Broad-spectrum viral membrane fusion inhibitors, as well as methods for preparing the same and their uses.

Abstract

This disclosure relates to lipopeptides or pharmaceutically acceptable salts, solvates, hydrates, complexes, chelates, non-covalent complexes or prodrugs thereof that are broad-spectrum viral membrane fusion inhibitors capable of strongly inhibiting human immunodeficiency virus and several other viruses and can be used to treat human immunodeficiency virus infections, several other viral infections, and diseases caused by these infections.

Description

[Technical Field]

[0001] This disclosure relates to the biomedical field and to lipopeptides (or pharmaceutically acceptable salts, solvates, hydrates, complexes, chelates, non-covalent complexes or prodrugs thereof), and further to polypeptides (or variants thereof). Lipopeptides (or pharmaceutically acceptable salts, solvates, hydrates, complexes, chelates, non-covalent complexes or prodrugs thereof) are broad-spectrum viral membrane fusion inhibitors capable of potently inhibiting human immunodeficiency virus and several other viruses, and can therefore be used to treat human immunodeficiency virus infections, several other viral infections, and diseases caused by these infections. [Background technology]

[0002] Infections caused by viruses such as human immunodeficiency virus (HIV), novel coronavirus (SARS-CoV-2), Middle East respiratory syndrome virus (MERS-CoV), Ebola virus (EBOV), Marburg virus (MARV), hepatitis B virus (HBV), influenza virus (IAV), rabies virus (RABV), varicella stomatitis virus (VSV), and respiratory syncytial virus (RSV) pose a serious threat to human health and social stability. Effective antiviral drugs, especially those with broad-spectrum antiviral effects, are severely lacking worldwide. Membrane fusion, including egg formation and the transport and release of intracellular substances, is a crucial biological phenomenon. Many viruses, including those mentioned above, infect host cells via membrane fusion pathways. The development of specific or broad-spectrum antiviral drugs targeting the general mechanisms of viral membrane fusion holds significant scientific and practical value.

[0003] HIV infection leads to acquired immunodeficiency syndrome (AIDS). Currently, there is no effective vaccine to prevent HIV, nor is there a drug that can completely cure HIV infection. Existing anti-HIV drugs include reverse transcriptase inhibitors, protease inhibitors, viral entry inhibitors, and integrase inhibitors, which act by inhibiting viral replication at various stages and play a major role in the treatment and prevention of AIDS. Widely used high-activity antiretroviral therapies ("cocktail" therapies) mainly consist of 3-4 reverse transcriptase inhibitors and protease inhibitors. However, due to the persistence of HIV infection, drugs need to be administered to patients for long periods, which easily leads to drug resistance and seriously affects the effectiveness of clinical treatment. Therefore, the development of new anti-HIV drugs has always been a crucial requirement for the prevention and control of AIDS.

[0004] HIV entry into host cells is mediated by a trimer envelope glycoprotein (Env) on its surface, which contains the surface subunit gp120 and the transmembrane subunit gp41. During the HIV infection process, gp120 first binds to the receptor CD4, and then to a co-receptor (such as CCR5 or CXCR4), which induces a conformational change in gp120, thereby exposing gp41 and activating its membrane fusion function. The N-terminal fusion peptide (FP) of gp41 is first inserted into the target cell membrane, and then the C-terminal heptad region (CHR) is reverse-bonded to the N-terminal heptad region (NHR) to form a stable 6-helix bundle (6-HB) structure, thereby bringing the viral membrane closer to the cell membrane for fusion, allowing HIV genetic material to enter the cell and resulting in infection. Crystal structure [1]The C-terminus of the NHR helix contains a deep, hydrophobic pocket, which has always been recognized as an important antiviral drug target. The N-terminal sequence "WMEWDREI" of the CHR helix is ​​the NHR pocket-binding domain (PBD), and its eight amino acids are located at positions a, b, c, d, e, f, g, and a of the CHR helix, respectively. Of these, the two W (tryptophan) amino acids at positions a and d, and the I (isoleucine) at position a are hydrophobic amino acids, which are inserted into the NHR pocket and mediate a broad hydrophobic effect, which is important for the formation and function of the viral fusion protein 6-HB.

[0005] Viral membrane fusion inhibitors (BFUs) have clear advantages in both treatment and prevention because they act in the early stages of viral replication by preventing the virus from entering target cells. However, currently, only one BFU is approved for clinical use by the U.S. Food and Drug Administration (FDA), and that is the HIV membrane fusion inhibitor enfuvirtide (also known as T20). As shown in Figure 1, T20 is a polypeptide derived from the gp41 CHR, consisting of 36 amino acids and not containing a PBD sequence, which exerts its antiviral effect by competitively binding to the NHR and inhibiting the formation of the virus's 6-HB. Because T20 has relatively low viral inhibitory activity and a relatively short half-life, it needs to be administered in large doses daily (90 mg twice daily). T20 readily induces drug resistance, thereby leading to the failure of clinical treatment. Therefore, the development of new HIV membrane fusion inhibitors is a constant source of interest both domestically and internationally.

[0006] Currently, research and development of HIV membrane fusion inhibitors are primarily focused on the role of PBDs, particularly using the early publicly available C34 polypeptide as a design template. [2] C34 contains PBD, and its amino acid sequence is recognized as the core sequence of CHR. Following T20, the original pharmaceutical company introduced the second-generation inhibitor T1249 and the third-generation inhibitor T2635. [3]Further designs have been developed, and both contain PBD sequences (see Figure 1). Both sifvirtide (SFT) and albuvirtide (ABT), which were initially developed in China, are designed based on the C34 sequence. [4][5] SFT is obtained by mutating 14 amino acids of C34 and adding serine (S) and glutamic acid (E) to the N-terminus and C-terminus, respectively, to enhance polypeptide stability and target binding ability. ABT is obtained by mutating only three amino acids of C34 and incorporating 3-maleimidopropanoic acid (MPA) into the lysine (K) side chain at position 13 to give it serum albumin binding ability. The antiviral activity of both SFT and ABT is not substantially improved compared to T20 and C34, but their biological half-lives are extended, which may reduce the frequency of administration. Modifying the C-terminus of the C34 polypeptide with a cholesterol molecule to form a C34-Chol lipopeptide can significantly improve antiviral activity and biological half-life. [6] .

[0007] The inventors' team has been engaged in the research and development of HIV membrane fusion inhibitors for many years and has designed several CHR polypeptide-based HIV membrane fusion inhibitors according to the structural and functional relationship of gp41. [7] Of these, LP-11 contains a PBD motif and its C-terminus is modified with a palmitic acid molecule; on the other hand, LP-98 does not contain a PBD motif and its C-terminus is modified with cholesterol. Compared to LP-11, LP-98 has significantly improved antiviral activity. However, the inventors also found that LP-98 has significantly reduced inhibitory activity against T20-resistant HIV strains, which consequently affects its drug viability. Therefore, continued research and development of novel viral membrane fusion inhibitor drugs is needed in the art to meet clinical needs.

[0008] Figure 1 shows diagrams of the sequence structures of T20, C34, T1249, T2635, SFT, ABT, C34-Chol, LP-11, and LP-98 mentioned above. [Overview of the project]

[0009] The technical challenge that this disclosure seeks to address is how to effectively inhibit HIV and several other viruses with membrane fusion capabilities. The inventors of this disclosure are dedicated to developing potent, broad-spectrum, and long-acting viral membrane fusion inhibitors over extended periods. In this study, the inventors have surprisingly discovered that, based on the CHR sequence of HIV, the introduction of a "rigid linker" and the substitution of several amino acids in the PBD can significantly enhance the antiviral activity of polypeptides.

[0010] Accordingly, in one embodiment, the present disclosure provides a lipopeptide or a pharmaceutically acceptable salt thereof, the lipopeptide being a viral membrane fusion inhibitor capable of inhibiting viruses such as HIV, SARS-CoV-2 and its mutants, MERS-CoV, EBOV, MARV, IAV, RABV, and VSV.

[0011] In another embodiment, the disclosure provides polypeptides or variants thereof, and conjugates comprising polypeptides or variants thereof.

[0012] In another embodiment, the disclosure provides isolated nucleic acids encoding polypeptides or variants thereof, vectors comprising isolated nucleic acids, and host cells comprising isolated nucleic acids or vectors.

[0013] In another embodiment, the disclosure provides a polymer formed from a lipopeptide or a pharmaceutically acceptable salt thereof, or from a polypeptide or a variant thereof.

[0014] In another embodiment, the Disclosure provides compositions comprising lipopeptides or pharmaceutically acceptable salts thereof, polypeptides or their variants, conjugates, isolated nucleic acids, vectors, host cells or polymers, and pharmaceutical compositions comprising lipopeptides or pharmaceutically acceptable salts thereof or polypeptides or their variants.

[0015] In another aspect, the disclosure provides the use of lipopeptides or pharmaceutically acceptable salts thereof, polypeptides or variants thereof in the manufacture of pharmaceuticals or the treatment of diseases. [Modes for carrying out the invention]

[0016] Detailed description of the invention The purpose and implementation of this disclosure are described in further detail below.

[0017] In the design of lipopeptide-based viral membrane fusion inhibitors, it is typically necessary to add a linker between the polypeptide sequence and an aliphatic group (e.g., fatty acids and cholesterol) to act as a linker arm. The expected binding site for the aliphatic group is the viral or cell membrane, which concentrates the polypeptide in the target region, thereby significantly improving the polypeptide's antiviral activity. Because polypeptides tend to form stable secondary structures and possess strong structural rigidity, flexible linkers are commonly selected for the connection between the polypeptide and the aliphatic group. This allows the polypeptide and the aliphatic group to form appropriate conformations and bind to their respective binding sites, while avoiding steric hindrance and other interactions, and allowing their respective effects to be fully realized. Common flexible linkers include combinations of glycine (G) and serine (S), e.g., (GGGGS)n or (GSGSG)n, where the size of n can be adjusted to increase or decrease the distance between domains. Another common flexible linker is small molecule polyethylene glycol (PEG)n, where n is mostly between 2 and 24. Currently, all viral membrane fusion inhibitor lipopeptides reported in the literature use flexible linkers. For example, the HIV membrane fusion inhibitor C34-Chol uses GSG, and LP-11 uses PEG8; in another example, the SARS-CoV-2 membrane fusion inhibitor IPB02V3 uses PEG8, while IPB24-IPB27 use PEG4, PEG5, PEG6, and PEG8, respectively. [8] Using EKL1C, GSG [9] Using the EK1C4, the Tandem GSGSG and PEG4[8] is used, and [SARS HRC -PEG4]2-chol is PEG4

[10] is used, etc.

[0018] The (EAAAK)n sequence that can form an α helix is a common rigid linker for preparing fusion proteins. It has internal hydrogen bonds, closely contacts the backbone of the peptide chain, is rigid and stable, thereby effectively separating domains. However, there is no precedent for using a rigid linker in currently prepared lipopeptide HIV membrane fusion inhibitors. The present disclosure prepares lipopeptides by using the rigid linker EAAAK sequence, and surprisingly finds that the rigid linker imparts a significant helical structure and strong antiviral activity to the polypeptide. In a specific example of the present disclosure, using the C34 polypeptide as a design template, lipopeptides C34-LP1 and C34-LP2 were first prepared through the GSGSG flexible linker and the EAAAK rigid linker respectively, and the results show that the EAAAK rigid linker helps to improve the anti-HIV activity of the lipopeptide. Furthermore, three new lipopeptides LP-121, LP-122 and LP-123 using EAAAK as a linker were designed, and EE and KK amino acid pairs were introduced at the corresponding positions b, c and f, g of the CHR helix to promote the formation of "salt bridge" structures at positions i and i+4. Unexpectedly, it was experimentally found that the LP-123 lipopeptide containing a shorter sequence has optimal anti-HIV activity.

[0019] The N-terminal amino acid WMEWDREI of the gp41 CHR helix is ​​the pocket-binding domain (PBD), where the two Ws at positions a and b of the CHR helix and the I at position a are inserted into the NHR pocket, mediating a broad hydrophobic effect, which plays a crucial role in the formation of the fusion protein 6-HB. Therefore, research and development of new-generation HIV membrane fusion inhibitors have focused primarily on the effects of the PBD, particularly using PBD-containing C34 polypeptides as design templates. Representative inhibitors in this field include T1249, T2635, SFT, ABT, and C34-Chol. While amino acid substitution is a common strategy for optimizing polypeptide inhibitors, in HIV membrane fusion inhibitors currently reported in the literature, the two Ws in the PBD sequence are used without exception, and there are no precedents for substituting W with other amino acids. To further improve the antiviral activity of the inhibitor, surprisingly, this disclosure has found that the antiviral activity of the HIV membrane fusion inhibitor can be significantly improved by substituting two hydrophobic W molecules in the PBD with two hydrophilic, low molecular weight tyrosine (Y) molecules. In a specific implementation process, LP-124 and LP-125 were prepared using the above-mentioned LP-122 and LP-123 lipopeptides as templates, respectively, and experiments verified that substituting W with Y significantly improved the antiviral activity of the inhibitor. Such a technical strategy changes the interaction between the PBD and the NHR pocket from a hydrophobic interaction to a hydrophilic interaction, providing a novel approach for the design of HIV membrane fusion inhibitors in this field.

