WO2015106673A1 - 新型抗hiv感染多肽及其利用 - Google Patents

新型抗hiv感染多肽及其利用 Download PDF

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WO2015106673A1
WO2015106673A1 PCT/CN2015/070570 CN2015070570W WO2015106673A1 WO 2015106673 A1 WO2015106673 A1 WO 2015106673A1 CN 2015070570 W CN2015070570 W CN 2015070570W WO 2015106673 A1 WO2015106673 A1 WO 2015106673A1
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polypeptide
seq
hiv
pharmaceutically acceptable
derivative
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PCT/CN2015/070570
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English (en)
French (fr)
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刘克良
蔡利锋
郑保华
张贵英
曹德强
姜喜凤
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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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Publication of WO2015106673A1 publication Critical patent/WO2015106673A1/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
    • 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
    • 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
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • 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
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention belongs to the field of biomedicine and relates to a polypeptide for inhibiting HIV infection and its use in preparing a medicament for preventing HIV infection or preventing or treating AIDS.
  • HIV Human immunodeficiency virus
  • Env contains a surface subunit gp120 and a transmembrane subunit gp41, and three Env forms a non-covalent complex embedded in the surface of the virus.
  • the surface subunit gp120 specifically recognizes and binds to receptors and co-receptors on the surface of target cells during virus-infected cells, and functions as a stable transmembrane subunit gp41, and releases gp41 at appropriate timing to initiate fusion;
  • the membrane subunit gp41 is a direct functional molecule that leads to viral-cell membrane fusion.
  • NHR N-terminal helix region
  • CHR C-terminal helix region
  • NHR-derived polypeptide was used as a fusion inhibitor
  • DP107 (aa 553-590) discovered by Wild et al. in 1992, and its activity of inhibiting viral infection was about 0.2-1 ⁇ M.
  • proteolytic cleavage product N36 (aa 546-581) was also used as a template for the design of NHR-derived polypeptides, and the original sequence was approximately 1 ⁇ M active.
  • Polypeptides derived directly from NHR also known as N-peptides
  • the N-peptide is easy to self-aggregate in solution, reducing its probability of binding to the target; in addition, since the NHR sequence is relatively conservative, the modification space is relatively small.
  • the exogenous NHR-derived polypeptide can directly bind to the endogenous CHR in the viral gp41 metastable structure to form an inactive six-helix structure; or competitively bind to endogenous NHR to form a coiled-coil core structure, hindering It forms a six-helix with normal function.
  • NHR In the helical structure of NHR, the seven residues contained in each two turns of the helix can be sequentially labeled as a-g positions. In the six helix, NHR self-polymerizes through its (a, d) hydrophobic residue to form a trimer complex helix, and binds to the peripheral CHR through its (e, g) residue. It is generally believed that in the solution state alone, the (a, d) residue of NHR can cause oligomerization to form a coiled composite helix, while the (e, g) site residue mainly causes oligomerization after oligomerization.
  • the spiral structure is agglomerated.
  • the N-peptide activity alone is relatively low, and its IC50 value for inhibiting virus-cell fusion is about 1-10 ⁇ M. According to reports in the literature, this may be related to the fact that it is prone to self-folding in solution, making the target binding active site less susceptible to exposure.
  • a chimeric protein is obtained by introducing a model peptide having a stable trimeric coiled-coil structure at the N- or C-terminus of NHR, so that it exhibits a stable trimer structure in a solution state.
  • the activity of the N peptide is greatly improved.
  • these chimeric proteins have large molecular weights and are difficult to synthesize, and there is still a certain distance as drug development.
  • the inventors After intensive research and creative labor, the inventors have surprisingly found that by retaining the length of the original N-peptide, by introducing a hydrophobic amino acid at the (a, d) site of the N-peptide, and in the N-peptide
  • the (e, g) site introduces a helical structural stability factor, a salt bridge, to obtain an N-peptide fusion inhibitor having a high fusion inhibitory activity with an IC50 value of 0.1 ⁇ M or less.
  • the amino acid residues of such polypeptide fusion inhibitors are It is a natural amino acid with obvious synthetic advantages. The following invention is thus provided:
  • a first aspect of the invention relates to a polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10:
  • W is tryptophan
  • N is asparagine
  • A is alanine
  • S is serine
  • K is lysine
  • L leucine
  • E glutamine
  • Q is glutamine
  • I is isoleucine
  • H histidine
  • M methionine
  • T is threonine
  • D is aspartic acid
  • R is arginine
  • Y is tyrosine
  • F is phenylalanine.
