WO2013127299A1 - 用于抑制hiv的多肽及其作用靶点 - Google Patents

用于抑制hiv的多肽及其作用靶点 Download PDF

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Publication number
WO2013127299A1
WO2013127299A1 PCT/CN2013/071625 CN2013071625W WO2013127299A1 WO 2013127299 A1 WO2013127299 A1 WO 2013127299A1 CN 2013071625 W CN2013071625 W CN 2013071625W WO 2013127299 A1 WO2013127299 A1 WO 2013127299A1
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Prior art keywords
polypeptide
hiv
pharmaceutically acceptable
derivative
acceptable salt
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PCT/CN2013/071625
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English (en)
French (fr)
Inventor
蔡利锋
刘克良
郑保华
王昆
姜喜凤
贾启燕
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中国人民解放军军事医学科学院毒物药物研究所
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Application filed by 中国人民解放军军事医学科学院毒物药物研究所 filed Critical 中国人民解放军军事医学科学院毒物药物研究所
Priority to CN201380004014.4A priority Critical patent/CN104039815A/zh
Publication of WO2013127299A1 publication Critical patent/WO2013127299A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C07K14/155—Lentiviridae, e.g. human immunodeficiency virus [HIV], visna-maedi virus or equine infectious anaemia virus
    • C07K14/16—HIV-1 ; HIV-2
    • C07K14/162—HIV-1 ; HIV-2 env, e.g. gp160, gp110/120, gp41, V3, peptid T, CD4-Binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Definitions

  • the present invention belongs to the field of biomedicine and relates to a polypeptide for inhibiting HIV and a target thereof. Background technique
  • Type 1 human immunodeficiency virus (HIV-1) is the causative agent of AIDS.
  • HIV-1 Type 1 human immunodeficiency virus
  • Env contains the surface subunit gpl20 and the transmembrane subunit gp41, and the three Env form 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.
  • NHR Gp41 N-terminal helix region
  • CHR C-terminal helix region
  • 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.
  • the exogenous CHR polypeptide binds to the NHR target to form an inactive six-helix structure, prevents endogenous active hexafiles from forming, inhibits viral-cell fusion and viral infection, and thus acts as a fusion inhibitor.
  • 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 contains the Try-Try-I le (WWI) 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 Nat iona l Academy of Sc iences of the Uni ted States of Amer ica, 1998. 95 (26) : p. 15613-15617. ).
  • WWI Try-Try-I le
  • 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 later discovered CP32.
  • C-peptide fusion inhibitors block viral infection by binding to their corresponding NHR targets; typical targets include N36 (US 6, 150, 088) and DP107
  • N36 and DP107 contain a common binding pocket and are also popular targets for small molecule fusion inhibitors.
  • fusion inhibitors do not contain a recognized pocket binding region necessary for maintaining high activity, and their sequences are different from existing fusion inhibitors T20, C34, CP32, etc., and are capable of inhibiting HIV virus, especially for existing ones. Fusion inhibitors produce drug-resistant HIV strains that have potential as anti-HIV-infected drugs.
  • the invention thus provided provides:
  • the invention according to one aspect of the invention relates to an (isolated) polypeptide, a derivative thereof or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises 11 amino acids in direct contact with the NHR of HIV-1 Residues, and two of the 11 amino acid residues are located at the N-terminus and C-terminus of the polypeptide, respectively.
  • direct contact herein refers to a polypeptide and HIV-1.
  • the spatial extension direction of the 11 amino acid residues is directed to NHR, and/or the amino acid residue directly interacts with the corresponding residue of NHR through hydrophobic interaction, electrostatic interaction or hydrogen bonding, Key sites for drug action (Chan, DC, CT Chutkowski, and PS Kim, Proceedings of the Nationala l Academy of Sc iences of the Uni ted States of Amer ica, 1998. 95 (26): p. 15613-15617. ). See Fig.l, where the shaded parts of the gray show these key points.
  • polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof according to any one of the present invention, wherein the sequence of the polypeptide is contained in the CHR of HIV-1; preferably, the polypeptide is 36 amino acids in length Residues.
  • polypeptide, derivative or pharmaceutically acceptable salt thereof according to any of the preceding claims, wherein the amino acid sequence of the polypeptide comprises WMEWDREI (SEQ ID NO: 24) or WASLWNWF (SEQ ID NO: 25); preferably The WMEWDREI or WASLWNWF is neither located at the N-terminus of the polypeptide nor at the C-terminus of the polypeptide.
  • sequences of the polypeptides are each contained in the CHR of HIV-1; more preferably, the sequences of the polypeptides are each included in SEQ ID NO: 27 below; in particular, contained in HIV-1 Among the 611 - 677 amino acids of CHR.
  • 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 shown in any one of SEQ ID NO: 1 to SEQ ID NO: 12, respectively.
  • the polypeptide sequences are shown in Table 1 below.
  • 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 methionine, T is threonine, D is aspartic acid, R is arginine, Y is Tyrosine and F are phenylalanine.
  • polypeptides 1-12 in Table 1 are also referred to as polypeptide 1 and polypeptide 2 polypeptides in the present invention.
  • CHR polypeptide 12 alternatively referred to as CHR polypeptide 1, CHR polypeptide 2 CHR polypeptide 12.
  • 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 [SEQ ID NO: 28], GEEE [SEQ ID NO: 29], etc.) having an amide group or an oligopeptide sequence such as 1 to 10 amino acid residues at the C-terminus, or lipophilicity A group (for example, a fatty acid chain having 3 to 20 carbon atoms, preferably a fatty acid chain having 8 to 16 carbon atoms), cholesterol, or the like.
  • EEE, KKK, GQAV [SEQ ID NO: 28], GEEE [SEQ ID NO: 29], etc. having an amide group or an oligopeptide sequence such as 1 to 10 amino acid residues at the C-terminus, or lipophilicity A group (for example, a fatty acid chain having 3 to 20 carbon atoms, preferably a fatty acid chain having 8 to 16 carbon atoms), cholesterol, or the like.
  • the N-terminus of the polypeptide is acetylated, and/or the C-terminal amidated.
  • Example 24 and Example 25 demonstrate that polypeptides 1-12 have high anti-HIV fusion inhibitor activity, indicating that HIV-1 gp41 CHR and its adjacent sequences are continuously present with highly active polypeptides, which are present in recognized CHRs.
  • An isolated high activity polypeptide sequence is an innovation.
  • 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.
  • 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.
  • the pharmaceutical composition can be according to methods known in the art. Method preparation.
  • 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.
  • 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.
  • a carrier 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.
  • 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.
  • 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, silic
  • 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.
  • various carriers known in the art can be widely used.
  • the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Ge luc i re, kaolin, talc, etc.; binders such as gum arabic, jaundice Glue, gelatin, ethanol, honey, liquid sugar, rice paste or batter; etc.; disintegrants, such as agar powder, dried starch, alginate, sodium dodecyl sulfate, decyl cellulose, ethyl cellulose, etc.
  • the drug delivery unit as a suppository, various carriers well known in the art can be widely used.
  • carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohols. Ester, gelatin, semi-synthetic glycerides, and the like.
  • 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.
  • an injectable preparation such as a solution, an emulsion, a lyophilized powder, and a suspension
  • all diluents conventionally used in the art for example, water, ethanol, polyethylene glycol, 1, 3 can be used.
  • - propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like can be used.
  • 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.
  • coloring agents 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.
  • composition as used herein is intended to include a product comprising specified quantities of each of the specified ingredients, as well as any product that results, directly or indirectly, from the specified combination of the specified ingredients.
  • 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 a polypeptide, a derivative thereof or a pharmaceutically acceptable salt thereof according to any of the invention for use in the manufacture of a medicament for the treatment and/or prophylaxis and/or adjuvant treatment of enveloped viral infections
  • 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.
  • the envelope virus infection is a disease or AIDS caused by HIV infection.
  • 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 may 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.
  • a polypeptide 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.
  • the dosage of the polypeptide of the present invention, or a derivative thereof, or a pharmaceutically acceptable salt thereof may be used in a mammal, especially a human.
  • Mg / kg body weight / day for example between 0. 01 - 10 mg / kg body weight / day.
  • polypeptide of the present invention can effectively prevent and/or treat and/or adjuvant the treatment of various diseases or conditions described herein.
  • 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 an (isolated) polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof, wherein the polypeptide has an amino acid sequence of any one of SEQ ID NO: 13 to SEQ ID NO: 23, respectively Shown.
  • polypeptide sequences are shown in Table 2 below.
  • 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 methionine, T is threonine, D is aspartic acid, R is arginine, Y is Tyrosine, F is phenylalanine, G is glycine Acid and V are proline.
  • polypeptides 13-23 in Table 2 are also referred to herein simply as polypeptide 13, polypeptide 14 polypeptide 23; or leg polypeptide 13, leg polypeptide 14 leg polypeptide 23.
  • NHR polypeptide sequences corresponding to sequences 1-12 for determining the binding target of the polypeptides in sequences 1-12.
  • the specific design is shown in Fig. 2, and the sequence of the polypeptide to which it is bound is listed in the grey font.
  • LAVERYLKDQQLLGIWGIS SEQ ID NO: 26
  • WTMHNWIQELSKNSWSANW 611 (SEQ ID NO: 27, in order to clearly show the binding relationship with the leg, the right end of the sequence 27 is the N-terminus, and the sequence direction should be read from the right-end w marked 611).
  • sequences are the interaction relationship between HIV-1 gp41 NHR and CHR estimated based on the existing crystal structure, and the underlined portion is a polypeptide sequence having an crystal structure.
  • the polypeptide 1-12 binds to the corresponding sequence in the polypeptide 13-23 to form a six-helix structure, indicating that the entire HIV-1 gp41 NHR sequence can be referred to as a target of a highly active fusion inhibitor, which is a recognized NHR formation.
  • the pocket is a combination of high activity fusion inhibitors and a prominent innovation.
  • a further aspect of the invention relates to the use of any one or more of the polypeptides 13-23 for screening for the treatment and/or prevention and/or adjuvant treatment of an enveloped virus-infected medicament; in particular, the enveloped viral infection A disease or AIDS caused by HIV infection.
  • the screening refers to screening as a target.
  • a further aspect of the invention relates to the use of any one or more of the polypeptides 13-23 for screening HIV fusion inhibitors or anti-HIV drugs.
  • the screening refers to screening as a target.
  • the two can serve as targets and ligands, thus exchanging the functions of the polypeptides 1-12 and 13-23 as targets and inhibitors.
  • designing targets on the basis of polypeptides 1-12, modifying polypeptides 13-23 to obtain highly active fusion inhibitors, and designing and developing drugs are also included in the design spirit of the present invention.
  • a further aspect of the invention relates to a nucleotide sequence encoding a polypeptide of any of the invention.
  • 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 an unnatural manner.
  • nucleic acid construct is synonymous with an expression cassette when the nucleic acid construct comprises all of the regulatory sequences necessary for expression of a coding sequence of the invention.
  • 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 open reading frame of the mRNA and a transcription termination sequence immediately downstream of the open reading frame of the 3' end of the mRNA.
  • 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.
  • control sequence is defined to include all components necessary or advantageous for expression of a peptide of the invention.
  • Each regulatory sequence may be naturally contained for the nucleic acid sequence encoding the polypeptide Or foreign.
  • These 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.
  • the regulatory sequences include promoters as well as termination signals for transcription and translation.
  • a linker-containing regulatory sequence can be provided 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.
  • linker-containing regulatory sequence can be provided 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.
  • operably linked is defined herein as a conformation wherein the regulatory sequences are located at appropriate positions relative to the coding sequence of the DM sequence such that the regulatory sequence
  • 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 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
  • 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, i.e., an mRNA untranslated region that is important for translation of the host cell.
  • the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. 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.
  • 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.
  • the native signal peptide coding region can be simply replaced with a foreign signal peptide coding region to enhance polypeptide secretion.
  • 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 a region located at the amino terminus of the polypeptide. Segment amino acid sequence.
  • 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.
  • 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.
  • regulatory sequences that modulate the expression of the polypeptide depending on the growth of the host cell.
  • regulatory systems are those that respond to chemical or physical stimuli, including in the presence of regulatory compounds, to open or shut down gene expression.
