WO2025167910A1 - Oral long-acting polypeptide hiv inactivator - Google Patents

Abstract

Provided are a novel long-acting HIV inactivating or inhibiting polypeptide that can be taken orally or has oral potential, a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant of the polypeptide, and a pharmaceutical composition, drug conjugate, drug complex, or fusion protein containing the above. Further, provided are a pharmaceutical use and a synthesis method of the polypeptide, the stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant of the polypeptide and the pharmaceutical composition, drug conjugate, drug complex, or fusion protein for treating or preventing diseases caused by HIV infection.

Description

An orally administrable long-acting polypeptide HIV inactivator

Cross-references

This application claims priority to PCT application No. PCT/CN2024/076010, filed on February 5, 2024. The entire contents of the prior application are deemed to be the disclosure contents of this application and are incorporated herein in their entirety.

Sequence Listing Reference

The sequence listing named DCF231808WO-Sequence Listing.xml, created on January 18, 2024, and having a size of 17,295 bytes, is hereby incorporated by reference in its entirety.

Technical Field

The present disclosure relates to the field of biomedicine, and in particular to polypeptide HIV virus inactivators and inhibitors.

Background Art

AIDS is a major infectious disease caused by infection with the human immunodeficiency virus (HIV). Since the first case of AIDS was discovered, the global death toll has exceeded 40 million; there are still 39 million infected people worldwide; and the number of new infections in 2023 will reach 1.3 million. AIDS has an extremely high ultimate mortality rate, is incurable, and lacks an effective vaccine. Drug treatment remains the only effective method. Based on research on the viral life cycle, five categories of anti-HIV drugs or HIV therapeutics have been developed: entry inhibitors, reverse transcriptase inhibitors, integrase inhibitors, protease inhibitors, and capsid inhibitors. Existing anti-HIV clinical drugs or HIV therapeutics can only work during the process of viral infection of host cells or after the virus enters the host cell. Therefore, they are only passive defenses and cannot actively attack the virus in the absence of host cells, directly inactivate free viral particles, and make the virus lose its infectious activity. Furthermore, the poor penetration of drugs into specific tissues and organs, such as the central nervous system, allows the virus to replicate at low levels in these tissues and organs. These inaccessible tissues and anatomical sites then become reservoirs of HIV. The existence of these reservoirs is a major factor in persistent HIV infection and the greatest obstacle to achieving complete HIV eradication and a cure for AIDS.

In the process of HIV invading host cells, the viral envelope glycoprotein (Env) plays an important role. HIV Env consists of the surface subunit gp120 and the transmembrane subunit gp41. The transmembrane subunit gp41 includes multiple functional regions in terms of sequence structure, namely: N-terminal fusion peptide region (FP), N-terminal repeat sequence region (NHR), C-terminal repeat sequence region (CHR), tryptophan-rich region, transmembrane region (TM), cytoplasmic tail region (CT), etc. Recently, researchers screened a 15-amino acid peptide F9170 (amino acid sequence: GWEALKYLWNLLQYW, SEQ ID NO: 5) from a peptide library. It not only exhibits effective HIV inhibitory activity, but also has the ability to inactivate HIV virus particles. Sequence analysis showed that the peptide originated from the lentiviral lytic peptide-3 (LLP-3) region in the gp41 CT domain. F9170's core mechanism of action may be through interaction with the lentiviral lytic peptide-1 region (LLP-1) within the gp41 CT domain, disrupting the integrity of the viral membrane and rendering the virus inactive. However, the F9170 peptide suffers from drugability drawbacks, such as a short in vivo half-life and the need for frequent injections, which severely limit its further clinical application. Therefore, there is an urgent need to develop HIV inactivators and inhibitors that are highly effective, safe, convenient to administer, and have high patient compliance.

Summary of the Invention

The present disclosure provides polypeptides that can be used as novel HIV inactivators and inhibitors, exhibiting one or more of the following advantages: ease of synthesis, low cost of synthesis, oral administration or potential for oral administration, high HIV inhibitory activity, efficient lysis of HIV virions, efficient inactivation of free HIV virions, long-lasting efficacy, effective entry into HIV anatomical reservoirs, and good biosafety. Compared to F9170, the polypeptides provided herein have significantly improved in vivo half-life, oral administration or potential for oral administration, and/or the ability to effectively enter HIV anatomical reservoirs.

In one aspect, the present disclosure provides a polypeptide or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, wherein the polypeptide comprises an amino acid sequence represented by any one of the following general formulas (I) to (IV), or an amino acid sequence represented by any one of the general formulas (I) to (IV) having 1, 2, 3, 4 or 5 conservative substitutions:
X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇ -X ₈ -X ₉ -X ₁₀ -X ₁₁ -X ₁₂ -X ₁₃ -X ₁₄ -W(I),
X ₂ -WEAX ₆ -KX ₉ -LWNX ₁₃ -LQYW(II),
GX ₂ -EALX ₆ -YLWNLLQYW(III),
GWEALKYLX ₉ -NLLX ₁₃ -YW(IV),

wherein _X1 represents any amino acid, preferably G or W, or an amino acid residue having similar properties thereto; _X2 , _X6 , _X9 , and _X13 are each independently a non-natural amino acid, ( _X2 ) and ( _X6 ), ( _X9 ) and ( _X13 ) are each covalently linked; _X3 represents any amino acid, preferably E or R, or an amino acid residue having similar properties thereto; _X4 represents any amino acid, preferably A or W, or an amino acid residue having similar properties thereto; _X5 represents any amino acid, preferably L or W, or an amino acid residue having similar properties thereto; _X7 represents any amino acid, preferably Y or R, or an amino acid residue having similar properties thereto; _X8 represents any amino acid, preferably L or W, or an amino acid residue having similar properties thereto; _X10 represents any amino acid, preferably N or R, or an amino acid residue having similar properties thereto; _X11 represents any amino acid, preferably L, W, or R, or an amino acid residue having similar properties thereto; ₁₂ represents any amino acid, preferably L or W, or an amino acid residue having similar properties; X ₁₄ represents any amino acid, preferably Y or R, or an amino acid residue having similar properties; the symbol "-" represents a peptide bond between amino acid residues. In some preferred embodiments, the polypeptides provided herein have at least 40%, 50%, 60%, 70%, 71%, 72%, 73%, 80%, or 90% sequence identity to F9170 (SEQ ID NO: 5).

In another aspect, the present disclosure provides a fusion protein comprising a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof. In one embodiment, the above-mentioned fusion protein optionally comprises another one or more proteins for treating or preventing HIV infection, such as an antibody against the intracellular segment of the HIV envelope protein, preferably an antibody against LLP1-GQ, such as an antibody against LLP1-GQ shown in SEQ ID NO: 4 (GACRAIRHIPRRIRQ).

In yet another aspect, the present disclosure provides a pharmaceutical composition comprising at least a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, and a pharmaceutically acceptable carrier or excipient. In one embodiment, the pharmaceutical composition may further comprise one or more other HIV therapeutic agents or HIV latent cell activators.

In yet another aspect, the present disclosure provides a drug conjugate comprising at least a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof. In one embodiment, the drug conjugate optionally comprises a carrier protein, such as serum albumin, immunoglobulin, ferritin, transferrin, α-2-macroglobulin, thyroxine-binding protein, and steroid-binding protein. In one embodiment, the drug conjugate optionally comprises one or more other HIV therapeutic agents or HIV latent cell activators.

In one aspect, the present disclosure provides a drug conjugate comprising at least a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof. In one embodiment, the drug conjugate optionally comprises a carrier protein, such as serum albumin, immunoglobulin, ferritin, transferrin, α-2-macroglobulin, thyroxine binding protein, and steroid binding protein. In one embodiment, the drug conjugate optionally comprises one or more other HIV therapeutic agents or HIV latent cell activators.