  • the oligopeptide sequence is an oligopeptide sequence consisting of 1-10 amino acid residues (eg EEE, KKK, GQAV, GEEE, etc.);
  • the lipophilic group is a fatty acid chain having 3 to 20 carbon atoms, more A fatty acid chain having 8 to 16 carbon atoms is preferred.
  • N-terminus of the polypeptide of the acetyl group (CH 3 CO-), and / or C-terminus amide group (-CONH 2).
  • a second aspect of the invention relates to a nucleic acid molecule encoding the polypeptide of any of the first aspects of the invention.
  • a third aspect of the invention relates to a recombinant vector comprising the nucleic acid molecule of the second aspect of the invention.
  • the vector is a cloning vector or an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector.
  • a fourth aspect of the invention relates to a recombinant cell comprising the recombinant vector of the third aspect of the invention.
  • a recombinant cell according to any one of the fourth aspects of the invention, wherein the cell is a prokaryotic cell (e.g., E. coli) or a eukaryotic cell (e.g., a yeast cell, a mammalian cell).
  • a prokaryotic cell e.g., E. coli
  • a eukaryotic cell e.g., a yeast cell, a mammalian cell.
  • a fifth aspect of the invention relates to a conjugate of the polypeptide of any of the first aspects of the invention, obtained directly or by covalent attachment of a linker to other molecules.
  • the other molecule is, for example, a molecule that enhances targeting (for example, an antibody), a molecule that increases stability or prolongs half-life (such as a PEG molecule), or other organism Active molecule.
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the polypeptide of any one of the first aspects of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a nucleic acid molecule of any of the second aspect of the invention, Or a recombinant vector according to any one of the third aspects of the invention, or a recombinant cell according to any one of the fourth aspects of the invention, or a conjugate of any of the fifth aspect of the invention; optionally further comprising pharmaceutically acceptable Carrier or excipient.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition of the present invention contains 0.1 to 90% by weight of any of the first aspects of the present invention.
  • a polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a nucleic acid molecule according to any one of the second aspects of the invention, or a recombinant vector according to any one of the third aspects of the invention, or any one of the fourth aspect of the invention A recombinant cell, or a conjugate of any of the fifth aspects of the invention.
  • Pharmaceutical compositions can be prepared according to methods known in the art.
  • polypeptide of any one of the first aspects of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, or the nucleic acid molecule of any of the second aspect of the invention, or the invention A recombinant vector according to any one of the three aspects, or a recombinant cell according to any one of the fourth aspects of the invention, or a conjugate according to any one of the fifth aspects of the invention, and one or more solid or liquid pharmaceutical excipients and/or Or an adjuvant is combined to form a suitable administration form or dosage form for human use.
  • a further aspect of the invention relates to a polypeptide according to any one of the first aspects of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a nucleic acid molecule according to any of the second aspects of the invention, or any of the third aspect of the invention
  • the recombinant vector, or the recombinant cell of any of the fourth aspects of the invention, or the conjugate of any of the fifth aspect of the invention for the preparation of a therapeutic and/or prophylactic and/or adjuvant treatment of an envelope-like viral infection Use in medicines for diseases.
  • the envelope virus is a human immunodeficiency virus (HIV), such as HIV-1.
  • HIV human immunodeficiency virus
  • the disease caused by the infection of the envelope virus is AIDS, such as type I AIDS.
  • a further aspect of the invention relates to a method of treating and/or preventing and/or adjunctively treating a disease caused by an envelope viral infection comprising administering to a subject in need thereof an effective amount of any one of the first aspects of the invention A polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a nucleic acid molecule according to any one of the second aspects of the invention, or a recombinant vector according to any one of the third aspects of the invention, or a recombination according to any one of the fourth aspects of the invention.
  • the step of the cell, or the conjugate of any of the fifth aspects of the invention comprising administering to a subject in need thereof an effective amount of any one of the first aspects of the invention A polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a nucleic acid molecule according to any one of the second aspects of the invention, or a recombinant vector according to any one of the third aspects of the invention, or a recombination according
  • the envelope virus is a human immunodeficiency virus (HIV), such as HIV-1.
