Y 2 3 LKLQ*E ZN*LQEB IK*LNE*Y 3 4 LKLQ*E ZK*LQEB IK*LNE*Y 4 5 LELQ*K ZN*LQEB IK*LNQ*Y 5 6 LELQ*K ZE*LQKB IK*LNQ*Y 6 7 LELQ*K ZN*LQEB IE*LNK*Y 7 8 LELQ*K ZE*LQKB IE*LNK*Y 8 9 LKLQXE ZKXLQEB IKXLNQX Y 9 10 LKLQBE ZKBLQEB IKBLNQB Y 10 C21DS6-5 ILKLQ*E ZN*LQEB IK*LNQ*Y 11 SARSHR2P DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL 12 EK1 SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL 13

[0182] In the sequence, "*" represents the special amino acid 2-amino-2-methyl-6-heptenic acid (S5), which is a non-natural amino acid; "Z" represents the special amino acid L-leucine (Nle); "X" represents the non-natural amino acid 2-amino-2-methyl-6-heptenic acid (S5) at that position, which is not cyclic; and "B" represents the special amino acid 2-aminoisobutyric acid (Aib).

[0183] By deleting isoleucine at the N-terminus of the C21DS6-5 peptide, introducing multiple EK mutations, and introducing the lipophilic palmitic acid group on the lysine side chain at the C-terminus of the peptide, a series of novel anti-SARS-CoV-2 lipopeptide molecules were obtained, as shown in Table 4.

[0184] Table 4 shows the prepared lipopeptides.

[0185]

[0186]

[0187] In the sequence, "*" represents the specific amino acid 2-amino-2-methyl-6-heptenic acid (S5), which is a non-natural amino acid; "Z" represents the specific amino acid L-leucine (Nle); "B" represents the specific amino acid 2-aminoisobutyric acid (Aib); "X" represents that the non-natural amino acid 2-amino-2-methyl-6-heptenic acid (S5) at this position is not cyclic; "C" represents the non-natural amino acid 2-amino-2-methyl-6-heptenic acid (S5) at this position is not cyclic; 16 "Represents palmitic acid; "(C 16 ")" indicates the introduction of a lipophilic palmitic acid group onto the side chain of the amino acid residue to which it is attached; "-" indicates a covalent bond.

[0188] To detect the inhibitory activity of the peptides and lipopeptides against coronavirus PsV infection, target cells (Caco-2 cells, American Type Culture Collection) were placed in 96-well plates at a density of 10⁴ cells per well one day prior to infection. PsV was mixed with an equal volume of the peptides and serially diluted with PBS at 37°C for 30 minutes. This mixture was then transferred to Huh-7 cells. After 12 hours, the medium was replaced, and the cells were cultured for another 48 hours. The cells were then washed with PBS, lysed with a lysis reagent (Promega), and the relative optical units (RLU) were measured using a luciferase assay kit.

[0189] The results are shown in Table 5. The results show that the novel membrane fusion inhibitor or lipopeptide of this invention exhibits low-micromolar antiviral activity against SARS-CoV-2. Furthermore, compared to the EK1 peptide already in clinical trials, the novel membrane fusion inhibitor or lipopeptide of this invention shows higher antiviral activity. The biological activity of the obtained lipopeptides is significantly improved compared to C21DS6-5. Among them, BC1-A lipopeptide shows the best inhibitory activity against SARS-CoV-2, with an EC50 value of 0.02 μM, nearly 45 times higher than C21DS6-5, and approximately 35 times higher than the EK1 peptide already in clinical trials. The other five lipopeptides, BC1-B, BC1-C, BC1-DB, BC1-DC, and BC1-DD, are 1.2-8.09 times more potent than C21DS6-5. In addition, BC1AS5B, through the substitution of Aib, achieves superior inhibitory activity compared to the control C21DS6-5.

