WO2013127300A1 - Polypeptide for use in inhibiting hiv, pharmaceutical composition of the polypeptide, and use thereof - Google Patents

Abstract

Disclosed are a polypeptide for use in inhibiting the HIV, a pharmaceutical composition comprising the polypeptide, and a use thereof. The polypeptide comprises the sequence as expressed by SEQ ID NO: 8 or by SEQ ID NO: 9. The polypeptide comprise neither a pocket-binding domain nor a lipid membrane-binding domain generally recognized as necessary for high activity polypeptide fusion inhibitors, and the polypeptide is of a reduced length, but the polypeptide exhibits an increased anti-HIV activity.

Description

 Polypeptide for inhibiting H I V, pharmaceutical composition thereof and use thereof

 The present invention belongs to the field of biomedicine and relates to a polypeptide for inhibiting HIV, a pharmaceutical composition thereof and use thereof. Background technique

 Type 1 human immunodeficiency virus (HIV-1) is the causative agent of AIDS. Currently, there are more than 30 million infected people worldwide, causing about 2 million deaths each year, and about 2 million new infected people every year, which is a serious threat to human health.

 HIV-1 infects host cells via its envelope glycoprotein (Env)-mediated viral-cell membrane fusion. Env contains the surface subunit gpl20 and the transmembrane subunit gp41, and three Env forms a non-covalent complex embedded in the surface of the virus. The surface subunit gpl20 is responsible for molecular recognition during viral infection of cells to find and access target cells, while functioning as a stable transmembrane subunit of gp41, and releasing gp41 at appropriate timing to initiate fusion; the transmembrane subunit gp41 is a virus- A direct functional molecule of cell membrane fusion.

 During viral cell fusion, there is a six-helix structure formed by the Gp41 N-terminal helix region (NHR) and the C-terminal helix region (CHR); the formation of this structure provides energy for virus-cell membrane fusion, and the virus-cell fusion is crucial. important. Drugs that block the formation of the helix can effectively inhibit HIV-cell membrane fusion and prevent viral infection and spread in the body, for the treatment of AIDS, known as fusion inhibitors.

The crystal structure shows that in the six helices, three spiral structures formed of NHR constitute the inner core, forming three grooves, and three CHRs are antiparallelly combined in the grooves. Exogenous CHR polypeptides can bind to NHR targets to form an inactive six-helix structure, prevent endogenous active hexafiles, inhibit viral-cell fusion and viral infection, and thus act as fusion inhibitors. The crystal structure reveals that the NHR contains a deeper pocket that has a critical interaction with the corresponding functional region of the CHR. This key binding region of CHR, called the pocket binding region, contains the Try-Try-Ile binding template and is considered to be the key to maintaining high activity peptide fusion inhibitors (Chan, DC, CT Chutkowski, and PS Kim, Proceedings of the National Academy of Sciences of the United States of America, 1998. 95(26): p. 15613-15617. ).

 Typical C-peptide fusion inhibitors include C34 (US 6,150,088) and its improved polypeptides, the first marketed fusion inhibitor T20 (US 5,464,933), and the later discovered CP32 (CN1793170, CN1955190). These C-peptide fusion inhibitors block viral infection by binding to their corresponding NHR targets; typical targets include N36 (US 6,150,088) and DP107 (US 5,656,480), which combine with C34 and CP32 to form a six-helix structure, N36 and DP107, respectively. It contains a common binding pocket and is a popular target for small molecule fusion inhibitors. Regarding the mechanism of action of T20, it is currently believed that the functional region formed by the 8 residues at the C-terminus and the lipid membrane are the key to ensuring the high activity of T20, which is called the lipid membrane binding region (Liu, SW, et al., Journal of Biological Chemistry, 2007. 282(13): p. 9612-9620. ). Since then, it has been generally accepted that high activity polypeptide fusion inhibition must contain at least one of the pocket binding region or the lipid membrane binding region, and some interhelical interactions are added to ensure high activity.

 Despite the availability of the fusion inhibitor T20 and other fusion inhibitors in clinical studies such as Sifuvirtide (CN1334122), the development of fusion inhibitors against resistant viruses has become a top priority due to the rapid emergence of resistant strains. At the same time, since the currently obtained highly active polypeptide fusion inhibitors are polypeptides of about 36 amino acid residues, the synthesis cost is relatively high, so that it is important to obtain a polypeptide having a short sequence and maintaining high anti-HIV activity. Summary of the invention

 Through intensive research and creative labor, the inventors unexpectedly obtained a polypeptide which binds very strongly to NHR, thereby developing a class of highly active fusion inhibitors which have different mechanisms of action from existing fusion inhibitors. Such fusion inhibitors do not contain the recognized pocket binding regions or lipid membrane binding regions necessary to maintain high activity, but maintain high activity through interactions between helical structures. Furthermore, the fusion inhibitor (polypeptide) of the present invention has a length of less than 31 residues and has a significant cost advantage. The following invention is thus provided:

One aspect of the invention relates to an (isolated) polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO: 8 or SEQ ID NO: Amino acid sequence. In the present invention, the inclusion of SEQ ID NO: 8 or SEQ ID NO: 9 means that the sequence of the polypeptide comprises or is SEQ ID NO: 8 or SEQ ID NO: 9 itself. In one embodiment of the invention, the polypeptide does not comprise SEQ ID NO: 3 itself. In one embodiment of the invention, the polypeptide does not comprise SEQ ID NO: 9 itself.

 The polypeptide, derivative or pharmaceutically acceptable salt thereof according to any of the preceding claims, wherein the polypeptide has a length of less than or equal to 31 amino acid residues; preferably, the polypeptide is further greater than or equal to 22 Amino acid. In one embodiment of the invention, the polypeptide has a length of less than or equal to 31 amino acid residues and greater than or equal to 26 amino acids. In one embodiment of the invention, the polypeptide is less than or equal to 29 amino acid residues in length and greater than or equal to 26 amino acids in length. In one embodiment of the invention, the polypeptide has a length of less than or equal to 31 amino acid residues and greater than or equal to 22 amino acids. In one embodiment of the invention, the polypeptide is less than or equal to 29 amino acid residues and greater than or equal to 22 amino acids in length. In one embodiment of the invention, the polypeptide is less than or equal to 26 amino acid residues in length and greater than or equal to 22 amino acids. In one embodiment of the invention, the polypeptide is less than or equal to 31 amino acid residues in length and greater than or equal to 29 amino acids in length. In one embodiment of the invention, the polypeptide is 31, 30, 29, 28, 27, 26, 25, 24, 23 or 22 amino acids in length. Preferably, the polypeptide is 26 amino acids in length.

 A polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof according to any of the invention, wherein the polypeptide is capable of binding to the NHR of HIV-1. Preferably, the sequences of the polypeptides are each included in the CHR of HIV-1.

 The polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof according to any one of the present invention, wherein the amino acid sequence of the polypeptide is as defined in any one of SEQ ID NOS: 1 - 2, 4 - 8, 10 - 12 The sequence is shown. The polypeptide sequences are shown in Table 1 below.

