MEMBRANE FUSION INHIBITORS FOR INHIBITING AIDS VIRUS AND ITS DRUG-RESISTANT STRAINS AND THEIR MEDICINAL USE

Abstract

Provided are membrane fusion inhibitors, derivatives thereof, pharmaceutical compositions thereof, and pharmaceutical uses thereof for inhibiting HIV and drug-resistant strains thereof.

Description

[Technical Field]

[0001] This application is based on and claims the benefit of priority from Chinese Application No. 202211410040.4, filed on November 11, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Technical Field The present application belongs to the field of biomedicine and relates to a membrane fusion inhibitor, a derivative thereof, or a pharmaceutical composition thereof for inhibiting human immunodeficiency virus and drug-resistant strains thereof, as well as medical uses thereof. [Background technology]

[0003] Background technology Acquired immunodeficiency syndrome (AIDS) is caused by infection with the human immunodeficiency virus (HIV), currently affecting approximately 38.4 million people worldwide (www.unaids.org). Because the development of an HIV vaccine has yet to be successful, drugs that block various stages of viral replication play an important role in the treatment and prevention of HIV infection. Drugs used in clinical treatment primarily include nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, viral entry inhibitors, and integrase inhibitors (www.fda.gov). Widely used and highly effective antiviral treatment regimens ("cocktail" therapy) primarily consist of three to four reverse transcriptase inhibitors and protease inhibitors. Because HIV infection is persistent, patients must receive drugs over a long period of time, and drug resistance is likely to develop, significantly impacting clinical treatment efficacy. Therefore, the development of new anti-HIV drugs is a constant and important need for the prevention and control of AIDS.

[0004] Entry of HIV into target cells is mediated by its surface envelope glycoprotein (Env), which is formed by the noncovalent binding of the surface subunit gp120 and the transmembrane subunit gp41. During HIV infection, sequential binding of gp120 to the cellular receptor CD4 and co-receptors (e.g., CCR5 or CXCR4) induces conformational changes in gp120, exposing gp41 and activating its membrane fusion function. The N-terminal fusion peptide (FP) of gp41 first inserts into the target cell membrane, and then the C-terminal heptad region (CHR) binds inversely to the N-terminal heptad region (NHR) to form a stable six-helix bundle (6-HB) structure, bringing the viral membrane into close proximity with the cellular membrane for fusion, allowing HIV genetic material to enter the cell and initiate infection. The structure reveals that the C-terminus of the NHR helix contains a deep hydrophobic pocket, while the N-terminal sequence of the CHR helix (WMEWDREI) is a pocket-binding domain (PBD). Two highly evolutionarily conserved tryptophans (W) and one isoleucine (I) are inserted into the NHR pocket, mediating a broad hydrophobic effect that plays an important role in the formation of the 6-HB structure and viral infection (Chan et al., 1997). The NHR hydrophobic pocket has always been recognized as an important drug target.

[0005] HIV membrane fusion inhibitors function at the early stage of viral replication, blocking viral entry into target cells and offering clear advantages for both treatment and prevention. However, only enfuvirtide (also known as T20) has been approved for clinical use by the U.S. Food and Drug Administration (FDA). T20 is a 36-amino acid polypeptide derived from the viral CHR and lacking the PBD sequence. It exerts its antiviral effect by competitively binding to the NHR and inhibiting viral 6-HB formation. T20 has relatively low viral activity, a relatively short half-life, the need for high daily doses, and a high susceptibility to drug resistance, which largely limit its clinical application. Therefore, the development of new HIV membrane fusion inhibitors has been a hot topic both domestically and internationally.

[0006] Currently, most research and development of membrane fusion inhibitors focuses on the effects of PBDs, particularly the C34 polypeptide (He, 2013), which was initially published as a design template. C34 contains a PBD, and its amino acid sequence is recognized as the core sequence of CHR. Sifuvirtide (SFT) and albuvirtide (ABT), which have been developed in China for some time, are both designed based on the C34 sequence. SFT was obtained by mutating 14 amino acids of C34, adding serine (S) and glutamic acid (E) to the N- and C-termini, respectively, to improve the polypeptide's stability and target binding ability. ABT was obtained by mutating only three amino acids of C34, incorporating 3-maleimidopropionic acid (MPA) into the side chain of lysine (K) at position 13, thereby conferring the ability to bind to serum albumin. The antiviral activity of SFT and ABT is not significantly improved compared to that of T20 and C34, but their biological half-lives are long, and ABT in particular can achieve therapeutic effects with once-weekly administration.

[0007] Our team has been engaged in the research and development of HIV membrane fusion inhibitors for many years and has designed several CHR polypeptide-based membrane fusion inhibitors based on the structure and function of gp41 (Xue et al., 2022). Among them, LP-98, like T20, does not contain a PBD sequence in its molecular structure. By removing the eight C-terminal amino acids of T20 and mutating 16 of the 28 retained amino acids to promote the formation of a "salt bridge" between the E and K amino acids, the stability of the polypeptide's helical structure has been significantly improved. Meanwhile, LP-98 becomes a lipopeptide by modifying the side chain of the C-terminal K with cholesterol. Research has shown that the average IC of LP-98, which inhibits the infection of a group of replicating HIV strains to various target cells, is 0.01. 50 The activity of LP-98 reached extremely low picomolar (pM) levels, a 273,368-fold increase compared to T20 and a 120,789-fold and 376,368-fold increase compared to the first-line AIDS treatments zidovudine (AZT) and lamivudine (3TC), respectively. The activity of LP-98 in inhibiting 36 representative internationally prevalent HIV-1 subtype pseudoviruses was 10,504-fold increased compared to T20 and 3,751-fold increased compared to AZT; LP-98 has extremely potent therapeutic and prophylactic effects when administered at low doses in a monkey infection model (Xue et al., 2022). Nevertheless, we also found that LP-98 exhibited significantly reduced inhibitory activity against T20-resistant HIV strains, thereby affecting its druggability.

[0008] A sequence comparison diagram of T20, C34, SFT, ABT and LP-98 is shown in FIG. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Chan, D.C., Fass, D., Berger, J.M., Kim, P.S., 1997. Core structure of gp41 from the HIV envelope glycoprotein. Cell 89, 263-273.

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Summary of the Invention

[0010] Summary The technical problem that the present application aims to solve is how to potently inhibit HIV and its drug-resistant strains. The present application focuses on the development of ultra-potent, broad-spectrum, and long-acting HIV membrane fusion inhibitors with novel structures that are expected to have great clinical development value and application prospects. Therefore, the present application provides membrane fusion inhibitors for inhibiting HIV and its drug-resistant strains, derivatives thereof, pharmaceutical compositions thereof, and pharmaceutical uses thereof.

[0011] The present application provides a lipopeptide, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof, or a derivative thereof.

[0012] In one embodiment, the lipopeptide has Formula I; Formula I: X1 - polypeptide P1-X2; is a compound represented by The polypeptide P1 is (a1) or (a2) or (a3): (a1) a polypeptide having the amino acid sequence "EXEELEKKIEELLKKAEEQQKKNEEELKKLEK"; (a2) a polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on (a1); (a3) a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to (a1), and having antiviral activity; and The terminal amino acid residue of polypeptide P1 is modified with a lipophilic compound group; X1 is an amino terminal protecting group of polypeptide P1; X2 is a carboxyl terminal protecting group of polypeptide P1.

[0013] Specifically, in polypeptide P1, X is isoleucine (I), valine (V) or leucine (L).

[0014] It is well known in the art that the amino acid at positions 2 (X), 9 (I), 16 (A), 23 (N), and 30 (L) in (a1) correspond to the amino acid at position a of the CHR helix, and the amino acid at positions 5 (L), 12 (L), 19 (Q), and 26 (E) in (a1) correspond to the amino acid at position d of the CHR helix. These amino acids at positions a and d are key amino acids for the formation of the 6-HB structure upon binding of the CHR to the NHR target sequence, and are not easily substituted with other amino acids due to their conserved sequence and function. In contrast, the amino acids at other positions in the CHR helix (positions b, c, e, f, and g) do not or barely interact directly with the NHR and are therefore easily substituted with other amino acids with no or only a slight effect on the antiviral activity of the polypeptide.

[0015] Therefore, the substitutions and / or deletions and / or additions are located at positions other than positions 2 (X), 9 (I), 16 (A), 23 (N), 30 (L), 5 (L), 12 (L), 19 (Q), and 26 (E) in (a1).

[0016] In another embodiment, the lipopeptide has Formula II; Formula II: X3 - polypeptide P2-X4; is a compound represented by The polypeptide P2 is (b1) or (b2) or (b3): (b1) a polypeptide having the amino acid sequence "EXEELEKKIEELLKKAEEQQKKNEEELKKLEKX"; (b2) A polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on (b1); (b3) a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to (b1), and having antiviral activity; and The terminal amino acid residue of polypeptide P2 is modified with a lipophilic compound group; X3 is an amino terminal protecting group of polypeptide P2; X4 is a carboxyl terminal protecting group of polypeptide P2.

[0017] Specifically, in polypeptide P2, "X" as the second amino acid residue is isoleucine (I), valine (V), or leucine (L), and "X" as the 33rd amino acid residue is lysine (K) or cysteine ​​(C). In some embodiments, in polypeptide P2, "X" as the second amino acid residue is isoleucine (I), and "X" as the 33rd amino acid residue is lysine (K) or cysteine ​​(C). In some embodiments, in polypeptide P2, "X" as the second amino acid residue is valine (V). In some embodiments, in polypeptide P2, "X" as the second amino acid residue is leucine (L). In some embodiments, in polypeptide P2, "X" as the 33rd amino acid residue is cysteine ​​(C). In some embodiments, in polypeptide P2, "X" as the 33rd amino acid residue is lysine (K).

[0018] It is well known in the art that the amino acid at positions 2 (X), 9 (I), 16 (A), 23 (N), and 30 (L) in (b1) correspond to the amino acid at position a of the CHR helix, and the amino acid at positions 5 (L), 12 (L), 19 (Q), and 26 (E) in (b1) correspond to position d of the CHR helix. These amino acids at positions a and d are key amino acids for the formation of the 6-HB structure upon binding of the CHR to the NHR target sequence, and are not easily substituted with other amino acids due to their conserved sequence and function. In contrast, the amino acids at other positions in the CHR helix (positions b, c, e, f, and g) do not or barely interact directly with the NHR and are therefore easily substituted with other amino acids with no or only a slight effect on the antiviral activity of the polypeptide.