  • HIV human immunodeficiency virus
  • the disease caused by the infection of the envelope virus is AIDS
  • the disease is, for example, type I AIDS.
  • a further aspect of the invention relates to a polypeptide according to any one of the first aspects of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a nucleic acid molecule according to any of the second aspects of the invention, or any of the third aspect of the invention
  • a further aspect of the invention relates to a polypeptide according to any one of the first aspects of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a nucleic acid molecule according to any of the second aspects of the invention, or any of the third aspect of the invention
  • the envelope virus is a human immunodeficiency virus (HIV), such as HIV-1.
  • HIV human immunodeficiency virus
  • the disease caused by the infection of the envelope virus is AIDS, such as type I AIDS.
  • a further aspect of the invention relates to a method of inhibiting HIV in vivo or in vitro comprising administering an effective amount of a polypeptide of any one of the first aspects of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a second of the invention
  • a nucleic acid molecule according to any one of the aspects, or a recombinant vector according to any one of the third aspects of the invention, or a recombinant cell of any one of the fourth aspects of the invention, or a conjugate of any of the fifth aspect of the invention.
  • the subject when used to inhibit HIV in vivo, is administered to a subject in need thereof, ie, a subject infected with HIV; when used to inhibit HIV in vitro, the subject is administered as needed Cells, cells that infect HIV.
  • a further aspect of the invention relates to a method of inhibiting HIV-1 Env-mediated cell fusion in vivo or in vitro comprising administering an effective amount of a polypeptide of any of the first aspects of the invention, a derivative, or a pharmaceutically acceptable salt thereof, or a nucleic acid molecule according to any one of the second aspects of the invention, or a recombinant vector according to any one of the third aspects of the invention, or a recombinant cell according to any one of the fourth aspects of the invention, or The step of the conjugate of any of the fifth aspects of the invention.
  • the subject when used to inhibit HIV in vivo, is administered to a subject in need thereof, ie, a subject infected with HIV; when used to inhibit HIV in vitro, the subject is administered as needed Cells, cells that infect HIV.
  • salts include: acid addition salts with inorganic acids or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; organic acids such as acetic acid, propionic acid, Caproic acid, cyclopentanoic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methylsulfonate Acid, ethanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, gluconic acid, glutamic acid,
  • compositions of the present invention comprise an effective amount of a polypeptide of the present invention, a derivative thereof, a pharmaceutically acceptable salt, a nucleic acid molecule, a recombinant vector, a recombinant cell, a conjugate, and one or more suitable pharmaceutically acceptable Carrier.
  • Pharmaceutical carriers herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human albumin, buffer substances such as phosphate, glycerin, sorbic acid, potassium sorbate, saturated plants.
  • Partial glyceride mixture of fatty acids, water, salt or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, fiber Substance, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax, lanolin.
  • composition of the compound of the present invention can be administered in any of the following ways: oral, spray inhalation, rectal administration, nasal administration, buccal administration, topical administration, parenteral administration, such as subcutaneous, intravenous, intramuscular, intraperitoneal, sheath Inside, intraventricular, intrasternal and intracranial injection or Enter, or use an explant reservoir. Among them, intravenous administration is preferred.
  • the pharmaceutical composition of the present invention can be administered in the form of a sterile injectable preparation, including sterile injectable water or an oil suspension or a sterile injectable solution.
  • a sterile injectable preparation including sterile injectable water or an oil suspension or a sterile injectable solution.
  • carriers and solvents which can be used include water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils may also be employed as a solvent or suspension medium such as a monoglyceride or a diglyceride.
  • the amount of a polypeptide of the invention administered to a subject will depend on the type and severity of the disease or condition and the characteristics of the subject, such as general health, age, sex, weight, and tolerance to the drug, and also The type of preparation and the mode of administration of the drug, as well as factors such as the dosing cycle or time interval. Those skilled in the art will be able to determine the appropriate dosage based on these and other factors.
  • the subject is a vertebrate, such as a mammal, such as a human, a monkey, a pig, a cow, a horse, a dog, a sheep, a mouse.
  • a mammal such as a human, a monkey, a pig, a cow, a horse, a dog, a sheep, a mouse.