In one embodiment, the pharmaceutical compositions, drug conjugates, drug couplings or fusion proteins disclosed herein can be independently prepared into dosage forms including tablets, pills, powders, suppositories, solutions, suspensions, emulsions, granules, tinctures, capsules, transdermal agents, aerosols, effervescent tablets, drops and lyophilized powders.

In one aspect, the present disclosure provides the use of a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, for preparing a medicament for treating or preventing diseases caused by HIV infection. In one embodiment, the dosage form of the medicament includes tablets, pills, powders, suppositories, solutions, suspensions, emulsions, granules, tinctures, capsules, transdermal formulations, aerosols, effervescent tablets, drops, and lyophilized powders.

In another aspect, the present disclosure provides the use of the polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, for use as a drug for treating or preventing diseases caused by HIV infection.

In another aspect, the present disclosure provides a method for treating or preventing a disease caused by HIV infection, comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof of the polypeptide, a pharmaceutical composition, drug conjugate, drug conjugate, or fusion protein disclosed herein. In one embodiment, the polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof of the polypeptide, a pharmaceutical composition, drug conjugate, drug conjugate, or fusion protein disclosed herein is administered by injection, cavity administration, respiratory tract administration, mucosal administration, or topical administration, preferably injection.

In yet another aspect, the present disclosure provides in vitro methods for any one or more of the following (a)-(e): (a) inhibiting HIV infection and/or replication, (b) inactivating HIV free virions, (c) preparing a preparation having in vitro activity to inhibit HIV infection, (d) eliminating viruses in HIV reservoirs or reducing the number of viruses in HIV reservoirs; (e) lysing HIV viral particles;

The methods include the use of a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof.

In one aspect, the present disclosure provides the use of a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, for in vitro use of any one or more of the following (a)-(e): (a) inhibiting HIV infection and/or replication, (b) inactivating HIV free virions, (c) preparing a preparation having in vitro HIV infection inhibitory activity, (d) eliminating viruses in HIV viral reservoirs or reducing the number of viruses in HIV viral reservoirs; (e) lysing HIV viral particles.

In one aspect, the present disclosure provides a method for generating a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof of the polypeptide, or a fusion protein disclosed herein, comprising synthesizing the polypeptide, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof of the polypeptide, or the fusion protein by a chemical method. In one embodiment, the chemical method can be a solid phase (e.g., Fmoc solid phase synthesis) or a liquid phase synthesis method. In one embodiment, the method for generating a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof of the polypeptide, or a fusion protein disclosed herein used herein is a method commonly used in the art.

The polypeptide provided by the present disclosure has at least one of the following obvious advantages: (1) it can directly kill free viruses; (2) it has only 15 amino acids, which is significantly shorter than other anti-HIV polypeptides, such as T20 (YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 6)), T2635 (TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL (SEQ ID NO: 7)), etc. Therefore, the polypeptide of the present invention is easy to synthesize and has low cost; (3) it has good biosafety; (4) compared with F9170 (amino acid sequence: GWEALKYLWNLLQYW, SEQ ID NO: 5), the polypeptide provided by the present invention can significantly prolong the half-life while maintaining the activity of inhibiting HIV replication and/or inactivating HIV free virions, which can significantly reduce the number of dosing times and improve patient compliance; (5) the polypeptide provided by the present invention is orally absorbable or has oral potential, which has significant advantages over other drugs of the same type and is expected to become the first orally absorbable peptide anti-HIV drug; (6) compared with F9170, the polypeptide provided by the present invention can effectively enter the HIV anatomical reservoir.

The polypeptides provided by the present disclosure will assist in the prevention and treatment of AIDS and the elimination or reduction of viruses in latent viral reservoirs, accelerating the application of new anti-HIV drugs and the development of new combined treatment strategies.

BRIEF DESCRIPTION OF THE DRAWINGS

The following provides a brief description of the drawings, which are provided to illustrate exemplary embodiments disclosed herein and not to limit these embodiments.

FIG1 is a synthetic schematic diagram of the double binding strategy construction in Example 1. FIG.

FIG2 is a mass spectrum of polypeptide D26 in Example 1.

FIG3 is a mass spectrum of polypeptide D1 in Example 1.

FIG4 is a mass spectrum of polypeptide F2 in Example 1.

FIG5 is a mass spectrum of polypeptide F6 in Example 1.

Figure 6 is a line graph of the blood drug concentrations at different time points after intravenous injection of D26, D1, F2, F6, or F9170 in Example 4. The solid circles represent D26, the solid squares represent D1, the solid equilateral triangles represent F2, the solid inverted triangles represent F6, and the solid diamonds represent F9170.

Figure 7 is a bar graph showing the area under the drug-time curve of mouse tissues and organs after intravenous injection of D26 or F9170 in Example 5, wherein mosaics represent D26 and solids represent F9170.

Figure 8 shows the drug-dose curve of 50 mg/kg D26 administered orally in Example 6. The F9170 plasma concentration is below the minimum detection limit, where the dotted line represents the lower limit of quantification.

Figure 9A is a graph showing the changes in body weight over time in male and female mice after injection with PBS, 20 mg/kg D26, and 100 mg/kg D26 in Example 7. Solid circles represent the PBS group, solid squares represent the 20 mg/kg D26 group, and solid triangles represent the 100 mg/kg D26 group.

Figure 9B shows the serum alanine aminotransferase levels of the mice in Example 7 at different times after injection with PBS, 20 mg/kg D26, and 100 mg/kg D26. The solid circles represent the PBS group, the solid squares represent the 20 mg/kg D26 group, and the solid triangles represent the 100 mg/kg D26 group.

Figure 9C shows the serum creatinine levels of the mice in Example 7 at different times after injection with PBS, 20 mg/kg D26, and 100 mg/kg D26. The solid circles represent the PBS group, the solid squares represent the 20 mg/kg D26 group, and the solid triangles represent the 100 mg/kg D26 group.

FIG10 is a mass spectrum of the polypeptide D26W1 in Example 1.

FIG11 is a mass spectrum of the polypeptide D26W4 in Example 1.

FIG12 is a mass spectrum of the polypeptide D26W5 in Example 1.

FIG13 is a mass spectrum of the polypeptide D26W8 in Example 1.

FIG14 is a mass spectrum of the polypeptide D26W11 in Example 1.

FIG15 is a mass spectrum of the polypeptide D26W12 in Example 1.

FIG16 is a mass spectrum of the polypeptide D26R3 in Example 1.

FIG17 is a mass spectrum of the polypeptide D26R7 in Example 1.

FIG18 is a mass spectrum of the polypeptide D26R10 in Example 1.

FIG19 is a mass spectrum of the polypeptide D26R11 in Example 1.

FIG20 is a mass spectrum of the polypeptide D26R14 in Example 1.

FIG21 is a mass spectrum of the polypeptide D26-2R in Example 1.

FIG22 is a mass spectrum of the polypeptide D26-3R in Example 1.

FIG23 is a mass spectrum of the polypeptide D26-4R in Example 1.

FIG24 is a mass spectrum of polypeptide D26-5R in Example 1.

DETAILED DESCRIPTION

Unless otherwise indicated, all numbers used in this specification and claims to indicate content, concentration, ratio, weight, particle size, percentage, technical effect, etc. should be understood as being modified by the term "about" or "approximately" in any case. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values.

Unless otherwise stated, the terms used herein have the usual understanding meaning to those skilled in the art. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained by the application, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or the manner understood by those skilled in the art. In general, the nomenclature used herein and the experimental procedures of organic chemistry, medicinal chemistry, biology described herein are well known in the art and are generally adopted in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meanings that are commonly understood by those of ordinary skill in the art of the application. Where there are multiple definitions for the terms used herein, unless otherwise stated, the definitions in this section shall prevail.

definition

As used herein, the expression "A and/or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B, and/or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C. The meanings of similar expressions can be deduced analogously.