[0019] Therefore, the substitutions and / or deletions and / or additions are located at positions other than positions 2 (X), 9 (I), 16 (A), 23 (N), 30 (L), 5 (L), 12 (L), 19 (Q), and 26 (E) in (b1).

[0020] Lipophilic compounds are lipid compounds such as cholesterol, cholesterol derivatives (e.g., cholesteryl hemisuccinate, 2-cholesteryl acetate, 2-cholesteryl propionate, 3-cholesteryl propionate, 2-cholesteryl butyrate, 2-cholesteryl isobutyrate, 3-cholesteryl butyrate, 3-cholesteryl isobutyrate, 4-cholesteryl butyrate, 2-cholesteryl valerate, 2-cholesteryl isovalerate, 3-cholesteryl valerate, 5-cholesteryl valerate, 2-cholesteryl caproate, 6-cholesteryl caproate, 2-cholesteryl enanthate, 7-cholesteryl enanthate, 2-cholesteryl caprylate, 8-cholesteryl caprylate, and cholesteryl bromoacetate), fatty acids containing 8 to 20 carbon atoms (e.g., octadecanoic acid or palmitic acid), dihydrosphingosine (DHS), or vitamin E.

[0021] Specifically, the lipophilic compound is cholesterol. In some embodiments, the lipophilic compound is cholesteryl hemisuccinate. In some embodiments, the lipophilic compound is cholesteryl bromoacetate. In some embodiments, the lipophilic compound is octadecanoic acid (stearic acid). In some embodiments, the lipophilic compound is palmitic acid. In some embodiments, the lipophilic compound is dihydrosphingosine. In some embodiments, the lipophilic compound is vitamin E.

[0022] When the terminal amino acid residue of a polypeptide is lysine, cholesterol modification of the terminal amino acid residue is specifically achieved by amidating the side chain amino group of the C-terminal lysine of the peptide chain.

[0023] When the terminal amino acid residue of a polypeptide is cysteine, cholesterol modification of the terminal amino acid residue is specifically achieved by carrying out a thioether-forming reaction between the side chain sulfhydryl group of the C-terminal cysteine ​​of the peptide chain and cholesteryl bromoacetate.

[0024] When the terminal amino acid residue of a polypeptide is lysine, the modification of the terminal amino acid residue with stearic acid is specifically achieved by amidating the side chain amino group of the C-terminal lysine of the peptide chain.

[0025] In some embodiments, the cholesterol is cholesteryl hemisuccinate, which undergoes an amidation reaction with the side chain amino group of lysine (K) in the linker to modify the polypeptide. It is well known in the art that cholesteryl bromoacetate, a polypeptide modification site, can also be used to modify the polypeptide by undergoing a thioether formation reaction with the side chain sulfhydryl group of cysteine ​​(C) in the linker.

[0026] X1 is any one selected from the group consisting of an acetyl (Ac) group, an amino (NH2) group, a maleoyl group, a succinyl group, a tert-butoxycarbonyl group, a benzyloxy group, another hydrophobic group, and a polymeric carrier group. In some embodiments, X1 is acetyl (Ac).

[0027] X2 is any one selected from the group consisting of an amino (NH2) group, a carboxyl group, a hydroxyl group, an amide group, a tert-butoxycarbonyl group, another hydrophobic group, and a polymeric carrier group. In some embodiments, X2 is amino (NH2).

[0028] X3 is any one selected from the group consisting of an acetyl (Ac) group, an amino (NH2) group, a maleoyl group, a succinyl group, a tert-butoxycarbonyl group, a benzyloxy group, another hydrophobic group, and a polymeric carrier group. In some embodiments, X3 is acetyl (Ac).

[0029] X4 is any one selected from the group consisting of an amino (NH2) group, a carboxyl group, a hydroxyl group, an amide group, a tert-butoxycarbonyl group, another hydrophobic group, and a polymeric carrier group. In some embodiments, X4 is amino (NH2).

[0030] The abbreviations for amino acids in the polypeptide (polypeptide P1 or polypeptide P2) have the meanings well known in the art. In addition to the amino acid abbreviations described above, the polypeptides of the present application further include A (alanine), Q (glutamine), N (asparagine), etc. The amino acids are L-amino acids, and one or more (e.g., 2 to 5, 2 to 4, or 2 to 3) amino acid residues in the polypeptide may be substituted with amino acids having chemically similar properties, such as L-amino acids, D-amino acids, artificially modified amino acids, or rare naturally occurring amino acids, to improve the bioavailability, stability, and / or antiviral activity of the polypeptide, where D-amino acids refer to amino acids corresponding to L-amino acids constituting proteins; artificially modified amino acids refer to common L-amino acids constituting proteins that have been modified by methods such as methylation and phosphorylation; and rare naturally occurring amino acids include rare proteinogenic amino acids and non-proteinogenic amino acids, such as 5-hydroxylysine, methylhistidine, γ-aminobutyric acid, and homoserine.

[0031] An amino acid in a polypeptide (polypeptide P1 or polypeptide P2) may be substituted, added, or deleted with one or more other amino acids, and the peptide still has the activity of potently inhibiting HIV. An amino acid substitution refers to replacing an amino acid residue at a certain position in the polypeptide sequence with another amino acid, preferably a conservative amino acid; an amino acid addition refers to inserting an additional amino acid residue at the N-terminus or C-terminus or other suitable position of the polypeptide sequence, and the inserted multiple amino acid residues may be fully or partially adjacent to each other, or may not be adjacent to each other; an amino acid deletion refers to removing one or more amino acid residues in the polypeptide sequence, provided that the modified polypeptide has the activity of inhibiting HIV.

[0032] The so-called "conservative amino acids" or "amino acid conservation" has a meaning well known in the art. For example, amino acids are classified into acidic amino acids, basic amino acids, and neutral amino acids according to the number of amino and carboxyl groups contained in the amino acid molecule, where acidic amino acids refer to E and D (aspartic acid), basic amino acids refer to K, R (arginine) and H (histidine), and neutral amino acids refer to A, L, I, V, C, Y (tyrosine), G (glycine), M (methionine), S (serine), T (threonine), F (phenylalanine), W (tryptophan) and P (proline). As another example, amino acids are classified into hydrophilic amino acids (D, E, H, K, Q, R, S, T) and hydrophobic amino acids (A, F, I, L, M, P, V, W, Y) according to their hydrophilicity and hydrophobicity. As 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; and aromatic amino acids refer to Y, F, and W. As another example, amino acids containing alcohol groups include S and T; aliphatic amino acids include L, I, V, and M; and cycloalkenyl-related amino acids include F, H, W, and Y. Those amino acids that have similar chemical properties, including size, shape, charge, and ability to form covalent or hydrogen bonds, are generally considered to be conservative amino acids.

[0033] Specifically, the polypeptide P1 is (c1) or (c2) or (c3) or (c4) or (c5) or (c6): (c1) a polypeptide having the sequence shown in SEQ ID NO: 1 in the sequence listing; (c2) a polypeptide having the sequence shown in SEQ ID NO: 5 in the sequence listing; (c3) a polypeptide having the sequence shown in SEQ ID NO: 6 in the sequence listing; (c4) a polypeptide having the sequence shown in SEQ ID NO: 9 in the sequence listing; (c5) a polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on any one of the polypeptides (c1) to (c4); (c6) A polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the polypeptides (c1) to (c4), and having antiviral activity. is.

[0034] Specifically, the polypeptide P1 is (c1) or (c2) or (c3) or (c4) or (c5): (c1) a polypeptide having the sequence shown in SEQ ID NO: 1 in the sequence listing; (c2) a polypeptide having the sequence shown in SEQ ID NO: 5 in the sequence listing; (c3) a polypeptide having the sequence shown in SEQ ID NO: 6 in the sequence listing; (c4) A polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on any one of the polypeptides (c1) to (c3); (c5) A polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the polypeptides (c1) to (c3), and having antiviral activity. is.

[0035] Specifically, the polypeptide P1 has the sequence shown in SEQ ID NO: 1, 5 or 6.

[0036] Specifically, the polypeptide P2 is (d1) or (d2) or (d3) or (d4): (d1) a polypeptide having the sequence shown in SEQ ID NO: 7 in the sequence listing; (d2) a polypeptide having the sequence shown in SEQ ID NO: 8 in the sequence listing; (d3) A polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on any one of the polypeptides (d1) to (d2); (d4) A polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the polypeptides (d1) to (d2), and having antiviral activity. is.

[0037] Specifically, the polypeptide P2 has the sequence shown in SEQ ID NO:7 or 8.

[0038] With over 10 years of research experience, the inventors' team has shown that polypeptides obtained by substitution, addition or deletion of amino acids in the parent polypeptide (polypeptide P1 or polypeptide P2) and having up to about 60%, 70%, 80%, 90% or 95% sequence identity can still have potent antiviral activity as inhibitors.

[0039] Therefore, the present application also provides (a1) or (a2) or (a3): (a1) a polypeptide having the amino acid sequence "EXEELEKKIEELLKKAEEQQKKNEEELKKLEK", where X is isoleucine, valine, or leucine; (a2) a polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on (a1); (a3) A polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to (a1), and having antiviral activity. or providing a polypeptide which is The polypeptide is (b1) or (b2) or (b3): (b1) a polypeptide having the amino acid sequence "EXEELEKKIEELLKKAEEQQKKNEEELKKLEKX", wherein "X" as the second amino acid residue is isoleucine, valine, or leucine, and "X" as the 33rd amino acid residue is lysine or cysteine; (b2) A polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on (b1); (b3) A polypeptide having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to (b1), and having antiviral activity. is.

[0040] In some embodiments, the polypeptide P1 is (c1) or (c2) or (c3) or (c4) or (c5) or (c6): (c1) a polypeptide having the sequence shown in SEQ ID NO: 1 in the sequence listing; (c2) a polypeptide having the sequence shown in SEQ ID NO: 5 in the sequence listing; (c3) a polypeptide having the sequence shown in SEQ ID NO: 6 in the sequence listing; (c4) a polypeptide having the sequence shown in SEQ ID NO: 9 in the sequence listing; (c5) a polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on any one of the polypeptides (c1) to (c4); (c6) A polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the polypeptides (c1) to (c4), and having antiviral activity. is.