  • the polypeptide involved in the present invention is 36-38 natural amino acid residues in length, and its activity is greatly improved compared with the original sequence, wherein the optimal active sequence is nearly 100-fold more active than the original sequence, reaching 39 nM.
  • These peptides have good application value as monomer fusion inhibitors directly derived from NHR, and their synthesis costs are greatly reduced, which has very good development prospects.
  • these polypeptides have good water solubility and can form a stable helical structure in a solution state, which is very advantageous for improving the water solubility of the drug and studying the mechanism of action of such drugs.
  • the abscissa is the wavelength (nm) and the ordinate is ⁇ (deg ⁇ cm 2 ⁇ dmol -1 ); wherein 1-7 are the numbers of the polypeptides, respectively.
  • the abscissa is temperature (°C), and the ordinate is ⁇ (deg ⁇ cm 2 ⁇ dmol -1 ); wherein 1-7 are the numbers of the polypeptides, respectively.
  • Env envelope glycoprotein
  • MALDI-TOF-MS Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry
  • NHR N-terminal heptad repeat
  • HIV Human immunodeficiency virus
  • the solid phase synthesis carrier Rink amide resin used in the examples is Tianjin Nankai Synthetic Co., Ltd.; the natural amino acids protected by HBTU, HOBt, DIEA and Fmoc are products of Shanghai Jier Biochemical Company and Chengdu Nuoxin Technology Co., Ltd.
  • N-methylpyrrolidone (NMP) is a product of ACROS; TFA, DMF, DCM are products of Beijing Bomai Technology Co., Ltd.; chromatographic pure acetonitrile is Fisher's product. Other reagents are domestically produced pure products if they are not described.
  • a standard Fmoc solid phase peptide synthesis method was employed. All polypeptide sequences are amidated at the C-terminus, acetylated N-terminus according to the conventions of polypeptide synthesis.
  • 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.
  • Microwave peptide synthesis amino acid: 0.2M Fmoc protected amino acid DMF solution; activator: 0.45M HBTU/HOBt DMF solution; activated base: 2M DIEA NMP solution; deprotecting agent: 20% v/v piperidine DMF solution ; blocking reagent: 20% v / v acetic anhydride in DMF solution.
  • Rink Amide resin 0.5g (0.25mm01) was placed in the reactor of CEM microwave peptide synthesizer, then amino acid, activator, activated base, deprotection reagent, 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 3 times with DMF, then shrunk with anhydrous methanol, and dried under vacuum at room temperature to obtain 2.05 g of a peptide resin.
  • the crude peptide obtained was purified by medium pressure or high pressure chromatography.
  • the column was a C8 column and the eluent was acetonitrile, water and a small amount of acetic acid. Specific operation steps: 1 g of crude peptide was weighed, 20 ml of water was added, 5 ml of acetonitrile was dissolved, and the membrane was filtered through a 0.22 ⁇ m microporous membrane filter, and the filtrate was applied.
  • the column was pre-equilibrated with 200 ml of 15% acetonitrile/water/0.1% TFA solution. After the sample was loaded, it was equilibrated with 200 ml of the same eluent.
  • the acetonitrile content was gradually increased to carry out component separation according to ultraviolet absorption.
  • the peaks were collected with different eluents.
  • the eluent components were separated by high-performance liquid phase and the same fractions of eluent were removed. Most of the solvents were removed by rotary evaporation, and the pure peptides were obtained by freeze-drying, ie, peptide 1, and the content of HPLC detection was >90%.
  • Polypeptide 1 was determined by MALDI-TOF-MS mass spectrometry (see Table 2).
  • polypeptide 2-10 was carried out in accordance with the method of Example 1.
  • the specific amino acid sequences of polypeptides 1-10 are shown in Table 1, respectively, which correspond to SEQ ID NO: 1-SEQ ID NO: 10, respectively. Its calculated molecular weight and measured molecular weight are shown in Table 2 below.
  • polypeptide samples used in the following Examples 11 - 12 were the polypeptides 1-10 synthesized in Example 1 - Example 10.
  • 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 CD4T-cell receptors and chemokine co-receptors CCR5 and CXCR4, which are recognized by HIV-1 Env.
  • the luciferase reporter gene is also transcribed in the cell, but the promoter of the gene is not contained, so the luciferase background expression of the cells alone 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 report luciferase in cells.