As used herein, the term "independently of one another" means that multiple events do not affect each other. For example, "X and Y are independently selected from any of a, b, c, d, e, f, and g" means that X can be any of a, b, c, d, e, f, and g, and Y can also be any of a, b, c, d, e, f, and g. The choice of X and the choice of Y can be the same or different, and the two do not interfere with each other.

As used herein, the terms "comprises" and "includes" mean that in addition to the listed elements, other elements are not excluded.

As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a certain position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or a certain position in each of the two polypeptides is occupied by lysine), then each molecule is identical at that position. The "identity" between two sequences, when expressed as a percentage, is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared × 100. For example, if 6 out of 10 positions of the two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have a total of 50% identity (3 out of a total of 6 positions match). Typically, comparisons are made when two sequences are aligned to produce maximum identity. Such an alignment can be achieved using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

As defined herein, the terms "polypeptide," "protein," "peptide," and "amino acid sequence" are used interchangeably herein to refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, it may contain modified amino acids or amino acid analogs, and may be interrupted by non-amino acid chemical moieties. The term also includes amino acid polymers that have been modified naturally or artificially, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a label or biologically active component. The term "peptide" includes two or more naturally occurring or synthetic amino acids linked by a covalent bond, such as an amide bond.

As used herein, unless otherwise indicated, the position numbering of a polypeptide or amino acid sequence is in the order from N-terminus to C-terminus, as is commonly practiced in the art. For example, the first amino acid from the N-terminus of the polypeptide or amino acid sequence is the first amino acid, the second amino acid from the N-terminus of the polypeptide or amino acid sequence is the second amino acid, the third amino acid from the N-terminus of the polypeptide or amino acid sequence is the third amino acid, and so on. As used herein, unless otherwise indicated, the left-hand end of a polypeptide or amino acid sequence is the N-terminus, i.e., the amino terminus, and the right-hand end is the C-terminus, i.e., the carboxyl terminus.

When used herein with respect to a peptide or polypeptide, "amino acid" and "amino acid residue" have the same meaning and refer to: when amino acids are linked by chemical bonds, some of their groups are lost due to their participation in the formation of the linking bond, and the remaining amino acid portion is the amino acid residue. The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis ( ^2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this article, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "amino acid" includes natural amino acids, as well as other "non-proteinogenic" α-amino acids/unnatural amino acids commonly used in the art of peptide chemistry to prepare analogs of natural peptides.

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

As used herein, the term "stereoisomer" refers to an isomer formed by having one or more stereogenic centers, and each isomeric center can exist in the form of R or S configuration or a combination thereof. Similarly, the polypeptides described herein may have one or more double bonds, and each double bond can exist in the form of E (trans) or Z (cis) configuration or a combination thereof. A specific stereoisomer, structural isomer, diastereomer, enantiomer or epimer should be understood to include all possible stereoisomers, structural isomers, diastereomers, enantiomers or epimers and mixtures thereof. Therefore, the polypeptides described herein include all configurationally different stereoisomers, structural isomers, diastereomers, enantiomers or epimers and their corresponding mixtures. Preferred structurally pure isomers of the polypeptides of the present invention are enantiomers or diastereomers. The techniques for converting a specific stereoisomer or maintaining a specific stereoisomer as it is, and the techniques for resolving a mixture of stereoisomers are well known in the art, and those skilled in the art can select an appropriate method according to the specific situation.

As used herein, a stapled peptide is a peptide in which a covalent bond is formed between the side chains of two amino acids in the peptide via an orthogonal reaction. A double-bridged stapled peptide is a peptide in which two independent covalent bonds are formed between the side chains of the amino acids in the peptide. Peptide stapling can be used to physically constrain the peptide to form or maintain a specific conformation (e.g., physically constraining the peptide to maintain its native alpha-helical state). Stapling can enhance the pharmaceutical properties of a peptide by helping to maintain the original structure required for the peptide to interact with a target molecule, increasing cell penetration, and/or protecting the peptide from proteolytic degradation.

As used herein, the term "functionally equivalent variant" refers to a polypeptide derived from a polypeptide sequence of the present invention that has been modified by amino acid substitution, deletion, or addition, or a derivative polypeptide having a certain sequence identity with a polypeptide of the present invention, provided that the derivative polypeptide retains at least 50%, at least 80%, or at least 100% of the function of the corresponding unmodified polypeptide. Functionally equivalent variants also include polypeptides having improved functional activity compared to the unmodified polypeptide.

Polypeptide, its stereoisomer, derivative, pharmaceutically acceptable salt, mixture or its function, etc. Variations of valence

The present disclosure provides a polypeptide or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, wherein the polypeptide comprises an amino acid sequence represented by any one of the following general formulae (I)-(IV), or an amino acid sequence represented by any one of the general formulae (I)-(IV) having 1, 2, 3, 4 or 5 conservative substitutions:
X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇ -X ₈ -X ₉ -X ₁₀ -X ₁₁ -X ₁₂ -X ₁₃ -X ₁₄ -W(I),
X ₂ -WEAX ₆ -KX ₉ -LWNX ₁₃ -LQYW(II),
GX ₂ -EALX ₆ -YLWNLLQYW(III),
GWEALKYLX ₉ -NLLX ₁₃ -YW(IV),

wherein _X1 represents any amino acid, preferably G or W, or an amino acid residue having similar properties thereto; _X2 , _X6 , _X9 , and _X13 are each independently a non-natural amino acid, ( _X2 ) and ( _X6 ), ( _X9 ) and ( _X13 ) are each covalently linked; _X3 represents any amino acid, preferably E or R, or an amino acid residue having similar properties thereto; _X4 represents any amino acid, preferably A or W, or an amino acid residue having similar properties thereto; _X5 represents any amino acid, preferably L or W, or an amino acid residue having similar properties thereto; _X7 represents any amino acid, preferably Y or R, or an amino acid residue having similar properties thereto; _X8 represents any amino acid, preferably L or W, or an amino acid residue having similar properties thereto; _X10 represents any amino acid, preferably N or R, or an amino acid residue having similar properties thereto; _X11 represents any amino acid, preferably L, W, or R, or an amino acid residue having similar properties thereto; ₁₂ represents any amino acid, preferably L or W, or an amino acid residue having similar properties; X ₁₄ represents any amino acid, preferably Y or R, or an amino acid residue having similar properties; the symbol "-" represents a peptide bond between amino acid residues. In some preferred embodiments, the polypeptides provided herein have at least 40%, 50%, 60%, 70%, 71%, 72%, 73%, 80%, or 90% sequence identity to F9170 (SEQ ID NO: 5).

In a preferred embodiment, the non-natural amino acids contained in the polypeptide disclosed herein or the stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof are independently selected from amino acids with olefin side chains, such as (S)-2-(4'-pentenyl)alanine (i.e., S5), (R)-2-(4'-pentenyl)alanine (i.e., R5), (S)-2-(7'-octenyl)alanine (i.e., S8), (R)-2-(7'-octenyl)alanine (i.e., R8) and 2-amino-2-(4'-pentenyl)-6-heptenoic acid (i.e., B5). More preferably, the non-natural amino acid is (S)-2-(4'-pentenyl)alanine (i.e., S5). In a preferred embodiment, the covalent bond formed between the non-natural amino acids includes a disulfide bond, a hydrocarbon chain, an intramolecular bond, a thioether bond, or a covalent bond formed by an azide-alkyne cycloaddition reaction. More preferably, the covalent bond is a covalent bond formed by an olefin metathesis reaction, for example, to form a full hydrocarbon chain bridge.