[0190] Table 5 Results of infection with SARS-CoV-2 pseudovirus

[0191] Name Structure EC 50 (μM) BC1 LKLQ*E ZN*LQEB IK*LNQ*Y-K(C 16 )]]> 0.75 BEC1 LKLQ*E ZN*LQEB IK*LNQ*Y-EAAAK-K(C 16 )]]> 5.07 BP4C1 LKLQ*E ZN*LQEB IK*LNQ*Y-PEG4-K(C 16 )]]> 2.10 BP8C1 LKLQ*E ZN*LQEB IK*LNQ*Y-PEG8-K(C 16 )]]> >10 BP12C1 LKLQ*E ZN*LQEB IK*LNQ*Y-PEG12-K(C 16 )]]> >10 BC1-A LKLQ*E ZK*LQEB IK*LNQ*Y-K(C 16 )]]> 0.02 BC1-B [CAT] LKLQ*E ZN*LQEB IK*LNE*Y-K(C 16 )]]> 0.11 BC1-C [CAT] LKQ*E ZK*LQEB IK*LNE*Y-K(C 16 )]]> 0.41 BC1-DA LELQ*K ZN*LQEB IK*LNQ*Y-K(C 16 )]]> 0.94 BC1-DB LELQ*K ZE*LQKB IK*LNQ*Y-K(C 16 )]]> 0.34 BC1-DC LELQ*K ZN*LQEB IE*LNK*Y--K(C 16 )]]> 0.26 BC1-DD LELQ* K ZE* LQKB IE* LNK* Y-K(C 16 )]]> 0.74 BC1AS5 LKLQXE ZKXLQEB IKXLNQX Y-K(C 16 )]]> 1.59 BC1AS5B LKLQBE ZKBLQEB IKBLNQB Y-K(C 16 )]]> 0.84 C21DS6-5 ILKLQ*E ZN*LQEB IK*LNQ*Y 0.89 SARSHR2P DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL 1.09 EK1 SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL 0.70

[0192] Example 3. In vitro and in vivo broad-spectrum antiviral experiment

[0193] 3.1 In vitro experiments

[0194] To detect the inhibitory activity of the peptide against coronavirus PsV infection, target cells (Caco-2 cells, American Type Culture Collection) were cultured at 10 μL per well one day before infection. 4 Cells were placed at a density of 100 cells per well in 96-well plates. PsV was mixed with an equal volume of peptide and serially diluted with PBS at 37°C for 30 minutes. This mixture was then transferred to Huh-7 cells. After 12 hours, the medium was changed, and the cells were cultured for another 48 hours. Cells were then washed with PBS, lysed with a lysis reagent (Promega), and the relative light units (RLU) were measured using a luciferase assay kit. The inhibition rate was calculated according to the following formula.

[0195]

[0196] Where X is the RLU value of a single well, and CC is the cell control group with only cells added. The mean RLU of the control group with added cells and pseudoviruses. The RLU is the average value for the virus control group. The experiment was repeated three times, and the data were fitted using a nonlinear regression four-parameter equation and the IC was calculated using GraphPadPrism software. 50 value.

[0197] Figure 1A The relationship between BC1-A concentration and the inhibition rate (%) of SARS-CoV-2 Omicron BA.5.2 pseudovirus infection was shown. Figure 1A As shown, the inhibition rate exhibited a significant upward trend with increasing BC1-A concentration. The IC50 of BC1-A was calculated. 50 The value was 0.15 μM, indicating that BC1-A has high inhibitory activity against OmicronBA.5.2.

[0198] 3.2 In vivo experiments

[0199] Male hACE2 transgenic mice (C57BL / 6J-[human ACE2-2A-CrERT2]) used in the experiment were provided by Cyagen (Suzhou) Biotechnology Co., Ltd. Mice were administered BC1-A (16 mg / kg) intranasally 30 minutes before or after intranasal inoculation with 30,000 pfu of SARS-CoV-2 virus. Euthanasia was performed on day 4 post-infection, and lung tissue was collected for the detection of viral RNA levels and pathological changes.

[0200] Figure 1B This diagram illustrates the administration of BC1-A peptide and SARS-CoV-2 infection. hACE2 transgenic C57BL / 6 mice (6-8 weeks old) in the prevention and untreated control groups were intranasally treated with either BC1-A peptide (16 mg / kg) or a vector control (1% DMSO + 99% H2O, 40 μL / mouse) 30 minutes before Omicron BA.5.2 infection. Mice in the treatment group received BC1-A peptide (16 mg / kg) intranasally 30 minutes after Omicron BA.5.2 challenge. Seven days after infection, the mice were sacrificed and tissues were collected.

[0201] Figure 1C The viral titer in the lungs of mice in each group was measured on day 7 post-infection. An asterisk indicates a significant difference, **p<0.01.

[0202] Figure 1DHistopathological examination of lung tissue from mice given or not given BC1-A. Lung tissue sections were stained with hematoxylin and eosin for histopathological examination. Graduation, 100 μm.

[0203] BC1-A showed high inhibitory activity against six SARS-CoV-2 Omeprone variants in vitro, as shown in Table 6.

[0204] like Figure 1A As shown in Figure D, BC1-A exhibits high inhibitory activity against the Omeprone variant BA.5.2 both in vivo and in vitro. BC1-A demonstrates excellent efficacy in the treatment and prevention of SARS-CoV-2 Omeprone infection.

[0205] Table 6. Results of in vivo infection with experimental variants of SARS-CoV-2 Omeprón.

[0206]

[0207] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures, or functionally equivalent variants thereof, said lipopeptide comprising: (1) an antiviral polypeptide, (2) a linker arm, (3) an amino acid residue, and (4) a lipophilic compound, wherein The connecting arm (2) may not be present; The amino acid residues in (3) are selected from K or C, or amino acid residues with similar properties.