[0041] In some embodiments, the polypeptide is (c1) or (c2) or (c3) or (c4) or (c5): (c1) a polypeptide having the sequence shown in SEQ ID NO: 1 in the sequence listing; (c2) a polypeptide having the sequence shown in SEQ ID NO: 5 in the sequence listing; (c3) a polypeptide having the sequence shown in SEQ ID NO: 6 in the sequence listing; (c4) A polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on any one of the polypeptides (c1) to (c3); (c5) a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the polypeptides (c1) to (c3), and having antiviral activity; or The polypeptide is (d1) or (d2) or (d3) or (d4): (d1) a polypeptide having the sequence shown in SEQ ID NO: 7 in the sequence listing; (d2) a polypeptide having the sequence shown in SEQ ID NO: 8 in the sequence listing; (d3) A polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on any one of the polypeptides (d1) to (d2); (d4) A polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the polypeptides (d1) to (d2), and having antiviral activity. is.

[0042] In some embodiments, the substitutions of amino acid residues described herein are conservative substitutions.

[0043] In this application, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the essential properties of a protein / polypeptide containing a certain 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 with 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., has similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been 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 uncharged polar 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, conservative substitution generally refers to the replacement of corresponding amino acid residues with other amino acid residues from the same side chain family.Methods for identifying conservative amino acid substitutions are well known in the art.

[0044] The lipopeptides described herein may contain one or more chiral centers and / or structures such as double bonds and therefore may exist as stereoisomers, including double bond isomers (e.g., geometric isomers), enantiomers (optical isomers), or diastereomers. Therefore, any chemical structure within the scope of the present description, whether partially or entirely comprised of the analogous structures, encompasses all possible enantiomers and diastereomers of the lipopeptide, including any one pure stereoisomer (e.g., pure geometric isomer, pure enantiomer, or pure diastereomer) and any one mixture of these stereoisomers. Those skilled in the art can also further resolve these racemic and stereoisomeric mixtures into their component enantiomers or stereoisomers by utilizing separation techniques or asymmetric synthesis methods. Lipopeptides include, but are not limited to, various optical isomers, racemic and / or other mixtures. In the above cases, single enantiomers or diastereomers, e.g., optically active isomers, can be obtained by asymmetric synthesis or racemic resolution. Resolution of the racemic mixture can be achieved by various methods, such as conventional recrystallization or chromatography using a resolving agent. Furthermore, lipopeptides also include cis and / or trans isomers having double bonds.

[0045] The lipopeptides of the present application include, but are not limited to, all of the various pharmaceutically acceptable forms of the lipopeptides, including various pharmaceutically acceptable salts, solvates, hydrates, complexes, chelates, non-covalent complexes, prodrugs, and any mixtures of the above forms based on the above substances.

[0046] Pharmaceutically acceptable salts of the present application include acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, methanesulfonate, borate, methyl bromide, bromide, methyl nitrate, calcium edetate, methyl sulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate, Acid salts include hydroxybenzoate, embonate, estolate, palmitate, ethanesulfonate, pantothenate, fumarate, phosphate / diphosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycolarsanilate, sulfate, hydroxybenzoate, diacetate, hydrabamine, succinate, hydrobromide, tannate, hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, triethiodide, lactate, and valerate. Depending on the application, pharmaceutically acceptable salts may be formed from cations such as sodium, potassium, and bismuth, or from bases such as ammonia, ethylenediamine, N-methyl-glutamine, 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, such as by reacting the free acid with an organic or inorganic base. In the presence of a basic group (e.g., an amino group), acid salts such as hydrochloride, hydrobromide, acetate, and pamoate can be used as pharmaceutical forms; in the presence of an acidic or alcoholic group, pharmaceutically acceptable esters such as acetate, maleate, and pivaloyloxymethyl, as well as esters known in the literature to improve solubility and hydrolysis, can be used as sustained-release pharmaceuticals or prodrug forms.

[0047] The present application also provides a conjugate comprising the polypeptide of the present application and a modification moiety. In some embodiments, the modification moiety is attached to the N-terminus or C-terminus of the polypeptide, optionally via a linker. In some embodiments, the modification moiety is a terminal protecting group. The terminal protecting group of the polypeptide includes an N-terminus protecting group and / or a C-terminus protecting group. As is well known, the N-terminus protecting group can be any one selected from the group consisting of an acetyl (Ac) group, an amino (NH2) group, a maleoyl group, a succinyl group, a tert-butoxycarbonyl group, a benzyloxy group, another hydrophobic group, and a polymeric carrier group. As is well known, the C-terminus protecting group can be any one selected from the group consisting of an amino (NH2) group, a carboxyl group, a hydroxyl group, an amide group, a tert-butoxycarbonyl group, another hydrophobic group, and a polymeric carrier group.

[0048] The present application also provides isolated nucleic acids encoding the polypeptides of the present application.

[0049] The present application also provides a vector comprising the isolated nucleic acid of the present application. Vectors useful for inserting target polynucleotides 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, phage, etc.

[0050] The present application also provides a host cell comprising the isolated nucleic acid and / or vector of the present application. Such host cells include, but are not limited to, prokaryotic cells such as 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 cell of the present application can also be a cell line.

[0051] The present application also provides (e1) or (e2) or (e3) or (e4) or (e5) or (e6): (e1) a multimer formed from any one of the lipopeptides; (e2) a multimer formed from any one of the pharmaceutically acceptable salts; (e3) a multimer formed from any one of the derivatives; (e4) a multimer formed from any one of the hydrates; (e5) a multimer formed from any one of the solvates; (e6) A multimer formed from any one of the polypeptides The present invention provides a multimer,

[0052] In some embodiments, the multimer comprises (e1) or (e2) or (e3): (e1) a multimer formed from any one of the lipopeptides; (e2) a multimer formed from any one of the pharmaceutically acceptable salts; (e3) A multimer formed from any one of the derivatives is.

[0053] The present application also provides a composition comprising a lipopeptide, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof, or a derivative thereof, or a polypeptide, or a conjugate, or an isolated nucleic acid, or a vector, or a host cell, or a multimer of the present application.

[0054] The present application also provides pharmaceutical compositions comprising a lipopeptide, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof, or a derivative thereof, or a polypeptide of the present application, optionally further comprising a pharmaceutically acceptable carrier. In some embodiments, the lipopeptide, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof, or a derivative thereof, or a polypeptide is present in an amount effective 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, SIV, and resistant strains thereof. In some embodiments, the HIV is HIV-1 and / or HIV-2. In some embodiments, the HIV is a T20-resistant virus. In some embodiments, the HIV is an LP-98-resistant virus. In some embodiments, the HIV is an LP-40-resistant virus. In some embodiments, the HIV is an LP-52-resistant virus. In some embodiments, the HIV is an SC29EK-resistant virus. In some embodiments, the HIV is an SC22EK-resistant virus. In some embodiments, the HIV is an MTSC22-resistant virus. In some embodiments, the HIV is an SFT-resistant virus. In some embodiments, the viral infection is an infection caused by a virus selected from the group consisting of a T20-resistant virus, an LP-40-resistant virus, an LP-52-resistant virus, an SC29EK-resistant virus, an SC22EK-resistant virus, an MTSC22-resistant virus, and an SFT-resistant virus. In some embodiments, the disease caused by a viral infection is AIDS.

[0055] The present application also relates to the use of any one of the above lipopeptides or any one of the above pharmaceutically acceptable salts or any one of the above derivatives or multimers, comprising (f1) or (f2) or (f3) or (f4): (f1) Use in the manufacture of a viral membrane fusion inhibitor; (f2) Use in the manufacture of a medicament for the prevention and / or treatment of a disease caused by a viral infection; (f3) Use as a viral membrane fusion inhibitor; (f4) Use in the prevention and / or treatment of diseases caused by viral infections The present invention provides the use, wherein

[0056] The present application also relates to the use of any one of the above polypeptides, (f1) or (f2) or (f3) or (f4): (f1) Use in the manufacture of a viral membrane fusion inhibitor; (f2) Use in the manufacture of a medicament for the prevention and / or treatment of a disease caused by a viral infection; (f3) Use as a viral membrane fusion inhibitor; (f4) Use in the prevention and / or treatment of diseases caused by viral infections The present invention provides the use, wherein

[0057] The present application also provides a product comprising any one of the above lipopeptides or any one of the above pharmaceutically acceptable salts or any one of the above derivatives or multimers, wherein the product has the function (g1) or (g2): (g1) Function as a viral membrane fusion inhibitor; (g2) The function of preventing and / or treating diseases caused by viral infections The product has the following structure:

[0058] The present application also provides a product comprising any one of the above polypeptides, having the function (g1) or (g2): (g1) Function as a viral membrane fusion inhibitor; (g2) The function of preventing and / or treating diseases caused by viral infections The product has the following structure:

[0059] The product may also include a carrier.

[0060] The present application also provides (g1) or (g2): (g1) Use as a viral membrane fusion inhibitor; (g2) Provided is a use of the product, which is a use for preventing and / or treating a disease caused by a viral infection.

[0061] The present application also provides products as viral membrane fusion inhibitors; or products for preventing and / or treating diseases caused by viral infections.

[0062] The present application also provides a method for treating or / and preventing a viral infection in an animal, comprising administering to a subject animal any one of the above lipopeptides, or any one of the above pharmaceutically acceptable salts, or any one of the above derivatives or multimers, thereby inhibiting the viral infection in the animal.

[0063] The present application also provides a method for treating and / or preventing a disease caused by a viral infection, comprising administering an effective amount of any one of the above-mentioned lipopeptides or a pharmaceutically acceptable salt thereof or a solvate thereof or a hydrate thereof or a derivative thereof or a polypeptide or a multimer or a pharmaceutical composition to a subject in need thereof.

[0064] Any one of the above viruses is HIV.

[0065] The virus is human immunodeficiency virus (HIV) and / or simian immunodeficiency virus (SIV).

[0066] In some embodiments, the virus is selected from the group consisting of HIV, SIV, and drug-resistant strains thereof.

[0067] Specifically, the HIV is HIV-1 and / or HIV-2.

[0068] Specifically, HIV is a T20 resistant virus.

[0069] Specifically, HIV is an LP-98 resistant virus.

[0070] Specifically, the virus is a T20-resistant virus and / or an LP-40-resistant virus and / or an LP-52-resistant virus and / or an SC29EK-resistant virus and / or an SC22EK-resistant virus and / or an MTSC22-resistant virus and / or an SFT-resistant virus.

[0071] In particular, any one of the above products may be a medicine or a vaccine.

[0072] In some embodiments, the HIV is a T20-resistant virus. In some embodiments, the HIV is an LP-40-resistant virus. In some embodiments, the HIV is an LP-52-resistant virus. In some embodiments, the HIV is an SC29EK-resistant virus. In some embodiments, the HIV is an SC22EK-resistant virus. In some embodiments, the HIV is an MTSC22-resistant virus. In some embodiments, the HIV is an SFT-resistant virus.