In one embodiment, the polypeptide provided by the present disclosure comprises an amino acid sequence described in any one of the following (a)-(t): (a) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence shown in SEQ ID NO: 1, preferably the amino acid sequence shown in SEQ ID NO: 1; (b) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence shown in SEQ ID NO: 2, preferably the amino acid sequence A(S5)EAL(S5)YL(S5)NLL(S5)YW shown in SEQ ID NO: 2, wherein the first and second S5 are covalently linked. and the third and fourth S5 are covalently bonded; (c) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence shown in SEQ ID NO: 3, preferably the amino acid sequence shown in SEQ ID NO: 3; (d) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence shown in SEQ ID NO: 10, preferably the amino acid sequence shown in SEQ ID NO: 10; (e) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 11. (f) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 12; (g) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 13; (h) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 14; : (i) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence of SEQ ID NO: 14, preferably the amino acid sequence of SEQ ID NO: 14; (j) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence of SEQ ID NO: 16, preferably the amino acid sequence of SEQ ID NO: 17; 6, preferably the amino acid sequence shown in SEQ ID NO: 16; (k) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence shown in SEQ ID NO: 17, preferably the amino acid sequence shown in SEQ ID NO: 17; (l) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 18, or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence shown in SEQ ID NO: 18, preferably the amino acid sequence shown in SEQ ID NO: 18; (m) an amino acid sequence having at least an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence shown in SEQ ID NO: 19; (n) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence shown in SEQ ID NO: 20; (o) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence having 1-5 conservative substitutions therefrom. an amino acid sequence, preferably the amino acid sequence set forth in SEQ ID NO: 21; (p) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence set forth in SEQ ID NO: 22; (q) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence set forth in SEQ ID NO: 23; (r) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence set forth in SEQ ID NO: 24; an amino acid sequence having at least 85% or at least 90% sequence identity or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence shown in SEQ ID NO: 24, preferably the amino acid sequence shown in SEQ ID NO: 24; (s) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence shown in SEQ ID NO: 8, preferably the amino acid sequence shown in SEQ ID NO: 8; (t) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence shown in SEQ ID NO: 9, preferably the amino acid sequence shown in SEQ ID NO: 9.

In one embodiment, the polypeptide provided by the present disclosure, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, has one or more of the following advantages: simple synthesis, low synthesis cost, oral administration or oral administration potential, antiviral activity that effectively inhibits HIV replication, activity that effectively inactivates HIV free virions, efficient lysis of HIV viral particles, long-lasting effect, effective entry into HIV anatomical reservoirs, and good biosafety. In one embodiment, the polypeptide provided by the present disclosure, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, has an improved half-life, oral administration or oral administration potential, and/or the ability to effectively enter HIV anatomical reservoirs compared to F9170.

As used herein, a "derivative" of a polypeptide refers to a product obtained by performing amino acid modification, conservative amino acid substitution and/or replacing hydrogen in amino acid residues on the basis of the polypeptide.

As used herein, the term "amino acid modification" includes, but is not limited to, N-terminal modification, C-terminal modification, and side chain modification. Modifications include, but are not limited to, hydroxylation, carboxylation, alkylation, acylation, phosphorylation, sulfonation, amidation, aldehyde formation, alcoholization, mercaptoethylation, esterification, and glycosylation.

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

As used herein, substitutions between amino acid residues with similar properties have little or no effect on the function, activity, or other biological properties of the peptide.

As used herein, the replacement of hydrogen in an amino acid residue may be that the hydrogen in the amino acid residue is replaced by any conventional substituent known in the art, including but not limited to alkyl, alkenyl, alkynyl, aromatic, alkoxy, carboxyl, aldehyde, carbonyl, hydroxyl, halogen, cyano, acyl, sulfonic acid, amino, thiol or nitro groups.

In one embodiment, the derivatives of the polypeptide provided by the present disclosure are selected from derivatives modified with maleimide, derivatives obtained by connecting an amino-terminal protecting group and/or a carboxyl-terminal protecting group to the amino-terminal, derivatives obtained by modification with protein or polyethylene glycol, derivatives obtained by connecting an oligopeptide or a lipophilic group or cholesterol to the amino-terminal and/or carboxyl-terminal, or derivatives obtained by substitution with amino acids having a D-type conformation, artificially modified amino acids, and rare amino acids existing in nature.

In one embodiment, the amino terminal protecting group provided by the present invention is selected from: H, a polymer derived from polyethylene glycol, a non-cyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted heteroarylalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group and R5-CO-, wherein R5 is selected from: H, a non-cyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted heterocyclic group and a substituted or unsubstituted heteroarylalkyl group, preferably, the amino terminal protecting group is selected from: H, acetyl (Ac), lauroyl, myristoyl and palmitoyl.

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

Pharmaceutical compositions, drug conjugates, drug couplings, fusion proteins

The present disclosure provides pharmaceutical compositions comprising at least a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, and a pharmaceutically acceptable carrier or excipient. In one embodiment, the pharmaceutical composition may further comprise one or more other HIV therapeutic agents or HIV latent cell activators.

HIV latent cell activators include histone deacetylase inhibitors (HDACi), BRD (bromodomain) protein inhibitors, protein kinase C (PKC) activators, positive transcription elongation factor b (p-TEFb) activators, DNA methyltransferase inhibitors (DNMTi), cytokines, etc. Anti-HIV drugs or HIV therapeutic agents include but are not limited to reverse transcriptase inhibitors, protease inhibitors, entry inhibitors, integration inhibitors, and maturation inhibitors. The above-mentioned reverse transcriptase inhibitors can be one or more of AZT, 3TC, ddI, d4T, ddT, TDF, Abacavir, Nevirapine Efavirenz and Delavirdine; the above-mentioned protease inhibitors can be one or more of Saquinavir mesylate, Idinavir, Ritonavir, Amprenavir, Kaletra and Nelfinavir mesylate; the above-mentioned invasion inhibitors can be one or more of Maraviroc, TAK-779, T20, T2635, Sifuvirtide, VIRIP, etc.; the above-mentioned integration inhibitors can be Raltegravir, etc.; the above-mentioned maturation inhibitors can be GSK3640254, Bevirimat, GSK3532795, GSK3640254, PF-46396, etc.; the above-mentioned integration inhibitors can be dolutegravir (DTG), elvitegravir (EVG), bictegravir (BIC) and cabotegravir (CAB), etc.

HIV therapeutic agents include, but are not limited to, abacavir, emtricitabine, lamivudine, tenofovir disoproxil fumarate, zidovudine, doravirine, efavirenz, etravirine, nevirapine, rilpivirine, atazanavir, darunavir, fosamprenavir, ritonavir, saquinavir, tipranavir, enfuvirtide, maraviroc, dolutegravir, raltegravir, ibalizumab, cobicistat, bictegravir, telafam, rilpivirine, elvitegravir, or lopinavir. As used herein, the term "pharmaceutical composition" refers to a mixture of a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof of the polypeptide, and other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and/or excipients. A pharmaceutical composition facilitates administration of the polypeptide, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof of the polypeptide to an organism. There are many ways in the art to administer a polypeptide, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, including but not limited to subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral, transdermal, pulmonary, ocular and topical administration.

In the present application, the pharmaceutical composition can be configured into a dosage form suitable for administration to a subject via a desired route of administration, including but not limited to tablets, capsules, caplets, pills, lozenges, powders, syrups, brews, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalants, creams, ointments, lotions, pastes, sprays, freeze-dried solutions, injections, and gels.