  • the promoter of the gene Both cells were cultured separately in DMEM containing 10% fetal bovine serum containing ampic/streptomycin double antibody at 37 ° C 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 50 ⁇ l per well was added to a 96-well cell culture plate (37,500/well), and 5% CO 2 was cultured at 37 ° C for 24 hours.
  • polypeptide or positive control sample (T20) is dissolved in phosphate buffer solution (PBS), or an appropriate amount of DMSO is added to fully dissolve, and the polypeptide concentration is determined by ultraviolet spectrometer at 280 nm. degree.
  • PBS phosphate buffer solution
  • the polypeptide solution was then diluted to the appropriate concentration and diluted moderately in a 96-well microtiter plate (Corning).
  • T20 Enfuvirtide, trade name Fuzeon sequence: (gp41aa 638-673)
  • the luciferase reporter gene kit (Promega) was taken out from the refrigerator, and the 5x cell lysate was diluted with double distilled water to 1 ⁇ lysate according to the amount, and placed at room temperature; the substrate was dissolved in the substrate buffer, and left at room temperature.
  • the detection conditions of the microplate reader (Molcular Devices M5 multi-plate reader) are set for use.
  • 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 air bubbles was avoided as much as possible; the substrate was rapidly added to the ELISA plate at 40 ⁇ l/well in the dark. Chemiluminescence was measured immediately 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 (IC50) is determined according to the concentration-chemiluminescence signal curve of the sample.
  • the IC50 value of the positive control sample should be stable within a certain range; in the ideal inhibition curve, the signal of the high concentration inhibitor should be close to the background signal, and the lowest concentration inhibitor The lower signal should be close to the saturated fused signal.
  • the cell fusion inhibitory activity of polypeptides 1-10 is shown in Table 3.
  • the positive control T20 has an IC 50 of 5.42 nM, which is consistent with the literature (Wild, CT, et al., Proceedings of the National Academy of Sciences of the United States of America). , 1994.91 (21): p.9770-9774.).
  • the results show that the polypeptide of the present invention has good HIV-1 Env-mediated cell fusion inhibitory activity and can be used as an HIV fusion inhibitor.
  • the present inventors studied the interaction of polypeptide 1-10 with gp41CHR by circular dichroism (CD).
  • the inventors selected C36MN, C36, and C36MC to correspond to the N38MC series (SEQ ID NO: 1-5) and N38Mut series (SEQ, respectively).
  • Example 1 For the preparation method, reference can be made to Example 1.
  • the circular dichroic instrument is a Bio-logic MOS450 spectrometer.
  • the CHR polypeptide will be assayed (ie C36MN, C36, C36MC) were dissolved in PBS, NHR polypeptide (ie, polypeptide 1-10) was dissolved in double distilled water, and the mother liquor concentration was determined according to the ultraviolet absorption of the solution at 280 nm; then diluted to prepare a 20 ⁇ M polypeptide PBS solution.
  • Formulating a sample of the polypeptide to be detected mixing C36MN and polypeptide 1-5 (or C36 and polypeptide 6-7) in a 1:1 volume ratio to obtain a mixed sample; if it is a separate polypeptide sample, 20 ⁇ M of the sample and the buffer solution are Mix 1:1. The sample was allowed to stand at 37 ° C for 30 min to allow sufficient reaction. The above experimental steps ensure that the concentration of the polypeptide in the sample remains the same.
  • the prepared sample was measured on a circular dichroic instrument with a scanning wavelength range of 190-260 nm, a wavelength interval of 1 nm, a scanning speed of 100 nm/min, and an average of three scans.
  • 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 solution secondary structure can be determined by the characteristic peak spectrum of the CD spectrum.
  • the mixed solution of all NHR-derived polypeptides and the mixed solution with the CHR-derived polypeptide showed negative peaks at 208 nm and 222 nm, suggesting that the solution state of the polypeptide is ⁇ -helical.
  • Different solutions show different heights of negative peaks, suggesting that the content of the helical structure is different.
  • the inventors also determined the thermal stability of the helical structures formed by polypeptides 1-5 and C36MN (and 6-7 and C36) by CD temperature scanning.