[0073] In some embodiments, the animal is a mammal, such as a human.

[0074] In some embodiments, the viral infection is an infection caused by a virus selected from the group consisting of HIV, SIV, and resistant strains thereof.

[0075] In some embodiments, the viral infection is an infection caused by a virus selected from the group consisting of a T20-resistant virus, an LP-40-resistant virus, an LP-52-resistant virus, an SC29EK-resistant virus, an SC22EK-resistant virus, an MTSC22-resistant virus, and an SFT-resistant virus.

[0076] In some embodiments, the disease caused by a viral infection is AIDS.

[0077] In some embodiments, the subject is a mammal, such as a human.

[0078] In practice, for the purpose of treating and / or preventing HIV infection, the lipopeptides of the present application can be administered to a patient directly as a pharmaceutical or in admixture with suitable carriers or additives.

[0079] The carrier material is preferably a pharmaceutically acceptable carrier material, including, but not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethylethyl cellulose, etc.), with water-soluble carrier materials being preferred. These materials can be used to prepare various dosage forms, including, but not limited to, tablets, capsules, drop pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. The dosage forms can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. A wide variety of carriers known in the art can be used to formulate unit dosage forms 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 moisturizing 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. Disintegrating agents and binders such as dry starch, alginate, agar powder, brown seaweed 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 tristearate, cocoa butter, and hardened oil; absorption promoters such as quaternary ammonium salts and sodium lauryl sulfate; lubricants such as talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be formulated into coated tablets such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layered or multi-layered tablets.A variety of carriers known in the art can be used to prepare unit dosage forms into pills.Examples of carriers include, for example, glucose, lactose, starch, cocoa butter, hardened vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin and talc, and other diluents and absorbents; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste and flour paste; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose and ethylcellulose.A variety of carriers known in the art can be used to prepare unit dosage forms into suppositories.Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohol, higher alcohol ester, gelatin and semi-synthetic glycerides. Any conventional diluent can be used to formulate the unit dosage form into an injectable preparation, such as a solution, emulsion, lyophilized powder, or suspension, for example, water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid ester. Furthermore, to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, or glycerin can be added to the injectable preparation, and conventional cosolvents, buffers, and pH adjusters can also be added. If necessary, colorants, preservatives, flavors, flavorings, sweeteners, and other materials can also be optionally added to the pharmaceutical preparation. The dosage form can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection; by intraluminal administration, such as rectal and vaginal administration; by respiratory administration, such as nasal administration; and by mucosal administration. Among the above administration routes, administration by injection is preferred.

[0080] The dosage of the lipopeptides or polypeptides of the present application will depend on various factors, such as, for example, the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the particular component used, the route of administration, and the frequency of administration, etc. The dose can be administered in a single unit dosage form or in multiple (e.g., 2, 3, or 4) unit dosage forms.

[0081] The lipopeptides or polypeptides of the present application may be used directly alone to treat or prevent HIV-infected patients, or may be used in combination with one or more antiviral drugs, including but not limited to reverse transcriptase inhibitors, protease inhibitors, entry inhibitors, integration inhibitors, and maturation inhibitors, to achieve the goal of improving overall therapeutic efficacy. The reverse transcriptase inhibitor may be one or more inhibitors selected from the group consisting of AZT, 3TC, ddI, d4T, ddT, TDF, abacavir, nevirapine, efavirenz, delavirdine, azuvudine, and ainuovirine; the protease inhibitor may be one or more inhibitors selected from the group consisting of saquinavir mesylate, indinavir, ritonavir, amprenavir, and nelfinavir mesylate; the entry inhibitor may be one or more inhibitors selected from the group consisting of maraviroc, TAK-779, T20, T2635, sifvirtide, and albuvirtide; and the integration inhibitor may be one or more inhibitors selected from the group consisting of raltegravir, dolutegravir, and elvitegravir.

[0082] The specific therapeutically effective dose level for an individual patient will depend on a variety of factors, including the disorder being treated and its severity; the activity of the specific active ingredient used; the specific composition used; the patient's age, weight, general health, sex, and dietary habits; the administration time, route of administration, and excretion rate of the specific active ingredient used; the duration of treatment; drugs used in combination or simultaneously with the specific active ingredient used; and similar factors well known in the pharmaceutical arts. For example, it is common practice in the art to start with a dose of the active ingredient lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Generally, the lipopeptides or polypeptides of the present application can be administered to mammals, particularly humans, at doses of 0.001 mg / kg body weight / day to 1000 mg / kg body weight / day, e.g., 0.01 mg / kg body weight / day to 100 mg / kg body weight / day, further e.g., 0.1 mg / kg body weight / day to 10 mg / kg body weight / day.

[0083] The lipopeptide of the present application has potent inhibitory activity against HIV-1, HIV-2, and SIV, and its inhibitory activity against T20-resistant and LP-98-resistant strains is particularly improved. Therefore, the lipopeptide of the present application is a broad-spectrum inhibitor and can be used to treat and prevent diseases caused by HIV-1, HIV-2, and SIV infection. The present application has great application value in the prevention and treatment of AIDS.

[0084] Previous studies have suggested that the hydrophobic pocket of the NHR helix of the HIV fusion protein gp41 is an important drug target, and that the eight-amino acid PBD sequence (W1MEW2DREI) at the N-terminus of the CHR helix plays an important role in the design of polypeptide HIV membrane fusion inhibitors. In particular, the interaction of two highly conserved tryptophans (designated W1 and W2) and one isoleucine (I) with the hydrophobic pocket is believed to be key for inhibitors to exert their antiviral activity by binding to the NHR target. Given the importance of the PBD sequence, this application focuses on the design and identification of potent HIV membrane fusion inhibitors containing PBD amino acids. Through systematic studies, we surprisingly discovered that lipopeptides containing only the two terminal amino acids, EI, of the PBD sequence significantly enhanced their antiviral activity, while the addition of the corresponding amino acid motifs, including W2 or W1, gradually reduced the antiviral activity of the lipopeptides. This important discovery provides innovative ideas and design strategies for the research and development of novel HIV membrane fusion inhibitor drugs. [Brief explanation of the drawings]

[0085] [Figure 1] FIG. 1 shows a sequence comparison of T20, C34, SFT, ABT and LP-98 in the background art. [Figure 2] FIG. 2 shows the results of Example 2 (identification of the sequence structure of lipopeptides and their activity against HIV-1). [Figure 3] Figure 3 shows the results of Example 3 (a graph showing the inhibitory effects of representative lipopeptides on various resistant HIV-1 strains, where the bold numbers in parentheses indicate the fold increase in activity of the lipopeptide compared to LP-98). [Figure 4] FIG. 4 shows the results of Example 4 (inhibitory effects of representative lipopeptides on HIV-2 and SIV). [Figure 5]FIG. 5 shows the results of Example 5 (helical structure characteristics of representative lipopeptides and their interactions with target sequences; A: α-helical content of lipopeptide inhibitors alone; B: helical stability of lipopeptide inhibitors alone; C: α-helical content of lipopeptide inhibitor complexes with N42 polypeptide; D: helical stability of lipopeptide inhibitor complexes with N42 polypeptide). DETAILED DESCRIPTION OF THE INVENTION

[0086] Sequence information Sequence information relevant to this application is shown in Table 1 below. [Table 1]

[0087] Detailed Description of the Preferred Embodiments The present application will be described in detail below with reference to specific embodiments, but the examples set forth herein are intended to illustrate the present application and not to limit the scope of the present application. The examples set forth below serve as guidelines for those skilled in the art to make modifications and are not intended to limit the scope of the present application in any way. Those skilled in the art can appropriately modify relevant parameters with reference to the present disclosure. Specifically, it should be noted that any similar substitutions or modifications are obvious to those skilled in the art and are all considered to be within the scope of the present application. The methods of the present application are described by preferred examples, but it is clear that those skilled in the art can achieve and apply the technology of the present application by making modifications or appropriate changes or combinations to the compounds and production methods described herein without departing from the content, spirit, and scope of the present application.

[0088] Unless otherwise specified, the experimental methods in the following examples are conventional methods according to the techniques or conditions described in the literature in the field or according to the product instructions. Materials, reagents, etc. used in the following examples are commercially available unless otherwise specified. Unless otherwise specified, quantitative tests in the following examples are performed in triplicate and the results are averaged. [Example]

[0089] Example 1. Preparation of Lipopeptides The following four lipopeptides were prepared: lipopeptide LP-101, which contains the two terminal amino acids E and I of the PBD (including one amino acid inserted into the NHR pocket); lipopeptide LP-102, which contains five amino acids W2EQKI (including two amino acids inserted into the NHR pocket); lipopeptide LP-103, which contains the five amino acids W2DREI (including two amino acids inserted into the NHR pocket); Lipopeptide LP-104 contains all eight PBD amino acids W1EEW2EKKI (including the three amino acids inserted into the NHR pocket). W1 refers to the first W from the N-terminus of the PBD, and W2 refers to the second W from the N-terminus of the PBD.

[0090] The antiviral activity of the above four lipopeptides was identified and further modified to obtain five other lipopeptides.

[0091] The following five lipopeptides were prepared: lipopeptide LP-105 (compared to lipopeptide LP-101, amino acid I inserted into the NHR pocket was replaced with amino acid V); lipopeptide LP-106 (compared to lipopeptide LP-101, amino acid I inserted into the NHR pocket was replaced with amino acid L); lipopeptide LP-107 (compared to lipopeptide LP-101, an amino acid K was added to the C-terminus of the peptide chain); lipopeptide LP-108 (compared to lipopeptide LP-101, an amino acid C was added to the C-terminus of the peptide chain); Lipopeptide LP-109 (compared to lipopeptide LP-101, the modifying group at the C-terminus of the peptide chain was replaced with stearic acid).

[0092] Lipopeptide LP-98 was prepared.

[0093] The nine lipopeptides prepared above, LP-98 to LP-108, each contained a cholesterol modification, and LP-109 contained a stearic acid modification. The cholesterol modifications of lipopeptides LP-98, LP-101, LP-102, LP-103, LP-104, LP-105, LP-106, and LP-107 were achieved by amidation of the side chain amino group of the C-terminal lysine of each peptide chain. The cholesterol modification of lipopeptide LP-108 was achieved by highly chemically selective thioether formation between the side chain sulfhydryl group of the C-terminal cysteine ​​of the peptide chain and cholesteryl bromoacetate. The amino termini of all ten lipopeptides were bound to acetyl (Ac) as the amino-terminal protecting group, and the carboxyl termini were bound to amino (NH) as the carboxy-terminal protecting group. The stearic acid modification of lipopeptide LP-109 was achieved by amidation of the side chain amino group of the C-terminal lysine of the peptide chain.