2. The lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof according to claim 1, wherein the (1) antiviral polypeptide is selected from bound peptides and unbound peptides, preferably from double-bridged bound peptides.

3. The lipopeptide according to claim 1 or 2, or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures, or functionally equivalent variants thereof, wherein the lipopeptide comprises the following amino acid sequence: (a) The amino acid sequence shown in Formula I, L-X1-L-Q-X2-X3-X4-X5-X6-L-Q-X7-X8-I-X9-X 10 -L-N-X 11 -X 12 -Y-X 13 -K-X 14 Formula I Where X1 represents E or K, or an amino acid residue with similar properties; X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n Furthermore, the side chains of X2 and X6, as well as X... 10 and X 12 The side chains are all connected by covalent bonds, or X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n And X2, X6, X 10 X 12 They do not form covalent bonds with each other, or X2, X6, X 10 X 12 Each is independently 2-aminoisobutyric acid (Aib); X3 represents K or E, or an amino acid residue with similar properties; X4 represents L-ortholeucine (Nle); X5 represents N, K, or E, or an amino acid residue with similar properties; X7 represents E or K, or an amino acid residue with similar properties; X8 represents 2-aminoisobutyric acid Aib; X9 represents E or K, or an amino acid residue with similar properties; X 11 It represents Q, E, or K, or amino acid residues with similar properties; X 13 This indicates a connecting arm, or that it does not exist. X 14 Indicates a lipophilic compound; The symbol "-" represents a covalent bond between residues; (b) An amino acid sequence having 1-5 conserved substitutions as described in Formula I; or (c) The sequence shown in Formula I is obtained by deleting 1-2 amino acids at the N-terminus or C-terminus.

4. The lipopeptide or its stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant according to any one of claims 1-3, wherein the S n The amino acids are independently selected from those with olefin side chains, preferably 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, more preferably, the S n It is 2-amino-2-methyl-6-heptenoic acid.

5. The lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures, or functionally equivalent variants according to any one of claims 1-4, wherein the non-natural amino acid S n The covalent bonds between the side chains are selected from disulfide bonds, hydrocarbon chains, intramolecular amide bonds, thioether bonds, and covalent bonds formed by azido-alkyne cycloaddition reactions.

6. The lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures, or functionally equivalent variants according to any one of claims 1-5, wherein the non-natural amino acid S n The covalent bonds between the side chains are formed through olefin metathesis reactions, and are preferably connected by all-hydrocarbon chain bridges.

7. The lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants according to any one of claims 1-6, when X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n Furthermore, the side chains of X2 and X6, as well as X... 10 and X 12 When the side chains are all covalently connected, S n It is 2-amino-2-methyl-6-heptenic acid; when X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n And X2, X6, X 10 X 12 When there are no covalent bonds between them, S n It is 2-amino-2-methyl-6-heptenoic acid.

8. The lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants according to any one of claims 1-7, wherein the connecting arm comprises a flexible or rigid connecting arm.

9. The lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof according to claim 8, wherein the linker comprises at least one of small molecule polyethylene glycol linkers, chain alkane linkers and oligopeptide linkers; preferably, the linker is selected from EAAAK, PEG4, PEG8 and PEG12.

10. The lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof according to any one of claims 1-9, wherein the lipophilic compound comprises fatty acids, cholesterol derivatives, hydroxycholesterol derivatives, dihydrosphingosine, or vitamin E.

11. The lipopeptide of claim 10, or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures, or functionally equivalent variants thereof, wherein the lipophilic compound comprises CH3(CH2). n COOH, where n is an integer from 1 to 30; preferably, the lipophilic compound is palmitic acid.