[0020] Furthermore, this disclosure prepares lipopeptide LP-126 based on LP-125 by replacing the first N-terminal amino acid T with a negatively charged amino acid E to further enhance the hydrophilic interaction and stability of the lipopeptide N-terminus. Additionally, lipopeptide LP-127 is prepared by reducing the eighth amino acid of LP-126 to E and substituting I at position a with the relatively less hydrophilic amino acid L (leucine); LP-128, containing only one Y, is prepared by removing the three N-terminal amino acids of LP-127. These novel inhibitors share common characteristics, including the presence of an EAAAK linker, a PBD motif with W replaced by Y, and abundant EE-KK salt bridge-forming amino acids. Regarding the amino acid sequence, only eight amino acids retain those of the original CHR sequence. Through specific experiments, these novel inhibitors have been found to possess potent anti-HIV activity. More unexpectedly, LP-127 and LP-128 have been found to effectively inhibit infection by pseudoviruses such as SARS-CoV-2, MERS-CoV, EBOV, MARV, IAV, and RABV, reflecting their broad-spectrum antiviral activity.

[0021] Figure 2 shows the sequence structures of LP-121, LP-122, LP-123, LP-124, LP-125, LP-126, LP-127, and LP-128. In the structural formulas of the lipopeptides shown in Figure 2, Ac represents the acetyl group, which is the amino-terminal protecting group of the polypeptide, and Chol represents the cholesterol molecule that modifies the C-terminus of the polypeptide. The modification is located on the side chain of the amino acid K at the C-terminus of the polypeptide. NH2 represents the amino group and is the carboxyl-terminal protecting group of the polypeptide.

[0022] This disclosure is at least in part based on the inventors' aforementioned unexpected discovery. Accordingly, in one embodiment, the disclosure relates to a lipopeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof comprising a polypeptide or a variant thereof, a modifying group linked to the C-terminus of the polypeptide or a variant thereof via a linker, and optionally a terminal protecting group of the polypeptide, (X1X2X3)mX4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 IX 16 X 17 LX 18 X 19 X 20 X 21 X 22 X 23 QQX 24 X 25 N(EX 26 )n Equation I In the formula, the polypeptide is represented by formula I; the modifying group is a lipophilic compound; and the linker is -(EAAAK)n1-, -(XP)n2-, -(EAAAK)n1-X 27 -or-(XP)n2-X 27 -and, X1 is T, E, or S; X2 is W or a hydrophilic amino acid; X3 is E, M, or Q; m is 0 or 1; X4 is E, A, or T; X5 is W or a hydrophilic amino acid; X6 is E or D; X7 is R, K, or Q; X8 is E, K, or A; X9 is L or I; X 10 is E, N, or A; X 11 is E or N; X 12 is L or Y; X 13 is E, T, or A; X 14 is K, S, R, or A; X 15 is K, L, R, or Q; X 16 is E, H, T, or Y; X 17 is E, S, R, or A; X 18is L or I; X 19 is K, E, or R; X 20 is K, E, Q, or A; X 21 is A or S; X 22 is E or Q; X 23 is E, N, or I; X 24 is K, E, or D; X 25 is K or R; X 26 is Q, E, R, A, or Y; n is either 0 or 1; X is any one amino acid; X 27 is either K or C; n1 is a natural number between 1 and 5; n² is a natural number between 1 and 5. Unlike polypeptides from which the mutant is derived from only one or a few (e.g., 1, 2, 3, 4, or 5) amino acid residue substitutions (e.g., conserved or non-conservative substitutions), the mutant provides a lipopeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex, or prodrug thereof that retains the biological function of the polypeptide from which the mutant is derived.

[0023] In some embodiments, X1 is T or E. In some embodiments, X1 is T. In some embodiments, X1 is E.

[0024] In some embodiments, X2 is W, D, E, H, K, Q, R, S, T, or Y. In some embodiments, X2 is W or Y. In some embodiments, X2 is Y. In some embodiments, X2 is W.

[0025] In some embodiments, X3 is E or M. In some embodiments, X3 is E. In some embodiments, X3 is M.

[0026] In some embodiments, X4 is E.

[0027] In some embodiments, X5 is W, D, E, H, K, Q, R, S, T, or Y. In some embodiments, X5 is W or Y. In some embodiments, X5 is Y. In some embodiments, X5 is W.

[0028] In some embodiments, X6 is E.

[0029] In some embodiments, X7 is R or K. In some embodiments, X7 is R. In some embodiments, X7 is K.

[0030] In some embodiments, X8 is E or K. In some embodiments, X8 is E. In some embodiments, X8 is K.

[0031] In some embodiments, X 10 is E or N. In some embodiments, X 10 In some embodiments, X 10 It is N.

[0032] In some embodiments, X 11 It is E.

[0033] In some embodiments, X 12 It is L.

[0034] In some embodiments, X 13 is E or T. In some embodiments, X 13 In some embodiments, X 13 It is T.

[0035] In some embodiments, X 14 is K or S. In some embodiments, X 14 is K. In some embodiments, X 14 S is.

[0036] In some embodiments, X 15is K or L. In some embodiments, X 15 is K. In some embodiments, X 15 It is L.

[0037] In some embodiments, X 16 is E or H. In some embodiments, X 16 In some embodiments, X 16 H is H.

[0038] In some embodiments, X 17 is E or S. In some embodiments, X 17 In some embodiments, X 17 S is.

[0039] In some embodiments, X 18 It is L.

[0040] In some embodiments, X 19 is K or E. In some embodiments, X 19 is K. In some embodiments, X 19 It is E.

[0041] In some embodiments, X 20 is K or E. In some embodiments, X 20 is K. In some embodiments, X 20 It is E.

[0042] In some embodiments, X 21 It is A.

[0043] In some embodiments, X 22 It is E.

[0044] In some embodiments, X 23 is E or N. In some embodiments, X 23 In some embodiments, X 23 It is N.

[0045] In some embodiments, X 24 is K or E. In some embodiments, X 24 is K. In some embodiments, X 24 It is E.

[0046] In some embodiments, X 25 is K.

[0047] In some embodiments, X 26 Q is Q.

[0048] In some embodiments, X is A, K, or E.

[0049] In some embodiments, m is 0. In some embodiments, m is 1.

[0050] In some embodiments, n is 0. In some embodiments, n is 1.

[0051] In some embodiments, X 27 is K. In some embodiments, X 27 C is C.

[0052] In some embodiments, n1 is a natural number between 1 and 4. In some embodiments, n1 is a natural number between 1 and 3. In some embodiments, n1 is 1 or 2. In some embodiments, n1 is 1. In some embodiments, n1 is 2. In some embodiments, n1 is 3. In some embodiments, n1 is 4. In some embodiments, n1 is 5.

[0053] In some embodiments, n2 is a natural number from 1 to 4. In some embodiments, n2 is a natural number from 1 to 3. In some embodiments, n2 is 1 or 2. In some embodiments, n2 is 1. In some embodiments, n2 is 2. In some embodiments, n2 is 3. In some embodiments, n2 is 4. In some embodiments, n2 is 5.

[0054] In some embodiments, X 25 is K, and the polypeptide is represented by Formula II, (X1X2X3)mX​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​10 X 11 X 12 X 13 X 14 X 15 IX 16 X 17 LX 18 X 19 X 20 X 21 X 22 X 23 QQX 24 KN(EX 26 )n Formula III In the formula, X1, X2, X3, m, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 26 The definitions of n and n are as described in any embodiment of this disclosure.

[0056] In some embodiments, X4 is E, and X 10 is E, and X 11 is E, and X 12 L is X 13 is E, and X 14 is K, and X 15 is K, and X 16 is E, and X 17 is E, and X 18 L is X 19 is K, and X 20 is K, and X 21 A is X 22 is E, and X 23 is E, and X 24 is K, and X 25 is K, and the polypeptide is represented by formula IV, (X1X2X3)m-EX5X6X7X8X9EELEKKIEELLKKAEEQQKKN(EX 26 )n Formula IV

[0057] In the formula, X1, X2, X3, m, X5, X6, X7, X8, X9, X 26 The definitions of , and n are as described in any embodiment of this disclosure.

[0058] In some embodiments, X4 is E, X6 is E, X7 is K, and X 10 is E, and X 11 is E, and X 12 L is X 13 is E, and X 14 is K, and X 15 is K, and X 16 is E, and X 17 is E, and X 18 L is X 19 is K, and X 20 is K, and X 21 A is X 22 is E, and X 23 is E, and X 24 is K, and X 25 is K, and the polypeptide is represented by formula V, (X1X2X3)m-EX5EKX8X9EELEKKIEELLKKAEEQQKKN(EX 26 )n Formula V

[0059] In the formula, X1, X2, X3, m, X5, X8, X9, X 26 The definitions of , and n are as described in any embodiment of this disclosure.

[0060] In some embodiments, the polypeptide terminal protecting group includes an N-terminal (amino-terminal) protecting group and / or a C-terminal (carboxy-terminal) protecting group. In some embodiments, the lipopeptides according to this disclosure include an N-terminal protecting group and a C-terminal protecting group. As is well known, the N-terminal protecting group may be any one selected from the group consisting of acetyl (Ac), amino (NH2), maleoil, succinyl, tert-butoxycarbonyl, benzyloxy, another hydrophobic group, and a polymeric support group. As is well known, the C-terminal protecting group may be any one selected from the group consisting of amino (NH2), carboxyl, hydroxyl, amide, tert-butoxycarbonyl, another hydrophobic group, and a polymeric support group. In some embodiments, the N-terminal protecting group is acetyl (Ac). In some embodiments, the C-terminal protecting group is amino (NH2).

[0061] The abbreviations for amino acids in polypeptides as disclosed herein have meanings well known in the art. For example, W is tryptophan, M is methionine, E is glutamic acid, D is aspartic acid, R is arginine, I isoleucine, N is asparagine, Y is tyrosine, T is threonine, S is serine, L is leucine, H is histidine, Q is glutamine, K is lysine, A is alanine, and G is glycine. In some embodiments, the amino acids are L-constituent amino acids, and one or more (e.g., 2-5, 2-4, or 2-3) amino acid residues in the polypeptide may also be replaced with amino acids having chemically similar properties, such as L-constituent amino acids, D-constituent amino acids, artificially modified amino acids, or naturally occurring rare amino acids, in order to improve the bioavailability, stability, and / or antiviral activity of the polypeptide, where D-constituent amino acids refer to amino acids corresponding to L-constituent amino acids that make up proteins; artificially modified amino acids refer to common L-constituent amino acids that make up proteins and are modified by methylation, phosphorylation, etc.; naturally occurring rare amino acids include rare amino acids that make up proteins and amino acids that do not make up proteins, such as 5-hydroxylysine, methylhistidine, γ-aminobutyric acid, and homoserine.

[0062] In some embodiments, the lipophilic compound is cholesterol (Chol), fatty acids, dihydrosphingosine (DHS), or vitamin E (tocopherol, Toc). In some embodiments, cholesterol includes cholesteryl hemisuccinate, 2-cholesteryl acetate, 2-cholesteryl propionic acid, 3-cholesteryl propionic acid, 2-cholesteryl butyrate, 2-cholesteryl isobutyrate, 3-cholesteryl butyrate, 3-cholesteryl isobutyrate, 4-cholesteryl butyrate, 2-cholesteryl valeric acid, 2-cholesteryl isovaleric acid, 3-cholesteryl valeric acid, 5-cholesteryl valeric acid, 2-cholesteryl caproic acid, 6-cholesteryl caproic acid, 2-cholesteryl enanthic acid, 7-cholesteryl enanthic acid, 2-cholesteryl caprylic acid, 8-cholesteryl caprylic acid, cholesteryl bromoacetate, and the like. In some embodiments, the fatty acid includes fatty acids containing 8 to 20 carbon atoms, such as octadecanoic acid or palmitic acid.

[0063] In some embodiments, the lipophilic compound is cholesterol (Chol). In some embodiments, the lipophilic compound is a fatty acid. In some embodiments, the lipophilic compound is dihydrosphingosine (DHS). In some embodiments, the lipophilic compound is vitamin E (tocopherol, Toc). In some embodiments, cholesterol is cholesteryl hemisuccinate. In some embodiments, the lipophilic compound is 2-cholesteryl acetate. In some embodiments, the lipophilic compound is 2-cholesteryl propionic acid. In some embodiments, the lipophilic compound is 3-cholesteryl propionic acid. In some embodiments, the lipophilic compound is 2-cholesteryl butyric acid. In some embodiments, the lipophilic compound is 2-cholesteryl isobutyric acid. In some embodiments, the lipophilic compound is 3-cholesteryl butyric acid. In some embodiments, the lipophilic compound is 3-cholesteryl isobutyric acid. In some embodiments, the lipophilic compound is 4-cholesteryl butyric acid. In some embodiments, the lipophilic compound is 2-cholesteryl valeric acid. In some embodiments, the lipophilic compound is 2-cholesteryl isovaleric acid. In some embodiments, the lipophilic compound is 3-cholesteryl valeric acid. In some embodiments, the lipophilic compound is 5-cholesteryl valeric acid. In some embodiments, the lipophilic compound is 2-cholesteryl caproic acid. In some embodiments, the lipophilic compound is 6-cholesteryl caproic acid. In some embodiments, the lipophilic compound is 2-cholesteryl enanthic acid. In some embodiments, the lipophilic compound is 7-cholesteryl enanthic acid. In some embodiments, the lipophilic compound is 2-cholesteryl caprylic acid. In some embodiments, the lipophilic compound is 8-cholesteryl caprylic acid. In some embodiments, the lipophilic compound is cholesteryl bromoacetate. In some embodiments, the fatty acid is a fatty acid containing 8 to 20 carbon atoms. In some embodiments, the lipophilic compound is octadecanoic acid. In some embodiments, the lipophilic compound is palmitic acid.