  • the specific method is as follows: the sample is diluted to 1 ⁇ M, added to the sample cell, the CD instrument program is set to temperature scanning, the detection wavelength is 220 nm, the scanning range is 20-90 degrees Celsius, and the program temperature is scanned and checked under stirring to obtain a CD signal with temperature curve ( See Figure 8 - Figure 14). A differential is calculated from the curve, and the Tm value is determined from the peak value of the first differential curve (see Table 4), that is, the thermal transition temperature of the sample.
  • Example 12 The inventors studied the interaction of polypeptides 1-8 with CHR-derived polypeptides by N-PAGE.
  • C36MN and C36 are respectively combined with N38MC series (polypeptide 1-5) and N38Mut series (polypeptides 6-8), and the sequence thereof is as shown in Example 12.
  • N38MC series polypeptide 1-5)
  • N38Mut series polypeptides 6-8
  • the sequence thereof is as shown in Example 12.
  • Example 1 For the preparation method, reference can be made to Example 1.
  • the original sequence of N38MC and N38 was used as a control, and the amino acid sequence thereof was as follows, and the preparation method was referred to Example 1.
  • N38MC Ac-Q QNNLLRA IEAQQHL LQLTVWG IKQLQAR ILAVERY LK-NH 2 (SEQ ID NO: 15)
  • the N-PAGE experiment was performed using a BG-Power 3500 multi-purpose electrophoresis apparatus (Beijing Baijing Biotechnology Co., Ltd.).
  • the polypeptides 1-8 were dissolved in double distilled water, C36MN and C36 were dissolved in PBS, and the mother liquor concentration was determined according to the ultraviolet absorption intensity at 280 nm; then, the polypeptide solution was diluted to prepare 200 ⁇ M.
  • C36MN and polypeptides 1-5 (C36 and polypeptide 6-7) were mixed in a 1:1 volume ratio to obtain a mixed sample; if it was a single polypeptide sample, 20 ⁇ M of the sample was mixed with the buffer solution at a ratio of 1:1.
  • the sample was allowed to stand at 37 ° C for 30 min to allow sufficient reaction.
  • the above experimental steps ensure that the concentration of the polypeptide in the sample remains the same. After the end, an equal volume of N-PAGE 2X loading buffer (Invitrogen) was added to each sample, and the mixture was mixed and used.
  • Separation gel (16%) was prepared: 8 ml of 30% acrylamide solution, 3.75 ml of 4X separation gel buffer, 3.1 ml of water, 100 ⁇ l of 10% AP solution, 10 ⁇ l of TEMED, and mixed by shaking. Carefully place the above solution between the two glass plates, leaving 1-2 cm of space on 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.
  • a concentrated gel (4%) was prepared: the n-butanol layer was discarded, the upper layer of the gel was carefully washed with deionized water, and the water was blotted with a filter paper.
  • 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.
  • the voltage is about 150V, the current is about 25mA, pre-electrophoresis for 20min, turn off the power.
  • Use a buffer to clean the sample tank use a sample gun or micro-syringe to slowly inject the sample solution into the bottom of the sample tank, turn on the power, start electrophoresis, and complete the electrophoresis after about 2 hours (visible band of bromophenol blue) For the leading edge, stop 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.