  • Other examples of regulatory sequences are those that enable gene amplification.
  • 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 prepare 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.
  • the nucleic acid sequence of the present invention can be expressed by inserting a nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate expression vector.
  • 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 DM 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.
  • the vector is a vector that, when introduced into a host cell, will integrate into the genome and replicate along with the integrated chromosome.
  • 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.
  • the vector of the invention contains one or more selectable 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.
  • Example of bacterial selection marker For example, the da l gene of Bacillus subtilis or Bacillus licheniformis, or the resistance marker of antibiotics such as ampicillin, kanamycin, chloramphenicol or tetracycline.
  • 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.
  • 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.
  • a further aspect of the invention relates to a recombinant host cell, which comprises the nucleic acid construct of any of the invention.
  • 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.
  • 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.
  • a bacterium e.g., an E. coli cell
  • yeast cell e.g., 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 recombinant host cells, for example, including: (a) under conditions suitable for the production of the peptide, culture containing A host cell having a nucleic acid construct comprising a nucleic acid sequence encoding the peptide; and (b) recovering the peptide.
  • 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.
  • 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.
  • 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 (eg ammonium sulfate precipitation), SDS-PAGE or extraction (see, eg, protein purification, edited by JC Janson and Lars Ryden, VCH Publ i shers, New York, 1989).
  • chromatography e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and Size exclusion chromatography
  • electrophoresis eg, preparative isoelectric focusing
  • differential solubility eg ammonium sulfate precipitation
  • SDS-PAGE or extraction see, eg, protein purification, edited by JC
  • 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.
  • leg or "HIV-1 leg” specifically refers to HIV-1 gp41 NHR; the term “CHR” or “HIV-1 CHR” specifically refers to HIV-1 gp41 CHR.
  • HIV fusion inhibitor includes, but is not limited to, a drug or agent that inhibits HIV (eg, inhibits HIV proliferation, infection, spread, etc.) or inhibits HIV-1 Env-mediated cell fusion.
  • effective amount includes a dose that can achieve treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
  • subject can refer to a patient or other polypeptide, derivative or pharmaceutically acceptable salt thereof according to any of the invention or a pharmaceutical composition according to any of the 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.
  • 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.
  • the polypeptide of the present invention is a novel anti-HIV infection polypeptide different from the existing fusion inhibitors T20, C34, CP32 and the like, has good anti-HIV fusion activity, and can inhibit HIV virus, especially resistance to existing fusion inhibitors.
  • Fig. 1 The CHR polypeptide sequence of the present invention, wherein the shaded portion is an amino acid residue in the CHR that is in direct contact with the NHR, and is a key site for drug action.
  • Fig. 2 Design of the NHR target sequence.
  • the shaded portion is the CHR polypeptide with which it acts.
  • the CHR polypeptide sequence is represented by an inverted sequence based on the antiparallel characteristics of the combination of the two.
  • Fig. 3 Characterization of CD and N-PAGE results of CHR polypeptide 2 interaction with NHR polypeptides, where:
  • Fig. 3 (A) is a CD spectrum of interaction between polypeptide 2 and polypeptide 22, wherein the square represents the CD spectrum of polypeptide 2, the circle represents the CD spectrum of polypeptide 22, and the upper triangle 2/22 represents the mixture of polypeptide 22 and polypeptide 2.
  • CD spectrum, lower triangle 2+22 indicates CD language superposition of polypeptide 22 and polypeptide 2 alone;
  • Fig. 3 (B) CD spectrum of interaction between polypeptide 2 and polypeptide 21, wherein the meaning of the legend is similar to that in Fig. 3 (A);
  • Fig. 3 (C) CD spectrum of interaction between polypeptide 2 and polypeptide 20, the meaning of which is similar to that in Fig. 3 (A);
  • Fig. 3 (D) CD spectrum of interaction between polypeptide 2 and polypeptide 19, the meaning of which is similar to that in Fig. 3 (A);
  • Fig. 3 (E) CD spectrum of interaction between polypeptide 2 and polypeptide 17, wherein the meaning of the legend is similar to that in Fig. 3 (A);
  • Fig. 4 Characterization of the CD spectrum and N-PAGE results of CHR polypeptide 3 interaction with NHR polypeptides, where:
  • Fig. 4 (A) is a CD spectrum of interaction between polypeptide 3 and polypeptide 21, wherein the square represents the CD spectrum of polypeptide 3, the circle ⁇ represents the CD spectrum of polypeptide 21, and the upper triangle 2/21 represents the mixture of polypeptide 21 and polypeptide 3.
  • CD spectrum, lower triangle 2+21 indicates CD language superposition of polypeptide 21 and polypeptide 3 alone;
  • Fig. 4 (B) CD spectrum of interaction between polypeptide 3 and polypeptide 20, wherein the meaning of the legend is similar to that in Fig. 4 (A);
  • Fig. 4 (C) CD spectrum of interaction between polypeptide 3 and polypeptide 19, wherein the meaning of the legend is similar to that in Fig. 4 (A);
  • Fig. 4 (D) CD spectrum of interaction between polypeptide 3 and polypeptide 18, wherein the meaning of the legend is similar to that in Fig. 4 (A);
  • Fig. 4 (E) CD spectrum of interaction between polypeptide 3 and polypeptide 17, wherein the meaning of the legend is similar to that in Fig. 4 (A);
  • Fig. 5 Characterization of the CD spectrum and N-PAGE results of the interaction of CHR polypeptide 4 with NHR polypeptides, wherein:
  • Fig. 5 (A) is a CD spectrum of interaction between polypeptide 4 and polypeptide 21, wherein the square represents the CD spectrum of polypeptide 4, the circle represents the CD spectrum of polypeptide 21, and the upper triangle 2/21 represents the mixture of polypeptide 21 and polypeptide 4. CD spectrum, lower triangle 2+21 indicates CD language superposition of polypeptide 21 and polypeptide 4 alone;
  • Fig. 5 (B) CD spectrum of interaction between polypeptide 4 and polypeptide 20, wherein the meaning of the legend is similar to that in Fig. 5 (A);
  • Fig. 5 (C) CD spectrum of interaction between polypeptide 4 and polypeptide 19, including the legend Meaning is similar to that in Fig. 5 (A);
  • Fig. 5 (D) CD spectrum of interaction between polypeptide 4 and polypeptide 18, wherein the meaning of the legend is similar to that in Fig. 5 (A);
  • Fig. 5 (E) CD spectrum of interaction between polypeptide 4 and polypeptide 17, wherein the meaning of the legend is similar to that in Fig. 5 (A);
  • Fig. 6 Characterization of the CD spectrum and N-PAGE results of CHR polypeptide 5 interaction with NHR polypeptides, where:
  • Fig. 6 (A) is a CD spectrum of interaction between polypeptide 5 and polypeptide 21, wherein the square represents the CD spectrum of polypeptide 5, the circle ⁇ represents the CD spectrum of polypeptide 21, and the upper triangle 2/21 represents the mixture of polypeptide 21 and polypeptide 5.
  • CD spectrum, lower triangle 2+21 indicates CD language superposition of polypeptide 21 and polypeptide 5 alone;
  • Fig. 6 (B) CD spectrum of interaction between polypeptide 5 and polypeptide 20, wherein the meaning of the legend is similar to that in Fig. 6 (A);
  • Fig. 6 (C) CD spectrum of interaction between polypeptide 5 and polypeptide 19, wherein the meaning of the legend is similar to that in Fig. 6 (A);
  • Fig. 6 (D) CD spectrum of interaction between polypeptide 5 and polypeptide 18, wherein the meaning of the legend is similar to that in Fig. 6 (A);
  • Fig. 6 (E) CD spectrum of interaction between polypeptide 5 and polypeptide 17, wherein the meaning of the legend is similar to that in Fig. 6 (A);
  • Fig. 7 Characterization of the CD spectrum and N-PAGE results of CHR polypeptide 6 interaction with NHR polypeptides, where:
  • Fig. 7 (A) is a CD spectrum of interaction between polypeptide 6 and polypeptide 19, wherein the square represents the CD spectrum of polypeptide 6, the circle represents the CD spectrum of polypeptide 19, and the upper triangle 6 VIII represents the mixture of polypeptide 6 and polypeptide 19.
  • CD spectrum, lower triangle 6+19 indicates superposition of CD language of polypeptide 6 and polypeptide 19 alone;
  • Fig. 7 (B) CD spectrum of interaction between polypeptide 6 and polypeptide 18, the meaning of which is similar to that in Fig. 7 (A);
  • Fig. 7 (C) CD spectrum of interaction between polypeptide 6 and polypeptide 17, wherein the meaning of the legend is similar to that in Fig. 7 (A);
  • Fig. 7 (D) CD spectrum of interaction between polypeptide 6 and polypeptide 16, wherein the meaning of the legend is similar to that in Fig. 7 (A);
  • Fig. 7 (E) CD spectrum of interaction between polypeptide 6 and polypeptide 15, wherein the meaning of the legend is similar to that in Fig. 7 (A);
  • Fig. 8 Characterization of the CD spectrum and N-PAGE results of the interaction of CHR polypeptide 3 with NHR polypeptide, wherein:
  • Fig. 8 (A) is a CD spectrum of interaction between polypeptide 7 and polypeptide 17, wherein the square represents the CD spectrum of polypeptide 7, the circle represents the CD spectrum of polypeptide 17, and the upper triangle 7 VIII represents the mixture of polypeptide 7 and polypeptide 17.
  • CD spectrum, lower triangle 7+17 indicates superposition of CD language of polypeptide 7 and polypeptide 17 alone;
  • Fig. 8 (B) CD spectrum of interaction between polypeptide 7 and polypeptide 16, wherein the meaning of the legend is similar to that in Fig. 8 (A);
  • Fig. 8 (C) CD spectrum of interaction between polypeptide 7 and polypeptide 15, wherein the meaning of the legend is similar to that in Fig. 8 (A);
  • AIDS Acquired Immure Deficiency Syndrome
  • AIDS Acquired Immune Deficiency Syndrome.
  • Ala Alanine, A
  • Env envelope glycoprotein
  • HIV Human immunodeficiency virus
  • Va l (Va l ine, V )
  • the solid phase synthesis carrier Rink amide resin used in the valine example is Tianjin Nankai Synthetic Co., Ltd.; HBTU, H0Bt, DIEA and Fmoc protected natural amino acids and D-type unnatural amino acids are Shanghai Jill Biochemical Company and Chengdu Nuoxin Technology Co., Ltd. products.
  • N-decylpyrrolidone (NMP) is a product of ACR0S; TFA is a product of Beijing Bomai Technology Co., Ltd.; DMF and DCM are products of South Korea's Samsung; and chromatographic pure acetonitrile is Fi sher. 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 acylated at the C-terminus and acetylated at the N-terminus (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/H0Bt/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, pure peptide content > 95%.
  • the matrix-assisted laser resolved time-of-flight mass spectrometry (MALDI-T0F-MS) was used to determine the molecular weight of the peptide.
  • microwave peptide synthesis conditions are as follows:
  • Amino acid 0. 2 M of DMF solution; Activator: 0. 45 M HBTU/HOBt in DMF solution; Activated base: 2M DIEA in NMP solution; Deprotecting agent: 20% v/v piperidine in DMF solution; 20% v/v acetic anhydride in DMF solution.
  • the Rink Amide resin was weighed in a reactor of a CEM microwave polypeptide synthesizer, and then the amino acid, the activator, the activated base, the deprotecting reagent, and the blocking reagent were prepared according to the above conditions.
  • the synthesis was carried out using a CEM microwave automatic peptide synthesizer. After completion, the peptide resin was washed 3 times with DMF, then shrunk with anhydrous decyl alcohol, and dried at room temperature under vacuum to obtain a peptide.
  • 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. Stirring was continued for 30 min.
  • the precipitate was filtered through a G4 sand filter suction funnel, washed repeatedly with cold diethyl ether for 3 times, and dried. Add 50 ml of double distilled water, 5 ml of acetonitrile to dissolve the solids thoroughly, suction filtration, and freeze-dry the filtrate to obtain the crude peptide 1.
  • the crude peptide obtained was purified by medium pressure or high pressure chromatography.