[0064] In some embodiments, the linker is -(EAAAK)n1-, where the definition of n1 is as described in any embodiment of this disclosure. In some embodiments, the linker is -(XP)n2-, where the definitions of X and n2 are as described in any embodiment of this disclosure. In some embodiments, the linker is -(EAAAK)n1-X 27 - and n2 and X 27 The definition is as described in any embodiment of this disclosure. In some embodiments, the linker is -(XP)n2-X 27 - and X, n2 and X 27 The definition of is as described in any embodiment provided herein.

[0065] Lipophilic compounds may be linked to the side chains of terminal amino acids in the linker, or they may be linked directly to the linker. Of these, fatty acids, dihydrosphingosine, and vitamin E can achieve modification of polypeptides by amidation with the amino side chain of lysine (Lys) in the linker; cholesterol can achieve modification of polypeptides by amidation with the amino side chain of lysine (Lys) in the linker, or by thioether formation with the sulfhydryl side chain of cysteine ​​(Cys) in the linker. In some specific embodiments, cholesterol is cholesteryl hemisuccinate, which achieves modification of polypeptides by amidation with the amino side chain of K in the linker. It is well known in the art that cholesteryl bromoacetate, as a polypeptide modifying group, can also achieve modification of polypeptides by thioether formation with the sulfhydryl side chain of cysteine ​​(C) in the linker.

[0066] In some embodiments, the lipopeptide has an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 11-19.

[0067] As is well known in the art, the chemical modification of polypeptides by the use of lipids to obtain so-called "lipopeptides" can increase the polypeptide's ability to target cell membranes and its antiviral activity, while also significantly improving the polypeptide's stability and biological half-life. Therefore, in the art as well, the polypeptides according to this disclosure have antiviral activity similar to that of lipopeptides. Based on this, in another aspect, this disclosure further provides polypeptides or variants thereof that are polypeptides or variants thereof described in any of the above embodiments of this disclosure. Specifically, the polypeptide comprises or consists of a sequence represented by formula I, formula II, formula III, formula IV, or formula V: (X1X2X3)mX4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 IX 16 X 17 LX 18 X 19 X 20 X 21 X 22 X 23 QQX 24 X 25 N(EX 26 )n Equation I (X1X2X3)mX4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 IX 16 X 17 LX 18 X 19 X 20 X 21 X 22 X 23 QQX 24 KN(EX 26 )n Formula II (X1X2X3)m-EX5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X15 IX 16 X 17 LX 18 X 19 X 20 X 21 X 22 X 23 QQX 24 KN(EX 26 )n Formula III (X1X2X3)m-EX5X6X7X8X9EELEKKIEELLKKAEEQQKKN(EX 26 )n Formula IV (X1X2X3)m-EX5EKX8X9EELEKKIEELLKKAEEQQKKN(EX 26 )n Formula V In the formula, X1, X2, X3, m, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 The definitions of n and n are as described in any embodiment of this disclosure.

[0068] In some embodiments, the lipopeptide has an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 21-29.

[0069] As is well known in the art, the amino acids corresponding to positions a and d of the CHR helix are crucial for the formation of the 6-HB structure by binding the CHR to the NHR target sequence, and they are sequence and function-conserved and therefore not readily substituted by other amino acids. In contrast, the amino acids corresponding to positions b, c, f, and g of the CHR helix have little to no direct interaction with the NHR and are therefore readily substituted by other amino acids with little to no effect on the antiviral activity of the polypeptide. Thus, amino acids in a polypeptide may be substituted, added, or deleted by one or more other amino acids, and the polypeptide may still have activity to inhibit HIV and / or other viruses. Amino acid substitution refers to the substitution of an amino acid residue at a given position in a polypeptide sequence with another amino acid, preferably a conserved amino acid, i.e., a conservative substitution; amino acid addition refers to the insertion of an additional amino acid residue(s) at the N-terminus, C-terminus, or other suitable position in a polypeptide sequence, the inserted amino acid residues may be entirely or partially contiguous, or discontinuous; amino acid deletion refers to the removal of one or more amino acid residues from a polypeptide sequence, insofar as the modified polypeptide has viral inhibitory activity.

[0070] The terms "conserved amino acids" or "conservation of amino acids" have a well-known meaning in this field. For example, amino acids are classified into acidic amino acids, basic amino acids, and neutral amino acids according to the number of amino groups and carboxyl groups contained in the amino acid molecule. Acidic amino acids refer to E and D, basic amino acids refer to K, R, and H (histidine), and neutral amino acids refer to A, L, I, V, C, Y, G, M, S, T, F (phenylalanine), W, and P (proline). As another example, amino acids are classified into hydrophilic amino acids (D, E, H, K, Q, R, S, T, Y) and hydrophobic amino acids (A, F, I, L, M, P, V, W) according to their hydrophilicity and hydrophobicity. As yet another example, hydrophilic uncharged amino acids refer to N, Q, S, and T; aliphatic uncharged amino acids refer to A, L, I, V, and G; nonpolar uncharged amino acids refer to C, M, and P; aromatic amino acids refer to Y, F, and W. In other examples, amino acids containing an alcohol group include S and T; aliphatic amino acids include L, I, V, and M; and cycloalkenyl-related amino acids include F, H, W, and Y. These amino acids, having similar size, shape, charge, and chemical properties, including the ability to form covalent or hydrogen bonds, are generally considered conserved amino acids.

[0071] In this disclosure, the term “conservative substitution” refers to an amino acid substitution that does not adversely affect or alter the essential properties of a protein / polypeptide, including its amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, for example, a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, generally speaking, a conservative substitution refers to the substitution of a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art.

[0072] Through more than 10 years of research experience, the inventors' team has demonstrated that polypeptides derived from amino acid substitutions, additions, or deletions in a parent polypeptide with up to approximately 30%, 40%, 50%, 60%, 70%, 80%, or 90% sequence identity can still possess potent antiviral activity as inhibitors. For example, in the case of the lipopeptides LP-126, LP-127, and LP-128 described above, only eight amino acids from the original CHR prototype are retained, with LP-126 and LP-127 having only 26.7% sequence identity to the amino acids of the original CHR prototype, and LP-128 having 29.6% sequence identity to the amino acids of the original CHR prototype. Therefore, any one of the polypeptides has approximately 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity and is a polypeptide with antiviral activity.

[0073] Regarding common rigid linkers used in the preparation of fusion proteins, in addition to the (EAAAK)n1 sequence which can form an α-helix, another common type of rigid linker is the proline (P)-rich sequence (XP)n2, where X is any amino acid, preferably alanine, lysine, or glutamic acid. Although the (XP)n2 sequence does not have a helical structure, the proline increases the rigidity of the backbone and allows for effective separation of domains. Therefore, in this disclosure, the (EAAAK)n1 linker can also be replaced with the (XP)n2 linker without significantly affecting the antiviral activity. Thus, the linker according to this disclosure may be either (EAAAK)n1 or (XP)n2. Furthermore, in order to link to lipophilic compounds, the linker may also contain one or more lysine (K) or cysteine ​​(C).

[0074] The lipopeptides described herein may contain one or more chiral centers and / or double bonds and such structures, and therefore may exist as stereoisomers including double bond isomers (e.g., geometric isomers), enantiomers (photoisomers), or diastereomers. Accordingly, any chemical structure within the scope described herein includes all possible enantiomers and diastereomers of lipopeptides, including any one pure stereoisomer (e.g., a pure geometric isomer, a pure enantiomer, or a pure diastereomer) and any one mixture of these stereoisomers, whether or not they partially or whole contain the similar structures described above. Those skilled in the art may further separate these racemates and mixtures of stereoisomers into enantiomers or stereoisomers of their constituents by utilizing separation techniques or asymmetric synthesis methods. Lipopeptides include, but are not limited to, various optical isomers, racemates, and / or other mixtures. In the above cases, a single enantiomer or diastereomer, such as an optically active isomer, can be obtained by asymmetric synthesis or racemic resolution. Racemic resolution can be achieved by various methods, such as routine recrystallization or chromatography using resolution aids. Furthermore, lipopeptides also include cis and / or trans isomers having double bonds.

[0075] The lipopeptides of this disclosure include, but are not limited to, all of the various pharmaceutically acceptable forms of lipopeptides. These pharmaceutically acceptable forms include various pharmaceutically acceptable salts, solvates, hydrates, complexes, chelates, non-covalent complexes, prodrugs based on the above substances, and any mixture of the above forms.

[0076] The pharmaceutically acceptable salts of this disclosure include acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, tartrate, mesylate, borate, methyl bromide, bromide, methyl nitrate, calcium edetate, methyl sulfate, camusylate, mucinate, carbonate, napsylate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, This includes pamoates, embonates, estruates, palmitates, esylates, pantothenates, fumarates, phosphates / diphosphates, gluceptates, polygalacturonates, glucons, salicylates, glutamates, stearates, glycolyl arsanilates, sulfates, hydroxybenzoates, acetates, hydravamin, succinates, hydrobroms, tannates, hydrochlorides, tartrates, hydroxynaphthates, theocrates, iodides, tosylates, triiodides, lactates, and valersates. Depending on the use, pharmaceutically acceptable salts may be formed from cations such as sodium, potassium, and bismuth, or from bases such as ammonia, ethylenediamine, N-methylglutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts can be prepared by standard methods, for example, by the reaction of a free acid with an organic or inorganic base. In the presence of a basic group (e.g., an amino group), acidic salts such as hydrochloride, hydrobromide, acetate, and pamoate may be used as drug forms; in the presence of an acidic or alcoholic group, pharmaceutically acceptable esters such as acetate, maleate, and pivaloyloxymethyl, as well as literature-known esters to improve solubility and hydrolysis, may be used as sustained-release agents or prodrugs.

[0077] In another embodiment, the disclosure also provides isolated nucleic acids encoding the polypeptides or variants thereof described above.

[0078] In another embodiment, the disclosure also provides vectors comprising isolated nucleic acids. Vectors useful for inserting a polynucleotide of interest are well known in the art and include, but are not limited to, cloning vectors and expression vectors. In one embodiment, the vector is, for example, a plasmid, cosmid, or phage.

[0079] In another aspect, the Disclosure also provides host cells comprising the isolated nucleic acids or vectors described above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli (E. coli) cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells such as mouse cells and human cells). The host cells of the Disclosure may also be cell lines.

[0080] In another embodiment, the disclosure also provides the lipopeptides, pharmaceutically acceptable salts thereof, solvates, hydrates, complexes, chelates, non-covalent complexes or prodrugs thereof, or polymers formed from the polypeptides thereof or their variants.

[0081] In another embodiment, the disclosure also provides conjugates comprising the polypeptides or their variants and modifying groups described above. In some embodiments, the modifying group is optionally linked to the N-terminus or C-terminus of the polypeptide or its variant via a linker. In some embodiments, the modifying group is a terminal protecting group. Terminal protecting groups of a polypeptide include an N-terminal protecting group and / or a C-terminal protecting group. As is well known, the N-terminal protecting group may be any one selected from the group consisting of acetyl (Ac), amino (NH2), maleoil, succinyl, tert-butoxycarbonyl, benzyloxy, another hydrophobic group, and a polymer carrier group. As is well known, the C-terminal protecting group may be any one selected from the group consisting of amino (NH2), carboxyl, hydroxyl, amide, tert-butoxycarbonyl, another hydrophobic group, and a polymer carrier group.

[0082] In another aspect, the Disclosure also provides compositions comprising the lipopeptides, pharmaceutically acceptable salts, solvates, hydrates, complexes, chelates, non-covalent complexes or prodrugs thereof, or the polypeptides or their variants, or the conjugates, or the isolated nucleic acids, or the vectors, or the host cells, or the polymers.

[0083] In another embodiment, the Disclosure also provides pharmaceutical compositions comprising the lipopeptides, pharmaceutically acceptable salts, solvates, hydrates, complexes, chelates, non-covalent complexes, or prodrugs thereof, and optionally further comprising pharmaceutically acceptable carriers and / or excipients. In some embodiments, the lipopeptides, pharmaceutically acceptable salts, solvates, hydrates, complexes, chelates, non-covalent complexes, or prodrugs thereof are present in amounts effective for treating viral infections or diseases caused by viral infections.

[0084] In another embodiment, the disclosure also provides pharmaceutical compositions comprising the polypeptide or a variant thereof, and optionally further comprising a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the polypeptide or a variant thereof is present in an amount effective for treating a viral infection or a disease caused by a viral infection.