 Table 1: Polypeptide sequences

 SEQ ID

 Amino acid sequence Name NO:

 1 IAEYAARIEALIRAAQEQQEKNEAALREL CP29C

2 IAEYAARIEALIRAAQEQQEKNEAALRELDK CP31C 3 INNYTSLIHSLIEESQNQQEKNEQELLEL CP29CW

4 NYAARIEALIRAAQEQQEKNEAELRELC CP5b

5 NYAARIEALIRAAQEQQEKNEAELREL CP5bl

6 NYAARIEALIRALQELQEKLEAILREL 267229

7 YAARIEALIRAAQEQQEKNEAALREL CP26C

8 IEALIRAAQEQQEKNEAALREL CP22C

9 LIRAAQEQQEKNEAALREL CP19C

10 YAAEIEALIRAAQEQQEKNEAALREL CP26C1

11 IEEYTKKIEELIKKSEEQQKKNEEELKKL CP29CEK

12 IEELIKKSEEQQKKNEEELKKL CP22CEK wherein the polypeptides numbered 3 and 9 in Table 1 are used as control samples in the present invention. Wherein amino acid is an abbreviation having the meanings well-known in the art, for example: W is tryptophan, N is asparagine, A is alanine, S is serine, K is lysine, L is leucine, E is valley Acid, Q is glutamine, I is isoleucine, H is histidine, M is 曱>, T is threonine, D is aspartic acid, R is arginine, Y is Tyrosine and F are phenylalanine.

 The polypeptides in Table 1 are sometimes referred to as polypeptide 1, polypeptide 2, and polypeptide, respectively, in the present invention.

3 polypeptide 12; or respectively referred to as sequence 1, sequence 2, sequence 3 sequence 12 . Wherein, sequence 3 is a native sequence, which means a natural polypeptide sequence taken from HIV-1 gp41 CHR.

 Without limiting the theory, Sequence 4, Sequence 5, Sequence 6, and Sequence 10 are based on Sequence 7, which are obtained by substituting or adding 1 or 2 or 3 amino acids.

 Without limiting the theory, the glutamate-lysine pair in Sequence 11, Sequence 12 forms a salt bridge within the polypeptide chain, which may be beneficial to increase the anti-HIV activity of the polypeptide.

 The polypeptide, derivative or pharmaceutically acceptable salt thereof according to any of the preceding claims, wherein the N-terminus of the polypeptide is linked to an acetyl group, an oligopeptide sequence, or a lipophilic group, and/or

An oligopeptide sequence (for example, EEE, KKK, GQAV, GEEE, etc.) having an amide group, an oligopeptide sequence, for example, 1 to 10 amino acid residues, or a lipophilic group (for example, a fatty acid chain having 3 to 20 carbon atoms) , wherein a fatty acid having 8 to 16 carbon atoms is preferred Chain), cholesterol, etc.

 The polypeptide, derivative or pharmaceutically acceptable salt thereof according to any of the preceding claims, wherein the polypeptide is N-terminally acetylated, and/or C-terminally amidated. Another aspect of the invention relates to a pharmaceutical composition comprising the polypeptide of any one of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof; optionally, the pharmaceutical composition further comprises pharmaceutically acceptable Carrier or excipient. Preferably, the pharmaceutical composition is an injection.

 The pharmaceutical composition of the present invention usually contains 0.1 to 90% by weight of the polypeptide of any one of the present invention, a derivative thereof or a pharmaceutically acceptable salt thereof. Pharmaceutical compositions can be prepared according to methods known in the art. When used for this purpose, the polypeptide of the present invention, a derivative thereof or a pharmaceutically acceptable salt thereof, may be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants, if desired, to be made human. The appropriate form of administration or dosage form will be employed.

The polypeptide of the present invention, a derivative thereof or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present invention may be administered in a unit dosage form, which may be enterally or parenterally, such as orally, muscle, subcutaneous, nasal cavity, Oral mucosa, skin, peritoneum or rectum. Formulations such as tablets, capsules, pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, lyophilized powders Injection, etc. It may be a general preparation, a sustained release preparation, a controlled release preparation, and various microparticle delivery systems. In order to form a unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, a diluent and an absorbent such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid. Aluminum, etc.; wetting agent and binder, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, gum arabic, gelatin paste, sodium carboxymethyl cellulose , shellac, thiol cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents, such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and tannic acid, calcium carbonate, polyoxyethylene, Sorbitol fatty acid ester, sodium dodecyl sulfate, decyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.; absorption promotion Agents such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants such as talc, silica, corn starch, stearates, boric acid, liquid paraffin, Polyethylene glycol and the like. Tablets may also be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer tablets and multilayer tablets. In order to prepare the administration unit into a pellet, various carriers known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin , ethanol, honey, liquid sugar, rice paste or batter; etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecyl sulfate, decyl cellulose, ethyl cellulose, and the like. In order to prepare the drug delivery unit as a suppository, various carriers well known in the art can be widely used. Examples of the carrier are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like. In order to form the administration unit into a capsule, the active ingredient of the polypeptide of the present invention, a derivative thereof or a pharmaceutically acceptable salt thereof is mixed with the above various carriers, and the mixture thus obtained is placed in a hard gelatin or soft. In the plastic bottle. The active ingredient, the polypeptide of the present invention, a derivative thereof or a pharmaceutically acceptable salt thereof can also be formulated into a microinjection, suspended in an aqueous medium to form a suspension, or can be incorporated into a hard capsule or used as an injection. In order to prepare the administration unit into an injection preparation such as a solution, an emulsion, a lyophilized powder injection and a suspension, all diluents conventionally used in the art, for example, water, ethanol, polyethylene glycol, 1, 3 may be used. - propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like. Further, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and a conventional cosolvent, a buffer, a pH adjuster or the like may be added.

 In addition, coloring agents, preservatives, perfumes, flavoring agents, sweeteners or other materials may also be added to the pharmaceutical preparations as needed.

 The dosage of the polypeptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the present invention depends on many factors such as the nature and severity of the disease to be prevented or treated, the sex or age of the patient or animal, Body weight and individual response, the specific active ingredients used, the route of administration and the number of doses administered. The above dosages may be administered in a single dosage form or divided into several, for example two, three or four dosage forms.

The term "composition" as used herein is meant to include a product comprising specified amounts of each of the specified ingredients, as well as any product produced directly or indirectly from a specified amount of each specified combination of ingredients. The actual dosage level of each active ingredient in the pharmaceutical compositions of the present invention can be varied so that the amount of active ingredient obtained is effective to the particular patient, and the composition and mode of administration provide the desired therapeutic response. The dosage level will be selected based on the activity of the particular active ingredient, the route of administration, the severity of the condition being treated, and the condition and past medical history of the patient to be treated. However, it is a practice in the art to dose the active ingredient from a level below that required to achieve the desired therapeutic effect, gradually increasing the dosage until the desired effect is achieved. A further aspect of the invention relates to the use of a polypeptide according to any of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prophylaxis and/or adjuvant treatment of an envelope-like viral infection. Specifically, the envelope virus infection is a disease or AIDS caused by HIV infection.

 A further aspect of the invention relates to a method of treating and/or preventing and/or adjunctively treating an envelope viral infection comprising administering to a subject an effective amount of a polypeptide of any of the invention, a derivative thereof or The step of its pharmaceutically acceptable salt. Specifically, the envelope virus infection is a disease or AIDS caused by HIV infection.