[0094] The structures of the 10 lipopeptides are shown below: LP-98: Ac-YEQKIEELLKKAEEQQKKNEEELKKLEK(chol)-NH2; LP-101: Ac-EIEELEKKIEELLKKAEEQQKKNEEELKKLEK(chol)-NH2; LP-102: Ac-WEQKIEELEKKIEELLKKAEEQQKKNEEELKKLEK(chol)-NH2; LP-103: Ac-WDREIEELEKKIEELLKKAEEQQKKNEEELKKLEK(chol)-NH2; LP-104: Ac-WEEWEKKIEELEKKIEELLKKAEEQQKKNEEELKKLEK(chol)-NH2; LP-105: Ac-EVEELEKKIEELLKKAEEQQKKNEEELKKLEK(chol)-NH2; LP-106: Ac-ELEELEKKIEELLKKAEEQQKKNEEELKKLEK(chol)-NH2; LP-107: Ac-EIEELEKKIEELLKKAEEQQKKNEEELKKLEKK(chol)-NH2; LP-108: Ac-EIEELEKKIEELLKKAEEQQKKNEEELKKLEKC(chol)-NH2; LP-109: Ac-EIEELEKKIEELLKKAEEQQKKNEEELKKLEKC(C18)-NH2. (chol) indicates that the C-terminal amino acid is modified with cholesterol, for example, cholesteryl hemisuccinate or cholesteryl bromoacetate. (C18) indicates that the C-terminal amino group is modified with stearic acid.

[0095] The amino acid sequences of the polypeptide peptide chains of the 10 lipopeptides are shown below (N-terminus to C-terminus): LP-98 (SEQ ID NO: 10): YEQKIEELLKKAEEQQKKNEEELKKLEK; LP-101 (SEQ ID NO: 1): EIEELEKKIEELLKKAEEQQKKNEEELKKLEK; LP-102 (SEQ ID NO: 2): WEQKIEELEKKIEELLKKAEEQQKKNEEELKKLEK; LP-103 (SEQ ID NO: 3): WDREIEELEKKIEELLKKAEEQQKKNEEELKKLEK; LP-104 (SEQ ID NO: 4): WEEWEKKIEELEKKIEELLKKAEEQQKKNEEELKKLEK; LP-105 (SEQ ID NO: 5): EVEELEKKIEELLKKAEEQQKKNEEELKKLEK; LP-106 (SEQ ID NO: 6): ELEELEKKIEELLKKAEEQQKKNEEELKKLEK; LP-107 (SEQ ID NO: 7): EIEELEKKIEELLKKAEEQQKKNEEELKKLEKK; LP-108 (SEQ ID NO: 8): EIEELEKKIEELLKKAEEQQKKNEEELKKLEKC; LP-109 (SEQ ID NO: 9): EIEELEKKIEELLKKAEEQQKKNEEELKKLEKC.

[0096] The lipopeptides can be prepared by any conventional method as described in the prior art.

[0097] Illustratively, the following methods can be used to prepare lipopeptides:

[0098] I. Chemical Reagents Required for the Preparation Process All chemical reagents, such as various Fmoc amino acids, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), N,N-dimethylformamide (DMF), hexahydropyridine (PIPE), ninhydrin, acetic anhydride (AcO), 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 used without further purification. Amino acid protected starting materials used in the synthesis of polypeptides include Fmoc-Lys(Dde)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Tyr(tBu)-OH. The abbreviations have their 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.

[0099] II. Synthesis of peptide resin Rink Amide MBHA resin was used as a carrier resin, which was sequentially coupled to the corresponding protected amino acids in the amino acid sequence of the polypeptide by Fmoc deprotection and coupling reactions to prepare the peptide resin.

[0100] 1. Coupling of the first protected amino acid to the backbone 0.3 mmol of the first protected amino acid Fmoc-Lys(Dde)-OH or Fmoc-Cys(Trt)-OH and 0.3 mmol of HOBt were dissolved in an appropriate amount of DMF; 0.3 mmol of DIC was then slowly added to the DMF solution of the protected amino acid under shaking, and the resulting mixture was reacted at room temperature for 5 minutes under shaking to obtain an activated protected amino acid solution, which was set aside for later use.

[0101] 0.1 mmol of Rink Amide MBHA resin (0.35 mmol / g × 0.3 g) was taken and deprotected (twice) with 25% PIPE / DMF solution (volume ratio) for 20 min, followed by washing and filtration to obtain the Fmoc-removed resin.

[0102] The activated first protected amino acid solution was added to the Fmoc-removed resin, and the coupling reaction was carried out for 60 min. After filtration and washing, the resin containing the first protected amino acid Fmoc-Lys(Dde) or Fmoc-Cys(Trt)-OH was obtained.

[0103] 2. Coupling of other protected amino acids to the backbone Using the same method as above to couple the first protected amino acid to the backbone, the corresponding other protected amino acids were sequentially coupled to the polypeptide to obtain a resin containing the backbone amino acids. Finally, the N-terminus was capped by acetylation with 0.3 mmol of AcO + 0.6 mmol of DIEA to complete the synthesis of the backbone. After each reaction in the above steps, the reaction was controlled by Kaiser test. If the condensation reaction of a certain amino acid was incomplete, the condensation was repeated once until the desired target peptide segment was obtained.

[0104] 3. Side chain coupling (1) The resin was treated with a 2% hydrazine hydrate / DMF solution (volume ratio) in as small a volume as possible to remove the side chain Dde protecting group of the C-terminal lysine (10 min, twice), and after filtration and washing, the Dde-removed resin was obtained and set aside for later use.

[0105] (2) Cholesterol modification of polypeptides The method for coupling cholesterol to lysine side chains is described in our published work (Xue et al., 2022). 0.3 mmol of cholesteryl hemisuccinate and 0.3 mmol of HOBt were dissolved in an appropriate amount of DMF; 0.3 mmol of DIC was then slowly added to the DMF solution of cholesteryl hemisuccinate and HOBt. The resulting mixture was allowed to react at room temperature for 5 minutes under shaking. The resulting solution containing cholesteryl hemisuccinate, HOBt, and DIC was added to the resulting Dde-removed resin, and the coupling reaction was carried out for 60 minutes. After filtration, washing, and drying, the peptide resin was obtained.

[0106] The method for coupling cholesterol to cysteine ​​side chains was described in a paper published by Dwyer et al. (2007). We routinely used this method in our laboratory to prepare cholesterol-modified lipopeptides, such as C34-Chol, LP-83, LP-86, and LP-97 (Xue et al., 2022; Zhu et al., 2019). First, we synthesized cholesteryl bromoacetate according to the technical route described in the paper. Then, it was grafted onto the polypeptide chain by means of a highly chemically selective thioether formation reaction between the side chain thiol group of the C-terminal cysteine ​​of the polypeptide and cholesteryl bromoacetate. After synthesizing the crude polypeptide product according to conventional methods, it was dissolved in pure DMSO, and 1 equivalent of cholesterol bromoacetate dissolved in a small amount of trifluoroacetic acid (TFA) was added to it. The pH was then adjusted to alkaline by adding pure diisopropylethylamine (DIEA). The reaction was monitored by RP-HPLC and was usually complete within 1 h.

[0107] (3) Stearoylation of polypeptides 0.3 mmol of stearoyl chloride and 0.6 mmol of DIEA were taken, dissolved in an appropriate amount of DMF, and slowly added to the Dde-removed resin obtained in step (1). The resulting mixture was reacted at room temperature under shaking for 60 minutes, and after filtration, washing and drying, the peptide resin was obtained.

[0108] III. Preparation of Crude Product The peptide resin was added to the cleavage reagent (15 mL / g resin) and mixed thoroughly. The resulting mixture was incubated at 30°C for 3 hours under shaking, thereby cleaving the target polypeptide from the resin and removing the side-chain protecting groups. The filtrate of the reaction mixture was collected. The resin was washed three more times with a small amount of TFA / DCM, and the combined filtrates were precipitated with anhydrous ether and centrifuged. The filter cake was further washed and precipitated twice with cold anhydrous ether, 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: HO: triisopropylsilane = 68.5:10:10:5:3.5:1 (v / v).

[0109] IV. Preparation of Crude Product The crude lipopeptide product was dissolved in water / acetonitrile. The resulting mixture was centrifuged to remove insoluble material and set aside for further use. Reverse-phase high-performance liquid chromatography was used for purification. The chromatography column used was an Agela C18 (10 μm, 100 Å, 50 × 250 mm) with a mobile phase consisting 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 eluted. The corresponding purified components were collected and directly lyophilized to remove the solvent, yielding a fluffy pure product of the polypeptide trifluoroacetate.

[0110] The pure polypeptide trifluoroacetate product was redissolved in water and acetonitrile, a large amount of anion exchange resin (in the form of acetate radicals) was added, the resulting mixture was stirred for 3 hours, filtered, the ion exchange resin was then rinsed with a water / acetonitrile mixed solvent, and the combined filtrate was lyophilized to obtain a fluffy crude polypeptide acetate product.

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

[0112] Example 2. Concept verification of novel lipopeptide structure In this example, the anti-HIV activity of novel lipopeptides was identified using an HIV pseudovirus system, and the superiority of the conceptual novel lipopeptides was verified using lipopeptide LP-98 as a control for the novel lipopeptides.

[0113] Test lipopeptide: lipopeptide LP-101, lipopeptide LP-102, lipopeptide LP-103, lipopeptide LP-104, lipopeptide LP-105, lipopeptide LP-106, lipopeptide LP-107, lipopeptide LP-108, LP-109 or lipopeptide LP-98 prepared in Example 1.

[0114] HIV-1 JRFLThe Env expression vector and backbone plasmid (pSG3 ΔEnv) and TZM-bl cells were described in the following publication: Xue, J., Chong, H., Zhu, Y., Zhang, J., Tong, L., Lu, J., Chen, T., Cong, Z., Wei, Q., He, Y., 2022. Efficient treatment and pre-exposure prophylaxis in rhesus macaques by an HIV fusion-inhibitory lipopeptide. Cell 185, 131-144 e118. HIV-1 JRFL The Env expression vector and the backbone plasmid (pSG3 ΔEnv) were co-transfected into HEK293T cells. The transfected cells were cultured for 48 hours in a cell culture incubator at 37°C and 5% CO2. The supernatant was then collected and filtered to obtain the filtrate. The resulting filtrate was used to express HIV-1. JRFL Virus solution containing pseudovirus particles (HIV-1 JRFL This was called the virus solution) and stored at -80°C for later use.