  • the column is a C18 column and the eluent is acetonitrile, water and a small amount of acetic acid.
  • the column was pre-equilibrated with 20% acetonitrile/water/0.1% glacial acetic acid solution 200 ml. After loading, the mixture was further equilibrated with 200 ml of the same eluent, and the eluent component was detected by HPLC.
  • the acetonitrile content was gradually increased according to the test results until the purified polypeptide peak was eluted.
  • the same fraction of the eluate was combined, and most of the solvent was removed by rotary evaporation, and the peptide was freeze-dried to obtain a content of >90% by HPLC.
  • Example 24 The preparation was carried out in accordance with the method of Example 1, except that the sequences of specific amino acids are shown in Tables 1 and 2, respectively. Its molecular weight and purity are shown in Table 3 above.
  • the polypeptide samples used in the following Examples 24 - 25 are the polypeptides synthesized in Examples 1 - 23 1 - 23
  • the present inventors performed activity assays on designed polypeptides using a HIV-1 Env mediated cell-cell fusion model.
  • the target cells are TZM-b l cells (US NIH AIDS Reagents and References Project, catalog number 8129), which express CD4 T-cell receptor and chemokine co-receptors CCR5 and CXCR4, which can be HIV-1.
  • Env recognizes that the luciferase reporter gene is also transcribed in the cell, but does not contain the promoter of the gene, so the luciferase background expression of the individual cells is very low.
  • Effector cells are HL2/3 cells (US NIH AIDS reagents and references) The item provided, catalog number 1294), which expresses HIV-1 ⁇ on its surface, attacks target cells by ⁇ , completes cell fusion, and also transcribes the promoter of the luciferase 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 were adherent cells, which were harvested by trypsin/EDTA digestion. 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 ⁇ l 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 DMS0, and the polypeptide concentration was measured at 280 nm using an ultraviolet spectrometer. The peptide solution was then diluted to the appropriate concentration and diluted moderately in a 96-well plate (Corning).
  • the fused cells were taken out, the medium was discarded, and washed twice with 200 ⁇ l/well PBS, and the washing solution was removed as much as possible; then, the lysate equilibrated to room temperature was added at 50 ⁇ l/well, and the cells were fully lysed by gently shaking for 5 minutes; The lysate was added to the 96-well chemiluminescence detection plate (Corning) at 40 ⁇ l/well, and the introduction of bubbles was avoided as much as possible; the substrate was rapidly added to the chemiluminescent ELISA plate at 40 ⁇ l/well in the dark. Immediately measure chemiluminescence on a microplate reader.
  • ratio > 5 indicates effective fusion.
  • 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 peptides 1-12 is shown in Table 4.
  • the positive control T20 has an IC50 of 2 ⁇ 0.5 mM, 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 ⁇ ).
  • the circular dichroic transmitter is the Bio log i c MOS450 spectrometer.
  • the CHR polypeptide was dissolved in PBS, the NHR polypeptide was dissolved in double distilled water, and the concentration was determined according to ultraviolet absorption at 280 nm; then 20 ⁇ M of the polypeptide solution was prepared.
  • the prepared sample was measured on a circular dichroic instrument with a scanning wavelength range of 190-260 nm, a wavelength interval of 0.5 nm, a scanning speed of 150 nm/min, and an average of 3 scans.
  • the buffer solution is first scanned to obtain a blank, and then the sample signal is scanned, and the blank signal is subtracted from the sample signal to obtain a CD signal.
  • the interaction of the two is determined by comparing the changes in the CD signal before and after mixing of the CHR and NHR polypeptides. Changes in the CD signal indicate that the two interact.
  • the inventors also determined the stability of the six helices formed by polypeptides 2 and 13-23 by CD temperature scanning.
  • the specific method is as follows: Dilute the sample to 1 ⁇ , add 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 the CD signal with temperature curve. . Calculate a differential according to the curve, determine the Tm value according to the peak value of the first differential curve (see Table 5), and the sample The thermal transition temperature.
  • the present inventors studied the interaction of polypeptide 2 with polypeptide 13-23 by non-denaturing polyacrylamide gel electrophoresis (N-PAGE), and completed using a BG-Power 3500 multi-electrophoresis apparatus (Beijing Baijing Biotechnology Co., Ltd.).
  • the experimental method is as follows:
  • NHR polypeptide and CHR polypeptide solution in a 1: 1 volume ratio to mix the sample; if it is a separate peptide sample, mix 200 ⁇ of the sample with the corresponding buffer solution 1: 1 .
  • the sample was allowed to stand at 37 °C for 30 min to allow sufficient reaction.
  • an equal volume of N-PAGE 2X loading buffer (Invitrogen) was added to each sample and mixed for use.
  • Electrophoresis 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.
  • Use a buffer to clean the sample tank use a sample gun or a micro syringe to slowly inject the sample solution into the bottom of the sample tank, turn on the power, and start electrophoresis. After about 2 hours, complete the electrophoresis (visible bromophenol blue strip)