When used in the above therapeutic and/or prophylactic or adjunctive treatment, a therapeutically and/or prophylactically effective amount of a polypeptide of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, may be used in pure form, or as a pharmaceutically acceptable ester or The form of the drug (in the presence of these forms) is applied. Alternatively, it can be administered as a pharmaceutical composition comprising a polypeptide of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients. It will be appreciated, however, that the total daily usage of a polypeptide of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention, is to be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a number of factors, including the disorder being treated and the severity of the disorder; the activity of the particular active ingredient employed; the particular composition employed. The age, weight, general health, sex and diet of the patient; the time of administration, the route of administration and the rate of excretion of the particular active ingredient employed; duration of treatment; in combination with or in combination with the particular active ingredient employed Drugs; and similar factors well known in the medical field. For example, it is the practice in the art to dose the active ingredient starting from a level lower than that required to achieve the desired therapeutic effect, gradually increasing the dosage until the desired effect is achieved. In general, the polypeptide of the present invention, a derivative thereof or a pharmaceutically acceptable salt thereof, for use in mammals, especially humans, may be administered at a dose of from 0.001 to 1000 mg/k body weight per day, for example from 0.01 to 100 mg/k body weight. /day, for example between 0.01 - 10 mg / kg body weight / day.

 The polypeptide of the present invention, a derivative thereof or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present invention can effectively prevent and/or treat and/or adjuvant the treatment of various diseases or diseases of the present invention. A further aspect of the invention relates to the use of a polypeptide according to any of the invention, a derivative thereof or a pharmaceutically acceptable salt thereof, in the preparation or as an HIV fusion inhibitor or an anti-HIV drug.

 A further aspect of the invention relates to a method of inhibiting HIV in vivo or in vitro comprising the step of using an effective amount of a polypeptide of any of the invention, a derivative thereof or a pharmaceutically acceptable salt thereof.

 A further aspect of the invention relates to a method of inhibiting HIV-1 Env-mediated cell fusion in vivo or in vitro comprising the use of an effective amount of a polypeptide of any of the invention, a derivative thereof or a pharmaceutically acceptable salt thereof A step of. A further aspect of the invention relates to a nucleotide sequence encoding a polypeptide of any of the invention.

 A nucleic acid construct comprising the nucleotide sequence of any one of the present invention; in particular, the nucleic acid construct is a recombinant vector; more specifically, the recombinant vector is a recombinant expression vector.

 The invention also relates to a nucleic acid construct comprising a nucleic acid sequence of the invention and one or more regulatory sequences operably linked thereto, which, under compatible conditions, directs the coding sequence in a suitable host cell expression. Expression is understood to include any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

"Nucleic acid constructs" are defined herein as single-stranded or double-stranded nucleic acid molecules that are isolated from a native gene, or modified to contain nucleic acid fragments that are combined and juxtaposed in a non-natural manner. When the nucleic acid construct comprises all of the regulatory sequences necessary for expression of a coding sequence of the invention, The term nucleic acid construct is synonymous with an expression cassette. The term "coding sequence" is defined herein as the portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product. The boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the 5' end of the mRNA reading frame and a transcription termination sequence immediately downstream of the mRNA 3, open reading frame. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

 The isolated nucleic acid sequence encoding the peptide of the present invention can be manipulated in a variety of ways to express the peptide. It may be desirable or necessary to process the nucleic acid sequence prior to insertion into the vector, depending on the expression vector. Techniques for modifying nucleic acid sequences using recombinant DNA methods are well known in the art.

 The term "control sequence" as used herein is defined to include all components necessary or advantageous for expression of a peptide of the invention. Each regulatory sequence may be naturally or foreign to the nucleic acid sequence encoding the polypeptide. Such regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequence should include a promoter and a termination signal for transcription and translation. In order to introduce a specific restriction site to link the regulatory sequence to the coding region of the nucleic acid sequence encoding the polypeptide, a regulatory sequence with a linker can be provided. The term "operably linked" is defined herein as a conformation wherein the regulatory sequences are located at appropriate positions relative to the coding sequence of the DNA sequence such that the regulatory sequences direct expression of the polypeptide.

 The control sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that is recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence that is transcriptionally active in the host cell of choice, including mutated, truncated and heterozygous promoters, which may be derived from extracellular or intracellular encoding homologous or heterologous to the host cell. The gene of the polypeptide.

 The control sequence may also be a suitable transcription termination sequence, i.e., a sequence that is recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that can function in the host cell of choice can be used in the present invention.

The control sequence may also be a suitable leader sequence, an untranslated region of mRNA that is important for translation of the host cell. A leader sequence operably linked to a nucleic acid sequence encoding a polypeptide 5, the end. Any leader sequence that can function in the host cell of choice can be used in the present invention.

 The control sequence may also be a signal peptide coding region which encodes an amino acid sequence linked to the amino terminus of the polypeptide which directs the entry of the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding region of the nucleic acid sequence may naturally contain a signal peptide coding region in which the translational reading frame is naturally joined to the coding region fragment of the secreted polypeptide. Alternatively, the 5' end of the coding region may contain a signal peptide coding region that is foreign to the coding sequence. When the coding sequence does not normally contain a signal peptide coding region, it may be necessary to add a foreign signal peptide coding region. Alternatively, the native signal peptide coding region can be simply replaced with a foreign signal peptide coding region to enhance polypeptide secretion. However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the host cell used can be used in the present invention.

 The control sequence may also be a propeptide coding region which encodes an amino acid sequence at the amino terminus of the polypeptide. The resulting polypeptide is referred to as a zymogen or propolypeptide. A propolypeptide is generally inactive and can be converted to a mature active polypeptide by cleavage of the propeptide from the propolypeptide by catalytic or autocatalytic.

 When there is a signal peptide and a propeptide region at the amino terminus of the polypeptide, the propeptide region is immediately adjacent to the amino terminus of the polypeptide, and the signal peptide region is adjacent to the amino terminus of the peptidomimetic region.

 It may also be desirable to add regulatory sequences that modulate the expression of the polypeptide depending on the growth of the host cell. Examples of regulatory systems are those that respond to chemical or physical stimuli, including in the presence of a regulatory compound, to open or shut down gene expression. Other examples of regulatory sequences are those that enable gene amplification. In these examples, the nucleic acid sequence encoding the polypeptide should be operably linked to a regulatory sequence.

The invention also relates to recombinant expression vectors comprising the nucleic acid sequences, promoters and transcriptional and translational termination signals of the invention. The various nucleic acids and regulatory sequences described above can be joined together to produce a recombinant expression vector which can include one or more convenient restriction sites for insertion or substitution of a nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequences of the invention can be expressed by inserting a nucleic acid sequence or a nucleic acid construct comprising the sequence into a suitable expression vector. Where an expression vector is prepared, the coding sequence can be placed in a vector for operably linked to appropriate expression control sequences. The recombinant expression vector can be any vector (e.g., a plasmid or virus) that facilitates recombinant DNA manipulation and expression of the nucleic acid sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell into which it will be introduced. The vector can be a linear or closed loop plasmid.