[0085] In some embodiments, the viral infection is an infection caused by a virus selected from the group consisting of HIV and its drug-resistant strains, SARS-CoV-2 and its mutant strains, MERS-CoV, EBOV, MARV, IAV, RABV, and VSV.

[0086] In some embodiments, the viral infection is an infection caused by HIV or a drug-resistant strain thereof.

[0087] In another embodiment, the Disclosure also provides a method for treating a viral infection or a disease caused by a viral infection, comprising administering an effective amount of the above lipopeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof, or the above polypeptide or a variant thereof, or the above pharmaceutical composition, to a subject requiring it. In another embodiment, the Disclosure also provides a method for inhibiting viral membrane fusion, comprising administering an effective amount of the above lipopeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof, or the above polypeptide or a variant thereof, or the above pharmaceutical composition, to a subject requiring it. In some embodiments, the viral infection is an infection caused by a virus selected from the group consisting of HIV and its drug-resistant strains, SARS-CoV-2 and its mutants, MERS-CoV, EBOV, MARV, IAV, RABV, and VSV. In some embodiments, the viral infection is an infection caused by HIV or its drug-resistant strain.

[0088] In another embodiment, the disclosure also provides the use of the above-mentioned lipopeptides or pharmaceutically acceptable salts, solvates, hydrates, complexes, chelates, non-covalent complexes or prodrugs thereof, or the above-mentioned polypeptides or variants thereof, in the manufacture of a pharmaceutical composition, wherein the pharmaceutical composition is used as a viral membrane fusion inhibitor or to treat a viral infection or a disease caused by a viral infection. In some embodiments, the viral infection is an infection caused by a virus selected from the group consisting of HIV and its drug-resistant strains, SARS-CoV-2 and its mutants, MERS-CoV, EBOV, MARV, IAV, RABV, and VSV. In some embodiments, the viral infection is an infection caused by HIV or its drug-resistant strains.

[0089] In another embodiment, the Disclosure also provides the use of the above-mentioned lipopeptides or pharmaceutically acceptable salts, solvates, hydrates, complexes, chelates, non-covalent complexes or prodrugs thereof, or the above-mentioned polypeptides or variants thereof, for use as viral membrane fusion inhibitors or for use in the treatment of viral infections or diseases caused by viral infections. In another embodiment, the Disclosure also provides the use of the above-mentioned polypeptides or variants thereof for use as viral membrane fusion inhibitors or for use in the treatment of viral infections or diseases caused by viral infections. In some embodiments, the viral infection is an infection caused by a virus selected from the group consisting of HIV and its drug-resistant strains, SARS-CoV-2 and its mutants, MERS-CoV, EBOV, MARV, IAV, RABV, and VSV. In some embodiments, the viral infection is an infection caused by HIV or its drug-resistant strains.

[0090] In another embodiment, the Disclosure also provides pharmaceutical compositions for inhibiting viral membrane fusion or for treating viral infections or diseases caused by viral infections, comprising the lipopeptides or pharmaceutically acceptable salts, solvates, hydrates, complexes, chelates, non-covalent complexes or prodrugs thereof, or the polypeptides or variants thereof. In some embodiments, the viral infection is an infection caused by a virus selected from the group consisting of HIV and its drug-resistant strains, SARS-CoV-2 and its mutants, MERS-CoV, EBOV, MARV, IAV, RABV, and VSV. In some embodiments, the viral infection is an infection caused by HIV or its drug-resistant strains.

[0091] In some embodiments, SARS-CoV-2 mutants include alpha mutants, beta mutants, gamma mutants, epsilon mutants, delta mutants, omicron mutants, and the like. In some embodiments, SARS-CoV-2 mutants include SARS-CoV-2 D614G and omicron BA4 / 5 mutants. In some embodiments, IAV includes influenza A virus, influenza B virus, and influenza C virus. In some embodiments, influenza A virus includes subtypes such as H1N1, H3N2, H5N1, and H7N9. In some embodiments, influenza A virus is H7N9.

[0092] In practice, the lipopeptides or polypeptides described herein may be administered to patients as pharmaceuticals, either directly or in combination with appropriate carriers or excipients, for the purpose of treating and / or preventing viral (e.g., HIV) infections or diseases caused by viral infections. Examples of carrier materials, though not limited to those described herein, include water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethylcellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (e.g., cellulose phthalate acetate, carboxymethylcellulose, etc.), with water-soluble carrier materials being preferred. Using these materials, various formulations can be prepared, though not limited to, tablets, capsules, dropper pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, and lyophilized powders for injection. The formulations may include conventional formulations, sustained-release formulations, controlled-release formulations, and various particulate delivery systems. A wide variety of carriers known in the art may be used to formulate the unit preparations into tablets. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; and wetting agents such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, gum arabic, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, and polyvinylpyrrolidone. Agents and binders; disintegrants, such as dried starch, arginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose and ethylcellulose; disintegration inhibitors such as sucrose, glyceryl tristeart, cocoa butter and hydrogenated oils; absorption enhancers, such as quaternary ammonium salts and sodium lauryl sulfate; lubricants, such as talc, silica, corn starch, steart, boric acid, liquid paraffin and polyethylene glycol.Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer or multilayer tablets. A wide variety of carriers known in the art can be used to formulate unit preparations into pills. Examples of carriers include, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, and wheat paste; and disintegrants such as agar powder, dried starch, arginate, sodium dodecyl sulfate, methylcellulose, and ethylcellulose. A wide variety of carriers known in the art can also be used to formulate unit preparations into suppositories. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To formulate the unit preparation into injectable preparations such as solutions, emulsions, lyophilized powders, and suspensions, all conventional diluents, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters, can be used. Furthermore, to prepare isotonic injectable preparations, appropriate amounts of sodium chloride, glucose, or glycerin may be added to the injectable preparation, and conventional cosolvents, buffers, and pH adjusters may also be added. In addition, colorants, preservatives, fragrances, flavorings, sweeteners, and other materials may be optionally added to the pharmaceutical preparation as needed. The above formulations can be administered by injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; intraluminal administration, including rectal and vaginal administration; respiratory administration, including nasal administration; and mucosal administration. Among the above routes of administration, administration by injection is preferred.

[0093] The dosage of the lipopeptides or polypeptides of this disclosure depends on various factors, such as the nature and severity of the disease being prevented or treated, the sex, age, weight and individual response of the patient or animal, the specific components used, the route of administration, and the frequency of administration. The above dosage may be administered in single-unit or multiple (e.g., two, three, or four) unit dosage forms.

[0094] The lipopeptides or polypeptides of this disclosure may be used alone or in combination with one or more antiviral agents to treat or prevent viral infections (e.g., HIV) or diseases caused by viral infections, in order to achieve the objective of improving the overall therapeutic effect. Antiviral agents include, but are not limited to, reverse transcriptase inhibitors, protease inhibitors, entry inhibitors, integration inhibitors, and maturation inhibitors. The above reverse transcriptase inhibitor may be one or more inhibitors selected from the group consisting of AZT, 3TC, ddI, d4T, ddT, TDF, abacavir, nevirapine, efavirenz, delavirdin, azuvudine, ainuobilin, etc.; the above protease inhibitor may be one or more inhibitors selected from the group consisting of saquinavir mesylate, indinavir, ritonavir, amprenavir, kaletra, nelfinavir mesylate, etc.; the above entry inhibitor may be one or more inhibitors selected from the group consisting of maraviroc, TAK-779, T20, T2635, shifvirtide, albuvirtide, etc.; the above integration inhibitor may be one or more inhibitors selected from the group consisting of raltegravir, dolutegravir, elvitegravir, etc.

[0095] The specific therapeutically effective dose level for any individual patient will depend on various factors, including the disorder being treated and its severity; the activity of the particular active ingredient used; the particular composition used; the patient's age, weight, overall health, sex, and diet; the timing of administration, route of administration, and excretion rate of the particular active ingredient used; the duration of treatment; drugs used in combination with the particular active ingredient used in combination or simultaneously; and similar factors well known in the medical field. For example, it is common practice in the art to start with a dosage of the active ingredient lower than the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, the lipopeptides or polypeptides of this disclosure can be administered to mammals, particularly humans, in dosages ranging from 0.001 mg / kg body weight / day to 1000 mg / kg body weight / day, for example, from 0.01 mg / kg body weight / day to 100 mg / kg body weight / day, and even further, for example, from 0.1 mg / kg body weight / day to 10 mg / kg body weight / day. [Brief explanation of the drawing]

[0096] [Figure 1] The diagram shows the sequence structures of T20, C34, T1249, T2635, SFT, ABT, C34-Chol, LP-11, and LP-98. Figure A shows the structure and function of the HIV-1 fusion protein gp41; Figure B shows the polypeptide, where the underlined sequence is the PBD, the three amino acids inserted into the NHR pocket are labeled in red, and the mutant amino acids are labeled in green; Chol represents a cholesterol molecule, and C16 represents a palmitic acid molecule. [Figure 2] This figure shows the sequence structure of viral membrane fusion inhibitors provided in the examples of this disclosure (including LP-121, LP-122, LP-123, LP-124, LP-125, LP-126, LP-127, and LP-128), where the underlined sequence is the PBD, the three amino acids inserted into the NHR pocket are labeled in red, and the mutant amino acid is labeled in green; Chol in parentheses represents a cholesterol molecule. [Figure 3]The examples provided in this disclosure illustrate the anti-HIV activity of the viral membrane fusion inhibitors, where A shows a comparison of the anti-HIV activity between C34-LP1 and C34-LP2; B shows a comparison of the anti-HIV activity of LP-121, LP-122 and LP-123, where the HIV-1 strains NL4-3 and JRFL used are pseudoviruses and SG3.1 is a replicating virus; the target cells used are TZM-bl cells. [Figure 4] Examples of the viral membrane fusion inhibitors provided in this disclosure show the anti-HIV activity, where A shows a comparison of the anti-HIV activity between LP-122 and LP-124; B shows a comparison of the anti-HIV activity between LP-123 and LP-125; and C shows a comparison of the anti-HIV activity between LP-126, LP-127 and LP-128. The HIV-1 strains used, NL4-3 and JRFL, are pseudoviruses, and SG3.1 is a replicating virus; the target cells used are TZM-bl cells. [Figure 5] The image shows a comparison of the anti-HIV activity of T20 and LP-127 and LP-128 provided in the examples of this disclosure, where A shows their inhibitory activity against replicated HIV-1 strains SG3.1, JR-CSF, and 89.6 infecting TZB-bI cells; B shows their inhibitory activity against replicated HIV-1 strains SG3.1, LAI.2, and 89.6 infecting MT-4 cells. [Figure 6] This document demonstrates the inhibitory activity of T20 and LP-123, LP-125, LP-127, and LP-128, provided in the examples of this disclosure, against representative pandemic HIV-1 pseudovirus strains. [Figure 7] This demonstrates the inhibitory activity of T20 and LP-123, LP-125, LP-127, and LP-128, provided in the examples of this disclosure, against cell fusion mediated by representative pandemic HIV-1 strains. [Figure 8] This document demonstrates the inhibitory activity of LP-98 and LP-123, LP-125, LP-127, and LP-128, as provided in the examples of this disclosure, against drug-resistant HIV-1 strains. [Figure 9]The broad-spectrum antiviral activity evaluation results of LP-127 and LP-128 provided in the examples of this disclosure are shown, and their inhibitory activity against SARS-CoV-2, MERS-CoV, EBOV, MARV, IAV(H7N9), RABV, and VSV was evaluated using pseudoviruses. [Figure 10] The in vitro cytotoxicity evaluation of LP-127 and LP-128 provided in the examples of this disclosure is shown. [Figure 11] The stability analysis results for LP-127 and LP-128 provided in the examples of this disclosure are shown below. [Figure 12] The secondary structure and thermal stability of the membrane fusion inhibitors provided in the examples of this disclosure, as detected by using circular dichroism (CD), are shown, where A shows the alpha-helix content (left figure) and thermal stability (left figure) of the lipopeptide alone; B shows the alpha-helix content (left figure) and thermal stability (left figure) of the complex formed between the lipopeptide and the target sequence polypeptide N44.

[0097] Sequence information Information regarding the sequences involved in this disclosure is provided in Table 1 below. [Table 1] [Examples]

[0098] This disclosure is described in detail below with reference to specific embodiments, and the examples provided herein are for illustrative purposes only and not to limit the scope of this disclosure. The examples provided below may serve as a guide for modifications by those skilled in the art, but are not intended to limit this disclosure. Those skilled in the art can refer to this disclosure to appropriately modify the relevant parameters. Specifically, it is necessary to demonstrate that all similar substitutions or modifications are obvious to those skilled in the art, and all of them are considered to be within the scope of this disclosure. While the methods of this disclosure have been described using preferred examples, it will be apparent that the techniques of this disclosure can be achieved and applied by those involved in this disclosure by modifying or appropriately changing or combining the compounds and preparation methods described herein without departing from the content, spirit and scope of this disclosure.

[0099] The experimental methods in the following examples are conventional methods unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. The materials, reagents, etc., used in the following examples are commercially available unless otherwise specified.