[0115] HIV-1 NL4-3 Virus solution (i.e., HIV-1 NL4-3 The preparation method of the virus solution containing pseudovirus particles is as follows: JRFL Env expression vectors were transfected into HIV-1 NL4-3 HIV-1 except that Env expression vector was substituted. JRFL The method for preparing the virus solution is basically the same as that for HIV-1. NL4-3 Pseudovirus (known in the literature as "HIV-1 NL4-3" is described in the following literature: Xue, J., Chong, H., Zhu, Y., Zhang, J., Tong, L., Lu, J., Chen, T., Cong, Z., Wei, Q., He, Y., 2022. Efficient treatment and pre-exposure prophylaxis in rhesus macaques by an HIV fusion-inhibitory lipopeptide. Cell 185, 131-144 e118.

[0116] Test virus solution: HIV-1 NL4-3 Virus solution or HIV-1 JRFL Virus solution.

[0117] 1. The test lipopeptides were dissolved in deionized water and then diluted with DMEM medium (using 3-fold dilutions) to obtain lipopeptide dilutions. Nine serial dilutions were set up for each test lipopeptide. 2. In a 96-well plate, lipopeptide diluent (50 μL / well) was added to drug wells, and DMEM medium (50 μL / well) was added to control wells. Three duplicate wells were set up for each well. 3. After step 2 is completed, add the test virus solution (50 μL / well, virus content 100 TCID 50 ) was added to a 96-well plate and incubated at room temperature for 30 minutes. 4. 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. Preparation of TZM-b1 cell suspension: TZM-b1 cells were resuspended in DMEM medium to a cell concentration of 10 × 10 4 DEAE-dextran was added to it so that the cells / mL and the DEAE-dextran concentration was 15 μg / mL. 5. After step 4, the supernatant was discarded and cell lysis solution (30 μL / well) was added. Cell lysis was carried out at room temperature for 15 minutes, and then luciferase assay substrate reagent was added. The relative luminescence units (RLU) were measured using a microplate luminometer, and an inhibition rate curve was generated to determine the half-maximal inhibitory concentration (IC) of the drug. 50 ) was calculated. Cell lysis solution: Promega, Catalog number: E1531. Luciferase assay substrate reagent: Promega, catalog number: E1501.

[0118] The results are shown in Figure 2.

[0119] Lipopeptide LP-101 inhibits HIV-1 in TZM-b1 cells NL4-3 Pseudoviruses and HIV-1 JRFL Pseudovirus infections were measured using the mean IC 50 The control lipopeptide LP-98 was able to potently inhibit HIV-1 at values ​​of 0.46 pM and 2.26 pM. NL4-3 Pseudoviruses and HIV-1 JRFL Pseudoviruses were tested for the mean IC 50 The inhibition was at values ​​of 1.07 pM and 2.58 pM.

[0120] Contrary to expectations, the antiviral activities of lipopeptides LP-102, LP-103, and LP-104 were significantly reduced. In particular, lipopeptide LP-104, which contains the full-length PBD sequence of 8 amino acids, showed a significantly reduced antiviral activity against HIV-1. NL4-3 Pseudoviruses and HIV-1 JRFL Pseudoviruses were each IC 50The lipopeptides inhibited the virion binding protein at 14.50 pM and 60.18 pM, respectively, which were approximately 32- and 27-fold less active than lipopeptide LP-101. The results showed that lipopeptides containing the two terminal amino acids E and I of the PBD had the highest antiviral activity, whereas lipopeptides containing more amino acids of the PBD had significantly reduced activity, revealing a relationship between the structure and function of lipopeptide inhibitors.

[0121] Lipopeptides LP-105 and LP-106 showed inhibitory activity similar to that of lipopeptide LP-101, suggesting that amino acid I inserted into the NHR hydrophobic pocket of the lipopeptides can be replaced with amino acid V or L without affecting the activity of the inhibitors.

[0122] Lipopeptides LP-107 and LP-108 also showed similar inhibitory activity to lipopeptide LP-101, suggesting that differences in cholesterol modification do not significantly affect the antiviral efficacy of the inhibitors.

[0123] Example 3. Inhibitory activity of novel lipopeptides against HIV-resistant strains HIV is prone to acquiring drug resistance, which can lead to treatment failure, making this a critical issue in drug research and development. In this example, we used an HIV pseudovirus system to evaluate the inhibitory activity of novel lipopeptides (e.g., lipopeptides LP-101 and LP-108) against multiple HIV membrane fusion inhibitor-resistant virus strains, and confirmed the druggability of the novel lipopeptides using lipopeptide LP-98 as a control.

[0124] The T20-resistant NL4-3 mutant was found to be a T20-induced HIV-1 NL4-3 These were mutant strains, particularly those containing the I37T, V38A, V38M, Q40H, N43K, D36S / V38M, I37T / N43K, or V38A / N42T mutations. NL4-3Virus (HIV-1 in the literature) NL4-3 The WT) and each of the above mutant strains are described in the following literature (see Table 3 in the literature): Chong, H., Yao, X., Zhang, C., Cai, L., Cui, S., Wang, Y., He, Y., 2012. Biophysical property and broad anti-HIV activity of albuvirtide, a 3-maleimimidopropionic acid-modified peptide fusion inhibitor. PloS, 7, e32599.

[0125] The LP-40-induced drug-resistant NL4-3 strain was found to be resistant to LP-40-induced HIV-1 NL4-3 These were mutant strains, specifically those containing the L33S, V38T, N42T, L33S / I37T, or L33S / V38A / N42T mutations. NL4-3 The virus (referred to in the literature as Pseudovirus NL4-3) and each of the above mutant strains are described in the following literature (see Tables 2 and 3 in the literature): Hu, Y., Yu, W., Geng, X., Zhu, Y., Chong, H., He, Y., 2022. In Vitro Selection and Characterization of HIV-1 Variants with Increased Resistance to LP-40, an Enfuvirtual-Based Lipopeptide Inhibitor. International Journal of Molecular Science 23.

[0126] LP-52-induced drug-resistant SIV mac239 The strains were SIV induced by LP-52. mac239 mutant strains, particularly those containing the V562A, V562M, V562A / E657G, V562M / E657G, V562A / S760G or V562M / S760G mutations. mac239Virus (Pseudovirus SIV in the literature) mac239 WT) and each of the above mutant strains are described in the following literature (see Table 2 in the literature): Yu, D., Xue, J., Wei, H., Cong, Z., Chen, T., Zhu, Y., Chong, H., Wei, Q., Qin, C., He, Y., 2020. Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques. Journal of Virology 94, e00384-00320.

[0127] The LP-52-induced drug-resistant NL4-3 strain was found to be a potent inhibitor of LP-52-induced HIV-1 NL4-3 These were mutant strains, particularly those containing the V547A / E646G or V547M / E646G mutations. NL4-3 Virus (Pseudovirus HIV-1 in the literature) NL4-3 The wild-type (WT) and each of the above mutant strains are described in the following literature (see Table 2 in the literature): Yu, D., Xue, J., Wei, H., Cong, Z., Chen, T., Zhu, Y., Chong, H., Wei, Q., Qin, C., He, Y., 2020. Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques. Journal of Virology 94, e00384-00320.

[0128] SC29EK-induced drug-resistant NL4-3 strain was found to be a resistant strain of SC29EK-induced HIV-1 NL4-3These were mutant strains, particularly those containing the E49A, N43K / E49A, Q39R / N43K / N126K or N43K / E49A / N126K mutations. NL4-3 Virus (HIV-1 in the literature) NL4-3 The wild type) and each of the above mutant strains are described in the following literature (see Table 1 in the literature): Wu, X., Liu, Z., Ding, X., Yu, D., Wei, H., Qin, B., Zhu, Y., Chong, H., Cui, S., He, Y., 2018. Mechanism of HIV-1 Resistance to an Electronically Constrained alpha-Helical Peptide Membrane Fusion Inhibitor. Journal of Virology 92, e02044-02017.

[0129] SC22EK-induced drug-resistant NL4-3 strain was found to be a resistant strain of HIV-1 induced by SC22EK. NL4-3 mutant strains, specifically those containing the E49K, N126K, or E49K / N126K mutations. NL4-3 Virus (HIV-1 in the literature) NL4-3 The HIV-1 wild type virus and each of the above mutant strains are described in the following literature (see Table 1 in the literature): Su, Y., Chong, H., Qiu, Z., Xiong, S., He, Y., 2015a. Mechanism of HIV-1 Resistance to Short-Peptide Fusion Inhibitors Targeting the Gp41 Pocket. Journal of Virology 89, 5801-5811.

[0130] The MTSC22-induced drug-resistant NL4-3 strain was found to be a resistant strain of MTSC22-induced HIV-1 NL4-3 mutant strains, specifically those containing the L57R, E136G, or L57R / E136G mutations. NL4-3Virus (HIV-1 in the literature) NL4-3 The wild type) and each of the above mutant strains are described in the following literature (see Table 1 in the literature): Su, Y., Chong, H., Xiong, S., Qiao, Y., Qiu, Z., He, Y., 2015b. Genetic Pathway of HIV-1 Resistance to Novel Fusion Inhibitors Targeting the Gp41 Pocket. Journal of Virology 89, 12467-12479.

[0131] The SFT-induced drug-resistant NL4-3 strain was shown to be a potent anti-HIV-1 inhibitor. NL4-3 HIV-1 is a mutant strain, specifically one containing the Q52R mutation. NL4-3 Virus (HIV-1 in the literature) NL4-3 The HIV-1 virus (WT) and each of the above mutant strains are described in the following literature (see Table 1 in the literature): Yu, D., Ding, X., Liu, Z., Wu, X., Zhu, Y., Wei, H., Chong, H., Cui, S., He, Y., 2018. Molecular mechanism of HIV-1 resistance to sifuvirtide, a clinical trial-approved membrane fusion inhibitor. The Journal of biological chemistry 293, 12703-12718.

[0132] Test virus solutions: virus solutions of each T20-resistant NL4-3 mutant strain, virus solutions of each LP-40-induced drug-resistant NL4-3 mutant strain, virus solutions of each LP-52-induced drug-resistant SIV mac239Virus solutions of mutant strains, virus solutions of each LP-52-induced drug-resistant NL4-3 mutant strain, virus solutions of each SC29EK-induced drug-resistant NL4-3 mutant strain, virus solutions of each SC22EK-induced drug-resistant NL4-3 mutant strain, virus solutions of each MTSC22-induced drug-resistant NL4-3 mutant strain, or virus solutions of SFT-induced drug-resistant NL4-3 mutant strain.