 The vector may be an autonomously replicating vector (i.e., a complete structure that exists extrachromosomally and can be replicated independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any mechanism to ensure self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, will integrate into the genome and replicate along with the integrated chromosome. Furthermore, a single vector or plasmid may be employed, or two or more vectors or plasmids, or transposons, which will generally contain all of the DNA that will be introduced into the genome of the host cell.

 Preferably, the vector of the present invention contains one or more selection markers for facilitating selection of transformed cells. A selectable marker is a gene whose product confers resistance to a biocide or virus, resistance to heavy metals, or confers auxotrophic prototrophy and the like. Examples of bacterial selection markers are the dal genes of Bacillus subtilis or Bacillus licheniformis, or the resistance markers of antibiotics such as ampicillin, kanamycin, chloramphenicol or tetracycline.

 Preferably, the vector of the present invention comprises an element which enables stable integration of the vector into the host cell genome, or which ensures that the vector autonomously replicates in the cell independently of the cell genome.

 In the case of autonomous replication, the vector may also contain an origin of replication enabling the vector to replicate autonomously in the host cell of interest. The origin of replication can be mutated to make it temperature sensitive in the host cell.

 More than one copy of the nucleic acid sequence of the invention can be inserted into the host cell to increase the yield of the gene product. An increase in the copy number of the nucleic acid sequence can be accomplished by inserting at least one additional copy of the sequence into the genome of the host cell, or by inserting an amplifiable selectable marker with the nucleic acid sequence, by culturing the cell in the presence of a suitable selection reagent, Cells containing an amplified copy of the selectable marker gene, thereby containing the additional copy nucleic acid sequence, are selected.

The procedures for ligating the above elements to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor , New York, 1989). A further aspect of the invention relates to a recombinant host cell comprising any of the invention The nucleic acid construct.

 The invention also relates to a recombinant host cell comprising a nucleic acid sequence of the invention useful for recombinant production of a polypeptide. A vector comprising the nucleic acid sequence of the present invention can be introduced into a host cell such that the vector is maintained as a chromosomal integrant or a self-replicating extra-chromosomal vector. The term "host cell" encompasses any progeny that differs from the parent cell due to mutations that occur during replication. The choice of host cell depends to a large extent on the polypeptide encoding gene and its source.

 The host cell may be a prokaryotic cell or a eukaryotic cell, such as a bacterium (e.g., an E. coli cell) or a yeast cell. The vector can be introduced into a host cell by techniques well known to those skilled in the art.

 The polypeptide of the present invention can be synthesized by artificial chemical synthesis, or can be expressed by a recombinant host cell, for example, comprising: (a) cultivating a host cell containing the nucleic acid construct under conditions suitable for producing the peptide, the nucleic acid construction The body comprises a nucleic acid sequence encoding the peptide; and (b) the peptide is recovered.

 In the preparation methods of the present invention, cells are cultured in a nutrient medium produced by a suitable polypeptide by methods known in the art. For example, small-scale or large-scale fermentation (including continuous, batch, batchwise or in a shake flask culture, laboratory or industrial fermentor) in a suitable medium, under conditions that permit expression and/or isolation of the polypeptide. Solid state fermentation) to culture cells. The cultivation is carried out in a suitable medium containing carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media may be provided by the supplier or may be prepared with reference to the disclosed compositions (e.g., as described in the catalogue of the American Type Culture Collection). If the polypeptide is secreted into the culture medium, the polypeptide can be recovered directly from the culture medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

 The polypeptide produced can be recovered by methods known in the art. For example, the polypeptide can be recovered from the culture medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

The polypeptides of the invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and Size exclusion chromatography), electrophoresis (eg, preparative isoelectric focusing), Differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE or extraction (see, for example, protein purification, edited by JC Janson and Lars Ryden, VCH Publishers, New York, 1989). In the present invention,

 The term "polypeptide" has its ordinary meaning as is well known to those skilled in the art and also includes derivatives, modifications and the like of the polypeptide.

 The term "HIV fusion inhibitor" includes, but is not limited to, a drug or agent that inhibits HIV (e.g., inhibits HIV proliferation, infection, transmission, etc.) or inhibits HIV-I Env-mediated cell fusion.

 The term "effective amount" includes dosages that can achieve treatment, prevention, alleviation, and/or alleviation of the disease or condition described herein in a subject.

 The term "subject" can refer to a patient or other pharmaceutical composition, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any of the present invention, for the treatment, prevention, alleviation and / or an animal, particularly a mammal, such as a human, a dog, a monkey, a cow, a horse, etc., which alleviates the disease or condition of the invention.

 The term "disease and/or condition" refers to a physical condition of the subject that is associated with the disease and/or condition of the present invention. Advantageous effects of the invention

 The present inventors systematically examined the interaction of HIV-1 gp41 CHR and NHR, and found that the improved 36-amino acid length gp41 CHR polypeptide has a strong interaction with NHR, but its anti-HIV activity is not correspondingly improved. Further research found that this stems from its own aggregation, which reduces the rate of binding to short-term exposed NHR targets (Cai, L., et al., Faseb Journal, 2012. 26.).

The polypeptides involved in the present invention are all less than 30 amino acid residues in length, and the shortest is only 22 peptides, but the activity is comparable to the current clinical use of T20. As a drug, these peptides have a much lower cost of synthesis and thus have a very good development prospect. At the same time, these peptides contain neither the pocket binding region necessary for stabilizing the six-helix structure nor the lipid membrane bound to the phospholipid membrane. The binding region (currently recognized as a high-activity peptide fusion inhibitor must have a pocket-binding region or a lipid-membrane binding region), the mechanism of action is significantly different from the existing fusion inhibitor C36 or T20, and thus is a novel fusion inhibitor. DRAWINGS

 Fig.l: CD spectrum of CHR polypeptide interacting with the corresponding CHR target. among them:

Fig.l (A) is a CD spectrum of interaction between polypeptide 1 and polypeptide N46, wherein the square represents the CD language of polypeptide 4, the circle represents the CD spectrum of polypeptide N46, and the upper triangle N46/4 represents the mixture of polypeptide N46 and polypeptide 4. CD spectrum, the lower triangle N46+4 indicates the superposition of the CD language of the polypeptide N46 and the polypeptide 4 separately scanned;

 Fig.l (B) CD spectrum of polypeptide 2 interacting with polypeptide N46, the meaning of which is similar to that in Fig.l (A);

 Fig.l (C) CD spectrum of interaction between polypeptide 4 and polypeptide N46, the meaning of which is similar to that in Fig.l (A);

 Fig.l (D) CD spectrum of peptide 5 interacting with polypeptide N46, the meaning of which is similar to that in Fig.l (A).

 Fig.l (E) CD spectrum of peptide 7 interacting with polypeptide N46, the meaning of which is similar to that in Fig.l (A).

 Fig.l (F) CD spectrum of peptide 8 interacting with polypeptide N46, the meaning of which is similar to that in Fig.l (A).

 Fig.l (G) The CD language in which peptide 9 interacts with polypeptide N46, and the meaning of the legend is similar to that in Fig.l (A).

 Fig.l (H) CD spectrum of polypeptide 10 interacting with polypeptide N46, the meaning of which is similar to that in Fig.l (A).