As used herein, the term "pharmaceutically acceptable salts" includes acid addition salts and base addition salts. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include, but are not limited to, acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate/carbonate, bisulfate/sulfate, borate, camphorsulfonate, citrate, cyclamates, edisylate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, 2-(4-hydroxybenzyl)benzoate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, 2-hydroxyethanesulfonate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, hexadecanoate, phosphate/hydrogenphosphate/dihydrogenphosphate, pyroglutamate, glucarate, stearate, salicylate, tannate, tartrate, toluenesulfonate, and trifluoroacetate. Suitable base addition salts are formed with bases which form non-toxic salts. Examples include, but are not limited to, aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. Hemi-salts of acids and bases, such as hemisulfate and hemicalcium salts, can also be formed. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002).

The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, which are involved in carrying or delivering the polypeptide of the present disclosure, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, within a subject or carrying or delivering the polypeptide of the present disclosure, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, to a subject so that it can perform its intended function. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate; Esters and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavorings; perfuming agents; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardening agents; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other nontoxic, compatible substances used in pharmaceutical formulations, or any combination thereof.

The present disclosure provides a drug conjugate comprising at least a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof of the polypeptide. In one embodiment, the drug conjugate optionally comprises a carrier protein, such as serum albumin, immunoglobulin, ferritin, transferrin, α-2-macroglobulin, thyroxine binding protein, and steroid binding protein. In one embodiment, the drug conjugate optionally comprises one or more other HIV therapeutic agents or HIV latent cell activators. In one embodiment, the drug conjugate is formed by linking a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof of the polypeptide to a carrier protein via a linker. In one embodiment, the drug conjugate is formed by linking a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof of the polypeptide to other HIV therapeutic agents or HIV latent cell activators via a linker. The linker can be a cleavable linker or a non-cleavable linker.

The present disclosure provides a drug conjugate comprising at least a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof of the polypeptide. In one embodiment, the drug conjugate optionally comprises a carrier protein, such as serum albumin, immunoglobulin, ferritin, transferrin, α-2-macroglobulin, thyroxine binding protein, and steroid binding protein. In one embodiment, the drug conjugate optionally comprises one or more other HIV therapeutic agents or HIV latent cell activators. In one embodiment, the drug conjugate is formed by covalently attaching a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof of the polypeptide to a carrier protein. In one embodiment, the drug conjugate is formed by covalently attaching a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof of the polypeptide to other HIV therapeutic agents or HIV latent cell activators.

The present disclosure provides a fusion protein comprising at least a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof of the polypeptide. In one embodiment, the above-mentioned fusion protein optionally comprises another one or more proteins for treating or preventing HIV infection, such as an antibody against the intracellular segment of the HIV envelope protein, preferably an antibody against LLP1-GQ, such as an antibody against LLP1-GQ shown in SEQ ID NO: 4 (GACRAIRHIPRRIRQ). In one embodiment, the polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof of the polypeptide comprised in the fusion protein is linked to another one or more proteins for treating or preventing HIV infection via a covalent bond.

As described in this article, LLP1-GQ is the structural domain of lentiviral lytic peptide-1 (LLP-1), which is amino acids 828-842 on LLP1 and has the sequence shown as GACRAIRHIPRRIRQ (SEQ ID NO:4).

In one embodiment, the pharmaceutical compositions, drug conjugates, drug couplings or fusion proteins disclosed herein can be independently prepared into dosage forms including tablets, pills, powders, suppositories, solutions, suspensions, emulsions, granules, tinctures, capsules, transdermal agents, aerosols, effervescent tablets, drops and lyophilized powders.

In one embodiment, the pharmaceutical compositions, drug conjugates, drug couplings or fusion proteins disclosed herein are administered in a variety of ways, including but not limited to, subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral, transdermal, pulmonary, ocular and topical administration.

Uses and methods

The present disclosure provides the use of a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, for the preparation of a medicament for treating or preventing a disease caused by HIV infection. In one embodiment, the dosage form of the medicament comprises tablets, pills, powders, suppositories, solutions, suspensions, emulsions, granules, tinctures, capsules, transdermal agents, aerosols, effervescent tablets, drops, and lyophilized powders. In one embodiment, the medicament is administered in a variety of ways, including, but not limited to, subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, pulmonary administration, ocular administration, and topical administration.

The present disclosure provides the use of the polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, as a drug for treating or preventing diseases caused by HIV infection.

The present disclosure provides a method for treating or preventing a disease caused by HIV infection, comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, a pharmaceutical composition, drug conjugate, drug conjugate, or fusion protein disclosed herein. In one embodiment, a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, a pharmaceutical composition, drug conjugate, drug conjugate, or fusion protein disclosed herein is administered by injection (including but not limited to subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection, etc.), cavity administration (including but not limited to digestive tract administration, genital tract administration, such as oral, enteral, rectal, vaginal, sublingual, etc.), respiratory tract administration (including but not limited to nasal, pulmonary), mucosal administration, or topical administration, preferably injection.

As used herein, the term "subject" includes animals, such as vertebrates, preferably mammals, such as dogs, cats, pigs, cows, sheep, horses, rodents (e.g., mice, rats, or guinea pigs), or primates (e.g., gorillas, chimpanzees, and humans).

As used herein, the term "treat," ...

As used herein, the term "therapeutically effective dose" refers to an amount that results in a benefit or treatment of a disease compared to a corresponding subject not receiving that amount, but is sufficiently low within the scope of sound medical judgment to avoid serious side effects. The therapeutically effective dose of the polypeptide described herein, or the stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, pharmaceutical composition, drug conjugate, drug conjugate or fusion protein will vary with the selected polypeptide, or the stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, pharmaceutical composition, drug conjugate, drug conjugate or fusion protein; route of administration; severity of the disease being treated; age, size, weight and physical condition of the patient being treated; medical history of the patient being treated; duration of treatment; nature of concurrent treatment; desired therapeutic effect and the like, but can still be determined by those skilled in the art in a routine manner.

As used herein, the term "prevention" refers to a method for blocking, reducing, inhibiting, preventing and/or delaying the occurrence of a disease or condition or symptom (e.g., respiratory diseases and symptoms, infection or autoimmune disease) in a subject, and a method for reducing the incidence of infectious diseases in the subject.

The present disclosure provides in vitro methods for any one or more of the following (a)-(e):

(a) inhibit HIV infection and/or replication,

(b) inactivate free HIV virions,

(c) preparing a preparation having in vitro HIV infection inhibitory activity,

(d) Eliminate HIV virus from the reservoir or reduce the number of HIV viruses in the reservoir,

(e) lysis of HIV virus particles;

The methods include the use of a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof.

The present disclosure provides for the use of a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, for any one or more of the following (a) to (e) in vitro:

(a) inhibit HIV infection and/or replication,

(b) inactivate free HIV virions,

(c) preparing a preparation having in vitro HIV infection inhibitory activity,

(d) Eliminate HIV virus from the reservoir or reduce the number of HIV viruses in the reservoir,

(e) Splitting HIV virus particles.

The present disclosure provides a method for generating a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or its functionally equivalent variant or fusion protein disclosed herein, which includes synthesizing the polypeptide, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or its functionally equivalent variant or fusion protein with a chemical method. In one embodiment, the chemical method can be a solid phase (e.g., Fmoc solid phase synthesis) or a liquid phase synthesis method. In one embodiment, the method for generating a polypeptide disclosed herein, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or its functionally equivalent variant or fusion protein disclosed herein used herein is a method commonly used in the art. In some embodiments, the polypeptide of the present invention can be chemically synthesized. For example, peptides can be synthesized using solid phase methods (Stewart, J.M. and Young, J.D., "Solid Phase Peptide Synthesis, 2nd ed.", (1984), Pierce Chemical Company, Rockford, Ill (1984)inois; Bodanzsky M.y Bodanzsky A., "The practice of Peptide Synthesis", (1994), Springer Verlag, Berlin; Lloyd Williams M. L., et al., "Solid Phase Peptide Synthesis, 2nd ed.", (1984), Pierce Chemical Company, Rockford, Ill (1984)inois); P.et al.,"Chemical Approaches to the Synthesis of Peptides and Proteins",(1997),CRC,Boca Raton,FL,USA), synthesis in solution, enzymatic synthesis (Kullmann W."Proteases as catalysts for enzymic syntheses of opioid peptides",(1980),J.Biol.Chem.,255(17),8234-8238) or any combination thereof.