[0133] Test lipopeptides: lipopeptide LP-101, lipopeptide LP-108, or lipopeptide LP-98 prepared in Example 1.

[0134] The method was the same as that in Example 2.

[0135] The results are shown in Figure 3.

[0136] The inhibitory activity of lipopeptide LP-101 against various T20-resistant NL4-3 mutant strains was increased 12- to 131-fold compared to lipopeptide LP-98, and the inhibitory activity of lipopeptide LP-108 was increased 14- to 304-fold compared to lipopeptide LP-98. The inhibitory activity of lipopeptide LP-101 against various LP-40-induced drug-resistant NL4-3 strains was increased 20- to 150-fold compared to lipopeptide LP-98, and the inhibitory activity of lipopeptide LP-108 was increased 9- to 250-fold compared to lipopeptide LP-98. The inhibitory activity of lipopeptide LP-101 against various LP-52-induced drug-resistant SIVmac239 strains was increased 71- to 758-fold compared to lipopeptide LP-98, and the inhibitory activity of lipopeptide LP-108 was increased 147- to 907-fold compared to lipopeptide LP-98. The inhibitory activity of lipopeptide LP-101 against two LP-52-induced drug-resistant NL4-3 strains was increased 106-fold or 46-fold compared to LP-98, and the inhibitory activity of lipopeptide LP-108 was increased 95-fold or 30-fold compared to lipopeptide LP-98. The inhibitory activity of lipopeptide LP-101 against various SC29EK-induced drug-resistant NL4-3 strains was increased 5-fold to 392-fold compared to lipopeptide LP-98, and the inhibitory activity of lipopeptide LP-108 was increased 4-fold to 429-fold compared to lipopeptide LP-98. The inhibitory activity of lipopeptide LP-101 against various SC22EK-induced drug-resistant NL4-3 strains was increased 3-fold to 11-fold compared to lipopeptide LP-98, and the inhibitory activity of lipopeptide LP-108 was increased 1-fold to 10-fold compared to lipopeptide LP-98. IC of lipopeptide LP-98 for inhibitory activity against various MTSC22-induced drug-resistant NL4-3 strains 50 Values ​​are IC of lipopeptide LP-101 50 The IC value was 0.4 to 2 times that of lipopeptide LP-108. 50 The inhibitory activity of lipopeptide LP-101 against the SFT-induced drug-resistant NL4-3 strain was increased 3-fold compared to lipopeptide LP-98, whereas lipopeptide LP-108 showed similar activity compared to lipopeptide LP-98.

[0137] Although T20, LP-40, and LP-52 did not have PBD amino acids, SC29EK, SC22EK, MTSC22, and SFT contained the complete PBD sequence, and in particular, MTSC22 further contained an N-terminal "MT hook" structure. Therefore, the above findings further shed light on the structure-function relationships of lipopeptides LP-101, LP-108, and LP-98 as inhibitors.

[0138] Example 4. Comparison of the inhibitory activity of lipopeptides against HIV-2 and SIV Test virus solution: HIV-2 ROD Virus solution, HIV-2 ST Virus solution, SIV mac239 Virus solution or SIV PBJ Virus solution.

[0139] HIV-2 ROD Viruses and HIV-2 ST Both viruses were infectious HIV-2 viruses (viral type: replicative type). mac239 Viruses and SIV PBJ Both viruses were SIV pseudoviruses (virus type: pseudotype). ROD Virus, HIV-2 ST Virus, SIV mac239 Viruses and SIV PBJ The viruses are described in the following publications (see Table S1 in the references): Xue, J., Chong, H., Zhu, Y., Zhang, J., Tong, L., Lu, J., Chen, T., Cong, Z., Wei, Q., He, Y., 2022. Efficient treatment and pre-exposure prophylaxis in rhesus macaques by an HIV fusion-inhibitory lipopeptide. Cell 185, 131-144 e118.

[0140] Test lipopeptides: lipopeptide LP-101, lipopeptide LP-108, or lipopeptide LP-98 prepared in Example 1.

[0141] The method was the same as that in Example 2.

[0142] The results are shown in Figure 4. Compared with lipopeptide LP-98, HIV-2 ROD Viruses and HIV-2 ST The inhibitory activity of lipopeptide LP-101 against viruses was increased by 2-fold and 2-fold, respectively, and SIV mac239 Viruses and SIV PBJ The inhibitory activity against the virus increased by 2-fold and 8-fold, respectively. ROD Virus, HIV-2 ST Viruses and SIV mac239 The inhibitory activity of lipopeptide LP-108 against the virus was comparable to that of lipopeptide LP-98, and the inhibitory activity of lipopeptide LP-108 against the virus was comparable to that of lipopeptide LP-98. PBJ Its inhibitory activity against the virus was increased by 8-fold compared to lipopeptide LP-98. Therefore, the lipopeptide of the present application has potent inhibitory activity against HIV-2 and SIV, further reflecting its broad-spectrum antiviral effect.

[0143] Example 5. Analysis of helical structure of lipopeptide and its binding stability To analyze the structural features of the lipopeptides of the present application and elucidate their mechanisms of action, we used circular dichroism (CD) to measure the secondary structure (α-helix) and helical stability (Tm value) of the lipopeptides LP-101 and LP-108 prepared in the Examples of the present application, as well as their complexes with target sequences. Lipopeptide LP-98 was used as a control.

[0144] I. Experimental Materials and Methods The NHR polypeptide, N42, corresponding to the NHR sequence of the gp41 fusion protein and used as a simulated target for lipopeptide inhibitors, was synthesized in the laboratory and is commonly used (see Xue et al., 2022). The N42 polypeptide is: Ac-STMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLT-NH2, and its amino acid sequence is set forth in SEQ ID NO: 11.

[0145] CD measurement: Lipopeptide LP-101, lipopeptide LP-108, lipopeptide LP-98, a mixture of N42 polypeptide and lipopeptide LP-101 (N42 / LP-101), a mixture of N42 polypeptide and lipopeptide LP-108 (N42 / LP-108), and a mixture of N42 polypeptide and lipopeptide LP-98 (N42 / LP-98) were dissolved in phosphate-buffered saline (PBS) at pH 7.2 to obtain final solutions with 10 μM lipopeptide and N42 polypeptide concentrations. Each solution was placed in a 37°C water bath for 30 min and then transferred to the corresponding cuvette. The molar ellipticity [θ]λ of the solution was scanned over the wavelength range of 195–270 nm using a JASCO spectropolarimeter (model J-815). Typical α-helical structures exhibited maximum negative peaks at 208 nm and 222 nm. Spectral values ​​were corrected by subtracting the PBS blank control. During calculations, the peak value of -33000°C.cm 2 .dmol -1 Using [θ] as the standard for 100% α-helical content, the percentage of α-helical content of the polypeptide was calculated according to the molar ellipticity of the solution at 222 nm. The solution was then 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 temperature-dependent changes in [θ] in the range of 20–98°C. The melting curves were smoothed, and the midpoint temperature (Tm) of the thermal dissociation transition was calculated using Origin software to reflect the degree of helical thermal stability.

[0146] II. Experimental Results and Analysis The results are shown in Figure 5.

[0147] The α-helical contents of lipopeptides LP-98 alone, LP-101 alone, and LP-108 alone were 49%, 62%, and 49%, respectively (Figure 5A), suggesting that lipopeptide LP-101 has high α-helicity. The Tm values ​​of lipopeptides LP-98 alone, LP-101 alone, and LP-108 alone were 48°C, 56°C, and 64°C, respectively (Figure 5B), suggesting that lipopeptide LP-108 had the highest helical stability, lipopeptide LP-101 had the second highest, and lipopeptide LP-98 had a relatively low helical stability.

[0148] The α-helical contents of the complexes formed with the N42 polypeptide and the lipopeptides LP-98, LP-101, and LP-108 were 73%, 82%, and 59%, respectively (Fig. 5C), and their Tm values ​​were 79°C, 88°C, and 89°C, respectively (Fig. 5D). The helicity of the N42 / LP-101 complex was relatively high, while that of the N42 / LP-108 complex was relatively low. However, the helical stability of the two complexes was comparable and significantly higher than that of the N42 / LP-98 complex.

[0149] The present application has been described in detail above. Those skilled in the art can carry out the present application in a wide range with equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present application and without needing to carry out undue experimentation. Although the present application provides specific examples, it is understood that the present application can be further modified. In general, according to the principles of the present application, the present application is intended to cover any changes, uses, or improvements to the present application, including changes made by routine techniques known in the art, that deviate from the scope disclosed in the present application. Some basic features can be applied according to the scope of the appended claims.

Claims

1. Formula I; Formula I:X 1 -polypeptide P1-X 2 ; is a compound represented by The polypeptide P1 is (a1) or (a2) or (a3): (a1) a polypeptide having the amino acid sequence "EXEELEKKIEELLKKAEEQQKKNEEELKKLEK"; (a2) a polypeptide having antiviral activity obtained by substitution and / or deletion and / or addition of amino acid residues based on (a1); (a3) a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to (a1), and having antiviral activity; and The terminal amino acid residue of polypeptide P1 is modified with a lipophilic compound group; X 1 is the amino terminal protecting group of polypeptide P1; X 2 is the carboxyl terminal protecting group of polypeptide P1 A lipopeptide, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof or a derivative thereof, characterized in that:

2. 2. The lipopeptide, its pharmaceutically acceptable salt, its solvate, its hydrate or its derivative according to claim 1, characterized in that in the polypeptide P1, X is isoleucine, valine or leucine.

3. Formula II; Formula II:X 3 -polypeptide P2-X 4 ; is a compound represented by The polypeptide P2 is (b1) or (b2) or (b3): (b1) a polypeptide having the amino acid sequence "EXEELEKKIEELLKKAEEQQKKNEEELKKLEKX"; (b2) A polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on (b1); (b3) a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to (b1), and having antiviral activity; and The terminal amino acid residue of polypeptide P2 is modified with a lipophilic compound group; X 3 is the amino terminal protecting group of polypeptide P2; X 4 is the carboxyl terminal protecting group of polypeptide P2 A lipopeptide, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof or a derivative thereof, characterized in that:

4. 4. The lipopeptide, its pharmaceutically acceptable salt, solvate, hydrate or derivative thereof according to claim 3, characterized in that in polypeptide P2, "X" as the second amino acid residue is isoleucine, valine, or leucine, and "X" as the 33rd amino acid residue is lysine or cysteine.