 Fig.l (I) CD spectrum of peptide 11 interacting with polypeptide N46, the meaning of which is similar to that in Fig.l (A).

 Fig.l (J) CD spectrum of polypeptide 12 interacting with polypeptide N46, the meaning of which is similar to that in Fig.l (A).

Fig. 2: CHR polypeptide (polypeptide 1, 2, 3, 5, 7, 8, 9, 10) with N38b Interaction of non-denaturing polyacrylamide gel electrophoresis results.

 Fig. 3: Fluorescence resonance energy transfer detection method The results of the interaction of CHR polypeptide 1, 7, 8 with the hydrophobic pocket region of the NHR target. detailed description

 The embodiments of the present invention are described in detail below with reference to the accompanying drawings. If no specific conditions are specified in the examples, they are carried out according to the general conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained commercially. The abbreviations used in the present invention have the following meanings:

 AIDS (Acquired Immure Deficiency Syndrome) AIDS, Acquired Immune Deficiency Syndrome.

 Ala ( Alanine, A ) Alanine

 Arg ( Arginine, R )

 Asn ( Asparagine, ) Asparagine

 Asp ( Asparticacid, D ) aspartic acid

 CD ( Cycurlar Dicroism ) circular dichroism

 DCM ( Dichloromethane ) Dichloromethane

 DMF (Ν,Ν-Dimethyl malonate) Dimercaptoamide

 DMSO dimethyl sulfoxide

 Env ( Envelope glycoprotein ) envelope glycoprotein

 ESI-MS ( Electronic spray ion mass spectroscop )

 Fmoc ( Fluorenylmethoxycarbonyl ) ^Methoxybutylation

 Gly ( Glycine, G ) glycine

 Gin ( Glutamine, Q ) Glutamine

 Glu ( Glutamic acid, E ) glutamic acid

6-HB (six-helix bundle) HBTU 2- ( 1H-1-hydroxybenzotriazole) -1,1,3,3-tetradecyl hexafluorophosphate

 His ( Histidine, H ) histidine

 HoBt ( 1-Hydroxyl benzotiazole anhydrous ) 1-based benzotriazine ITC ( Isothermal Titration Calorimetr ) isothermal titration heat

 NHR ( N-terminal heptad repeat ) N-terminal heptad repeat

 CHR (C-terminal heptad repeat) C-terminal hepta repeat

 HIV ( Human immunodeficiency virus )

 HIV-1 type 1 human immunodeficiency virus

 HPLC (high performance liquid chromatograph)

He ( Isoleucine, I ) Isoleucine

 Leu ( Leucine, L ) Bright Acid

 Met ( Methionine, M ) 曱 曱

 Nal norleucine

 Lys ( Lysine, K ) Lysine

 Phe ( Phenylalanine, F ) Phenylalanine

 Ser ( Serine, S ) Serine

 TFA (Trifluoroacetic acid ) trifluoroacetic acid

 Thr ( Threonie, T ) Threonine

 Tyr ( Tyrosine, Y )

 Val (Valine, V ) proline

 The solid phase synthetic carrier Rink amide resin used in the examples is Tianjin Nankai Synthetic Co., Ltd.; HBTU, HOBt, DIEA and Fmoc protected natural amino acids and D-type unnatural amino acids are products of Shanghai Jill Biochemical Co., Ltd. and Chengdu Nuoxin Technical Co., Ltd. . N-decylpyrrolidone (NMP) is a product of ACROS; TFA is a product of Beijing Bomai Technology Co., Ltd.; DMF and DCM are products of Bomaijie; and chromatographic pure acetonitrile is a product of Fisher Company. Other reagents are domestically produced pure products if they are not described. Example 1: Preparation of polypeptide 1

A standard Fmoc solid phase peptide synthesis method was employed. All polypeptide sequences are in accordance with the polypeptide Conventional synthesis will amidate the C-terminus, N-terminal acetylation (as known to those skilled in the art, these modifications have no fundamental effect on polypeptide activity). Rink Amide resin was selected and the peptide was extended from the C-terminus to the N-terminus. The condensing agent is HBTU/HOBt/DIEA. The deprotecting agent is a piperidine/DMF solution. The lysing agent is TFA, and the crude peptide is dissolved in water and stored in lyophilization. Separation and purification by medium pressure liquid chromatography or HPLC, the pure peptide content is >95%. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) was used to determine the molecular weight of the polypeptide.

 The microwave peptide synthesis conditions are as follows:

 Amino acid: 0.2 M DMF solution; Activator: 0.45 M HBTU/HOBt DMF solution; Activated base: 2M DIEA in NMP solution; Deprotecting agent: 20% v/v piperidine in DMF solution; Blocking reagent: 20% v /v A solution of acetic anhydride in DMF.

 Rink Amide resin 0.5g (0.25 mmol) was weighed into the reactor of CEM microwave peptide synthesizer, and then amino acid, activator, activated alkali, deprotection reagent and blocking reagent were prepared according to the above conditions, and then CEM microwave was used. Automated peptide synthesizer for synthesis. After completion, the peptide resin was washed with DMF for 3 times, then shrunk with anhydrous decyl alcohol, and dried under vacuum at room temperature to obtain 2.05 g of a peptide resin.

 Cleavage of Peptide Resin: The above synthesized peptide resin was weighed and placed in a 250 ml eggplant-shaped flask, ice-cooled, and electromagnetically stirred. The lysate was prepared by adding lg peptide resin to 10 ml (volume ratio: trifluoroacetic acid: ethanediol: m-phenol: water = 95:1:2:2). TFA needs to be cooled in advance by ice bath for 30 minutes or used in the refrigerator in advance. The prepared lysate was added to the peptide resin under ice bath conditions, electromagnetically stirred, the resin turned orange-red, and reacted for 30 minutes under ice bath conditions, then the ice bath was removed, and the reaction was continued at room temperature for 90 minutes to complete the reaction. 200 ml of cold diethyl ether was added to the reactor under vigorous stirring, and a white precipitate was precipitated, and stirring was continued for 30 min. The precipitate was filtered through a G4 sand filter funnel, washed repeatedly with cold diethyl ether for 3 times, and dried. 50 ml of double distilled water and 10 ml of acetonitrile were added to dissolve the solid sufficiently, and the mixture was filtered, and the filtrate was freeze-dried to obtain 1.03 g of a crude peptide.

The crude peptide obtained was purified by medium pressure or high pressure chromatography. The column is a C8 column and the eluent is acetonitrile, water and a small amount of acetic acid. Specific procedure: Weigh the crude peptide lg, add 20ml of water, dissolve 5ml of acetonitrile, centrifuge for 10min at 3000rpm, and take the supernatant for loading. The column was pre-equilibrated with 200 ml of 15% acetonitrile/water/0.1% glacial acetic acid solution. After loading, the mixture was further equilibrated with 200 ml of the same eluent, and the eluent component was detected by HPLC. Gradually increase according to the test results The acetonitrile content is until the purified polypeptide peak is eluted. The same fractions of the eluate were combined, and most of the solvent was removed by rotary evaporation, and the peptide was freeze-dried to obtain a purity of >95% by HPLC.

 The pure peptide was determined by MALDI-TOF-MS mass spectrometry (see Table 2 below).