  • the belt is the leading edge and stops after a certain distance from the sample hole.
  • Dyeing Remove the gel and wash it three times with double distilled water for 5 minutes each. The gel was covered with BioRadG250 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.
  • Table 5 Tm values of the six helices formed by polypeptides 2 and 13-23
  • Example 26 show that CD signal changes occur when CHR polypeptide 3 is mixed with NHR polypeptides 21, 20, 19, 18, 17 (Fig. 4 A-E), demonstrating their interaction.
  • Tm value The thermal stability (Tm value) of the six helix formed by polypeptide 3 and polypeptide 21, 20, 19, 18, 17 is shown in Table 6.
  • Example 27 show that CD signal changes (F i g. 5 A-E ) occur when CHR polypeptide 4 is mixed with NHR polypeptides 21, 20, 19, 18, 17 to demonstrate their interaction.
  • Tm value The thermal stability (Tm value) of the six helix formed by polypeptide 4 and polypeptide 21, 20, 19, 18, 17 is shown in Table 7.
  • Table 7 Tm values of the six helices formed by peptide 4 and NHR polypeptide
  • Example 28 show that CD signal changes occur when CHR polypeptide 5 is mixed with NHR polypeptides 21, 20, 19, 18, 17 (Fig. 6 A-E), demonstrating their interaction.
  • Tm value The thermal stability (Tm value) of the six helix formed by polypeptide 5 and polypeptide 21, 20, 19, 18, 17 is shown in Table 8.
  • Table 8 Tm values of the six helices formed by peptide 5 and NHR polypeptides
  • Example 29 show that CD signal changes occur when CHR polypeptide 6 is mixed with NHR polypeptides 19, 18, 17, 16, and 15 (Fig. 7 A-E), demonstrating their interaction.
  • Tm value The thermal stability (Tm value) of the six-helix formed by polypeptide 6 and polypeptides 19, 18, 17, 16, and 15 is shown in Table 9.
  • Table 9 Tm values of the six helices formed by polypeptide 6 and NHR polypeptide
  • Example 30 show that CD signal changes (Fi g. 8 A-C ) occur when CHR polypeptide 7 is mixed with NHR polypeptides 17, 16, 15 to demonstrate their interaction.
  • Tm value The thermal stability (Tm value) of the six helix formed by peptide 7 and polypeptide 15, 16, 17 is shown in the table.

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Abstract

公开了一种抑制HIV的多肽及其作用靶点，所述多肽包含11个与HIV-1的NHR直接接触的氨基酸残基，其中两个氨基酸残基分别位于多肽的N末端和C末端。还公开了所述多肽的作用靶点及医药用途。该多肽不含公认的保持高活性必需的口袋结合区，其能够抑制HIV病毒特别是对已有融合抑制剂产生耐药性的HIV毒株，能用于抗HIV感染。

Description

用于抑制 H I V的多肽及其作用靶点 技术领域
本发明属于生物医药领域， 涉及用于抑制 HIV的多肽及其作用靶 点。 背景技术
1型人免疫缺陷病毒（ HIV-1 )是艾滋病的病原体， 目前全球有超 过 3000万感染者， 每年导致约 200万人死亡， 并且每年还新增约 200 万感染者， 严重威胁人类的健康。
HIV-1 通过其包膜糖蛋白 （Env )介导的病毒 -细胞膜融合感染宿 主细胞。 Env包含表面亚基 gpl20和跨膜亚基 gp41， 三个 Env形成非 共价复合体镶嵌在病毒表面。 表面亚基 gpl20负责病毒感染细胞过程 中的分子识别以找到和接近靶细胞， 同时起着稳定跨膜亚基 gp41 功 能， 并在适当时机释放出 gp41 以启动融合； 跨膜亚基 gp41是病毒- 细胞膜融合的直接功能分子。
病毒细胞融合过程中有一由 gp41 N-端螺旋区（ NHR )和 C-端螺旋 区 （CHR ) 形成的六螺旋结构； 该结构的形成为病毒-细胞膜融合提供 能量，对病毒-细胞融合至关重要。 能够阻止六螺旋形成的药物则可以 有效地抑制艾滋病毒-细胞膜融合从而阻止病毒感染和体内传播，用于 艾滋病治疗， 称为融合抑制剂。
晶体结构显示， 在六螺旋中， 三个由 NHR形成的螺旋结构构成内 核， 形成三个沟槽， 三个 CHR反平行结合在沟槽中。 外源 CHR多肽可 结合在 NHR靶点中形成无活性的六螺旋结构， 阻止内源的活性六螺旋 体生成， 抑制病毒-细胞融合和病毒感染， 从而用作融合抑制剂。 晶体 结构揭示 NHR中含有一个较深的口袋， 与 CHR的相应功能区有关键相 互作用。这个 CHR关键结合区称为口袋结合区，其中含有 Try-Try-I le ( WWI )结合模板，被认为是维持高活性多肽融合抑制剂的关键（ Chan, D. C. , C. T. Chutkowski, and P. S. Kim, Proceedings of the Nat iona l Academy of Sc iences of the Uni ted States of Amer ica, 1998. 95 (26) : p. 15613-15617. ) 。
典型的 C-肽融合抑制剂包括 C34 ( US 6, 150， 088 )及其改进多肽、 首个上市融合抑制剂 T20 ( US 5, 464, 933 ) 、 以及后来发现的 CP32
( CN1793170, CN1955190 ) 。 这些 C-肽融合抑制剂通过与其对应 NHR 靶点结合阻止病毒感染；典型的靶点包括 N36( US 6, 150, 088 )和 DP107
( US 5, 656, 480 ) ， 分别与 C34和 CP32结合形成六螺旋结构， 其中 N36和 DP107 中含有一个共同的结合口袋， 也是小分子融合抑制剂的 热门靶点。
尽管有上市的融合抑制剂 T20和其它临床研究中的融合抑制剂如 S ifuvi rt ide ( CN1334122 ) ， 但由于抗药病毒株的快速出现， 使得针 对抗性病毒的融合抑制剂研发成为当务之急， 开发针对新靶点的药物 是解决药物抗性的有效途径； 确定的新靶点还可用于新的小分子融合 抑制剂的设计和开发。 发明内容
本发明人经过深入的研究和创造性的劳动， 出乎意料地得到了与 NHR 结合非常强的多肽， 从而开发出一类与已有融合抑制剂作用机制 不同的高活性融合抑制剂。 该类融合抑制剂 （本发明的多肽）不含公 认的保持高活性必需的口袋结合区， 其序列不同于已有的融合抑制剂 T20、 C34、 CP32等， 能够抑制 HIV病毒特别是对已有融合抑制剂产生 耐药性的 HIV毒株， 具有作为抗 HIV感染药物的潜力。 由此提供了下 述发明： 本发明的一个方面涉及一种 （分离的） 多肽、 其衍生物或其可药 用盐， 其中， 所述多肽包含 11个与 HIV - 1的 NHR直接接触的氨基酸 残基， 并且这 11 个氨基酸残基中的两个分别位于多肽的 N末端和 C 末端。
本领域人员可以理解，术语 "直接接触"在此是指在多肽与 HIV-1 gp41 NHR形成的六螺旋结构里， 所述 11个氨基酸残基的空间伸展方 向指向 NHR, 和 /或所述氨基酸残基通过疏水作用、 静电作用或氢键与 NHR的对应残基直接作用， 是药物作用的关键位点（Chan, D. C. , C. T. Chutkowski, and P. S. Kim, Proceedings of the Nat iona l Academy of Sc iences of the Uni ted States of Amer ica, 1998. 95 (26)： p. 15613-15617. ) 。 如 Fig.l , 其中灰色阴影部分示出了这些关键位点。
根据本发明任一项所述的多肽、 其衍生物或其可药用盐， 其中， 所述多肽的序列均包含在 HIV - 1的 CHR中； 优选地，所述多肽的长度 为 36个氨基酸残基。
根据本发明任一项所述的多肽、 其衍生物或其可药用盐， 其中， 所述多肽的氨基酸序列包含 WMEWDREI ( SEQ ID NO: 24 ) 或 WASLWNWF ( SEQ ID NO: 25 ) ； 优选地， 所述 WMEWDREI或 WASLWNWF既不位于 多肽的 N末端， 也不位于多肽的 C末端。
优选地，所述多肽的序列分别均包含在 HIV - 1的 CHR中； 更优选 地，所述多肽的序列分别均包含在下文中的 SEQ ID NO: 27中；具体地， 包含在 HIV - 1的 CHR的第 611 - 677个氨基酸中。
根据本发明任一项所述的多肽、 其衍生物或其可药用盐， 其中， 所述多肽的氨基酸序列分别如 SEQ ID NO: 1至 SEQ ID NO: 12中任一 序列所示。 多肽序列见下面的表 1。
表 1: 多肽序列
序 SEQ ID
氨基酸序列
号 NO:
1 WNASWSNKSLEQ IWNHMTWMEWDRE INNYTSLIHSL 1
2 WSNKSLEQ IWNHMTWMEWDRE I NYTSLIHSLIEES 2
3 KSLEQIWNHMTWMEWDREINNYTSLIHSLIEESQNQ 3
4 QIWN MTWMEWDRE INNYTSLIHSLI EESQNQQEKN 4
5 NNMTWMEWDRE INNYTSLIHSLI EESQNQQEKNEQE 5 6 WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL 6
7 WDREINNYTSLIHSLIEESQNQQEKNEQELLELDKW 7
8 INNYTSLIHSLIEESQNQQEKNEQELLELDKWASLW 8
9 YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF 9
10 IHSL IEESQNQQEKNEQELLELDKWASLWNWF I TW 10
11 L I EESQNQQEKNEQELLELDKWASLWNWF I TNWLW 11
12 SQNQQEKNEQELLELDKWASLWNWFDI TWNLWY I KL 12
其中氨基酸是缩写具有本领域公知的含义， 例如： W为色氨酸、 N 为天冬酰胺、 A为丙氨酸、 S为丝氨酸、 K为赖氨酸、 L为亮氨酸、 E为谷 氨酸、 Q为谷氨酰胺、 I为异亮氨酸、 H为组氨酸、 M为曱硫氨酸、 T为苏 氨酸、 D为天冬氨酸、 R为精氨酸、 Y为酪氨酸、 F为苯丙氨酸。
表 1中的多肽 1-12在本发明中也简称为多肽 1、 多肽 2 多肽