[0100] Example 1: Preparation of viral membrane fusion inhibitor lipopeptides The cholesterol modifications of the 10 lipopeptides prepared in the examples of this disclosure (C34-LP1, C34-LP2, LP-121, LP-122, LP-123, LP-124, LP-125, LP-126, LP-127, and LP-128) were each achieved by an amidation reaction between cholesteryl bromoacetate and the amino group of the C-terminal lysine (K) side chain of the peptide chain, the specific methods of which are described in literature published by the inventors' laboratory. [7] In the examples of this disclosure, the amino terminus of all lipopeptides was bonded with acetyl (Ac) as an amino terminus protecting group, and the carboxyl terminus was bonded with amino (NH2) as a carboxy terminus protecting group.

[0101] I. Chemical reagents required for the preparation process All chemical reagents, including various Fmoc amino acids, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), N,N-dimethylformamide (DMF), piperidine (PIPE), ninhydrin, acetic anhydride (Ac2O), N,N-diisopropylethylamine (DIEA), hydrazine hydrate, cholesteryl hemisuccinate, trifluoroacetic acid (TFA), ethanedithiol (EDT), thioanisole (TA), triisopropylsilane (TIPS), and phenol, were purchased from major chemical reagent suppliers and were not further purified before use. The amino acid protection raw materials used in polypeptide synthesis include Fmoc-Lys(Dde)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Met-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, and Fmoc-Ala-OH. The abbreviations in these terms have well-known meanings; for example, Fmoc is 9-fluorenylmethoxycarbonyl, Dde is 1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl, Boc is t-butoxycarbonylacyl, tBu is t-butyl, OtBu is t-butoxy, Trt is trityl, and Pbf is (2,3-dihydro-2,2,4,6,7-pentamethylbenzofuran-5-yl)sulfonyl.

[0102] II. Synthesis of Peptide Resins Using link amide MBHA resin as a carrier resin, peptide resins were prepared by sequentially coupling the corresponding protecting amino acids in the amino acid sequence of the polypeptide with Fmoc deprotection and coupling reactions.

[0103] 1. Coupling of the first protective amino acid to the main chain. 0.3 mmol of the first protective amino acid Fmoc-Lys(Dde)-OH and 0.3 mmol of HOBt were taken and dissolved in an appropriate amount of DMF; 0.3 mmol of DIC was then taken and slowly added to the DMF solution of the protective amino acid under shaking, and the resulting mixture was reacted at room temperature under shaking for 5 minutes to obtain an activated protective amino acid solution, which was set aside for later use.

[0104] 0.1 mmol of Link Amido MBHA resin (0.35 mmol / g × 0.3 g) was taken and deprotected with 25% PIPE / DMF solution (volume ratio) for 20 minutes (twice), then washed and filtered to obtain the Fmoc-removed resin.

[0105] The activated first protective amino acid solution was added to the Fmoc removal resin, and a coupling reaction was carried out for 60 minutes. After filtration and washing, a resin containing the first protective amino acid Fmoc-Lys(Dde) was obtained.

[0106] 2. Coupling of other protective amino acids to the main chain. Using the same method as described above for coupling the first protective amino acid to the main chain, the corresponding other protective amino acids of the polypeptide were sequentially coupled to obtain a resin containing the main chain amino acids. Finally, the N-terminus was capped by acetylation with 0.3 mmol of Ac2O and 0.6 mmol of DIEA to complete the synthesis of the main chain. After each of the above steps, the reaction was controlled by a Kaiser test, and if the condensation reaction of a certain amino acid was incomplete, the condensation was repeated once until the desired target peptide segment, i.e., the polypeptide described herein, was obtained.

[0107] 3. Cholesterol coupling of lysine side chains The resin was treated with the smallest possible volume of 2% hydrazine hydrate / DMF solution (by volume) to remove the Dde protecting group from the C-terminal lysine side chain (10 minutes, twice). After filtration and washing, a Dde-removed resin was obtained and set aside for later use. 0.3 mmol of cholesteryl hemisuccinate and 0.3 mmol of HOBt were taken and dissolved in an appropriate amount of DMF. 0.3 mmol of DIC was then taken and slowly added to the DMF solution of cholesteryl hemisuccinate and HOBt, and the resulting mixture was reacted at room temperature under shaking for 5 minutes. The prepared solution containing cholesteryl hemisuccinate, HOBt, and DIC was added to the obtained Dde-removed resin, and a coupling reaction was carried out for 60 minutes. After filtration, washing, and drying, a peptide resin was obtained.

[0108] III. Preparation of Crude Products The peptide resin described above was taken and added to a cleavage reagent (15 mL / g resin) and mixed thoroughly. The resulting mixture was reacted at 30°C for 3 hours with shaking to cleave the target polypeptide from the resin and remove the side chain protecting groups. The filtrate of the reaction mixture was collected. The resin was further washed three times with a small amount of TFA / DCM, and after combining the filtrates, anhydrous ether was added to precipitate the combined filtrate, which was then centrifuged. The filtration cake was further washed, precipitated twice with cold anhydrous ether, and then dried under vacuum to obtain a white powder, i.e., the crude lipopeptide product; the cleavage reagent had the following composition: trifluoroacetic acid:1,2-ethanedithiol:thioanisole:phenol:H2O:triisopropylsilane = 68.5:10:10:5:3.5:1 (v / v).

[0109] IV. Preparation of the pure product The crude lipopeptide product described above was taken, dissolved with water / acetonitrile, and the resulting mixture was centrifuged to remove insoluble substances and set aside for later use. Reverse-phase high-performance liquid chromatography was used for purification. The chromatography column used was Agela C18 (10 μm, 100 Å, 50 × 250 mm), and the mobile phase consisted of mobile phase A (0.05% TFA and 2% acetonitrile in water) and mobile phase B (90% acetonitrile in water). The mobile phase flow rate was 25 mL / min, and the UV detection wavelength was 220 nm. The crude product solution was loaded onto the chromatography column and gradient elution was performed to recover the corresponding purified components, which were then directly freeze-dried to remove the solvent and obtain a pure product of the polypeptide trifluoroacetate in a bubbly state.

[0110] The pure product of the polypeptide trifluoroacetate was redissolved in water and acetonitrile, a large amount of anion exchange resin (in the form of an acetate radical) was added, the resulting mixture was stirred for 3 hours, filtered, the ion exchange resin was rinsed with a water / acetonitrile mixed solvent, the filtrates were combined and freeze-dried to obtain a pure product of polypeptide acetate in a fluffy state.

[0111] The chemical structure of the synthesized lipopeptide was characterized by MALDI-TOF mass spectrometry, and its purity was determined by analytical high-performance liquid chromatography (Agela C18-4.6×250mm, flow rate: 1 mL / min). The results showed that the synthesized lipopeptide had a purity of over 95%.

[0112] Example 2: Effect of EAAAK rigid linker on the anti-HIV activity of membrane fusion inhibitors In this example, C34-LP1 (containing a GSGSG flexible linker) and C34-LP2 (containing an EAAAK rigid linker) were compared in terms of anti-HIV activity. The materials and methods used were described in the literature published by the inventors' laboratory. [7] The viruses used were described as follows: pseudoviruses of HIV-1 strains NL4-3 and JRFL, and replicated viruses (live viruses) of HIV-1 strain SG 3.1.

[0113] 1. Experimental Method (1) Virus preparation: HEK293T cells were co-transfected with the HIV-1 backbone plasmid pSG3Δenv using plasmids expressing NL4-3 or JRFL envelope protein (Env); HEK293T cells were also transfected with molecular clones encoding SG3.1. The transfected cells were cultured for 48 hours in a cell culture incubator at 37°C and 5% CO2, and the supernatant was collected. The filtrate was collected by filtration to obtain a virus solution containing NL4-3 pseudovirus, JRFL pseudovirus, or SG3.1 live virus. After titration, the solution was stored at -80°C for later use.

[0114] (2) The test lipopeptides were dissolved in deionized water and then diluted in DMEM medium in 3-fold serial dilutions to obtain lipopeptide dilutions. Nine dilutions were prepared for each test lipopeptide.

[0115] (3) In a 96-well plate, lipopeptide dilution (50 μL / well) was added to the drug wells, and DMEM medium (50 μL / well) was added to the control wells. Each well was set up as three double wells. Then, 100 TCID 50 The virus solution (adjusted to 50 μL / well) was added and incubated at room temperature for 30 minutes.

[0116] (4) Resuspend TZM-b1 cells in DMEM medium until the cell concentration is approximately 10 × 10 4 DEAE-dextran was added to achieve a cell / mL concentration of 15 μg / mL. After step (3) was completed, the TZM-b1 cell suspension (100 μL / well) was added to a 96-well plate and cultured for 48 hours in a cell culture incubator at 37°C and 5% CO2.

[0117] (5) Discard the cell culture supernatant and add 30 μL of cell lysate (Promega, catalog number: E1531) to each well. After lysing the cells at room temperature for 15 minutes, add the luciferase assay substrate reagent (Promega, catalog number: E1501). The relative luminescence units (RLU) were measured using a microplate luminometer, and an inhibition rate curve was created to determine the maximum half dose inhibitory concentration (IC) of the drug. 50 ) was calculated.

[0118] 2. Experimental Results and Analysis The results are shown in Figure 3A. The results showed that C34-LP1 caused NL4-3 pseudovirus and JRFL pseudovirus to infect TZM-bl cells at mean IC50s of 55 pM and 69.4 pM, respectively. 50 While C34-LP2 inhibits NL4-3 pseudovirus and JRFL pseudovirus infection of TZM-bl cells at mean IC50s of 20.23 pM and 19.9 pM, respectively. 50 Inhibits C34-LP1 at an average IC of 15.44 pM, which inhibits live SG3.1 viruses from infecting MT-4 cells. 50 While C34-LP2 inhibits the infection of SG3.1 live viruses in MT-4 cells at an average IC of 3.2 pM, C34-LP2 inhibits the infection of MT-4 cells at an average IC of 3.2 pM. 50 The results showed that inhibition occurred at a numerical level. In comparison, the inhibitory activity of C34-LP2 against NL4-3 pseudovirus and JRFL pseudovirus was approximately 3 times and 4 times higher, respectively, than that of C34-LP1; the inhibitory activity of C34-LP2 against live SG3.1 virus was approximately 5 times higher than that of C34-LP1. The experimental results indicated that the EAAAK rigid linker helps improve the anti-HIV activity of membrane fusion inhibitors.

[0119] Example 3: Design of membrane fusion inhibitors and identification of antiviral activity By directly comparing the anti-HIV activities of C34-LP1 and C34-LP2, it was confirmed that the use of the EAAAK rigid linker can improve the antiviral effect of lipopeptide membrane fusion inhibitors. An example of this disclosure further prepared lipopeptides LP-121, LP-122, and LP-123, using EAAAK as a linker and further introducing EE and KK amino acid pairs to corresponding positions b, c, f, and g of the CHR helix, respectively, to promote the formation of a "salt bridge" structure. The anti-HIV activity of LP-121, LP-122, and LP-123 was measured using the method of Example 2. The results are shown in Figure 3B. The results showed the IC50 of LP-121 against NL4-3 pseudovirus, JRFL pseudovirus, and SG3.1 live virus. 50 The values ​​were 51.98 pM, 42.39 pM, and 9.88 pM, respectively; IC of LP-122 against NL4-3 pseudovirus, JRFL pseudovirus, and SG3.1 live virus. 50 The values ​​were 22.45 pM, 41.55 pM, and 9.05 pM, respectively; IC of LP-123 against NL4-3 pseudovirus, JRFL pseudovirus, and SG3.1 live virus. 50 The values ​​were 18.05 pM, 14.59 pM, and 3.68 pM, respectively. In comparison, the LP-123 lipopeptide containing the shorter sequence showed optimal anti-HIV activity, while the LP-121 lipopeptide with an extended N-terminus and the LP-122 lipopeptide with an extended C-terminus showed reduced anti-HIV activity. This unexpected result clarifies the relationship between the structure and function of membrane fusion inhibitors.

[0120] Example 4: Technological strategies for further optimizing membrane fusion inhibitors To further improve the antiviral activity of membrane fusion inhibitors, this disclosure creatively prepares membrane fusion inhibitor lipopeptides LP-124 and LP-125. Two hydrophobic amino acids W in the N-terminal pocket-binding domain (PBD) of the polypeptide sequences of LP-122 and LP-123 were replaced with two hydrophilic and low molecular weight amino acids Y to facilitate the conversion of the interaction between the inhibitor and the NHR target in the pocket domain from highly hydrophobic to hydrophilic.

[0121] Similarly, the anti-HIV activity of LP-124 and LP-125 was determined and compared for analysis using the experimental method of Example 2.

[0122] The results are shown in Figures 4A and 4B. The results showed the IC of LP-124 against NL4-3 pseudovirus, JRFL pseudovirus, and SG3.1 live virus. 50 The values ​​were 2.07 pM, 5.93 pM, and 1.06 pM, respectively, indicating an increase of approximately 11, 7, and 9 times, respectively, compared to the activity of its template LP-122. IC50 of LP-125 against NL4-3 pseudovirus, JRFL pseudovirus, and SG3.1 live virus. 50 The values ​​were 3.43 pM, 5.78 pM, and 1.11 pM, respectively, which represent increases of approximately 5 times, 3 times, and 3 times, respectively, compared to the activity of its template LP-123.