 Table 2: Molecular Weight and Purity of Polypeptides

Example 2 - 12: Preparation of polypeptide 2 - 12

 The preparation was carried out in accordance with the method of Example 1, and the specific amino acid sequences are shown in Table 1, respectively. Its molecular weight and purity are shown in Table 2 above. The polypeptide samples used in the following Examples 13 - 18 were the polypeptides 1 - 12 synthesized in Examples 1 - 12. Example 13: Anti-HIV-1 cell-cell fusion activity assay of polypeptide

The present inventors performed activity assays on designed polypeptides using a HIV-1 Env mediated cell-cell fusion model. The target cells are TZM-bl cells (US NIH AIDS Reagents and References Project, catalog number 8129), which express CD4 T-cell receptors and chemotaxis on their surface. The factor co-receptors CCR5 and CXCR4 are recognized by HIV-1 Env, and the luciferase reporter gene is also transcribed intracellularly, but does not contain the promoter of the gene, so the luciferase background expression of the individual cells is very low. The effector cells are HL2/3 cells (US NIH AIDS Reagents and References Project, catalog number 1294), which express HIV-1 Env on the surface, attack target cells by Env, complete cell fusion, and also fluoresceinase in cells. The promoter of the reporter gene. Both cells were cultured separately in DMEM containing 10% fetal bovine serum containing ampic/streptomycin double antibody at 37 degrees in an incubator containing 5% CO ₂ . Both cells are adherent cells, which are harvested or passaged after digestion with trypsin/EDTA. Cells were counted using a cell counting plate.

The TZM-bl target cells were adjusted to a concentration of 750,000/ml with a medium, and added to a 96-well cell culture plate (37,500/well) at 50 μl per well, and cultured for 24 hours at 5% C0 ₂ at 37 °C.

 The polypeptide or positive control sample (T20 or C34) was dissolved in phosphate buffered saline (PBS) or dissolved in an appropriate amount of DMSO to determine the polypeptide concentration at 280 nm using an ultraviolet light. The polypeptide solution was then diluted to the appropriate concentration and diluted moderately in a 96-well microtiter plate (Corning).

 Prepare 1.5 million / ml of HL2 / 3 effector cells.

Add 20 μl/well of the diluted inhibitor to the TZM-bl cells cultured the day before, then add 50 μl/well of the prepared HL2/3 effector cells; one of the 96-well cell culture plates was replaced with PBS instead. The agent was used to determine the saturation fusion signal, and the other line was used to replace the HL2/3 cells with DMEM medium for background signal determination. Incubate for 5% C0 ₂ at 37 degrees for 6-8 hours to fully fuse.

 The luciferase reporter gene kit (Promega) was taken out of the refrigerator, and the 5x cell lysate was diluted with double distilled water to lx lysate according to the amount, and placed at room temperature; the substrate was dissolved in the substrate buffer at room temperature. Place; At the same time, set the detection conditions of the microplate reader (Molcular Devices M5) to be set aside.

The fused cells were taken out, the medium was discarded, and washed twice with 200 μl/well PBS, and the washing solution was removed as much as possible; then, the lysate equilibrated to room temperature was added at 50 μl/well, and the cells were fully lysed by gently shaking for 5 minutes; Add lysate to 96μl chemiluminescence at 40μ1/well In the measurement of the plate (Corning), the introduction of bubbles should be avoided as much as possible during the sample loading; the substrate was quickly added to the plate for chemiluminescence at 40 μl/well in the dark, and the chemiluminescence was immediately measured on a microplate reader.

The effectiveness of target cell and effector cell fusion was determined based on the ratio of saturated fusion signal to background signal, with a ratio > 5 indicating efficient fusion. The concentration of the semi-inhibitor (IC ₅ ) was determined from the concentration-chemiluminescence signal curve of the sample, and the IC _{5 of the} positive control sample. The value should be stable within a certain range; in the ideal inhibition curve, the signal under the high concentration inhibitor should be close to the background signal, and the signal at the lowest concentration inhibitor should be close to the saturated fusion signal.

The cell fusion inhibitory activity of polypeptide 1-12 is shown in Table 3. The positive control T20 has an IC ₅₀ of 11.6 ± 4·0ηΜ, which is consistent with the literature 4 (Wild, CT, et al., Proceedings of the National Academy of Sciences of the United States of America, 1994. 91(21): p. 9770-9774. ).

 Table 3: Cellular cell-cell fusion inhibitory activity

The results show that the polypeptide of more than 22 amino acid residues of the present invention has good HIV-1 Env-mediated cell fusion inhibitory activity, and can be used as an HIV fusion inhibitor, and the natural order Columns of SEQ ID NO: 3 and less than 22 residues have poor activity of SEQ ID NO: 9. Example 14: Laboratory Adaptation of Polypeptides to HIV-1 _TTTR Infection Inhibition Experiment

 Laboratory supplies

 Peptides 1 - 9; wherein the polypeptides 1, 2, 7, and 8 with better activity were selected as samples to be tested, and polypeptides 3 and 9 were used as controls.

 The laboratory adapts to the strain HIV-1IIIB (see literature Pan, C., L. Cai, et al. (2009). "Combinations of the First and Next Generations of Human Immunodeficiency Virus (HIV) Fusion Inhibitors Exhibit a Highly Potent Synergistic Effect Against Enfuvirtide-Sensitive and -Resistant HIV Type 1 Strains." Journal of Virology 83(16): 7862-7872. Other available HIV-1 strains can also be used, resulting in similar results to this example)

 2. Experimental method

 The activity of polypeptides 1-9 was examined using a laboratory-adapted virus strain HIV-1IIIB infection inhibition assay. In 200 ul of RPMI1640 medium, 100 TCID50 (50% tissue culture infected amount) of HIVIIIB strain was used to infect 104/ml MT-2 cells, and different inhibitors were added at a concentration gradient. After overnight culture, the culture supernatant was discarded. Replace with fresh medium. On the fourth day after infection, lOOul of the culture supernatant was taken from each well, and an equal volume of 5% Triton X-100 was added, and the p24 antigen was quantitatively detected by ELISA. Specific experimental methods can also be found in Jiang, S., et al., Antimicrobial Agent and Chemotherapy, 2004. 48(11): p. 4349-4359.

 3. Experimental results

 As shown in Table 4 below.

 Table 4: HIV infection inhibitory activity of peptides

ID IC ₅₀ (nM)

 SEQ ID NO 1 3.5±0.2

 SEQ ID NO 2 26±1.6

 SEQ ID NO 3 >2000

SEQ ID NO 7 34 ± 0.9 SEQ ID NO: 8 1160±460

 SEQ ID NO: 9 >2000

 T20 68±22

 The results showed that the HIV-1 inhibitory activities of 1, 2, and 7 were superior to the clinical drug T20, while the activity of 22 peptide 8 was relatively low, and the controls 3 and 9 were inactive, consistent with the cell-cell fusion activity. Example 15: Study of peptide 1-12 and NHR targets by circular dichroism

 Experimental purpose

 The interaction of 1-12 with the gp41 NHR target was studied in circular dichroism (CD).