12; 或者称为 CHR多肽 1、 CHR多肽 2 CHR多肽 12。
根据本发明任一项所述的多肽、 其衍生物或其可药用盐， 其中， 所述多肽的 N末端连接乙酰基、 寡肽序列、 或亲脂性基团， 和 /或
C末端连接酰胺基、寡肽序列如 1 - 10个氨基酸残基的寡肽序列（例 如 EEE、 KKK、 GQAV [SEQ ID NO: 28]、 GEEE [SEQ ID NO: 29]等） 、 或 亲脂性基团（例如含有 3到 20个碳原子的脂肪酸链，其中优选含 8-16 个碳原子的脂肪酸链） 、 胆固醇等。
所述多肽的 N末端乙酰化， 和 /或 C末端酰胺化。
本发明实施例 24和实施例 25证明， 多肽 1-12具有高的抗 HIV 融合抑制剂活性， 表明 HIV-1 gp41 CHR及其相邻序列连续存在高活性 多肽，这对公认的 CHR中存在几个孤立的高活性多肽序列是一种创新。 本发明的另一方面涉及一种药物组合物， 其包含本发明任一项所 述的多肽、 其衍生物或其可药用盐； 可选地， 所述药物组合物还包含 药学上可接受的载体或辅料。 优选地， 所述药物组合物为注射剂。
通常本发明药物组合物含有 0. 1 - 90重量％的本发明任一项所述 的多肽、 其衍生物或其可药用盐。 药物组合物可根据本领域已知的方 法制备。 用于此目的时， 如果需要， 可将本发明的多肽、 其衍生物或 其可药用盐与一种或多种固体或液体药物赋形剂和 /或辅剂结合，制成 可作为人用的适当的施用形式或剂量形式。
本发明的多肽、 其衍生物或其可药用盐或者本发明的药物组合物 可以以单位剂量形式给药， 给药途径可为肠道或非肠道， 如口服、 肌 肉、 皮下、 鼻腔、 口腔粘膜、 皮肤、 腹膜或直肠等。 给药剂型例如片 剂、 胶嚢、 滴丸、 气雾剂、 丸剂、 粉剂、 溶液剂、 混悬剂、 乳剂、 颗 粒剂、 脂质体、 透皮剂、 口含片、 栓剂、 冻干粉针剂等。 可以是普通 制剂、 緩释制剂、 控释制剂及各种微粒给药系统。 为了将单位给药剂 型制成片剂， 可以广泛使用本领域公知的各种载体。 关于载体的例子 是， 例如稀释剂与吸收剂， 如淀粉、 糊精、 硫酸钙、 乳糖、 甘露醇、 蔗糖、 氯化钠、 葡萄糖、 尿素、 碳酸钙、 白陶土、 微晶纤维素、 硅酸 铝等； 湿润剂与粘合剂， 如水、 甘油、 聚乙二醇、 乙醇、 丙醇、 淀粉 浆、 糊精、 糖浆、 蜂蜜、 葡萄糖溶液、 阿拉伯胶浆、 明胶浆、 羧曱基 纤维素钠、 紫胶、 曱基纤维素、 磷酸鉀、 聚乙烯吡咯烷酮等； 崩解剂， 例如干燥淀粉、 海藻酸盐、 琼脂粉、 褐藻淀粉、 碳酸氢钠与枸橼酸、 碳酸钙、 聚氧乙烯、 山梨糖醇脂肪酸酯、 十二烷基磺酸钠、 曱基纤维 素、 乙基纤维素等； 崩解抑制剂， 例如蔗糖、 三硬脂酸甘油酯、 可可 脂、 氢化油等； 吸收促进剂， 例如季铵盐、 十二烷基硫酸钠等； 润滑 剂， 例如滑石粉、 二氧化硅、 玉米淀粉、 硬脂酸盐、 硼酸、 液体石蜡、 聚乙二醇等。 还可以将片剂进一步制成包衣片， 例如糖包衣片、 薄膜 包衣片、肠溶包衣片， 或双层片和多层片。 为了将给药单元制成丸剂， 可以广泛使用本领域公知的各种载体。 关于载体的例子是， 例如稀释 剂与吸收剂， 如葡萄糖、 乳糖、 淀粉、 可可脂、 氢化植物油、 聚乙烯 吡咯烷酮、 Ge luc i re、 高岭土、 滑石粉等； 粘合剂如阿拉伯胶、 黄蓍 胶、 明胶、 乙醇、 蜂蜜、 液糖、 米糊或面糊等； 崩解剂， 如琼脂粉、 干燥淀粉、 海藻酸盐、 十二烷基磺酸钠、 曱基纤维素、 乙基纤维素等。 为了将给药单元制成栓剂， 可以广泛使用本领域公知的各种载体。 关 于载体的例子是， 例如聚乙二醇、 卵磷脂、 可可脂、 高级醇、 高级醇 的酯、 明胶、 半合成甘油酯等。 为了将给药单元制成胶嚢， 将有效成 分本发明的多肽、 其衍生物或其可药用盐与上述的各种载体混合， 并 将由此得到的混合物置于硬的明明胶嚢或软胶嚢中。 也可将有效成分 本发明的多肽、 其衍生物或其可药用盐制成微嚢剂， 混悬于水性介质 中形成混悬剂， 亦可装入硬胶嚢中或制成注射剂应用。 为了将给药单 元制成注射用制剂， 如溶液剂、 乳剂、 冻干粉针剂和混悬剂， 可以使 用本领域常用的所有稀释剂， 例如， 水、 乙醇、 聚乙二醇、 1, 3-丙二 醇、 乙氧基化的异硬脂醇、 多氧化的异硬脂醇、 聚氧乙烯山梨醇脂肪 酸酯等。 另外， 为了制备等渗注射液， 可以向注射用制剂中添加适量 的氯化钠、 葡萄糖或甘油， 此外， 还可以添加常规的助溶剂、 緩冲剂、 pH调节剂等。
此外， 如需要， 也可以向药物制剂中添加着色剂、 防腐剂、 香料、 矫味剂、 甜味剂或其它材料。
本发明的多肽、 其衍生物或其可药用盐或者本发明的药物组合物 的给药剂量取决于许多因素， 例如所要预防或治疗疾病的性质和严重 程度， 患者或动物的性别、 年龄、 体重及个体反应， 所用的具体活性 成分， 给药途径及给药次数等。 上述剂量可以单一剂量形式或分成几 个， 例如二、 三或四个剂量形式给药。
本文所用的术语 "组合物" 意指包括包含指定量的各指定成分的 产品，以及直接或间接从指定量的各指定成分的组合产生的任何产品。
可改变本发明药物组合物中各活性成分的实际剂量水平， 以便所 得的活性成分的量能有效针对具体患者， 并且组合物和给药方式得到 所需的治疗反应。 剂量水平须根据具体活性成分的活性、 给药途径、 所治疗病况的严重程度以及待治疗患者的病况和既往病史来选定。 但 是， 本领域的做法是， 活性成分的剂量从低于为得到所需治疗效果而 要求的水平开始， 逐渐增加剂量， 直到得到所需的效果。 本发明的再一方面涉及本发明任一项所述的多肽、 其衍生物或其 可药用盐在制备治疗和 /或预防和 /或辅助治疗包膜类病毒感染的药物 中的用途； 具体地， 所述包膜类病毒感染为 HIV感染所致疾病或艾滋 病。
本发明的再一方面涉及一种治疗和 /或预防和 /或辅助治疗包膜类 病毒感染的方法，包括给与受试者有效量的本发明任一项所述的多肽、 其衍生物或其可药用盐的步骤。 具体地， 所述包膜类病毒感染为 HIV 感染所致疾病或艾滋病。
当用于上述治疗和 /或预防或辅助治疗时， 治疗和 /或预防有效量 的本发明的多肽、 其衍生物或其可药用盐可以以纯形式应用， 或者以 药学可接受的酯或前药形式（在存在这些形式的情况下）应用。 或者， 可以以含有本发明的多肽、 其衍生物或其可药用盐与一种或多种药物 可接受赋形剂的药物组合物给药。 但应认识到， 本发明的多肽、 其衍 生物或其可药用盐或者本发明的药物组合物的总日用量须由主诊医师 在可靠的医学判断范围内作出决定。 对于任何具体的患者， 具体的治 疗有效剂量水平须根据多种因素而定， 所述因素包括所治疗的障碍和 该障碍的严重程度； 所采用的具体活性成分的活性； 所采用的具体组 合物； 患者的年龄、 体重、 一般健康状况、 性别和饮食； 所采用的具 体活性成分的给药时间、 给药途径和排泄率； 治疗持续时间； 与所采 用的具体活性成分组合使用或同时使用的药物； 及医疗领域公知的类 似因素。 例如， 本领域的做法是， 活性成分的剂量从低于为得到所需 治疗效果而要求的水平开始， 逐渐增加剂量， 直到得到所需的效果。 一般说来， 本发明的多肽、 其衍生物或其可药用盐用于哺乳动物特别 是人的剂量可以介于 0. 001 - 1000 mg/kg体重 /天， 例如介于 0. 01 - 100 mg/kg体重 /天， 例如介于 0. 01 - 10 mg/kg体重 /天。
本发明的多肽、 其衍生物或其可药用盐或者本发明的药物组合物 可以有效地预防和 /或治疗和 /或辅助治疗本发明所述的各种疾病或病 症。 本发明的再一方面涉及本发明任一项所述的多肽、 其衍生物或其 可药用盐在制备或作为 HIV融合抑制剂或者抗 HIV药物中的用途。 本发明的再一方面涉及一种在体内或体外抑制 HIV的方法，包括使 用有效量的本发明任一项所述的多肽、其衍生物或其可药用盐的步骤。
本发明的再一方面涉及一种在体内或体外抑制 HIV-1 Env介导的 细胞融合的方法， 包括使用有效量的本发明任一项所述的多肽、 其衍 生物或其可药用盐的步骤。 本发明的再一方面涉及一种 （分离的） 多肽， 其衍生物或其可药 用盐， 其中， 所述多肽的氨基酸序列分别如 SEQ ID NO: 13至 SEQ ID NO: 23中任一序列所示。
多肽序列见下面的表 2。
表 2: 多肽序列
Figure imgf000009_0001
其中氨基酸是缩写具有本领域公知的含义， 例如： W为色氨酸、 N 为天冬酰胺、 A为丙氨酸、 S为丝氨酸、 K为赖氨酸、 L为亮氨酸、 E为谷 氨酸、 Q为谷氨酰胺、 I为异亮氨酸、 H为组氨酸、 M为甲硫氨酸、 T为苏 氨酸、 D为天冬氨酸、 R为精氨酸、 Y为酪氨酸、 F为苯丙氨酸、 G为甘氨 酸、 V为缬氨酸。
表 2中的多肽 13-23在本发明中也简称为多肽 13、多肽 14 多 肽 23; 或者称为腿多肽 13、 腿多肽 14 腿多肽 23。
根据 HIV-1 gp41核心部分晶体结构以及 gp41中 NHR和 CHR的反 平行结合特征，本发明人设计了与序列 1-12对应的 NHR多肽序列，用 于确定序列 1-12中多肽的结合靶点。 具体设计见 Fig.2， 图中以灰色 字体列出了与之结合的多肽序列。
NHR:
527/
LAVERYLKDQQLLGIWGIS ( SEQ ID NO: 26 )
CHR (反向） ：
677n
IYWLW
WTMHNWIQELSKNSWSANW611 ( SEQ ID NO: 27 , 为了清楚地表示出与腿 的结合对应关系，序列 27的右端为 N末端，序列方向应从右端的标为 611的 w读起） 。
上面的序列 （ SEQ ID NO: 26和 SEQ ID NO: 27 ) 为根据已有晶体 结构推测的 HIV-1 gp41 NHR和 CHR的相互作用关系， 下划线部分为已 有晶体结构的多肽序列。
本发明实施中，多肽 1-12与多肽 13-23中对应序列结合形成六螺 旋结构， 显示整个 HIV-1 gp41 NHR序列都可称为高活性融合抑制剂的 靶点， 这对公认的 NHR形成的口袋是高活性融合抑制剂的结合靶点是 一个突出的创新。
本发明的再一方面涉及多肽 13 - 23中任意一个或多个多肽在筛选 治疗和 /或预防和 /或辅助治疗包膜类病毒感染药物中的用途；具体地， 所述包膜类病毒感染为 HIV感染所致疾病或艾滋病。具体地，所述筛选 是指作为靶点筛选。
本发明的再一方面涉及多肽 13 - 23中任意一个或多个多肽在筛选 HIV融合抑制剂或者抗 HIV药物中的用途。 具体地， 所述筛选是指作为 靶点筛选。 本发明实施中， 根据 HIV-l gp41 NHR和 CHR间的相互作用， 二者 可互为靶点和配基，因此将 1-12和 13-23中多肽作为靶点和抑制剂的 功能互换。 具体地， 在多肽 1-12基础上设计靶点， 对多肽 13-23进行 改造得到高活性融合抑制剂， 进行药物设计和开发， 也包含在本发明 的设计精神内。
本发明的再一方面涉及编码本发明中任一项所述多肽的核苷酸序 列。
一种核酸构建体， 其包含本发明任一项所述的核苷酸序列； 具体 地， 所述核酸构建体为重组载体； 更具体地， 所述重组载体为重组表 达载体。
本发明还涉及包含本发明所述核酸序列及与之可操作连接的 1或 多个调控序列的核酸构建体， 所述调控序列在其相容条件下能指导编 码序列在合适的宿主细胞中进行表达。 表达应理解为包括多肽生产中 所涉及的任何步骤， 包括， 但不限于转录、 转录后修饰、 翻译、 翻译 后修饰和分泌。
"核酸构建体" 在文中定义为单链或双链核酸分子， 它们分离自 天然基因， 或者经修饰而含有以非天然方式组合和并列的核酸片段。 当核酸构建体包含表达本发明所述编码序列必需的所有调控序列时， 术语核酸构建体与表达盒同义。 术语 "编码序列" 在文中定义为核酸 序列中直接确定其蛋白产物的氨基酸序列的部分。 编码序列的边界通 常是由紧邻 mRNA 5' 端开放读码框上游的核糖体结合位点（对于原核 细胞）和紧邻 mRNA 3' 端开放读码框下游的转录终止序列确定。 编码 序列可以包括， 但不限于 DNA、 cDNA和重组核酸序列。
可以以多种方式操作编码本发明所述肽的分离的核酸序列， 使其 表达所述肽。 可能期望或必须在插入载体之前对核酸序列进行加工， 这取决于表达载体。 应用重组 DNA方法修饰核酸序列的技术为本领域 所熟知。
本文中术语 "控制序列" 定义为包括表达本发明肽所必需或有利 的所有组分。 每个调控序列对于编码多肽的核酸序列可以是天然含有 的或外来的。 这些调控序列包括， 但不限于， 前导序列、 多聚腺苷酸 化序列、 前肽序列、 启动子、 信号序列和转录终止子。 最低限度， 调 控序列要包括启动子以及转录和翻译的终止信号。 为了导入特定的限 制位点以便将调控序列与编码多肽的核酸序列的编码区进行连接， 可 以提供带接头的调控序列。 术语 "可操作连接" 在文中定义为这样一 种构象， 其中调控序列位于相对 DM序列之编码序列的适当位置， 以 使调控序列指导多肽的表达。