The various embodiments described above for the polypeptides of the present disclosure, or stereoisomers, derivatives, pharmaceutically acceptable salts, mixtures or functionally equivalent variants thereof, are also applicable to the pharmaceutical compositions, drug conjugates, drug conjugates, fusion proteins, uses and methods of the present disclosure (as long as they are not inherently inconsistent with each other), and the various embodiments formed by such combination are considered part of the present disclosure.

Example

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

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

Example 1. Synthesis and purification of polypeptides

The peptides D26, D26W1, D26W4, D26W5, D26W8, D26W11, D26W12, D26R3, D26R7, D26R10, D26R11, D26R14, D26-2R, D26-3R, D26-4R, D26-5R, D1, F2, and F6 provided herein were synthesized using a CEM Libery Blue microwave peptide synthesizer using the Fmoc solid-phase synthesis method (SPSS). Rink Amide Resin was used as the solid phase support with a resin loading of 0.53 mmol/g. Oxyma/DIC was used as the condensation reagent, and a 20% piperidine solution was used as the deprotection reagent. After each condensation reaction, the mixture was washed three times with DMF solution.

The ratio of amino acid usage to Rink Amide Resin usage was 6:1. The amino acids used were as follows: Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, and the unnatural amino acid S5. The coupling of amino acids was performed according to the peptide sequence and instrument operating procedures. If isoleucine or leucine is present in the peptide sequence, the amino acids following isoleucine, leucine, and the amino acids following them are condensed twice by default. Special amino acids, such as S5 in this experimental sequence, are also condensed twice by default. The peptide-resin with amino acid linkage was removed, and after removing the terminal Fmoc, acetic anhydride/DIEA = 1/1 was added and stirred for 30 minutes to complete acetylation.

When single-bridging F2 and F6, the acetylated peptide-resin undergoes an olefin metathesis reaction without deprotection to form a hydrocarbon chain connecting bridge. To synthesize a double-bridged hydrocarbon chain-stapled peptide D26 and D1, the peptide resin after synthesis of the first two S5 amino acids is removed and the olefin metathesis reaction is repeated without deprotection to perform the first "staple" reaction. The coupling of the remaining amino acids is continued, and after completion of solid-phase synthesis, the peptide resin is subjected to the above deprotection, acetylation, and second "staple" reaction. The specific process is shown in Figure 1.

The sequenced peptide was then cleaved by adding 10 mL of cleavage buffer (trifluoroacetic acid/ethanedithiol/water/m-methyl ether/benzcresol = 8.25/0.25/0.5/0.5/0.5, v/v) per 0.1 mmol of peptide-resin. The mixture was ice-bathed for 30 minutes and then stirred at room temperature for 3 hours. Finally, anhydrous ether was added to precipitate the peptide, which was then washed and dissolved in a mixture of acetonitrile and water. The crude peptide was transferred to a centrifuge tube and lyophilized to obtain the crude peptide.

A small amount of the crude peptide was dissolved in water and acetonitrile and analyzed using an RP-C8 column (250 mm × 4.6 mm, 5 μm) with a solvent system of water and acetonitrile (Phase A: 0.1% CF ₃ COOH/H ₂ O; Phase B: 1% CF ₃ COOH/90% CH ₃ CN/10% H ₂ O). Fractions corresponding to the main peak were collected and their molecular weights were determined by MALDI-TOF-MS to confirm the correctness of the peptide structure. The sequences and verification results of the generated peptides D26, D26W1, D26W4, D26W5, D26W8, D26W11, D26W12, D26R3, D26R7, D26R10, D26R11, D26R14, D26-2R, D26-3R, D26-4R, D26-5R, D1, F2, and F6 are shown in Table 1 and Figures 2-5 and 10-24. In Table 1, * ^1-4 represent S5, with * ¹ and * ² forming an all-hydrocarbon chain bridge, and * ³ and * ⁴ forming an all-hydrocarbon chain bridge.

Table 1 Polypeptide sequences

For peptide purification, the crude peptide was dissolved in water and acetonitrile, filtered through a 0.45 μm organic filter membrane, and then analyzed and purified using LC-20A high-performance preparative liquid chromatography. The chromatographic column was a C8 preparative column (250 mm × 30 mm, 5 μm) with a binary gradient elution. The solvent system consisted of water and acetonitrile (Phase A: 0.1% _CF₃COOH / _H₂O ; Phase B: 1% _CF₃COOH /90% _CH₃CN /10% _H₂O ). The dual wavelength channels were 210 nm and 254 nm, and the injection volume was 5 mL. Fractions of the target peak were collected and analyzed for purity using reversed-phase HPLC. The purified peptide was then lyophilized. Purification gradient conditions were as follows: 30-50% B (0-5 min); 50-70% B (5-10 min); 70-90% B (10-20 min); 90-100% B (20-30 min); 100% B (30-40 min), flow rate 15 mL/min, detection at 210 nm. Purity was analyzed using the aforementioned RP-C8 column (250 mm × 4.6 mm, 5 μm) with the same mobile phase ratio and the following gradient conditions: 10-50% B (0-5 min); 50-70% B (5-10 min); 70-90% B (10-15 min); 90-100% B (15-20 min); 100% B (25-30 min), flow rate 1 mL/min, detection at 210 nm. HPLC purity was greater than 95.0%.

Example 2. Anti-HIV-1 activity experiment

The infection inhibitory activity of laboratory-adapted HIV-1X4 strain IIIB and HIV-1R5 strain Bal (from NIH AIDS Reagent Program, catalog numbers 398 and 510, respectively) was determined.

50 μL of PBS solution containing 4.9, 19.5, 78.1, 312.5, 1250 and 5000 nM peptides D26, D26W1, D26W4, D26W5, D26W8, D26W11, D26W12, D26R3, D26R7, D26R10, D26R11, D26R14, D26-2R, D26-3R, D26-4R, D26-5R, D1, F2, F6 and F9170 was mixed with 50 μL of approximately 100 times 50% tissue culture infectious dose ( _TCID50 ) of live HIV-1 virus and incubated at 37°C for 30 min. Then, 100 μL of MT- ² cells (for X4 viruses) or CEMx174 5.25M7 cells (for R5 viruses) (MT-2 cells from the NIH AIDS Reagent Program, Catalog No. 237; M7 cells from Sigma-Aldrich, Catalog No. 94022543) at 2 × 10 5 cells/mL were added and incubated overnight at 37°C. The supernatant was replaced with fresh RPMI-1640 medium containing 10% fetal bovine serum (FBS), and the cells were cultured for a further 3 days. 50 μL of the culture supernatant was collected and mixed with an equal volume of 5% (v/v) Triton X-100. The p24 antigen in the final mixture was detected by ELISA. Briefly, these mixtures were added to a plate coated with 5 μg/mL of anti-HIV immunoglobulin (HIV Ig) from the NIH AIDS Reagent Program. Anti-p24 mAb 183 (cultured from the Anti-HIV-1 p24 Hybridoma cell line from the NIH AIDS Reagent Program, Catalog No. 1513), rabbit anti-mouse IgG-HRP (Dako) (Dako Denmark HRP-modified rabbit anti-mouse monoclonal antibody, Catalog No. P0260), and substrate 3,3,5,5-TMB (Sigma-Aldrich, Catalog No. 860336) were added sequentially. Absorbance at 450 nm (A450) was measured using a multi-detection microplate reader (Ultra 384, Tecan). The half-inhibitory concentration was calculated using Calcusyn software (Biosoft), and graphs were plotted using GraphPad software.