5. the lipophilic compound is cholesterol, a cholesterol derivative (e.g., cholesteryl hemisuccinate, 2-cholesteryl acetate, 2-cholesteryl propionate, 3-cholesteryl propionate, 2-cholesteryl butyrate, 2-cholesteryl isobutyrate, 3-cholesteryl butyrate, 3-cholesteryl isobutyrate, 4-cholesteryl butyrate, 2-cholesteryl valerate, 2-cholesteryl isovalerate, 3-cholesteryl valerate, 5-cholesteryl valerate, 2-cholesteryl caproate, 6-cholesteryl caproate, 2-cholesteryl enanthate, 7-cholesteryl enanthate, 2-cholesteryl caprylate, 8-cholesteryl caprylate, and cholesteryl bromoacetate), a fatty acid containing 8 to 20 carbon atoms (e.g., octadecanoic acid or palmitic acid), dihydrosphingosine (DHS), or vitamin E; Preferably, the lipophilic compound is cholesterol; Preferably, the lipophilic compound is cholesteryl hemisuccinate; Preferably, the lipophilic compound is cholesteryl bromoacetate; Preferably, the lipophilic compound is stearic acid. The lipopeptide according to any one of claims 1 to 4, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof or a derivative thereof.

6. X 1 is any one selected from the group consisting of an acetyl group, an amino group, a maleoyl group, a succinyl group, a tert-butoxycarbonyl group, a benzyloxy group, another hydrophobic group, and a polymeric carrier group; X 2 is any one selected from the group consisting of an amino group, a carboxyl group, a hydroxyl group, an amide group, a tert-butoxycarbonyl group, another hydrophobic group, and a polymeric carrier group; X 3 is any one selected from the group consisting of an acetyl group, an amino group, a maleoyl group, a succinyl group, a tert-butoxycarbonyl group, a benzyloxy group, another hydrophobic group, and a polymeric carrier group; X 4 is any one selected from the group consisting of an amino group, a carboxyl group, a hydroxyl group, an amide group, a tert-butoxycarbonyl group, another hydrophobic group, and a polymeric carrier group. The lipopeptide according to any one of claims 1 to 5, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof or a derivative thereof.

7. The polypeptide P1 is (c1) or (c2) or (c3) or (c4) or (c5): (c1) a polypeptide having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 9 in the sequence listing; (c2) a polypeptide having the sequence shown in SEQ ID NO: 5 in the sequence listing; (c3) a polypeptide having the sequence shown in SEQ ID NO: 6 in the sequence listing; (c4) A polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on any one of the polypeptides (c1) to (c3); (c5) A polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the polypeptides of (c1) to (c3), and having antiviral activity. and; The polypeptide P2 is (d1) or (d2) or (d3) or (d4): (d1) a polypeptide having the sequence shown in SEQ ID NO: 7 in the sequence listing; (d2) a polypeptide having the sequence shown in SEQ ID NO: 8 in the sequence listing; (d3) a polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on any one of the polypeptides (d1) to (d2); (d4) A polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the polypeptides (d1) to (d2), and having antiviral activity. is The lipopeptide according to any one of claims 1 to 6, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof or a derivative thereof.

8. (a1) or (a2) or (a3): (a1) a polypeptide having the amino acid sequence "EXEELEKKIEELLKKAEEQQKKNEEELKKLEK", where X is isoleucine, valine, or leucine; (a2) a polypeptide having antiviral activity obtained by substitution and / or deletion and / or addition of amino acid residues based on (a1); (a3) a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to (a1), and having antiviral activity; or (b1) or (b2) or (b3): (b1) a polypeptide having the amino acid sequence "EXEELEKKIEELLKKAEEQQKKNEEELKKLEKX", wherein "X" as the second amino acid residue is isoleucine, valine, or leucine, and "X" as the 33rd amino acid residue is lysine or cysteine; (b2) A polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on (b1); (b3) A polypeptide having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to (b1), and having antiviral activity. and; Preferably, (c1) or (c2) or (c3) or (c4) or (c5): (c1) a polypeptide having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 9 in the sequence listing; (c2) a polypeptide having the sequence shown in SEQ ID NO: 5 in the sequence listing; (c3) a polypeptide having the sequence shown in SEQ ID NO: 6 in the sequence listing; (c4) A polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on any one of the polypeptides (c1) to (c3); (c5) a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the polypeptides (c1) to (c3), and having antiviral activity; or (d1) or (d2) or (d3) or (d4): (d1) a polypeptide having the sequence shown in SEQ ID NO: 7 in the sequence listing; (d2) a polypeptide having the sequence shown in SEQ ID NO: 8 in the sequence listing; (d3) a polypeptide having antiviral activity obtained by substituting and / or deleting and / or adding amino acid residues based on any one of the polypeptides (d1) to (d2); (d4) A polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the polypeptides (d1) to (d2), and having antiviral activity. A polypeptide characterized in that:

9. A conjugate comprising the polypeptide of claim 8 and a modification site, For example, the modification site is attached to the N-terminus or C-terminus of the polypeptide, optionally via a linker; For example, the modification site is a terminal protecting group. The conjugate.

10. An isolated nucleic acid encoding the polypeptide of claim 8.

11. A vector comprising the isolated nucleic acid of claim 10.

12. A host cell comprising the isolated nucleic acid of claim 10 and / or the vector of claim 11.

13. (e1) or (e2) or (e3) or (e4) or (e5): (e1) a multimer formed from the lipopeptide according to any one of claims 1 to 7; (e2) a multimer formed from the pharmaceutically acceptable salt of any one of claims 1 to 7; (e3) a multimer formed from the derivative according to any one of claims 1 to 7; (e4) a multimer formed from the hydrate according to any one of claims 1 to 7; (e5) A multimer formed from the solvate according to any one of claims 1 to 7; or A multimer formed by the polypeptide of claim 8. A multimer.

14. 14. A composition comprising the lipopeptide according to any one of claims 1 to 7, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof or a derivative thereof, or the polypeptide according to claim 8, or the conjugate according to claim 9, or the isolated nucleic acid according to claim 10, or the vector according to claim 11, or the host cell according to claim 12, or the multimer according to claim 13.

15. A pharmaceutical composition comprising the lipopeptide according to any one of claims 1 to 7, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof or a derivative thereof, or the polypeptide according to claim 8, optionally further comprising a pharmaceutically acceptable carrier, Preferably, the lipopeptide, its pharmaceutically acceptable salt, solvate, hydrate or derivative thereof, or polypeptide is present in an amount effective to treat 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, SIV, and resistant strains thereof; Preferably, the HIV is HIV-1 and / or HIV-2; Preferably, the HIV is a T20 resistant virus; Preferably, the HIV is an LP-98 resistant virus; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of a T20-resistant virus, an LP-40-resistant virus, an LP-52-resistant virus, an SC29EK-resistant virus, an SC22EK-resistant virus, an MTSC22-resistant virus, and an SFT-resistant virus; Preferably, the disease caused by a viral infection is AIDS. The pharmaceutical composition.

16. (f1) or (f2) or (f3) or (f4): (f1) Use in the manufacture of a viral membrane fusion inhibitor; (f2) Use in the manufacture of a medicament for the prevention and / or treatment of a disease caused by a viral infection; (f3) Use as a viral membrane fusion inhibitor; (f4) Use in the prevention and / or treatment of diseases caused by viral infections; and Preferably, the viral infection is an infection caused by a virus selected from the group consisting of HIV, SIV, and resistant strains thereof; Preferably, the HIV is HIV-1 and / or HIV-2; Preferably, the HIV is a T20 resistant virus; Preferably, the HIV is an LP-98 resistant virus; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of a T20-resistant virus, an LP-40-resistant virus, an LP-52-resistant virus, an SC29EK-resistant virus, an SC22EK-resistant virus, an MTSC22-resistant virus, and an SFT-resistant virus; Preferably, the disease caused by a viral infection is AIDS. Use of the lipopeptide according to any one of claims 1 to 7, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof or a derivative thereof, or the polypeptide according to claim 8 or the multimer according to claim 13.

17. Function (g1) or (g2): (g1) Function as a viral membrane fusion inhibitor; (g2) the ability to prevent and / or treat diseases caused by viral infections; and Preferably, the viral infection is an infection caused by a virus selected from the group consisting of HIV, SIV, and resistant strains thereof; Preferably, the HIV is HIV-1 and / or HIV-2; Preferably, the HIV is a T20 resistant virus; Preferably, the HIV is an LP-98 resistant virus; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of a T20-resistant virus, an LP-40-resistant virus, an LP-52-resistant virus, an SC29EK-resistant virus, an SC22EK-resistant virus, an MTSC22-resistant virus, and an SFT-resistant virus; Preferably, the disease caused by a viral infection is AIDS. A product comprising the lipopeptide according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a hydrate thereof, or a derivative thereof, or the polypeptide according to claim 8, or the multimer according to claim 13.

18. 1. A method for treating or / and preventing a viral infection in an animal, comprising: Administering to a subject animal an effective amount of the lipopeptide according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a hydrate thereof, or a derivative thereof, or the polypeptide according to claim 8, or the multimer according to claim 13, or the pharmaceutical composition according to claim 15; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of HIV, SIV, and resistant strains thereof; Preferably, the HIV is HIV-1 and / or HIV-2; Preferably, the HIV is a T20 resistant virus; Preferably, the HIV is an LP-98 resistant virus; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of a T20-resistant virus, an LP-40-resistant virus, an LP-52-resistant virus, an SC29EK-resistant virus, an SC22EK-resistant virus, an MTSC22-resistant virus, and an SFT-resistant virus; Preferably, the animal is a mammal, e.g., a human. The method.

19. 1. A method for treating and / or preventing a disease caused by a viral infection, comprising: Administering an effective amount of the lipopeptide according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof, or a derivative thereof, or the polypeptide according to claim 8, or the multimer according to claim 13, or the pharmaceutical composition according to claim 15 to a subject in need thereof; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of HIV, SIV, and resistant strains thereof; Preferably, the HIV is HIV-1 and / or HIV-2; Preferably, the HIV is a T20 resistant virus; Preferably, the HIV is an LP-98 resistant virus; Preferably, the viral infection is an infection caused by a virus selected from the group consisting of a T20-resistant virus, an LP-40-resistant virus, an LP-52-resistant virus, an SC29EK-resistant virus, an SC22EK-resistant virus, an MTSC22-resistant virus, and an SFT-resistant virus; Preferably, the disease caused by a viral infection is AIDS; Preferably, the subject is a mammal, e.g., a human. The method.

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