调控序列可以是合适的启动子序列， 即可被表达核酸序列的宿主 细胞识别的核酸序列。启动子序列含有介导多肽表达的转录调控序列。 启动子可以是在所选宿主细胞中有转录活性的任何核酸序列， 包括突 变的、 截短的和杂合的启动子， 可以得自编码与宿主细胞同源或异源 的胞外或胞内多肽的基因。
调控序列还可以是合适的转录终止序列， 即能被宿主细胞识别从 而终止转录的一段序列。 终止序列可操作连接在编码多肽的核酸序列 的 3， 末端。 在所选宿主细胞中可发挥功能的任何终止子都可以用于 本发明。
调控序列还可以是合适的前导序列， 即对宿主细胞的翻译十分重 要的 mRNA非翻译区。前导序列可操作连接于编码多肽的核酸序列的 5， 末端。在所选宿主细胞中可发挥功能的任何前导序列均可用于本发明。
调控序列还可以是信号肽编码区， 该区编码一段连在多肽氨基端 的氨基酸序列， 能引导编码多肽进入细胞分泌途径。 核酸序列编码区 的 5， 端可能天然含有翻译读框一致地与分泌多肽的编码区片段自然 连接的信号肽编码区。 或者， 编码区的 5， 端可含有对编码序列是外 来的信号肽编码区。当编码序列在正常情况下不含有信号肽编码区时， 可能需要添加外来信号肽编码区。 或者， 可以用外来的信号肽编码区 简单地替换天然的信号肽编码区以增强多肽分泌。 但是， 任何能引导 表达后的多肽进入所用宿主细胞的分泌途径的信号肽编码区都可以用 于本发明。
调控序列还可以是肽原编码区， 该区编码位于多肽氨基末端的一 段氨基酸序列。所得多肽被称为酶原或多肽原。 多肽原通常没有活性， 可以通过催化或自我催化而从多肽原切割肽原而转化为成熟的活性多 肽。
在多肽的氨基末端即有信号肽又有肽原区时， 肽原区紧邻多肽的 氨基末端， 而信号肽区则紧邻肽原区的氨基末端。
添加能根据宿主细胞的生长情况来调节多肽表达的调控序列可能 也是需要的。调控系统的例子是那些能对化学或物理刺激物（包括在有 调控化合物的情况下）作出反应，从而开放或关闭基因表达的系统。调 控序列的其他例子是那些能使基因扩增的调控序列。 在这些例子中， 应将编码多肽的核酸序列与调控序列可操作连接在一起。
本发明还涉及包含本发明核酸序列、 启动子和转录及翻译终止信 号的重组表达载体。 可以将上述各种核酸和调控序列连接在一起来制 备重组表达载体， 该载体可以包括 1或多个方便的限制位点， 以便在 这些位点插入或取代编码多肽的核酸序列。 或者， 可以通过将核酸序 列或包含该序列的核酸构建体插入适当表达载体而表达本发明所述核 酸序列。 制备表达载体时， 可使编码序列位于载体中以便与适当的表 达调控序列可操作连接。
重组表达载体可以是任何便于进行重组 DM操作并表达核酸序列 的载体（例如质粒或病毒）。 载体的选择通常取决于载体与它将要导入 的宿主细胞的相容性。 载体可以是线性或闭环质粒。
载体可以是自主复制型载体（即存在于染色体外的完整结构，可独 立于染色体进行复制）， 例如质粒、 染色体外元件、微小染色体或人工 染色体。 载体可包含保证自我复制的任何机制。 或者， 载体是一个当 导入宿主细胞时， 将整合到基因组中并与所整合到的染色体一起复制 的载体。 此外， 可应用单个载体或质粒， 或总体包含将导入宿主细胞 基因组的全部 DNA的两个或多个载体或质粒， 或转座子。
优选本发明所述载体含有 1 或多个便于选择转化细胞的选择标 记。选择标记是这样一个基因，其产物赋予对杀生物剂或病毒的抗性、 对重金属的抗性， 或赋予营养缺陷体原养型等。 细菌选择标记的例子 如枯草芽孢杆菌或地衣芽孢杆菌的 da l基因， 或者抗生素如氨苄青霉 素、 卡那霉素、 氯霉素或四环素的抗性标记。
优选本发明所述载体包含能使载体稳定整合到宿主细胞基因组 中， 或保证载体在细胞中独立于细胞基因组而进行自主复制的元件。
就进行自主复制的情况而言， 载体还可以包含复制起点， 使载体 能在目标宿主细胞中自主地复制。 复制起点可以带有使其在宿主细胞 中成为温度敏感型的突变。
可以向宿主细胞插入 1个以上拷贝的本发明核酸序列以提高该基 因产物的产量。 该核酸序列的拷贝数增加可以通过将该序列的至少 1 个附加拷贝插入宿主细胞基因组中， 或者与该核酸序列一起插入一个 可扩增的选择标记， 通过在有合适选择试剂存在下培养细胞， 挑选出 含有扩增拷贝的选择性标记基因、从而含有附加拷贝核酸序列的细胞。
用于连接上述各元件来构建本发明所述重组表达载体的操作是本 领域技术人员所熟知的（参见例如 Sambrook等， 分子克隆实验室手册， 第二版， 冷泉港实验室出版社， 冷泉港， 紐约， 1989)。 本发明的再一方面涉及一种重组宿主细胞， 其本发明任一项所述 的核酸构建体。
本发明还涉及包含可用来重组生产多肽的本发明所述核酸序列的 重组宿主细胞。 可将包含本发明之核酸序列的载体导入宿主细胞， 从 而使该载体以上述染色体整合体或自我复制的染色体外载体形式得以 维持。 术语 "宿主细胞" 涵盖任何由于复制期间发生的突变而与亲本 细胞不同的后代。 宿主细胞的选择很大程度上取决于多肽编码基因及 其来源。
宿主细胞可以是原核细胞或者真核细胞， 例如细菌 （如大肠杆菌 细胞）或酵母细胞。 可以通过本领域技术人员熟知的技术将载体导入 宿主细胞。
本发明的多肽可以人工化学合成， 也可以通过重组宿主细胞进行 蛋白质的表达， 例如， 包括： （a)在适于产生所述肽的条件下， 培养含 有核酸构建体的宿主细胞，该核酸构建体包含编码所述肽的核酸序列； 和（b)回收该肽。
在本发明所述制备方法中， 用本领域已知方法在合适多肽产生的 营养培养基中培养细胞。 例如， 可以在合适的培养基中， 在允许多肽 表达和 /或分离的条件下，通过摇瓶培养、 实验室或工业发酵罐中小规 模或大规模发酵（包括连续、分批、分批加料或固态发酵）来培养细胞。 在包含碳和氮源以及无机盐的合适的培养基中， 采用本领域已知的步 骤进行培养。 合适的培养基可由供应商提供或者可以参照公开的组成 (例如， 美国典型培养物保藏中心的目录中所述）来制备。 如果多肽被 分泌到培养基中，则可以直接从培养基中回收多肽。如果多肽不分泌， 可以从细胞裂解物中回收。
可以用本领域已知方法回收所产生的多肽。 例如， 可以通过常规 操作（包括， 但不限于离心、 过滤、 抽提、 喷雾干燥、 蒸发或沉淀）从 培养基中回收多肽。
可以通过各种本领域已知的操作来纯化本发明所述多肽， 这些操 作包括， 但不限于层析（例如， 离子交换层析、 亲和层析、 疏水作用层 析、 层析聚焦、 和大小排阻层析）、 电泳（例如， 制备性等电点聚焦）、 差示溶解度（例如硫酸铵沉淀）、 SDS-PAGE 或抽提（参见例如， 蛋白质 纯化， J. C. Janson 和 Lars Ryden编， VCH Publ i shers, New York, 1989)。 本发明中，
术语 "多肽" 具有本领域人员公知的一般含义， 并且还包括多肽 的衍生物、 修饰物等等。
术语 "腿"或 "HIV-1腿"具体是指 HIV-1 gp41 NHR; 术语 "CHR" 或 "HIV-1 CHR" 具体是指 HIV-1 gp41 CHR。
术语 "HIV融合抑制剂" 包括但不限于： 抑制 HIV (例如抑制 HIV 增殖、 感染、 传播等） 或者抑制 HIV-1 Env介导细胞融合的药物或者 试剂。 术语 "有效量" 包括可在受试者中实现治疗、 预防、 减轻和 /或緩 解本发明所述疾病或病症的剂量。
术语 "受试者" 可以指患者或者其它接受本发明任一项所述的多 肽、 其衍生物或其可药用盐或者本发明任一项所述的药物组合物以治 疗、预防、 减轻和 /或緩解本发明所述疾病或病症的动物， 特别是哺乳 动物， 例如人、 狗、 猴、 牛、 马等。
术语 "疾病和 /或病症"是指所述受试者的一种身体状态， 该身体 状态与本发明所述疾病和 /或病症有关。 发明的有益效果
本发明的多肽是与已有的融合抑制剂 T20、 C34、 CP32等序列相异 的新抗 HIV感染多肽， 具有良好的抗 HIV融合活性， 能够抑制 HIV病 毒特别是对已有融合抑制剂产生耐药性的 HIV毒株。 附图说明
Fig. 1： 本发明的 CHR多肽序列， 其中， 阴影部分为 CHR中与 NHR 直接接触的氨基酸残基， 是药物作用的关键位点。
Fig. 2： NHR靶点序列的设计。 阴影部分为与其作用的 CHR多肽。 根据二者结合的反平行特征， CHR多肽序列以反转序列表示。
Fig. 3: 表征 CHR多肽 2与 NHR多肽相互作用的 CD谱和 N-PAGE 结果， 其中：
Fig. 3 ( A )是多肽 2与多肽 22相互作用的 CD谱， 其中方块表示 多肽 2的 CD谱， 圓圏表示多肽 22的 CD谱， 上三角 2/22表示多肽 22 和多肽 2混合后的 CD谱，下三角 2+22表示多肽 22和多肽 2单独扫描 的 CD语叠加；
Fig. 3 ( B ) 多肽 2与多肽 21相互作用的 CD谱， 其中的图例的含 义与 Fig. 3 ( A ) 中做类似理解；
Fig. 3 ( C ) 多肽 2与多肽 20相互作用的 CD谱， 其中的图例的含 义与 Fig. 3 ( A ) 中做类似理解； Fig.3 (D) 多肽 2与多肽 19相互作用的 CD谱， 其中的图例的含 义与 Fig.3 (A) 中做类似理解；
Fig.3 (E) 多肽 2与多肽 17相互作用的 CD谱， 其中的图例的含 义与 Fig.3 (A) 中做类似理解；
Fig.3 ( F ) 多肽 2与 NHR多肽相互作用的 N-PAGE结果。
Fig.4: 表征 CHR多肽 3与 NHR多肽相互作用的 CD谱和 N-PAGE 结果， 其中：
Fig.4 (A)是多肽 3与多肽 21相互作用的 CD谱， 其中方块表示 多肽 3的 CD谱， 圓圏表示多肽 21的 CD谱， 上三角 2/21表示多肽 21 和多肽 3混合后的 CD谱，下三角 2+21表示多肽 21和多肽 3单独扫描 的 CD语叠加；
Fig.4 (B) 多肽 3与多肽 20相互作用的 CD谱， 其中的图例的含 义与 Fig.4 (A) 中做类似理解；
Fig.4 (C) 多肽 3与多肽 19相互作用的 CD谱， 其中的图例的含 义与 Fig.4 (A) 中做类似理解；
Fig.4 (D) 多肽 3与多肽 18相互作用的 CD谱， 其中的图例的含 义与 Fig.4 (A) 中做类似理解；
Fig.4 (E) 多肽 3与多肽 17相互作用的 CD谱， 其中的图例的含 义与 Fig.4 (A) 中做类似理解；
Fig.4 (F) 多肽 3与 NHR多肽相互作用的 N-PAGE结果。
Fig.5: 表征 CHR多肽 4与 NHR多肽相互作用的 CD谱和 N-PAGE 结果， 其中：
Fig.5 (A)是多肽 4与多肽 21相互作用的 CD谱， 其中方块表示 多肽 4的 CD谱， 圓圏表示多肽 21的 CD谱， 上三角 2/21表示多肽 21 和多肽 4混合后的 CD谱，下三角 2+21表示多肽 21和多肽 4单独扫描 的 CD语叠加；
Fig.5 (B) 多肽 4与多肽 20相互作用的 CD谱， 其中的图例的含 义与 Fig.5 (A) 中做类似理解；
Fig.5 (C) 多肽 4与多肽 19相互作用的 CD谱， 其中的图例的含 义与 Fig.5 (A) 中做类似理解；
Fig.5 (D) 多肽 4与多肽 18相互作用的 CD谱， 其中的图例的含 义与 Fig.5 (A) 中做类似理解；
Fig.5 (E) 多肽 4与多肽 17相互作用的 CD谱， 其中的图例的含 义与 Fig.5 (A) 中做类似理解；
Fig.5 (F) 多肽 4与 NHR多肽相互作用的 N-PAGE结果。
Fig.6: 表征 CHR多肽 5与 NHR多肽相互作用的 CD谱和 N-PAGE 结果， 其中：
Fig.6 (A)是多肽 5与多肽 21相互作用的 CD谱， 其中方块表示 多肽 5的 CD谱， 圓圏表示多肽 21的 CD谱， 上三角 2/21表示多肽 21 和多肽 5混合后的 CD谱，下三角 2+21表示多肽 21和多肽 5单独扫描 的 CD语叠加；
Fig.6 (B) 多肽 5与多肽 20相互作用的 CD谱， 其中的图例的含 义与 Fig.6 (A) 中做类似理解；
Fig.6 (C) 多肽 5与多肽 19相互作用的 CD谱， 其中的图例的含 义与 Fig.6 (A) 中做类似理解；
Fig.6 (D) 多肽 5与多肽 18相互作用的 CD谱， 其中的图例的含 义与 Fig.6 (A) 中做类似理解；
Fig.6 (E) 多肽 5与多肽 17相互作用的 CD谱， 其中的图例的含 义与 Fig.6 (A) 中做类似理解；
Fig.6 (F) 多肽 5与 NHR多肽相互作用的 N-PAGE结果。
Fig.7: 表征 CHR多肽 6与 NHR多肽相互作用的 CD谱和 N-PAGE 结果， 其中：
Fig.7 (A)是多肽 6与多肽 19相互作用的 CD谱， 其中方块表示 多肽 6的 CD谱， 圓圏表示多肽 19的 CD谱， 上三角 6八 9表示多肽 6 和多肽 19混合后的 CD谱， 下三角 6+19表示多肽 6和多肽 19单独扫 描的 CD语叠加；
Fig.7 (B) 多肽 6与多肽 18相互作用的 CD谱， 其中的图例的含 义与 Fig.7 (A) 中做类似理解； Fig.7 (C) 多肽 6与多肽 17相互作用的 CD谱， 其中的图例的含 义与 Fig.7 (A) 中做类似理解；