The results are shown in Table 2, which demonstrate that D26, D26W1, D26W4, D26W5, D26W8, D26W11, D26W12, D26R3, D26R7, D26R10, D26R11, D26R14, D26-2R, D26-3R, D26-4R, D26-5R, D1, F2, and F6 can effectively inhibit the replication of laboratory-adapted strains HIV-1IIIB and HIV-1Bal, reaching or even exceeding the antiviral activity of F9170.

Table 2 Virus inhibition activity of compounds

Example 3. HIV-1 free virion inactivation experiment

Mix 50 μL of 200-fold TCID50 HIV-1X4 strain IIIB and HIV-1R5 strain Bal with 50 μL of PBS solutions containing peptides D26, D1, F2, F6, and F9170 at concentrations of 4.9, 19.5, 78.1, 312.5, 1250, and 5000 nM, respectively, and incubate at 4°C for 1 hour. Add 15% PEG-6000 at a final concentration of 3% at 25 μL/well and incubate at 4°C for 1 hour. Centrifuge the peptide solution at 4°C, 13,000 rpm/min, for 30 minutes, and discard the supernatant. Resuspend the virus in 3% PEG-6000 containing 10 mg/mL BSA and centrifuge at 4°C, 13,000 rpm/min. Discard any residual supernatant. Repeat this step once. Resuspend the virus in 100 μL/well of serum-free medium and add 3×10 ⁴ /well of the corresponding target cells diluted in complete medium. A cell control (cells only, without virus or drug) and a virus control (drug-free, with virus and corresponding target cells) were also included. Culture the cells in a 37°C, 5% CO 2 incubator. On day 4, collect 50 μL/well of the supernatant and add 50 μL/well of 5% Triton-100 to lyse the virus. The virus inactivation activity of the compounds was determined by detecting p24 antigen in the supernatant using a double-antibody sandwich ELISA.

The results are shown in Table 3, which show that D26, D1, F2, and F6 can effectively inactivate the laboratory-adapted strains HIV-1IIIB and HIV-1Bal, and their activity is comparable to that of F9170.

Table 3 Virus inactivation activity of compounds

Example 4. Determination of half-life in rats

Eighteen Sprague-Dawley rats (female:male ratio = 1:1) (purchased from Vital River Laboratories) were randomly divided into three groups and injected intravenously with 1 mg/kg of D26, D1, F2, F6, D26W1, D26W4, D26W5, D26W8, D26W11, D26W12, or F9170, respectively, based on their body weight. Blood samples (100 μL) were collected from the orbits at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 8 d after injection. The supernatant was collected by centrifugation at 3000 rpm/min at 4°C for 20 min and stored at −20°C. The sample concentrations were determined by LC-MS (Shimadzu, Japan). The half-life of the peptide in mice was calculated using the non-compartmental model module in the software Phoenix WinNonlin (version 8.2, Certara, U.S.A.).

The results are shown in Table 4 and Figure 6, showing that the half-life of F9170 is only 3.89h, while the half-life of D26 in rats is significantly extended to 86.6h. The half-lives of F2 and F6 modified by the single-bridge binding strategy are basically the same as those of F9170. D1, D26W1, D26W4, D26W5, D26W8, D26W11, D26W12, D26R3, D26R7, D26R10, D26R11, D26R14, D26-2R and D26-3R polypeptides, which are modified using the same double-bridge binding strategy as D26, have half-lives that are 2-26 times higher than those of F9170. It reflects the unique advantages of double-bridged stapled peptides such as D26, D1, D26W1, D26W4, D26W5, D26W8, D26W11, D26W12, D26R3, D26R7, D26R10, D26R11, D26R14, D26-2R and D26-3R.

Table 4 In vivo half-life of compounds

Example 5. Rat tissue exposure experiment

Thirty Sprague-Dawley rats were randomly divided into two groups and injected intravenously with 1 mg/kg of D26 or F9170, respectively, based on body weight. Tissues and organs (brain, testis, spleen, small intestine, and cervical lymph nodes) were collected from three mice at 5 minutes, 2 hours, 4 hours, 6 days, and 12 days after injection and stored at -20°C. Tissue homogenates were prepared and the concentrations of D26 or F9170 in the tissues were determined by LC-MS _. The area under the curve (AUC) for D26 and F9170 in each tissue was calculated using the non-compartmental modeling module in Phoenix WinNonlin (version 8.2, Certara, USA).

The results are shown in Table 5 and Figure 7, which show that the AUC of D26 in HIV anatomical reservoirs such as the brain, spleen, small intestine, cervical lymph nodes and testicles is much higher than that of F9170, indicating that D26 can better distribute in the anatomical reservoirs and kill HIV therein.

Table 5 AUC of compounds in various tissues

Example 6. Oral absorption experiment

Twelve SD rats were randomly divided into two groups and administered 50 mg/kg D26 or F9170 by oral gavage. 100 μL of orbital blood samples were collected 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 8 days after oral gavage. The supernatant was centrifuged at 3000 rpm/min at 4°C for 20 minutes and stored at -80°C until the sample concentration was determined by LC-MS.

The results are shown in Figure 8 , which show that no signal was detected in the blood samples of the F9170 group, but a clear signal was detected in the D26 group, which lasted for at least 5 days. This indicates that D26 can be absorbed into the blood through the gastrointestinal tract and has certain oral potential.

Example 7. Safety evaluation

Eighteen 6-8 week old BALB/c mice (purchased from SBEF (Beijing) Biotechnology Co., Ltd.) were randomly divided into three groups and injected intravenously with 20 mg/kg, 100 mg/kg and an equal volume of PBS according to the body weight of the mice. The body weight changes of the mice were monitored at different times after injection, and blood samples were collected from the mice. The serum transaminase and serum creatinine levels in the serum were determined using serum transaminase and serum creatinine assay kits (NJJCBIO).

The results are shown in Figures 9A-C, which show that at doses of 20 mg/kg and 100 mg/kg, there was no significant difference in body weight change, serum transaminase, and serum creatinine levels between the mice and the PBS group, demonstrating the good in vivo safety of D26.

Claims (12)