12. The lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof according to any one of claims 1-11, wherein the lipopeptide comprises the sequence shown in any one of (a)-(m), a sequence having 1-5 conserved substitutions with the sequence shown in any one of (a)-(m), or the sequence shown in any one of (a)-(m) with 1-2 amino acids deleted from the N-terminus or C-terminus: (a)L-K-L-Q-X2-E-Z-N-X6-L-Q-E-B-I-K-X 10 -L-N-Q-X 12 -Y-K-C 16 ; (b)L-K-L-Q-X2-E-Z-N-X6-L-Q-E-B-I-K-X 10 -L-N-Q-X 12 -Y-E-A-A-A-K-K-C 16 ; (c)L-K-L-Q-X2-E-Z-N-X6-L-Q-E-B-I-K-X 10 -L-N-Q-X 12 -Y-PEG4-K-C 16 ; (d) LKLQ-X2-EZN-X6-LQEBIKX 10 -LNQX 12 -Y-PEG8-KC 16 4 (e)LKLQ-X2-EZN-X6-LQEBIKX 10 -LNQX 12 -Y-PEG12-KC 16 ; (f)L-K-L-Q-X2-E-Z-K-X6-L-Q-E-B-I-K-X 10 -L-N-Q-X 12 -Y-K-C 16 ; (g)L-K-L-Q-X2-E-Z-N-X6-L-Q-E-B-I-K-X 10 -L-N-E-X 12 -Y-K-C 16 ; (h)L-K-L-Q-X2-E-Z-K-X6-L-Q-E-B-I-K-X 10 -L-N-E-X 12 -Y-K-C 16 ; (i)L-E-L-Q-X2-K-Z-N-X6-L-Q-E-B-I-K-X 10 -L-N-Q-X 12 -Y-K-C 16 ; (j)L-E-L-Q-X2-K-Z-E-X6-L-Q-K-B-I-K-X 10 -L-N-Q-X 12 -Y-K-C 16 ; (k)L-E-L-Q-X2-K-Z-N-X6-L-Q-E-B-I-E-X 10 -L-N-K-X 12 -Y-K-C 16 ; (l)L-E-L-Q-X2-K-Z-E-X6-L-Q-K-B-I-E-X 10 -L-N-K-X 12 -Y-K-C 16 ; (m)LKLQBEZKBLQEBIKBLNQBY-K-C16; in, X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n Furthermore, the side chains of X2 and X6, as well as X... 10 and X 12 The side chains are all covalently connected, or X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n And X2, X6, X 10 X 12 They do not form covalent bonds with each other. Preferably, when X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n Furthermore, the side chains of X2 and X6, as well as X... 10 and X 12 When the side chains are all covalently connected, S n It is 2-amino-2-methyl-6-heptenic acid; when X2, X6, X 10 X 12 Each is an independent non-natural amino acid S n And X2, X6, X 10 X 12 When there are no covalent bonds between them, S n It is 2-amino-2-methyl-6-heptenoic acid; "Z" represents the special amino acid L-ortholeucine (Nle). "B" represents the special amino acid 2-aminoisobutyric acid Aib; "PEG4" represents polyethylene glycol, "PEG8" represents polyethylene glycol octadecyl glycol, and "PEG12" represents polyethylene glycol dodecyl glycol. C 16 "Represents palmitic acid; The symbol "-" represents a covalent bond between residues; Preferably, C 16 It is connected to K through its side chain.

13. The lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof according to any one of claims 1-12, wherein the lipopeptide comprises the sequence shown in any one of SEQ ID NOs: 14-27, a sequence having 1-5 conserved substitutions with the sequence shown in any one of SEQ ID NOs: 14-27, a sequence in any one of SEQ ID NOs: 14-27 with 1-2 amino acids deleted at the N-terminus or C-terminus, or a functional variant thereof.

14. The lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof according to any one of claims 1-13, wherein the derivatives of the lipopeptide comprise amino acid-modified derivatives.

15. The lipopeptide or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof according to claim 14, wherein the amino acid modification comprises replacing one or more of the L-type amino acids with D-type amino acids, artificially modified amino acids or rare amino acids in nature, C-terminal modification of amino acids and N-terminal modification of amino acids.

16. An antiviral polypeptide or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, wherein the antiviral polypeptide comprises the amino acid sequence of any one of (a)-(f): (a) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 5, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO:

5. (b) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 6, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO: 6; (c) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 7, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO: 7; (d) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 8, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO:

8. (e) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 9, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO:

9. (f) An amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having 1-5 conserved substitutions with the amino acid sequence shown in SEQ ID NO: 10, or a functional variant thereof, preferably the amino acid sequence shown in SEQ ID NO:

10.

17. A pharmaceutical composition comprising a lipopeptide or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof according to any one of claims 1-15, or an antiviral polypeptide or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof according to claim 16, and a pharmaceutically acceptable carrier.

18. A pharmaceutical composition comprising a lipopeptide or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof according to any one of claims 1-15, an antiviral polypeptide or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof according to claim 16, or a pharmaceutical composition according to claim 17, and a second therapeutic agent.

19. The use of any lipopeptide of claims 1-15 or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof, the antiviral polypeptide of claim 16 or its stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof, the pharmaceutical composition of claim 17, or the pharmaceutical composition of claim 18 in the preparation of a medicament for treating or preventing viral infections.

20. The application according to claim 19, wherein the virus comprises a coronavirus, preferably selected from SARS-CoV, MERS-CoV and SARS-CoV-2.

21. A method for preparing a lipopeptide or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof according to any one of claims 1-15, comprising (1) synthesizing the antiviral polypeptide, (2) optionally, linking the antiviral polypeptide to a linker arm, (3) loading amino acid residues, and (4) conjugating a lipophilic compound.

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