Fig.7 (D) 多肽 6与多肽 16相互作用的 CD谱， 其中的图例的含 义与 Fig.7 (A) 中做类似理解；
Fig.7 (E) 多肽 6与多肽 15相互作用的 CD谱， 其中的图例的含 义与 Fig.7 (A) 中做类似理解；
Fig.7 (F) 多肽 6与 NHR多肽相互作用的 N-PAGE结果。
Fig.8: 表征 CHR多肽 3与 NHR多肽相互作用的 CD谱和 N-PAGE 结果， 其中：
Fig.8 (A)是多肽 7与多肽 17相互作用的 CD谱， 其中方块表示 多肽 7的 CD谱， 圓圏表示多肽 17的 CD谱， 上三角 7八 7表示多肽 7 和多肽 17混合后的 CD谱， 下三角 7+17表示多肽 7和多肽 17单独扫 描的 CD语叠加；
Fig.8 (B) 多肽 7与多肽 16相互作用的 CD谱， 其中的图例的含 义与 Fig.8 (A) 中做类似理解；
Fig.8 (C) 多肽 7与多肽 15相互作用的 CD谱， 其中的图例的含 义与 Fig.8 (A) 中做类似理解；
Fig.8 (D) 多肽 7与 NHR多肽相互作用的 N-PAGE结果。 具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述， 但是本领 域技术人员将会理解， 下列实施例仅用于说明本发明， 而不应视为限 定本发明的范围。 实施例中未注明具体条件者， 按照常规条件或制造 商建议的条件进行。 所用试剂或仪器未注明生产厂商者， 均为可以通 过市购获得的常规产品。 在本发明中使用的缩写具有下面的意义：
AIDS ( Acquired Immure Deficiency Syndrome ) 艾滋病， 获得 性免疫缺陷综合症。 Ala (Alanine, A) 丙氨酸
Arg ( Arginine, R ) 精氨酸
Asn ( Asparagine, ) 天冬醜胺
Asp ( Aspart icacid, D ) 天冬氛酸
DCM ( Dichlorome thane ) 二氯曱烷
DMF (N, N-Dimethyl malonate) 二曱基曱酰胺
Env ( Envelope glycoprotein ) 包膜糖蛋白
ESI -MS ( Electronic spray ion mass spectroscopy ) 电喷盾语
Fmoc ( Fluorenylmethoxycarbonyl ) 药曱氧欺基
Gly (Glycine, G) 甘氨酸
Gin ( Glutamine, Q) 谷醜胺
Glu ( Glutamic acid, E ) 谷氛酸
6-HB ( six-helix bundle ) 六螺旋体
HBTU 2- ( 1H-1-羟基苯并三唑） -1， 1， 3， 3-四曱基六氟磷酸 His ( Histidine, H)组氨酸
HoBt ( 1-Hydroxyl benzot iazole anhydrous ) 1_ 基笨并三氮 峻
NHR ( N-terminal heptad repeat ) N -端七联重复序列
CHR ( C-terminal heptad repeat ) C -端七联重复序列
HIV ( Human immunodeficiency virus ) 人免疫缺陷病毒
HIV-1 1型人免疫缺陷病毒
HPLC (high performance liquid chromatography ) 高效液相 色谱
lie ( Isoleucine, I) 异亮氨酸
Leu ( Leucine, L ) 亮氛酸
Met (Methionine, M) 曱疏氨酸
Nal 正亮氨酸
Lys (Lysine, K) 赖氨酸
Phe ( Phenylalanine, F) 苯丙氛酸 Ser ( Ser ine, S ) 丝氨酸
TFA ( Tr if luoroacet ic acid ) 三氟乙酸
Thr ( Threonie , T ) 苏氨酸
Tyr ( Tyros ine , Y ) 赂氛酸
Va l ( Va l ine, V ) 缬氨酸 实施例所用固相合成载体 Rink 酰胺树脂为天津南开合成责任有 限公司产品； HBTU、 H0Bt、 DIEA及 Fmoc保护的天然氨基酸和 D型的 非天然氨基酸为上海吉尔生化公司及成都诺新技术责任公司产品。 N- 曱基吡咯烷酮（NMP )为 ACR0S公司产品； TFA为北京博迈科技有限公 司产品； DMF、 DCM为韩国三星公司产品； 色谱纯乙腈为 Fi sher公司 产品。 其它试剂如无说明均为国产分析纯产品。 实施例 1: 多肽 1的制备
采用标准的 Fmoc 固相多肽合成方法。 所有多肽序列均 C-端为酰 胺化， N-端为乙酰化 （本领域人员知晓， 这些修饰对多肽活性没有根 本性影响） 。 选用 Rink Amide树脂， 肽类由 C-端向 N-端延长。 缩合 剂为 HBTU/H0Bt/DIEA。 脱保护剂为哌啶 /DMF溶液。 裂解剂为 TFA, 粗 肽水溶解后冻干保存。用中压液相色谱法或 HPLC进行分离纯化， 纯肽 含量〉 95%。 基盾辅助激光解析飞行时间质谱（ MALDI-T0F-MS )确定多 肽分子量。
微波多肽合成条件如下：
氨基酸： 0. 2 M的 DMF溶液； 活化剂： 0. 45M HBTU/HOBt 的 DMF 溶液； 活化碱： 2M DIEA的 NMP溶液； 脱保护剂： 20% v/v哌啶的 DMF 溶液； 封闭试剂： 20% v/v乙酸酐的 DMF溶液。
称取 Rink Amide树脂 0. 5g ( 0. 25 mmo l ) 置入 CEM微波多肽合成 仪的反应器中， 然后将氨基酸、 活化剂、 活化碱、 脱保护试剂、 封闭 试剂按上述条件配好后， 用 CEM微波全自动多肽合成仪进行合成。 完 成后肽树脂用 DMF洗涤 3遍后用无水曱醇收缩， 室温真空干燥， 得肽 树脂 2. 05g
肽树脂的裂解： 将上述合成好的肽树脂称量后放入 250ml茄形瓶 中， 冰浴， 电磁搅拌。 按 lg肽树脂加入 10ml的量配置裂解液（体积 比： 三氟乙酸： 乙二硫醇： 间苯酚： 水 =82. 8: 10: 5: 2. 5 ) TFA 需预 先冰浴降温 30min或者预先放于；水箱中使用。 将配制好的裂解液加入 到冰浴条件下的肽树脂中， 电磁搅拌， 树脂变橙红色， 冰浴条件下反 应 30min， 然后撤冰浴， 室温下再继续反应 90min使反应完成。 剧烈 搅拌下向反应器中加入冷乙醚 200ml，析出白色沉淀，继续搅拌 30min; 用 G4的砂芯抽滤漏斗滤出析出物， 用冷乙醚反复洗涤 3遍， 晾干。 加 入双蒸水 50ml， 乙腈 5ml 使固体充分溶解， 抽滤， 滤液冻干得粗肽 1. 03g
所得粗肽用中压或高压色谱进行纯化。 色谱柱为 C18柱， 洗脱液 为乙腈， 水及少量乙酸。 具体操作步骤： 称取粗肽 lg， 加水 20ml， 乙 腈 5ml溶解， 3000转 /分钟下离心 10min， 取上清液上样。 色谱柱预先 用 15%乙腈 /水 /0. 1%冰乙酸溶液 200ml平衡， 上样后继续用 200ml 同 样洗脱液平衡， 高效液相检测洗脱液成分。 根据检测结果逐步升高乙 腈含量， 直至所纯化的多肽峰被洗脱出来。 合并同组分洗脱液， 旋转 蒸发除去大部分溶剂， 冻干得纯肽， HPLC检测含量〉 90%
纯肽经 MALDI-TOF-MS质语确定其分子量 （见下面的表 3 )
表 3: 多肽的分子量和纯度
多肽序号 对应的实验编号 测定分子量 纯度％
1 LKC058 4563. 4 98. 8
2 LKC121 4540. 6 83. 3
3 LKC087 4523. 9 95. 9
4 LKC060 4565. 7 98. 4
5 LKC088 4524. 8 96. 9
6 LKC025 4532. 8 97. 6
7 LKC090 96. 3
8 LKC062 4386. 6 88. 7 9 LKC012 4492. 2 91. 9
10 LKC091 4543. 5 96. 8
11 LKC064 4619. 2 97. 4
12 LKC065 4652. 9 98. 6
13 LKC063 4107. 6 98. 0
14 LKC118 4090. 0 93. 1
15 LKC048 4385. 9 90. 0
16 LKC061 4365. 0 96. 5
17 LKC094 4420. 8 95. 8
18 LKC026 95. 4
19 LKC119 94. 1
20 LKC059 4540. 3 96. 6
21 LKC120 4427. 6 83. 6
22 LKC132 4328. 5 97. 9
23 LKC057 4500. 1 96. 5 实施例 2-23: 多肽 2-23的制备
参照实施例 1中的方法进行制备， 除了具体氨基酸的序列分别如 表 1和表 2中所示。 其分子量和纯度如上面的表 3所示。 下面的实施例 24 - 25所用多肽样品为实施例 1 - 23所合成的多肽 1 - 23
实施例 24 : 多肽的抗 HIV-1融合活性检测
本发明人用 HIV-1 Env介导的细胞-细胞融合模型对设计的多肽进 行活性测定。靶细胞为 TZM-b l细胞（美国 NIH艾滋病试剂和参照物项 目提供， 目录号为 8129 ) ， 其表面表达 CD4 T-细胞受体和趋化因子辅 助受体 CCR5和 CXCR4 , 可被 HIV-1 Env识别， 同时细胞内还转录荧光 素酶报告基因， 但不含该基因的启动子， 因此单独细胞的荧光素酶背 景表达量很低。 效应细胞为 HL2/3细胞（美国 NIH艾滋病试剂和参照 物项目提供， 目录号为 1294 ) ， 其表面表达 HIV-1 Εην, 由 Εην进攻 靶细胞， 完成细胞融合， 同时细胞内还转录荧光素酶报告基因的启动 子。 两种细胞先在含有氨苄 /链霉素双抗的含 10%胎牛血清的 DMEM中， 37度下在含有 5% C02的培养箱中单独培养。 两种细胞均为贴壁细胞， 细胞胰酶 /EDTA消化后收获传代。 细胞用细胞计数板计数。
将 TZM-bl靶细胞用培养基调整到浓度为 75万 /ml, 以每孔 50μ1 加入 96孔细胞培养板中 （ 3.75万 /孔） ， 5% C02， 37度下培养 24小 时。
将多肽或阳性对照样品（ T20或 C34 )溶于磷酸緩冲溶液生理盐水 (PBS) 中， 或加入适量 DMS0使充分溶解， 用紫外光谱仪在 280nm处 测定多肽浓度。 然后将多肽溶液稀释到适当的浓度， 在 96 孔酶标板 (Corning) 中等比稀释。
配制 150万 /ml的 HL2/3效应细胞。
将 20μ1/孔的等比稀释的抑制剂加入前一天培养的 TZM-bl 细胞 中， 然后加入 50μ1/孔的配制好的 HL2/3效应细胞； 96孔细胞培养板 的其中一排用 PBS替代抑制剂用于测定饱和融合信号， 另一排用 DMEM 培养基替代 HL2/3细胞用于测定背景信号。 5% C02， 37度下培养 6-8 小时使之充分融合。
将荧光素酶报告基因的试剂盒（Promega)从冰箱中取出， 将 5x 细胞裂解液按照用量用双蒸水稀释至 lx裂解液， 室温下放置； 用底物 緩冲液溶解底物， 室温下放置； 同时将酶标仪（Molcular Devices M5 多功能酶标仪）检测条件设置好备用。
将融合好的细胞取出， 弃去培养基， 用 200μ1/孔 PBS洗两次， 尽 量去除清洗液； 然后以 50μ1/孔加入平衡到室温的裂解液， 轻轻震动 下反应 5min使细胞充分裂解； 将裂解液以 40μ1/孔加入 96孔化学发 光检测用酶标板板（Corning)中， 加样时尽量避免引入气泡； 避光下 将底物以 40μ1/孔迅速加入化学发光用酶标板中， 立即在酶标仪上测 定化学发光。
根据饱和融合信号和背景信号的比值确定靶细胞和效应细胞的有 效融合， 比值〉 5 表明有效融合。 根据样品的浓度 -化学发光信号曲线 确定其半抑制剂浓度（ IC5。）， 阳性对照样品的 IC5。值应稳定在一定范 围； 理想的抑制曲线中高浓度抑制剂下信号应接近背景信号， 最低浓 度抑制剂下信号应接近饱和融合信号。
多肽 1-12的细胞融合抑制活性列于表 4， 阳性对照 T20的 IC50 为 2±0·5 nM， 与文献 4艮道相符 （Wild, C. T. , et al. , Proceedings of the National Academy of Sciences of the United States of America, 1994. 91 (21): p. 9770-9774· ) 。
表 4: 多肽的细胞融合抑制活性
Figure imgf000025_0001
表 4的结果显示， 多肽 1-12均具有 HIV-1融合抑制活性，特别是 其中 2-10的 IC5。 <46nM， 达到了临床前活性水平， 具有成为新的融合 抑制剂的候选药物的潜力。 实施例 25:圓二色谱（ CD )和非变性聚丙烯酰胺凝胶电泳（ N-PAGE ) 表征多肽 2与多肽 13-23的相互作用
1. 实验目的