[0123] Furthermore, in the examples of this disclosure, three lipopeptides LP-126, LP-127, and LP-128 were prepared, and their anti-HIV activity was determined using the same method as described above. The results are shown in Figure 4C. The results show the IC50 activity of LP-126, LP-127, and LP-128 against NL4-3 pseudovirus. 50 The values ​​were 3.85 pM, 5.01 pM, and 3.27 pM, respectively; ICs of LP-126, LP-127, and LP-128 against JRFL pseudovirus. 50 The values ​​were 6.08 pM, 5.63 pM, and 4.97 pM, respectively; IC of LP-126, LP-127, and LP-128 against SG3.1 live virus. 50 The values ​​were shown to be 1.65 pM, 2.31 pM, and 1.75 pM, respectively.

[0124] These results demonstrate that substituting the W amino acid with the Y amino acid in the PBD sequence of membrane fusion inhibitors can significantly enhance antiviral activity.

[0125] Example 5: Comparison and analysis of the anti-HIV activity of LP-127, LP-128, and T20. T20 is currently the only membrane fusion inhibitor approved by the U.S. Food and Drug Administration (FDA) for clinical use in the treatment of HIV-1 infection. In this study, we further evaluated the anti-HIV activity of LP-127 and LP-128 using multiple replicated HIV-1 strains and compared their inhibitory activity to that of T20.

[0126] 1. Experimental Method The method for preparing replicated HIV-1 strains SG3.1, JRCSF, 89.6, and LAI.2 is the same as that described in Example 2, which was obtained by transfecting HEK293T cells with molecular clone plasmids. The specific method is described in the literature published by the inventors' laboratory. [7] The examples in this disclosure not only determined the activity of membrane fusion inhibitors that inhibit these viruses from infecting TZB-bI cells (the method was the same as in Example 2), but also determined the activity of membrane fusion inhibitors that inhibit these replicated HIV-1 strains from infecting MT-4 cells (the specific experimental method is as follows):

[0127] (1) The lipopeptides were gradually diluted in a 96-well plate by setting up three repeating wells and nine gradients, and then 200 TCID 50 Virus and approximately 4 x 10 4 Individual MT-4 cells were added to each well in sequence.

[0128] (2) The plates were placed in a 37°C, 5% CO2 incubator for 4 hours, then centrifuged at 200g for 5 minutes at room temperature, and the supernatant was discarded. The cells were washed once more with 200 μl / well of RPMI-1640 culture medium, and then cultured in 200 μl / well of RPMI-1640 complete culture medium.

[0129] (3) On day 4, the 96-well plate was centrifuged at 200 g for 5 minutes at room temperature, the supernatant was taken at 30 μl / well and transferred to a new 96-well plate, and 120 μl / well of 1% Triton-X100 solution was added to inactivate the virus. The dilution factor was determined using the positive control well and initial concentration for P24 antigen quantification. P24 antigen quantification was performed using a 96-well microplate reader for measurement, according to the procedure described in the kit specifications. Then, each culture supernatant was diluted to the appropriate concentration and the P24 antigen quantification assay was performed. The inhibition curves for each drug were fitted by using a 4-parameter logistic equation in nonlinear regression analysis, and IC 50 The value was calculated.

[0130] 2. Experimental Results and Analysis The results are shown in Figure 5.

[0131] In TZM-bl cells, LP-127, LP-128, and T20 showed IC50 concentrations of 2.04 pM, 0.68 pM, and 2911.83 pM, respectively. 50 The values ​​inhibit SG3.1, resulting in ICs of 10.38 pM, 7.47 pM, and 5599 pM, respectively. 50 The JR-CSF was inhibited by these values, resulting in ICs of 9.09 pM, 8.56 pM, and 15841.67 pM, respectively. 50 The inhibitory activity of LP-127 and LP-128 against SG3.1 was 1427-fold and 4282-fold higher, respectively, than that of T20; their inhibitory activity against JR-CSF was 539-fold and 750-fold higher than that of T20, and their inhibitory activity against 89.6 was 1743-fold and 1851-fold higher, respectively, than that of T20.

[0132] In MT-4 cells, LP-127, LP-128, and T20 showed IC50 concentrations of 0.69 pM, 0.4 pM, and 1133.7 pM, respectively. 50 The values ​​inhibit SG3.1, resulting in IC levels of 0.27 pM, 0.08 pM, and 745.77 pM, respectively. 50 The values ​​inhibit LAI.2, resulting in ICs of 1.45 pM, 0.83 pM, and 4801.5 pM, respectively.50 The inhibitory activity of LP-127 and LP-128 against SG3.1 was 1643 times and 2834 times higher, respectively, than that of T20; the inhibitory activity against LAI.2 was 2762 times and 9322 times higher, respectively, than that of T20; and the inhibitory activity against 89.6 was 3311 times and 5785 times higher, respectively, than that of T20.

[0133] Example 6: Broad-spectrum anti-HIV activity of membrane fusion inhibitors of the present disclosure HIV is highly variable and has evolved into many subtypes and recombinant viruses. While HIV-1 subtypes A, B, and C are the main viruses causing the AIDS pandemic, recombinant viruses A / E and B / C are the dominant viruses in China. In this study, we further evaluated the broad-spectrum anti-HIV activity of the membrane fusion inhibitors disclosed herein through a group of cell fusion experiments using pseudoviruses of 12 representative global pandemic HIV-1 strains, and compared and analyzed their activity with that of T20.

[0134] 1. Experimental Method The materials and methods used are described in the literature published by the inventors' laboratory. [7] The preparation method for HIV-1 pseudovirus and the antiviral experiments were the same as those described in Example 2. Cell-cell fusion inhibitory activity mediated by the HIV-1 envelope protein (Env) was detected by using cell fusion experiments based on a DSP fluorescence reporting system.

[0135] (1) Spread 293T cells (effector cells) in a 96-well plate (approximately 1.5 × 10⁻¹⁰ 4 Cells / wells), CXCR4 / CCR5 and DSP 8-11 Target cells 293FT (approximately 1.5 × 10) that stably express this gene. 4 After spreading the cells ( / mL) onto a 10 cm cell culture dish, the cells were cultured at 37°C and under 5% CO2 conditions.

[0136] (2) After 16 hours of incubation, HIV-1 Env expression plasmid and DSP 1-7 Plasmids were simultaneously transfected into 293T effector cells in a 1:1 ratio.

[0137] (3) After 24 hours of transfection, the membrane fusion inhibitor was dissolved in deionized water, diluted to the initial concentration in DMEM medium, and added to effector cells (50 μL / well) in 96-well plates in 3-fold serial dilutions, resulting in 9 dilutions and 3-well sets. The control group was treated with DMEM medium without the inhibitor. The plates were incubated at 37°C and 5% CO2 for 1 hour.

[0138] (4) Resuspend 293FT cells until the cell concentration is approximately 30 × 10 4 The solution was adjusted to the desired cell / mL ratio, and EnduRen live cell substrate (Promega) was added in a 1:4000 ratio and thoroughly mixed. The mixture was incubated at 37°C and 5% CO2 for 30 minutes.

[0139] (5) 293FT cells were added to the HIV-1 effector cell wells at a rate of 100 μL / well. The plate was centrifuged at 300 g for 1 minute to ensure complete contact between the effector cells and target cells. After culturing the mixture at 37°C for 1 hour, luciferase activity (relative luminescence units, RLU) was detected, and an inhibition rate curve was created to determine the maximum half-dose inhibitory concentration (IC) of the drug. 50 ) was calculated.

[0140] 2. Experimental Results and Analysis Figure 6 shows the inhibition results of the membrane fusion inhibitors described herein against infection with representative pandemic HIV-1 pseudovirus strains. The T20 polypeptide inhibited 12 pseudoviruses with an average IC50 of 2.09 pM. 50 While inhibiting at a certain value, the four lipopeptides LP-123, LP-125, LP-127, and LP-128 inhibited 12 pseudoviruses at average IC50 levels of 27.39 pM, 4.22 pM, 4.89 pM, and 3.19 pM, respectively. 50Inhibition was achieved by value. In comparison, the inhibitory activity of LP-123, LP-125, LP-127, and LP-128 against 12 pseudoviruses was 732 times, 4752 times, 4101 times, and 6286 times higher, respectively, than the inhibitory activity against T20.

[0141] Figure 7 shows the inhibition results of the membrane fusion inhibitors of this disclosure against cell fusion mediated by a representative pandemic HIV-1 strain, Env. T20 polypeptide inhibited 12 viral membrane fusions with an average IC50 of 11214.53 pM. 50 While inhibiting the fusion of 12 viruses at average IC50 levels, the four lipopeptides LP-123, LP-125, LP-127, and LP-128 inhibited the membrane fusion of 12 viruses at average IC50 levels of 24.04 pM, 11.9 pM, 4.97 pM, and 4.02 pM, respectively. 50 The inhibitory activity of LP-123, LP-125, LP-127, and LP-128 against membrane fusion of 12 viruses was 466, 942, 2256, and 2790 times higher, respectively, than that of T20.

[0142] Example 7: Inhibitory activity of the membrane fusion inhibitors of this disclosure against T20-resistant virus strains. Another important biological characteristic of HIV is its susceptibility to drug resistance, leading to treatment failure, which is a critical issue to address in drug research and development. In this study, the antiviral activity of the membrane fusion inhibitors described herein was evaluated using a high T20-resistant mutant strain, and their inhibitory activity was compared and analyzed with that of lipopeptide LP-98.

[0143] 1. Experimental Method Based on the HIV-1 strain NL4-3, a group of 12 T20-resistant mutant strains (L33S, I37T, V38A, V38M, Q40H, N43K, L33S / I37T, G36S / V38M, I37T / N43K, V38A / N42T, L33S / V38A / N42T, N43K / E49A / N126K) with single, double, or triple mutations was constructed (the construction method is described in the literature published by the inventors' laboratory). [7]

[11]

[12] The method for producing mutant pseudoviruses and the antiviral experiments were the same as those described in Example 2.

[0144] 2. Experimental Results and Analysis The results are shown in Figure 8. The results showed that LP-98 eliminated 12 drug-resistant mutants with an average IC of 2663.27 pM. 50 While inhibiting at a certain value, LP-123, LP-125, LP-127, and LP-128 inhibited 12 drug-resistant mutants with average IC50 values ​​of 55.51 pM, 6.29 pM, 7.02 pM, and 9.13 pM, respectively. 50 The inhibitory activity was demonstrated by the values. By comparison, the inhibitory activity of LP-123, LP-125, LP-127, and LP-128 against 12 drug-resistant mutant strains was 48-fold, 423-fold, 379-fold, and 292-fold higher, respectively, than that of LP-98. Therefore, the membrane fusion inhibitors of this disclosure exhibit potent inhibitory activity against T20-resistant virus strains, reflecting a significant advantage in terms of pharmacoagulation.

[0145] Example 8: Broad-spectrum inhibitory activity of the membrane fusion inhibitors of this disclosure against other viruses In this study, the inhibitory activity of LP-127 and LP-128 against SARS-CoV-2 and several other viruses was evaluated using a pseudovirus system.

[0146] 1. Experimental Method Methods for preparing pseudoviruses are well-established techniques in the art and can be routinely used by those skilled in the art. Methods for preparing SARS-CoV-2 D614G and BA4 / 5 mutant strains, MERS-CoV, IAV(H7N9), and VSV pseudoviruses, as well as antiviral experiments, are described in literature published by the inventors' laboratory.

[13]

[14]

[15] The preparation of EBOV, MARV, and RABV pseudoviruses obtained by co-transfecting HEK293T cells with a plasmid encoding the viral envelope protein together with the HIV-1 backbone plasmid pNL4-3.luc.R is also described in the above literature. The steps of the antiviral experiment were substantially consistent with the method described in Example 2, with the main differences being the type of target cell and the amount of pseudovirus used. The target cell for SARS-CoV-2 pseudovirus was 293T / ACE2; the target cell for MERS-CoV, EBOV, and MARV pseudoviruses was Huh-7; the target cell for H7N9 pseudovirus was MDCK; and the target cell for RABV and VSV pseudoviruses was HEK293T. The amount of each pseudovirus used was 1000 TCID. 50 That was the case.

[0147] 2. Experimental Results and Analysis The results are shown in Figure 9. Surprisingly, lipopeptides LP-127 and LP-128 were able to effectively inhibit infection by two SARS-CoV-2 mutant strains, with LP-127 and LP-128 exhibiting IC50s of 27.4 nM and 25.12 nM, respectively. 50 The D614G mutant was inhibited at these values; IC25 levels were 26.56 nM and 23.51 nM, respectively. 50 The inventors found that these values ​​inhibited the Omicron BA4 / 5 mutant. LP-127 and LP-128 inhibited IC50 at 382.72 nM and 455.67 nM, respectively. 50 These values ​​inhibited MERS-CoV; ICs of 61.68 nM and 274.47 nM, respectively. 50 The EBOV is inhibited by the values ​​of 143.98 nM and 534.44 nM ICs, respectively. 50 The values ​​inhibit MARV; ICs of 414.87 nM and 352.6 nM respectively. 50 The values ​​inhibit H7N9; ICs of 1731nM and 1130nM respectively. 50 The RABV is inhibited by the values ​​of 1509nM and 1490nM ICs, respectively. 50 The value inhibited VSV.