 2. Experimental materials

 Peptide 1 - 12;

 Using N46 as a target, according to the core crystal structure of HIV-1 gp41, N46 is a component of the six-helix core of HIV-1 gp41 and binds tightly to the six-helix peripheral polypeptide containing polypeptide 3 (Lu, Μ·, et al. Nature Structural Biology (1995) 2(12): 1075-1082), is the core target of fusion inhibitors, and its sequence is as follows:

Ac-TLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGI KQLQARIL-CONH ₂ , (except for C-terminal amidation and N-terminal acetylation, the remaining amino acid sequence is represented as SEQ ID NO: 13)

 For the preparation of the above polypeptide, reference may be made to Pan, C" et al., Journal of Virology, 2009. 83(16): p. 7862-7872.

 The circular dichroic instrument is a Biologic MOS450 spectrometer.

 3. Experimental methods

 The CHR polypeptide was dissolved in PBS, N46 was dissolved in double distilled water, and the concentration was determined by ultraviolet absorption at 280 nm; then 20 μL of the polypeptide PBS solution was prepared.

Formulation of the polypeptide sample to be tested: Mix Ν46 and polypeptide 1-12 in a 1:1 volume ratio to obtain a mixed sample; if it is a separate polypeptide sample, mix 20 μΜ of the sample with the buffer solution at 1:1. The sample was allowed to stand at 37 degrees for 30 minutes to allow sufficient reaction. The above experimental steps ensure that the concentration of the polypeptide in the sample remains the same. The prepared sample is measured on a circular dichroic instrument, and the scanning wavelength range of the instrument is

190-260nm, wavelength interval is lnm, scanning speed is 100nm/min, and scanning is performed 4 times. The buffer is first scanned to obtain a blank, then the sample signal is scanned, and the blank signal is subtracted from the sample signal to obtain a CD signal.

 The interaction between the CHR and NHR peptides before and after mixing was determined by comparing the CD signal changes.

 Further, the inventors also measured the six-spiral stability of 1-12 and N46 by CD temperature scanning. The specific method is as follows: Dilute the sample to 1 μΜ, add to the sample cell, set the CD instrument program to temperature scan, detect the wavelength 220nm, scan range 20-90 degrees Celsius, perform program temperature scanning and check under stirring to get CD signal with temperature curve . A differential is calculated from the curve, and the Tm value is determined based on the peak value of the first differential curve, and the thermal transition temperature of the sample.

 3. Experimental results

 (1) A change in the CD signal indicates an interaction between the two. Changes in CD signal were observed in the mixture of peptides 1-12 and N46, indicating that they all acted on gp41 NHR and inhibited the fusion of HIV and human cells by forming an inactive six-helix structure with NHR (Fig.l, A - J ).

 (2) The Tm values are shown in Table 4 below.

 Table 4: Tm values of peptides

 No. Tm(°C )

 SEQ ID NO: 1 77.0

 SEQ ID NO: 2 79.2

 SEQ ID NO: 3 59.8

 SEQ ID NO: 4 63.4

 SEQ ID NO: 5 60.0

 SEQ ID NO: 7 65.6

 SEQ ID NO: 8 60.6

 SEQ ID NO: 10 67.0

 SEQ ID NO: 11 74.5

SEQ ID NO: 12 46.0 As can be seen from Table 4, the thermostableness of the six-helix formed by the polypeptide of the present invention and N46 is higher than that of the native sequence 3, indicating that these polypeptides can bind to NHR to form a stable structure to prevent the formation of active intermediates of viral proteins, and block the virus- Cell membrane fusion, which is used to treat AIDS by inhibiting viral infection. Example 16: Isothermal titration calorimetry study of peptide 1-12 and NHR targets

 Experimental purpose

 The thermodynamics of the action of peptides 1-12 and N46 were analyzed by isothermal titration calorimetry.

 2. Experimental materials

 Peptide 1 - 12.

 3. Experimental methods

 Isothermal titration calorimetry was performed on a MicroCal iTC200 (GE). The polypeptide 1-12 was dissolved in Tris-Ac buffer, and then dropped into N46 dissolved in exactly the same buffer solution, 2 μl each, at intervals of 120 s, and 20 drops were co- titrated. The thermodynamics and constants used to calculate the combination of the software after the measurement include the combined equivalence ratio N, the binding constant, the molar binding enthalpy, and the molar binding entropy.

 4. Experimental results

 The results of the measurements are shown in Table 5.

 Table 5: Peptide and NHR target data

The results show that the polypeptide of the present invention has a good binding ability to the NHR target. Example 17: Non-denaturing polyacrylamide gel electrophoresis (N-PAGE) study of the interaction of polypeptides 1-12 with NHR targets Experimental purpose

The effects of polypeptides 1-12 and N38b were studied by N-PAGE, which is a polypeptide sequence optimized from N46 according to the crystal structure of HIV-1 _g p41. N38b has one less positive amino acid residue than N46, and it is easier to form a negatively charged complex with our designed peptide for non-denaturing electrophoretic analysis. The amino acid sequence of N38b is given below.

 2. Experimental materials

 Peptide 1 - 12;

 N38b, the sequence is as follows:

 Ac-QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQ - CONH2, (wherein the C-terminal amidation and N-terminal acetylation, the remaining amino acid sequence portion is represented as SEQ ID NO: 14).

 2. Experimental method

 N-PAGE was completed using the BG-Power 3500 Multi-purpose Electrophoresis System (Beijing Baijing Biotechnology Co., Ltd.).

 Peptides 1-12 were dissolved in PBS, N38b was dissolved in double distilled water, and the concentration was determined by ultraviolet absorption at 280 nm; then 200 μM of the polypeptide solution was prepared. N38b and polypeptide 1-12 were mixed in a ratio of 1:1 to obtain a mixture of the two samples; if it is a separate polypeptide sample, a 200 μΜ sample was mixed with the buffer solution at 1:1. The sample was allowed to stand at 37 °C for 30 min to allow sufficient reaction. At the end, an equal volume of N-PAGE 2X loading buffer (Invitrogen) was added to each sample and mixed for use.

 Prepare the separation gel (16%): 30% acrylamide solution 8 ml, 4X separation gel buffer 3.75 ml, water 3.1 ml, 10% AP solution 100 μΐ, TEMED 10 μΐ, shake mixing. Carefully place the above solution between the two glass plates, leaving a space of l-2 cm in the upper layer to fill the concentrated gel, and carefully add water-saturated n-butanol using a syringe. The gel was polymerized in about 30 minutes.

Prepare concentrated gel (4%): Discard the n-butanol layer, carefully wash the upper layer of the gel with deionized water, and blot the water with filter paper. Add 1 ml of 30% acrylamide solution, 2 ml of 4X concentrated gel buffer, 4.8 ml of water, add 100 μM of 10% AP solution, TEMED 10 μΐ, and mix by shaking. Fill the glass plate with the above solution and insert the loading comb. After waiting for the gel polymerization, the sample comb is pulled out and installed according to the description of the electrophoresis device. The upper tank is the negative electrode and the lower tank is the positive electrode. Dilute 10X Tris-Gly running buffer to IX and fill the capacitor. The voltage is about 150V, the current is about 25mA, pre-power, swimming for 20min, turn off the power. Use a buffer to clean the sample tank, use a sample gun or a micro-syringe to gently inject the sample solution into the bottom of the sample tank, turn on the power, start electrophoresis, and complete the electrophoresis in about 2 hours (visible bromophenol blue) The strip is the leading edge and stops after a certain distance from the sample hole.