用圓二色谱（CD )等方法研究 CHR多肽 2与 NHR多肽 13-23的相 互作用。
2. 实验材料
多肽 2、 13-23。
圓二色傳仪为 Bio log i c MOS450谱仪。
3. 实验方法
将测定 CHR多肽溶于 PBS中， NHR多肽溶于双蒸水中，根据 280nm 下紫外吸收确定浓度； 然后配制 20 μΜ的多肽溶液。
配制要检测的多肽样品： 将 NHR多肽和 CHR多肽以 1: 1体积比混 合物的二者混合样品； 如果是单独多肽样品， 将 20 μΜ的样品用相应 緩冲溶液 1: 1混合。样品在 37度下放置 30min使充分反应。上述实验 步骤可确保样品中的多肽浓度保持一致。
将配制好的样品在圓二色谱仪上测定， 仪器扫描波长范围为 190-260nm, 波长间隔为 0. 5nm， 扫描速度为 150nm/min， 扫描 3次进 行平均。 先用緩冲溶液样品扫描得到空白， 然后扫描样品信号， 将空 白信号从样品信号中扣除得到 CD信号。
通过比较 CHR和 NHR多肽混合前和混合后的 CD信号变化来确定二 者的相互作用。 CD信号变化表明两者具有相互作用。
本发明人还通过 CD温度扫描测定多肽 2与 13-23形成的六螺旋稳 定性。 具体方法如下： 将样品稀释到 1 μΜ， 加入样品池， 将 CD仪器 程序设为温度扫描， 检测波长 220nm， 扫描范围 20-90摄氏度， 搅拌 下进行程序温度扫描和检查得到 CD信号随温度变化曲线。根据曲线计 算一次微分， 根据一次微分曲线的峰值确定 Tm值（见表 5 ) ， 及样品 的热转变温度。
本发明人用非变性聚丙烯酰胺凝胶电泳（N-PAGE)研究多肽 2与 多肽 13-23的相互作用，使用 BG-Power3500多用电泳仪（北京百晶生 物技术有限公司） 完成。 实验方法如下：
将 200μΜ的 NHR多肽和 CHR多肽溶液以 1: 1体积比混合， 是为 混合样品； 如果是单独多肽样品， 将 200 μΜ 的样品用相应緩冲溶液 1: 1混合。 样品在 37度下放置 30min使充分反应。 结束后， 每个样品 中加入等体积的 N-PAGE 2X上样緩冲液（ Invitrogen公司） ， 混匀后 待用。
配制分离胶（20%) : 30%丙烯酰胺溶液 10 ml, 4X分离胶緩冲液 3.75 ml, 水 1.24 ml, 10% AP (过 υ酸铵） 溶液 100 μΐ, TEMED
(Ν,Ν,Ϊ ,Ϊ -四曱基乙二胺） 10 μΐ, 震荡混合。 小心将上述溶液， 两层玻璃板之间， 上层留有 l-2cm的空间以便灌制浓缩胶， 再使用注 射器小心加入水饱和正丁醇。 凝胶在 30分钟左右完成聚合。
配制浓缩胶（4%) : 将正丁醇层弃去， 用去离子水小心清洗凝胶 上层， 用滤纸吸干水。 加入 30%丙烯酰胺溶液 1 ml, 4X浓缩胶緩冲液 2 ml, 水 4.8ml， 加入 10% AP溶液 100 μΐ, TEMED 10 μΐ, 震荡混合。 用上述溶液注满玻璃板， 插入上样梳子。
电泳： 等待凝胶聚合后将上样梳子拔出，按照电泳装置说明安装， 上槽为负极， 下槽为正极。 将 10X Tris-Gly电泳緩冲液稀释到 IX， 注满电容槽。 电压约 150V, 电流约为 25mA，预电泳 20min， 关掉电源。 使用緩冲液清洗上样槽， 使用加样枪或微量注射器将所配样品溶液緩 慢地注入加样槽底部， 打开电源， 开始电泳， 约 2小时后， 完成电泳 (可视溴酚蓝的条带为前沿， 距离上样孔一定距离后停止） 。
染色： 取下凝胶， 用双蒸水洗三遍， 各 5分钟。 加入 BioRadG250 染色液覆盖凝胶， 染色 1小时。 弃去染色液， 加入双蒸水脱色三遍， 各 10分钟。 使用平板扫描仪或者凝胶成像系统扫描染色后的凝胶。
通过比较电泳的染色条带来确定 NHR多肽 和 CHR多肽的相互作 用。 3. 实验结果
( 1 )如 Fig.3A-E, CHR多肽 2与 NHR多肽 22、 21、 20、 19、 18、 17混合后都出现 CD信号变化， 证明了它们之间的相互作用。
( 2) Tm值如表 5所示。
表 5: 多肽 2与 13-23形成的六螺旋的 Tm值
Figure imgf000028_0001
( 3) 非变性聚丙烯酰胺凝胶电泳的结果显示 （Fig.3 F) ： 多肽 17、 18、 19、 20、 21、 22与多肽 2的各混合溶液在电泳中均表现出不 同于多肽 2 单独溶液的新条带， 证明多肽 17、 18、 19、 20、 21、 22 与多肽 2之间均存在明确的相互作用。 实施例 26:圓二色谱（ CD)和非变性聚丙烯酰胺凝胶电泳（ -PAGE ) 表征多肽 3与多肽 13-23的相互作用
参照实施例 25中的方法进行实验。
实施例 26结果显示， CHR多肽 3与 NHR多肽 21、 20、 19、 18、 17 混合后都出现 CD信号变化 （Fig.4 A-E) ， 证明了它们之间的相互作 用。
多肽 3与多肽 21、 20、 19、 18、 17形成六螺旋的热稳定性（ Tm 值） 见表 6。
表 6: 多肽 2与 NHR多肽形成的六螺旋的 Tm值
多肽序号 实验编号 Tm ( °C )
17 LKC094 44.0
18 LKC026 54.0 19 LKC119 52. 9
20 LKC059 65. 9
21 LKC120 59. 7
22 LKC132 36. 1
多肽 3与 13-23相互作用的 N-PAGE结果显示 （ Fig. 4 F ) ， 多肽 17、 18、 19、 20、 21与多肽 3的各混合溶液在电泳中均表现出不同于 多肽 3单独溶液的新条带， 证明多肽 17、 18、 19、 20、 21 与多肽 3 之间均存在明确的相互作用。 实施例 27 :圓二色谱（ CD )和非变性聚丙烯酰胺凝胶电泳（ -PAGE ) 表征多肽 4与多肽 13-23的相互作用
参照实施例 25中的方法进行实验。
实施例 27结果显示， CHR多肽 4与 NHR多肽 21、 20、 19、 18、 17 混合后都出现 CD信号变化 （F i g. 5 A-E ) ， 证明了它们之间的相互作 用。
多肽 4与多肽 21、 20、 19、 18、 17形成六螺旋的热稳定性（ Tm 值） 见表 7。
表 7 : 多肽 4与 NHR多肽形成的六螺旋的 Tm值
Figure imgf000029_0001
多肽 4与 NHR多肽相互作用的 N-PAGE结果显示 （ F ig. 5 F ) ， 多 肽 17、 18、 19、 20、 21与多肽 4的各混合溶液在电泳中均表现出不同 于多肽 4单独溶液的新条带， 证明多肽 17、 18、 19、 20、 21与多肽 4 之间均存在明确的相互作用。 实施例 28:圓二色谱（ CD)和非变性聚丙烯酰胺凝胶电泳（ N-PAGE) 表征多肽 5与多肽 13-23的相互作用
参照实施例 25中的方法进行实验。
实施例 28结果显示， CHR多肽 5与 NHR多肽 21、 20、 19、 18、 17 混合后都出现 CD信号变化 （Fig.6 A-E) ， 证明了它们之间的相互作 用。
多肽 5与多肽 21、 20、 19、 18、 17形成六螺旋的热稳定性（ Tm 值） 见表 8。
表 8: 多肽 5与 NHR多肽形成的六螺旋的 Tm值
Figure imgf000030_0001
多肽 5与 NHR多肽相互作用的 N-PAGE结果显示 （ Fig.6 F ) ， 多 肽 17、 18、 19、 20、 21与多肽 5的各混合溶液在电泳中均表现出不同 于多肽 5单独溶液的新条带， 证明多肽 17、 18、 19、 20、 21与多肽 5 之间均存在明确的相互作用。 实施例 29:圓二色谱（ CD)和非变性聚丙烯酰胺凝胶电泳（ -PAGE ) 表征多肽 6与多肽 13-23的相互作用
参照实施例 25中的方法进行实验。
实施例 29结果显示， CHR多肽 6与 NHR多肽 19、 18、 17、 16、 15 混合后都出现 CD信号变化 （Fig.7 A-E) ， 证明了它们之间的相互作 用。
多肽 6与多肽 19、 18、 17、 16、 15形成六螺旋的热稳定性（ Tm 值） 见表 9。 表 9 : 多肽 6与 NHR多肽形成的六螺旋的 Tm值
Figure imgf000031_0001
多肽 6与 NHR多肽相互作用的 N-PAGE结果显示 （ F ig. 7 F ) ， 多 肽 15、 16、 17、 18、 19与多肽 6的各混合溶液在电泳中均表现出不同 于多肽 6单独溶液的新条带， 证明多肽 15、 16、 17、 18、 19与多肽 6 之间均存在明确的相互作用。 实施例 30:圓二色谱（CD )和非变性聚丙烯酰胺凝胶电泳（N-PAGE ) 表征多肽 7与多肽 13-23的相互作用
参照实施例 25中的方法进行实验。
实施例 30结果显示， CHR多肽 7与 NHR多肽 17、 16、 15混合后 都出现 CD信号变化（Fi g. 8 A-C ) ， 证明了它们之间的相互作用。
多肽 7与多肽 15、 16、 17形成六螺旋的热稳定性（Tm值）见表
10。
表 10: 多肽 7与 NHR多肽形成的六螺旋的 Tm值
Figure imgf000031_0002
多肽 7与 NHR多肽相互作用的 N-PAGE结果显示 （ F ig. 8 D ) ， 多 肽 15、 16、 17与多肽 7的各混合溶液在电泳中均表现出不同于多肽 7 单独溶液的新条带， 证明多肽 15、 16、 17与多肽 7之间均存在明确的 相互作用。 尽管本发明的具体实施方式已经得到详细的描述， 本领域技术人 员将会理解。 根据已经公开的所有教导， 可以对那些细节进行各种修 改和替换， 这些改变均在本发明的保护范围之内。 本发明的全部范围 由所附权利要求及其任何等同物给出。

Claims

权 利 要 求
1. 一种分离的多肽、 其衍生物或其可药用盐， 其中， 所述多肽包 含 11个与 HIV - 1的 HR直接接触的氨基酸残基，并且这 11个氨基酸 残基中的两个分别位于多肽的 N末端和 C末端。
2. 根据权利要求 1所述的多肽、 其衍生物或其可药用盐， 其中， 所述多肽的序列分别均包含在 HI V - 1的 CHR中；优选地，所述多肽的 长度为 36个氨基酸残基。
3. 根据权利要求 1或 2所述的多肽、其衍生物或其可药用盐，其 中， 所述多肽的氨基酸序列包含 WMEWDREI或 WASLWNWF; 优选地， 所 述 WMEWDREI或 WASLWNWF既不位于多肽的 N末端， 也不位于多肽的 C 末端。
4. 根据权利要求 1所述的多肽、 其衍生物或其可药用盐， 其中， 所述多肽的氨基酸序列分别如 SEQ ID NO: 1至 SEQ ID NO: 12中任一 序列所示。
5. 一种分离的多肽、 其衍生物或其可药用盐， 其中， 所述多肽的 氨基酸序列分别如 SEQ ID NO: 13至 SEQ ID NO: 23中任一序列所示。
6. 根据权利要求 4或 5所述的多肽、其衍生物或其可药用盐，其 中， 所述多肽的 N 末端连接乙酰基、 寡肽序列、 或亲脂性基团， 和 / 或
C末端连接酰胺基、 寡肽序列、 或亲脂性基团； 优选地，
所述多肽的 N末端乙酰化， 和 /或 C末端酰胺化。
7. 一种药物组合物，其包含权利要求 1至 6中任一项所述的多肽、 其衍生物或其可药用盐； 可选地， 所述药物组合物还包含药学上可接 受的载体或辅料。
8. 权利要求 1至 6中任一项所述的多肽、其衍生物或其可药用盐 在制备治疗和 /或预防和 /或辅助治疗包膜类病毒感染的药物中的用 途； 具体地， 所述包膜类病毒感染为 HIV感染所致疾病或艾滋病。
9. 权利要求 1至 6中任一项所述的多肽、其衍生物或其可药用盐 在制备或作为 HIV融合抑制剂或者抗 HIV药物中的用途。
10. 一种在体内或体外抑制 HIV的方法，包括使用有效量的权利要 求 1至 6中任一项所述的多肽、 其衍生物或其可药用盐的步骤。
11. 一种在体内或体外抑制 HIV-1 Env介导的细胞融合的方法， 包 括使用有效量的权利要求 1至 6中任一项所述的多肽、 其衍生物或其可 药用盐的步骤。
12. 一种治疗和 /或预防和 /或辅助治疗包膜类病毒感染的方法， 包括给与受试者有效量的权利要求 1至 6中任一项所述的多肽、 其衍生 物或其可药用盐的步骤； 具体地，所述包膜类病毒感染为 HIV感染所致 疾病或艾滋病。
13. 权利要求 1至 6中任一项所述的多肽在筛选治疗和 /或预防和 / 或辅助治疗包膜类病毒感染药物中的用途； 具体地， 所述包膜类病毒 感染为 HIV感染所致疾病或艾滋病。
14. 权利要求 1至 6中任一项所述的多肽在筛选 HIV融合抑制剂或 者抗 HIV药物中的用途。
15. 编码权利要求 1至 6中任一项所述多肽的核苷酸序列。
16. 一种核酸构建体， 其包含权利要求 15所述的核苷酸序列； 具 体地， 所述核酸构建体为重组载体； 更具体地， 所述重组载体为重组 表达载体。
17. 一种重组宿主细胞， 其包含权利要求 16所述的核酸构建体。
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