[0148] The experimental results described above demonstrate that LP-127 and LP-128 can not only potently inhibit various HIV-1 subtypes and T20-resistant virus strains, but can also effectively inhibit several other viruses, indicating that they are broad-spectrum antiviral candidate drugs.

[0149] Example 9: Analysis of in vitro cytotoxicity of lipopeptides LP-127 and LP-128 To clarify the antiviral activity specificity and drug potential of the membrane fusion inhibitors LP-127 and LP-128 described herein, their in vitro cytotoxicity was further evaluated in this case.

[0150] 1. Experimental Method In vitro cytotoxicity was detected using the CCK-8 cell proliferation / toxicity assay kit (manufacturer: Abbkine, catalog number: KTC 011001). The specific procedure was as follows: (1) In a 96-well plate, the test lipopeptide was diluted with a 3-fold gradient, and 9 dilutions and 3 double wells were used for each dilution; each well contained 100 μL of lipopeptide solution; a DMEM medium control well (100 μL per well) was set up; (2) Approximately 10 × 10⁶ units were added to the 96-well plate from step (1). 4 (3) A suspension of test cells (TZM-bl, MT-4, MDCK, Huh-7, Hep-2, HEK293T, 293T / ACE2) at a cell / mL concentration was added to 100 μL / well and incubated at 37°C under 5% CO2 conditions for 48 hours; (4) CCK-8 solution was added to each well at 20 μL and the plate was incubated in an incubator for 2 hours; then the absorbance at 450 nm (OD450) was measured with a microplate reader. The inhibition rate curve was fitted using GraphPad Prism software to determine the maximum half-dose cytotoxic concentration (CC) of the drug. 50 ) was calculated.

[0151] 2. Experimental Results and Analysis The results are shown in Figure 10. The results showed that LP-127 produced an average CC of 15.68 μM in seven test cells. 50showed having, and the CC on TZM-bl cells 50 was 10.21 μM, and the CC on MT-4 cells 50 was 15.45 μM, and the CC on MDCK cells 50 was 12.89 μM, and the CC on Huh-7 cells 50 was 31.28 μM, and the CC on Hep-2 cells 50 was 16.46 μM, and the CC on HEK293T cells 50 was 9.07 μM, and the CC on 293T / ACE2 cells 50 was 14.42 μM. The results showed that LP-128 had an average CC of 19.91 μM in 7 test cells 50 showed having, and the CC on TZM-bl cells 50 was 7.49 μM, and the CC on MT-4 cells 50 was 28.22 μM, and the CC on MDCK cells 50 was 18.05 μM, and the CC on Huh-7 cells 50 was 40.28 μM, and the CC on Hep-2 cells 50 was 12.48 μM, and the CC on HEK293T cells 50 was 8.72 μM, and the CC on 293T / ACE2 cells 50 was 24.1 μM.

[0152] CC 50 / IC 50 analysis showed that both LP-127 and LP-128 had very high therapeutic selection indices (SI).

[0153] Example 10 Stability Study of Lipopeptides LP-127 and LP-128 To further investigate the drugability of the membrane fusion inhibitors of the present disclosure, in this example, LP-127 and LP-128 were left standing at room temperature or 37 °C for a long time, incubated with human serum, or digested with protease, and then the stability was evaluated by detecting changes in antiviral activity.

[0154] 1. Experimental Method Thermal stability experiment: After an aqueous solution of LP-127 or LP-128 at a concentration of 300 μM was placed at room temperature or 37 °C for various periods, their activities that inhibit the infection of NL4-3 pseudovirus to TZM-bl cells were determined by the same method as in Example 2.

[0155] Human serum stability experiment: 20% human serum was mixed with LP-127 or LP-128 at a final concentration of 150 μm; after the resulting mixture was incubated at 37 °C for 0, 5, 30, 60, 120, 180 or 240 minutes, their activities that inhibit the infection of NL4-3 pseudovirus to TZM-bl cells were determined by the same method as in Example 2.

[0156] Digestion by protease: LP-127 or LP-128 was mixed with protease K, trypsin or α-chymotrypsin (products of Sigma-Aldrich, catalog numbers: P2308, T4799, and C4129 respectively) at a ratio of 20:1 (the final concentrations were 2 mg / mL and 0.1 mg / mL respectively); after the resulting mixture was incubated at 37 °C for 0, 30, 60, 120, 180 or 240 minutes, their activities that inhibit the infection of NL4-3 pseudovirus to TZM-bl cells were determined by the same method as in Example 2.

[0157] 2. Experimental results and analysis The results are shown in Figure 11. Compared with the untreated lipopeptide (when the incubation time is 0), after treatment with different temperatures, human serum, or three proteases for various periods, there were no significant changes in the antiviral activities of LP-127 and LP-128, indicating that they are very stable.

[0158] Example 11 Helical structure characteristics of the membrane fusion inhibitor of the present disclosure and analysis of its binding stability To analyze the structural characteristics of the membrane fusion inhibitors of this disclosure and investigate their mechanisms of action, circular dichroism (CD) techniques were used to determine the secondary structure (α-helix) and thermal stability of LP-121, LP-122, LP-123, LP-124, LP-125, LP-126, LP-127, and LP-128, as well as their complexes with their target sequence N44.

[0159] 1. Experimental Method The CD determination method, as well as the synthesis and preparation of polypeptide N44 (Ac-TVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL-NH2), derived from the gp41 NHR sequence and serving as a simulated target for the inhibitor, are described in the literature published by the inventors.

[16] It was described.

[0160] The test lipopeptide and a mixture of N44 and the lipopeptide were each dissolved in phosphate-buffered saline (PBS) with a pH of 7.2 to obtain solutions with final concentrations of 10 μM of both the lipopeptide and the N44 polypeptide. Each solution was placed in a 37°C water bath for 30 minutes and then transferred to the corresponding cuvette. The change in the molar ellipticity [θ]λ of the solution in the wavelength range of 195–270 nm was scanned using a JASCO spectropolarimeter (model J-815). Typical α-helical structures could show maximum negative peaks at 208 nm and 222 nm. Spectral values ​​were corrected by subtracting the PBS blank control. During calculations, -33000°C.cm 2 .dmol -1 The peak value was used as a standard for 100% α-helix content, and the percentage of α-helix content of the polypeptide was calculated according to the molar ellipticity of the solution at 222 nm. Subsequently, the solution was added to the corresponding cuvette for thermal stability detection, and the CD temperature control module was adjusted to scan the polypeptide solution at a rate of 2°C / min to detect the temperature-dependent change of [θ]222 in the range of 20–98°C. Melting curves were plotted and smoothed, and the midpoint temperature value (Tm) of the thermal dissociation transition was calculated using Origin software to reflect the degree of helix thermal stability.

[0161] 2. Experimental Results and Analysis The results are shown in Figure 12.

[0162] As shown in Figure 12A, the α-helix content of LP-121, LP-122, LP-123, LP-124, LP-125, LP-126, LP-127, or LP-128 individually was 69%, 65%, 56%, 64%, 78%, 51%, 57%, or 66%, respectively, indicating that each lipopeptide inhibitor itself possesses high helicality, with LP-125 exhibiting the highest helicality. However, their Tm values ​​could not be accurately calculated according to the melting curve.

[0163] As shown in Figure 12B, the α-helix content of mixtures of LP-121, LP-122, LP-123, LP-124, LP-125, LP-126, LP-127, or LP-128 with N44 was 81%, 81%, 79%, 79%, 89%, 66%, 68%, or 84%, respectively, and their Tm values ​​were >98°C, >98°C, >98°C, 86°C, 91°C, 92°C, 92°C, or 90°C, respectively, indicating that the lipopeptide inhibitors can interact with N44 to form more stable α-helix structures, particularly the three lipopeptides (LP-121, LP-122, LP-123) having PBDs containing amino acid W. The results also showed that substituting W with Y could reduce the binding ability of the PBD, but lipopeptides containing Y still had relatively high binding stability (Tm > 86°C).

[0164] The present disclosure has been described in detail above. Without departing from the spirit and scope of the present disclosure and without the need to perform unnecessary experiments, those skilled in the art can implement the present disclosure over a wide range of parameters, concentrations, and conditions. While the present disclosure provides specific examples, it will be understood that the present disclosure may be further modified. In general, according to the principles of the present disclosure, it is intended to cover any modifications, uses, or improvements to the present disclosure, including modifications made using known, everyday art in the art, which deviate from the scope disclosed herein. Several basic features may apply according to the appended claims.

[0165] References: [1]. Chan DC, Kim PS. HIV entry and its inhibition. Cell 1998, 93: 681-684. [2]. He Y. Synthesized peptide inhibitors of HIV-1 gp41-dependent membrane fusion. Curr Pharm Des 2013, 19: 1800-1809. [3]. Dwyer JJ, Wilson KL, Davison DK, Freel SA, Seedorff JE, Wring SA, et al. Design of helical, oligomeric HIV-1 fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus. Proc Natl Acad Sci USA 2007, 104: 12772-12777. [4]. He Y, Xiao Y, Song H, Liang Q, Ju D, Chen X, et al. Design and evaluation of sifuvirtide, a novel HIV-1 fusion inhibitor. J Biol Chem 2008, 283: 11126-11134. [5]. Chong H, Yao X, Zhang C, Cai L, Cui S, Wang Y, et al. Biophysical property and broad anti-HIV activity of albuvirtide, a 3-maleimimidopropionic acid-modified peptide fusion inhibitor. PLoS One 2012, 7: e32599. [6]. Ingallinella P, Bianchi E, Ladwa NA, Wang YJ, Hrin R, Veneziano M, et al. Addition of a cholesterol group to an HIV-1 peptide fusion inhibitor dramatically increases its antiviral potency. Proc Natl Acad Sci U S A 2009, 106: 5801-5806. [7]. Xue J, Chong H, Zhu Y, Zhang J, Tong L, Lu J, et al. Efficient treatment and pre-exposure prophylaxis in rhesus macaques by an HIV fusion-inhibitory lipopeptide. Cell2022, 185: 131-144 e118. [8]. Yu D, Zhu Y, Jiao T, Wu T, Xiao X, Qin B, et al. Structure-based design and characterization of novel fusion-inhibitory lipopeptides against SARS-CoV-2 and emerging variants. Emerg Microbes Infect 2021, 10: 1227-1240. [9]. Zhou J, Xu W, Liu Z, Wang C, Xia S, Lan Q, et al. A highly potent and stable pan-coronavirus fusion inhibitor as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases. Acta Pharm Sin B 2021.

[10] . Outlaw VK, Bovier FT, Mears MC, Cajimat MN, Zhu Y, Lin MJ, et al. Inhibition of Coronavirus Entry In Vitro and Ex Vivo by a Lipid-Conjugated Peptide Derived from the SARS-CoV-2 Spike Glycoprotein HRC Domain. mBio 2020,11:e01935-01920.

[11] . 11. Hu Y, Yu W, Geng X, Zhu Y, Chong H, He Y. In Vitro Selection and Characterization of HIV-1 Variants with Increased Resistance to LP-40, Enfuvirtide-Based Lipopeptide Inhibitor. Int J Mol Sci2022, 23.

[12] . Wu X, Liu Z, Ding X, Yu D, Wei H, Qin B, et al. Mechanism of HIV-1 Resistance to an Electronically Constrained alpha-Helical Peptide Membrane Fusion Inhibitor. J Virol 2018, 92: e02044-02017.

[13] . Yu D, Zhu Y, Yan H, Wu T, Chong H, He Y. Pan-coronavirus fusion inhibitors possess potent inhibitory activity against HIV-1, HIV-2, and simian immunodeficiency virus. Emerg Microbes Infect 2021, 10: 810-821.

[14] . Zhu Y, Hu Y, Liu N, Chong H, He Y. Potent inhibition of diverse Omicron sublineages by SARS-CoV-2 fusion-inhibitory lipopeptides. Antiviral Res 2022, 208: 105445.

[15] . Zhu Y, Yu D, Hu Y, Wu T, Chong H, He Y. SARS-CoV-2-derived fusion inhibitor lipopeptides exhibit highly potent and broad-spectrum activity against divergent human coronaviruses. Signal Transduct Target Ther 2021, 6: 294.

[16] . Zhu Y, Ding X, Yu D, Chong H, He Y. The Tryptophan-Rich Motif of HIV-1 gp41 Can Interact with the N-Terminal Deep Pocket Site: New Insights into the Structure and Function of gp41 and Its Inhibitors.J Virol 2019, 94: effff1234.