 The gel was removed and washed three times with double distilled water for 5 minutes each. The gel was covered with BioRad G250 staining solution and stained for 1 hour. The staining solution was discarded and decolorized three times with double distilled water for 10 minutes each. The stained gel is scanned using a flatbed scanner or gel imaging system.

 4. Experimental results

 As shown in Fig. 2.

 The results showed that: the polypeptide sequences 1, 2, 3, 5, 8, and 10 can form a new band with the target peptide N38b, and the peptides 7 and 9 can bind to the N38b to disappear the original C-peptide band, indicating that these polypeptides Both can interact with N38b. Example 18: Fluorescence Resonance Energy Transfer Detection Method for the Interaction of Peptides 1-12 with the Hydrophobic Pockets of NHR Targets

 Experimental purpose

 The interaction of 1-12 with the gp41 NHR target was studied by fluorescence resonance energy transfer detection (FRET).

 2. Experimental materials

 The probe peptide CP2-LY covering the pocket binding region and the target peptide env2.0 binding pair were used to detect the strength of the polypeptide 1-12 to compete for binding to the target peptide. The sequences of CP2-LY and env2.0 are as follows:

Ac-MTWBEWDREIBNYTSLIC-CONH ₂ (CP2, wherein in addition to C-terminal amidation and N-terminal acetylation, the remaining amino acid sequence portion is represented as SEQ ID NO: 15)

Ac- MTWBEWDREIBNYTSLIC(LY)-CONH ₂ (CP2-LY, in which in addition to C-terminal amidation and dye fluorescent iodine acetamide dipotassium salt and N-terminal acetylation, The remaining amino acid sequence portion is represented as SEQ ID NO: 16)

Bpy-GQAVEAQQHLLQLTVWGIKQLQARILAVEKK-CONH ₂ (env2.0, except for C-terminal amidation and N-terminal Bpyylation, the remaining amino acid sequence portion is represented as SEQ ID NO: 17)

 Wherein B is α-aminoisobutyric acid, LY is a dye of Lucifer yellow iodoacetamide dipotassium salt, and Bpy is 2,2 '-dipyridine-5,-carboxylic acid. The preparation method of €?2 ¥ and 61^2.0 can be referred to €31, , 61 31., Antimicrobial Agent and Chemotherapy, 2007. 51(7): p. 2388-2395.

The buffer used in the experiment was a Tris-Ac (pH 7.0) buffer containing 0.01% tween 20. Env2.0 was dissolved in a buffer, and its concentration was determined based on ultraviolet absorption at 290 nm. 2 mM ammonium ferrous sulfate solution was prepared in buffer; env2.0 and ammonium ferrous sulfate solution were mixed in a ratio of 3:1 to prepare Fe (Env2.0) ₃ solution for use. CP2-LY was dissolved in a buffer and its concentration was determined based on ultraviolet absorption at 425 nm.

 3. Experimental methods

 Different sample solutions were mixed in a 96-well round bottom plate (Costar 3799, Corning Incorporation, USA) and transferred to a 384-well black plate (Greiner Bio-one 781096) using a Spectra Max M5 seed meter (Molecular Devices, USA ) , the fluorescence signal intensity was measured at 540 nm of the excitation light at 425 nm.

Determination of the binding strength (Kd) of CP2-LY and env2.0: 10 μL of CP 1-LY solution containing 10 μL of buffer and 1 μM per well in a 384-well plate and a starting concentration of 100 μΜ 10 μL of a 2-fold diluted Fe(Env2.0) ₃ solution. Repeat the test three times.

Peptide competition for binding to target peptide intensity (Ki): 10 μL of 1 μM CP2-LY solution per well in a 384-well plate, 10 μM Fe(Env2.0) ₃ solution 10 μ L, 10 μL of the stepwise 2-fold dilution of the inhibitor polypeptide at a starting concentration of 100 μΜ. Using CP2 as a control, each sample was tested in triplicate.

 4. Experimental results

 As shown in Fig. 3.

The results showed that, unlike the control polypeptide CP2, the peptides 1, 7, and 8 showed no inhibition on the binding of CP2-LY and env2.0. Because the combination of CP2-LY and env2.0 is mainly generation The interaction between the NHR-CHR of the pocket binding region is shown, thus demonstrating that the active site of action of polypeptides 1, 7 and 8 is not a hydrophobic pocket region on the NHR.

Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations may be made to those details in accordance with the teachings of the invention, which are within the scope of the invention. The full scope of the invention is indicated by the appended claims and any equivalents thereof.

Claims

Rights request

An isolated polypeptide, a derivative thereof or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

The polypeptide, the derivative thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein the polypeptide has a length of less than or equal to 31 amino acid residues; preferably, the polypeptide has a length greater than or equal to 22 amino acids.

The polypeptide, the derivative thereof, or a pharmaceutically acceptable salt thereof, according to claim 2, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 1 - 2, 4 - 8, 10 - 12, respectively. The sequence is shown.

The polypeptide, derivative or pharmaceutically acceptable salt thereof according to claim 3, wherein the N-terminus of the polypeptide is linked to an acetyl group, an oligopeptide sequence, or a lipophilic group, and/or

 The C-terminus is attached to an amide group, an oligopeptide sequence, or a lipophilic group.

The polypeptide, a derivative thereof or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the polypeptide is N-terminally acetylated, and/or C-terminally amidated.

6. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 5, a derivative thereof, or a pharmaceutically acceptable salt thereof; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable Carrier or excipient.

7. Use of a polypeptide according to any one of claims 1 to 5, a derivative thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prophylaxis and/or adjuvant treatment of an enveloped viral infection; The envelope virus infection is a disease or AIDS caused by HIV infection.

The polypeptide according to any one of claims 1 to 5, a derivative thereof or a pharmaceutically acceptable salt thereof Use in the preparation or as an HIV fusion inhibitor or anti-HIV drug.

A method of inhibiting HIV in vivo or in vitro, comprising the step of using an effective amount of the polypeptide of any one of claims 1 to 5, a derivative thereof, or a pharmaceutically acceptable salt thereof.

A method of inhibiting HIV-1 Env-mediated cell fusion in vivo or in vitro, comprising using an effective amount of the polypeptide of any one of claims 1 to 5, a derivative thereof, or a pharmaceutically acceptable salt thereof step.

11. A nucleotide sequence encoding the polypeptide of any one of claims 1 to 3.

A nucleic acid construct comprising the nucleotide sequence of claim 11; specifically, the nucleic acid construct is a recombinant vector; more specifically, the recombinant vector is a recombinant expression vector.

13. A recombinant host cell comprising the nucleic acid construct of claim 12.

14. A method of treating and/or preventing and/or adjunctively treating an enveloped viral infection, comprising administering to a subject an effective amount of the polypeptide of any one of claims 1 to 5, a derivative thereof, or The step of pharmaceutically acceptable salt; specifically, the envelope virus infection is a disease or AIDS caused by HIV infection.