A polypeptide or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, wherein the polypeptide comprises an amino acid sequence represented by any one of the following general formulas (I) to (IV), or an amino acid sequence represented by any one of the general formulas (I) to (IV) having 1 to 5 conservative substitutions:
X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇ -X ₈ -X ₉ -X ₁₀ -X ₁₁ -X ₁₂ -X ₁₃ -X ₁₄ -W(I),
X ₂ -WEAX ₆ -KX ₉ -LWNX ₁₃ -LQYW(II),
GX ₂ -EALX ₆ -YLWNLLQYW(III),
GWEALKYLX ₉ -NLLX ₁₃ -YW(IV), wherein _X1 represents any amino acid, preferably G or W or an amino acid residue having similar properties thereto; X ₂ , X ₆ , X ₉ , and X ₁₃ are independently unnatural amino acids, and (X ₂ ) and (X ₆ ), (X ₉ ) and (X ₁₃ ) are covalently bonded to each other; _X3 represents any amino acid, preferably E or R or an amino acid residue having similar properties thereto; _X4 represents any amino acid, preferably A or W or an amino acid residue having similar properties thereto; _X5 represents any amino acid, preferably L or W or an amino acid residue having similar properties thereto; X ₇ represents any amino acid, preferably Y or R or an amino acid residue having similar properties thereto; X ₈ represents any amino acid, preferably L or W or an amino acid residue having similar properties thereto; X ₁₀ represents any amino acid, preferably N or R or an amino acid residue having similar properties thereto; X ₁₁ represents any amino acid, preferably L, W or R or an amino acid residue having similar properties thereto; X ₁₂ represents any amino acid, preferably L or W or an amino acid residue having similar properties thereto; X ₁₄ represents any amino acid, preferably Y or R or an amino acid residue having similar properties thereto; The symbol "-" indicates a peptide bond between amino acid residues. The polypeptide according to claim 1, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, wherein the polypeptide comprises an amino acid sequence represented by the following general formula (V), or an amino acid sequence of the general formula (V) having 1-5 conservative substitutions: GX ₂ -X ₃ -ALX ₆ -X ₇ -LX ₉ -X ₁₀ -X ₁₁ -LX ₁₃ -X ₁₄ -W(V), wherein X ₂ , X ₆ , X ₉ , and X ₁₃ are independently non-natural amino acids, and (X ₂ ) and (X ₆ ), and (X ₉ ) and (X ₁₃ ) are covalently bonded; _X3 represents any amino acid, preferably E or R or an amino acid residue having similar properties thereto; X ₇ represents any amino acid, preferably Y or R or an amino acid residue having similar properties thereto; X ₁₀ represents any amino acid, preferably N or R or an amino acid residue having similar properties thereto; X ₁₁ represents any amino acid, preferably L or R or an amino acid residue having similar properties thereto; X ₁₄ represents any amino acid, preferably Y or R or an amino acid residue having similar properties thereto; The symbol "-" indicates a peptide bond between amino acid residues. The polypeptide according to claim 1 or 2, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, Preferably, the non-natural amino acids are independently selected from amino acids with olefin side chains, such as (S)-2-(4'-pentenyl)alanine (S5), (R)-2-(4'-pentenyl)alanine (i.e., R5), (S)-2-(7'-octenyl)alanine (S8), (R)-2-(7'-octenyl)alanine (i.e., R8) and 2-amino-2-(4'-pentenyl)-6-heptenoic acid (i.e., B5), more preferably, the non-natural amino acid is (S)-2-(4'-pentenyl)alanine (i.e., S5); Preferably, the covalent bond connection includes a disulfide bond, a hydrocarbon chain, a molecular lactam bond, a thioether bond, or a covalent bond connection formed by an azide-alkyne cycloaddition reaction. More preferably, the covalent bond connection is a covalent bond connection formed by an olefin metathesis reaction, such as a full hydrocarbon chain connection bridge. The polypeptide according to any one of claims 1 to 3, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixed compound or functionally equivalent variant thereof, wherein the polypeptide comprises the amino acid sequence of any one of the following (a) to (t): (a) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 1; (b) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having 1-5 conservative substitutions to the amino acid sequence of SEQ ID NO: 2, preferably the amino acid sequence A(S5)EAL(S5)YL(S5)NLL(S5)YW as set forth in SEQ ID NO: 2, wherein the first and second S5 positions are covalently bonded, and the third and fourth S5 positions are covalently bonded; (c) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 3; (d) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 10; (e) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having 1-5 conservative substitutions to the amino acid sequence of SEQ ID NO: 11, preferably the amino acid sequence of SEQ ID NO: 11; (f) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 12; (g) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 13; (h) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 14; (i) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 15; (j) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 16; (k) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 17; (l) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 18; (m) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 19; (n) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 20; (o) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 21; (p) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 22; (q) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 23; (r) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 24; (s) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having 1-5 conservative substitutions therefrom, preferably the amino acid sequence of SEQ ID NO: 8; (t) an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having 1-5 conservative substitutions with the amino acid sequence shown in SEQ ID NO: 9, preferably the amino acid sequence shown in SEQ ID NO: 9. The polypeptide or the stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof according to any one of claims 1 to 4 has one or more of the following advantages: simple synthesis, low synthesis cost, oral administration or oral administration potential, antiviral activity that effectively inhibits HIV replication, efficient lysis of HIV virus particles, efficient inactivation of HIV free virus particles, long-lasting effect, effective entry into HIV anatomical reservoirs, and good biosafety; preferably, compared with F9170, the polypeptide or the stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof has an improved half-life, can be orally administered or has oral administration potential, and/or can effectively enter HIV anatomical reservoirs. The polypeptide according to any one of claims 1 to 5, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture, or functionally equivalent variant thereof, wherein the derivative is selected from a derivative modified with maleimide, a derivative with an amino-terminal protecting group attached to the amino terminus and/or a carboxyl-terminal protecting group attached to the carboxyl terminus, a derivative modified with protein or polyethylene glycol, a derivative with an oligopeptide or a lipophilic group or cholesterol attached to the amino terminus and/or the carboxyl terminus, or a derivative obtained by substitution with amino acids having a D-form conformation, artificially modified amino acids, and rare amino acids existing in nature. The polypeptide according to claim 6, or the stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, wherein the amino terminal protecting group is selected from the group consisting of: H, a polymer derived from polyethylene glycol, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted heteroarylalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group and R ₅ -CO-, wherein R ₅ is selected from the group consisting of: H, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted heterocyclic group and a substituted or unsubstituted heteroarylalkyl group, preferably, the amino terminal protecting group is selected from the group consisting of: H, acetyl (Ac), lauroyl, myristoyl and palmitoyl; The carboxyl terminal protecting group is selected from: -NR ₃ R ₄ , -OR ₃ and -SR ₃ , wherein R ₃ and R ₄ are independently selected from: H, a polymer derived from polyethylene glycol, a non-cyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted heteroarylalkyl group, a substituted or unsubstituted aryl group, and a substituted or unsubstituted arylalkyl group. Preferably, the carboxyl terminal protecting group is selected from: -NR ₃ R ₄ and -OR ₃ , wherein R ₃ and R ₄ are independently selected from: H, methyl, ethyl, hexyl, dodecyl and hexadecyl. A pharmaceutical composition, a drug conjugate, a drug conjugate or a fusion protein, wherein the pharmaceutical composition comprises the polypeptide according to any one of claims 1 to 7, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant of the polypeptide, and a pharmaceutically acceptable carrier or excipient, and optionally further comprises one or more other HIV therapeutic agents or HIV latent cell activators; the drug conjugate or drug conjugate comprises the polypeptide according to any one of claims 1 to 7, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant of the polypeptide, The fusion protein comprises a polypeptide according to any one of claims 1 to 7, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, and optionally comprises one or more other proteins for treating or preventing HIV infection, such as an antibody against the intracellular segment of the HIV envelope protein, preferably an antibody against LLP1-GQ. A method for treating or preventing a disease caused by HIV infection, comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide according to any one of claims 1 to 7, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, or the pharmaceutical composition, drug conjugate, drug coupling or fusion protein according to claim 8. The method of claim 9, wherein the polypeptide, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, the pharmaceutical composition, the drug conjugate, the drug coupling or the fusion protein is administered in combination with one or more other HIV therapeutic agents, wherein the one or more other HIV therapeutic agents include reverse transcriptase inhibitors, protease inhibitors, entry inhibitors, integration inhibitors and maturation inhibitors. In vitro methods for any one or more of the following (a)-(e): (a) inhibit HIV infection and/or replication, (b) inactivate free HIV virions, (c) preparing a preparation having in vitro HIV infection inhibitory activity, (d) Eliminate HIV virus from the reservoir or reduce the number of HIV viruses in the reservoir, (e) lysis of HIV virus particles; The method comprises using the polypeptide according to any one of claims 1 to 7, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof. A method for producing the polypeptide according to any one of claims 1 to 7, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, or the fusion protein according to claim 8, comprising chemically synthesizing the polypeptide, or a stereoisomer, derivative, pharmaceutically acceptable salt, mixture or functionally equivalent variant thereof, or the fusion protein.