Claims

1. A lipopeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof comprising a polypeptide or a variant thereof, a modifying group linked to the C-terminus of the polypeptide or the variant thereof via a linker, and optionally a terminal protecting group of the polypeptide, (X １ X ２ X ３ )mX ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ IX １６ X １７ LX １８ X １９ X ２０ X ２１ X ２２ X ２３ QQX ２４ X ２５ N(EX ２６ )n Equation I In the formula, the polypeptide is represented by formula I, the modifying group is a lipophilic compound, and the linker is -(EAAAK)n １ -, -(XP)n ２ -, -(EAAAK)n １ -X ２７ - or - (XP)n ２ -X ２７ - and; X １ is T, E, or S; X ２ is a W or hydrophilic amino acid; X ３ is E, M, or Q; m is either 0 or 1; X ４ is E, A, or T; X ５ is a W or hydrophilic amino acid; X ６ is either E or D; X ７ is R, K, or Q; X ８ is E, K, or A; X ９ is L or I; X １０ is E, N, or A; X １１ is either E or N; X １２ is either L or Y; X １３ is E, T, or A; X １４ is K, S, R, or A; X １５ is K, L, R, or Q; X １６ is E, H, T, or Y; X １７ is E, S, R, or A; X １８ is L or I; X １９ is K, E, or R; X ２０ is K, E, Q, or A; X ２１ is A or S; X ２２ is either E or Q; X ２３ is E, N, or I; X ２４ is K, E, or D; X ２５ is either K or R; X ２６ is Q, E, R, A, or Y; n is either 0 or 1; X is any one amino acid; X ２７ is K or C; n １ are natural numbers from 1 to 5; n ２ are natural numbers from 1 to 5; Unlike the polypeptide from which the variant is derived, which is derived only by substitutions of one or more (e.g., one, two, three, four, or five) amino acid residues (e.g., conservative or non-conservative substitutions), the lipopeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex, or prodrug thereof retains the biological function of the polypeptide from which the variant is derived.

2. A lipopeptide according to claim 1, or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex, or prodrug thereof, characterized by one or more of the following features: -X １ is either T or E; -X ２ is W, D, E, H, K, Q, R, S, T, or Y; -X ３ is either E or M; -X ４ E is; -X ５ is W, D, E, H, K, Q, R, S, T, or Y; -X ６ E is; -X ７ is R or K; -X ８ is either E or K; -X １０ is either E or N; -X １１ E is; -X １２ L is; -X １３ is either E or T; -X １４ is either K or S; -X １５ is either K or L; -X １６ is either E or H; -X １７ is either E or S; -X １８ L is; -X １９ is either K or E; -X ２０ is either K or E; -X ２１ A is; -X ２２ E is; -X ２３ is either E or N; -X ２４ is either K or E; -X ２５ is K; -X ２６ Q is; -X is A, K, or E.

3. A lipopeptide according to claim 1, or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex, or prodrug thereof, characterized by one or more of the following features: -X ２ is W or Y, preferably Y; -X ３ E is; -X ５ is W or Y, preferably Y; -X ７ is K; -X １０ E is; -X １３ E is; -X １４ is K; -X １５ is K; -X １６ E is; -X １７ E is; -X １９ is K; -X ２０ is K; -X ２３ E is; -X ２４ It is K.

4. The polypeptide is represented by formula I, formula III, formula IV, or formula V, (X １ X ２ X ３ )mX ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ IX １６ X １７ LX １８ X １９ X ２０ X ２１ X ２２ X ２３ QQX ２４ KN(EX ２６ )n Formula II (X １ X ２ X ３ )m-EX ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ IX １６ X １７ LX １８ X １９ X ２０ X ２１ X ２２ X ２３ QQX ２４ KN(EX ２６ )n Formula III (X １ X ２ X ３ )m-EX ５ X ６ X ７ X ８ X ９ EELEKKIEELLKKAEEQQKKN(EX ２６ )n Formula IV (8 １ 8 ２ 8 ３ m-X ５ 5.8 ８ 8 ９ EEEKKIEELLKKKEEQQQK68(EX ２６ )n Formula V In the formula, X １ , X ２ , X ３ , m, X ４ , X ５ , X ６ , X ７ , X ８ , X ９ , X １０ , X １１ , X １２ , X １３ , X １４ , X １５ , X １６ , X １７ , X １８ , X １９ , X ２０ , X ２１ , X ２２ , X ２３ , X ２４ , X ２６ The definitions of n are as described in any one of claims 1 to 3. A lipopeptide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex, or prodrug thereof.

5. The aforementioned lipophilic compound is cholesterol (for example, cholesteryl hemisuccinate, 2-cholesteryl acetate, 2-cholesteryl propionic acid, 3-cholesteryl propionic acid, 2-cholesteryl butyrate, 2-cholesteryl isobutyrate, 3-cholesteryl butyrate, 3-cholesteryl isobutyrate, 4-cholesteryl butyrate, 2-cholesteryl valeric acid, 2-cholesteryl isovaleric acid, 3-cholesteryl valeric acid, 5-cholesteryl valeric acid, 2-cholesteryl caproic acid, 6-cholesteryl A lipopeptide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof, wherein the lipopeptide is lylcaproic acid, 2-cholesteryl enanthic acid, 7-cholesteryl enanthic acid, 2-cholesteryl caprylic acid, 8-cholesteryl caprylic acid, and cholesteryl bromoacetate), a fatty acid (e.g., a fatty acid containing 8 to 20 carbon atoms), dihydrosphingosine, or vitamin E.

6. The lipopeptide according to claim 5, or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof, wherein the fatty acid containing 8 to 20 carbon atoms is palmitic acid or octadecanoic acid.

7. The lipopeptide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the lipopeptide has an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 11 to 19.

8. A polypeptide or variant thereof comprising, or consisting of, a sequence represented by formula I: (X １ X ２ X ３ )mX ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ IX １６ X １７ LX １８ X １９ X ２０ X ２１ X ２２ X ２３ QQX ２４ X ２５ N(EX ２６ )n Equation I During the ceremony, X １ These are T, E, and S; X ２ is a W or hydrophilic amino acid; X ３ is E, M, or Q; m is either 0 or 1; X ４ is E, A, or T; X ５ is a W or hydrophilic amino acid; X ６ is either E or D; X ７ is R, K, or Q; X ８ is E, K, or A; X ９ is L or I; X １０ is E, N, or A; X １１ is either E or N; X １２ is either L or Y; X １３ is E, T, or A; X １４ is K, S, R, or A; X １５ is K, L, R, or Q; X １６ is E, H, T, or Y; X １７ is E, S, R, or A; X １８ is L or I; X １９ is K, E, or R; X ２０ is K, E, Q, or A; X ２１ is A or S; X ２２ is either E or Q; X ２３ is E, N, or I; X ２４ is K, E, or D; X ２５ is either K or R; X ２６ is Q, E, R, A, or Y; n is either 0 or 1; Unlike the polypeptide from which the variant originates, the variant retains the biological function of the polypeptide from which the variant originates, such as a polypeptide or a variant thereof.

9. A polypeptide or variant thereof according to claim 8, characterized by one or more of the following features: -X １ is either T or E; -X ２ is W, D, E, H, K, Q, R, S, T, or Y; -X ３ is either E or M; -X ４ E is; -X ５ is W, D, E, H, K, Q, R, S, T, or Y; -X ６ E is; -X ７ is R or K; -X ８ is either E or K; -X １０ is either E or N; -X １１ E is; -X １２ L is; -X １３ is either E or T; -X １４ is either K or S; -X １５ is either K or L; -X １６ is either E or H; -X １７ is either E or S; -X １８ L is; -X １９ is either K or E; -X ２０ is either K or E; -X ２１ A is; -X ２２ E is; -X ２３ is either E or N; -X ２４ is either K or E; -X ２５ is K; -X ２６ Q is the answer.

10. A polypeptide or variant thereof according to claim 8, characterized by one or more of the following features: -X ２ is W or Y, preferably Y; -X ３ E is; -X ５ is W or Y, preferably Y; -X ７ is K; -X ８ is either E or K; -X １０ E is; -X １３ E is; -X １４ is K; -X １５ is K; -X １６ E is; -X １７ E is; -X １９ is K; -X ２０ is K; -X ２３ E is; -X ２４ It is K.

11. The polypeptide is represented by formula II, formula III, formula IV, or formula V, (X １ X ２ X ３ )mX ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ IX １６ X １７ LX １８ X １９ X ２０ X ２１ X ２２ X ２３ QQX ２４ KN(EX ２６ )n Formula II (X １ X ２ X ３ )m-EX ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ IX １６ X １７ LX １８ X １９ X ２０ X ２１ X ２２ X ２３ QQX ２４ KN(EX ２６ )n Formula III (X １ X ２ X ３ )m-EX ５ X ６ X ７ X ８ X ９ EELEKKIEELLKKAEEQQKKN(EX ２６ )n Formula IV (8 １ 8 ２ 8 ３ m-X ５ 5.8 ８ 8 ９ EEEKKIEELLKKKEEQQQK68(EX ２６ )n Formula V In the formula, X １ , X ２ , X ３ , m, X ４ , X ５ , X ６ , X ７ , X ８ , X ９ , X １０ , X １１ , X １２ , X １３ , X １４ , X １５ , X １６ , X １７ , X １８ , X １９ , X ２０ , X ２１ , X ２２ , X ２３ , X ２４ , X ２６ The definitions of n are as described in any one of claims 8 to 10. A polypeptide or a variant thereof according to any one of claims 8 to 10.

12. The polypeptide or a variant thereof according to any one of claims 8 to 10, wherein the lipopeptide has an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs. 21 to 29.

13. A conjugate comprising a polypeptide or a variant thereof according to any one of claims 8 to 12 and a modifying group, For example, the modifying group may be optionally linked to the N-terminus or C-terminus of the polypeptide or its variant via a linker; For example, the modifying group is a terminal protecting group, a conjugate.

14. An isolated nucleic acid encoding a polypeptide or a variant thereof according to any one of claims 8 to 12.

15. A vector comprising an isolated nucleic acid as described in claim 14.

16. A host cell comprising the isolated nucleic acid described in claim 14 and / or the vector described in claim 15.

17. A polymer formed from a lipopeptide according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex, or prodrug thereof, or from a polypeptide according to any one of claims 8 to 10, or a variant thereof.

18. A composition comprising a lipopeptide according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex, or prodrug thereof, or a polypeptide according to any one of claims 8 to 12, or a variant thereof, or a conjugate according to claim 13, or an isolated nucleic acid according to claim 14, or a vector according to claim 15, or a host cell according to claim 16, or a polymer according to claim 17.

19. A pharmaceutical composition comprising a lipopeptide according to any one of claims 1 to 7 or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof, or a polypeptide according to any one of claims 8 to 10 or a variant thereof, and optionally further comprising a pharmaceutically acceptable carrier and / or excipient; Preferably, the lipopeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof, or the polypeptide or its variant, is present in an amount effective for treating a viral infection or a disease caused by a viral infection; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of HIV and its drug-resistant strains, SARS-CoV-2 and its mutant strains, MERS-CoV, EBOV, MARV, IAV, RABV, and VSV; Preferably, the viral infection is an infection caused by HIV or a drug-resistant strain thereof, in the pharmaceutical composition.

20. A method for inhibiting viral membrane injection or for treating a viral infection or a disease caused by a viral infection, comprising administering an effective amount of a lipopeptide according to any one of claims 1 to 7 or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof, or a polypeptide according to any one of claims 8 to 10 or a variant thereof, or a pharmaceutical composition according to claim 19, to a subject requiring such treatment; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of HIV and its drug-resistant strains, SARS-CoV-2 and its mutant strains, MERS-CoV, EBOV, MARV, IAV, RABV, and VSV; Preferably, the viral infection is an infection caused by HIV or a drug-resistant strain thereof, in a method.

21. The use of a lipopeptide according to any one of claims 1 to 7 or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof, or a polypeptide according to any one of claims 8 to 10 or a variant thereof, in the manufacture of a pharmaceutical composition, wherein the pharmaceutical composition is used as a viral membrane fusion inhibitor or for treating a viral infection or a disease caused by a viral infection; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of HIV and its drug-resistant strains, SARS-CoV-2 and its mutant strains, MERS-CoV, EBOV, MARV, IAV, RABV, and VSV; Preferably, the viral infection is an infection caused by HIV or a drug-resistant strain thereof.

22. A lipopeptide according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof, or a polypeptide according to any one of claims 8 to 10, for use as a viral membrane fusion inhibitor, or for use in the treatment of a viral infection or a disease caused by a viral infection; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of HIV and its drug-resistant strains, SARS-CoV-2 and its mutant strains, MERS-CoV, EBOV, MARV, IAV, RABV, and VSV; Preferably, the viral infection is an infection caused by HIV or a drug-resistant strain thereof, and the lipopeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof, or polypeptide or variant thereof.

23. A pharmaceutical composition for inhibiting viral membrane fusion or for treating a viral infection or a disease caused by a viral infection, comprising a lipopeptide according to any one of claims 1 to 7 or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, non-covalent complex or prodrug thereof, or a polypeptide according to any one of claims 8 to 10 or a variant thereof; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of HIV and its drug-resistant strains, SARS-CoV-2 and its mutant strains, MERS-CoV, EBOV, MARV, IAV, RABV, and VSV; Preferably, the viral infection is an infection caused by HIV or a drug-resistant strain thereof, in the pharmaceutical composition.

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