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Abstract

本发明属于生物医药领域，提供了用于抑制HIV感染的多肽，所述多肽包含选自SEQ ID NO：1-10所示的氨基酸序列，还提供了所述多肽在制备抗HIV感染或预防或治疗艾滋病的药物中的用途。本发明的多肽为直接衍生于NHR的单体融合抑制剂，其活性较原始序列提高，合成成本低。

Description

新型抗HIV感染多肽及其利用 技术领域
本发明属于生物医药领域，涉及用于抑制HIV感染的多肽及其在制备抗HIV感染或预防或治疗艾滋病的药物中的用途。
背景技术
人免疫缺陷病毒(HIV)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。目前世界范围内HIV感染主要以1型(HIV-1)为主。
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gp120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gp120在病毒感染细胞过程中可以特异识别并结合于靶细胞表面的受体和辅助受体，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是导致病毒-细胞膜融合的直接功能分子。
在病毒细胞融合过程中，gp41的N-端螺旋区(NHR)和C-端螺旋区(CHR)通过折叠形成了一个发夹状的六螺旋结构；该结构的形成为病毒-细胞膜融合提供了能量，对病毒-细胞融合至关重要。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。能够阻止HIV-1六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。
最初NHR衍生多肽作为融合抑制剂的发现是1992年Wild等发现的DP107(aa 553-590)，其抑制病毒感染的活性在0.2-1μM左右。另外，蛋白水解酶切产物N36(aa 546-581)也作为NHR衍生多肽设计的模板，原始序列的活性约为1μM。直接衍生于NHR的多肽(也称为N肽)抗融合活性比衍生于CHR的多肽要差很多，主要原因可能是单分子 的N肽在溶液中容易自身聚集，降低了其与靶点的结合几率；另外由于NHR序列比较保守，改造空间相对较小。外源NHR衍生多肽可直接与病毒gp41亚稳态结构中的内源CHR结合，形成无活性的六螺旋结构；或与内源NHR竞争性的结合形成复合螺旋(coiled-coil)内核结构，阻碍其形成有正常功能的六螺旋。
在NHR的螺旋结构中，每两圈螺旋所包含的七个残基可以依次标记为a-g位。在六螺旋中，NHR通过其(a，d)位疏水残基自聚形成三聚体复合螺旋，并通过其(e，g)位残基与外围的CHR结合。一般认为，在单独存在的溶液状态下，NHR的(a，d)位残基可以使其发生寡聚形成卷曲复合螺旋，而(e，g)位点残基则主要使寡聚后的复合螺旋结构发生沉聚。单独的N肽活性均比较低，其抑制病毒-细胞融合的IC50值约为1-10μM。根据文献报道，这可能与其在溶液中容易发生自身折叠从而使得靶结合活性位点不易暴露有关。
在克服NHR多肽自聚方面，一般通过在NHR的N或C末端引入有稳定三聚体卷曲螺旋结构的模型肽，得到嵌合蛋白，使其在溶液状态下呈现稳定的三聚体结构，可大大改善N肽的活性。但是这些嵌合蛋白分子量大，合成难度较高，作为药物开发仍存在一定距离。
发明内容
本发明人经过深入的研究和创造性的劳动，令人惊奇地发现，在保留原始N肽长度的基础上，通过在N肽的(a，d)位点引入疏水性氨基酸，并在N肽的(e，g)位点引入螺旋结构稳定因素-盐桥，可以得到单体具有0.1μM以下IC50值的有高融合抑制活性的N肽融合抑制剂，这类多肽融合抑制剂的氨基酸残基均为天然氨基酸，具有明显的合成优势。由此提供了下述发明：
本发明的第一方面涉及一种多肽、其衍生物、或其可药用盐，其中，所述多肽包含选自SEQ ID NO：1～SEQ ID NO：10所示的氨基酸序列：
QWKNLLEAIKAQQELLKLTVEGIKQLQERIKAVERYLK(SEQ ID NO：1)；
QINNLLRAIEALQHLLQLLVWGIKQLQARILAVERYLK(SEQ ID NO：2)；
QWNNLLEAIKALQELLKLLVEGIKQLQERIKAVERYLK(SEQ ID NO：3)；
QWKNLLEAIKALQELLKLLVEGIKQLQERIKAVERYLK(SEQ ID NO：4)；
WKNLLEAIKALQELLKLLVEGIKQLQERIKAVERYL(SEQ ID NO：5)；
LLSGIVQQINNLLRAIEALQHLLQLIVWGIKQLQARIL(SEQ ID NO：6)；
LLKGIVEQIKKLLEAIKALQELLKKIVEGIKWLQERIL(SEQ ID NO：7)；
LLKGIVEQIKNLLEAIKALQELLKLIVEGIKWLQERIK(SEQ ID NO：8)；
LWEQIRKLLSGIVQQINNLLRAIEALQHLLQKIVEGIK(SEQ ID NO：9)；
LWKQIRELLKGIVEQIKNLLEAIKALQELLKLIVEGIK(SEQ ID NO：10)。
其中氨基酸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。
根据本发明第一方面任一项所述的多肽、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、亲脂性基团、或胆固醇等；
优选地，所述寡肽序列为1-10个氨基酸残基组成的寡肽序列(例如EEE、KKK、GQAV、GEEE等)；
优选地，所述亲脂性基团为含有3到20个碳原子的脂肪酸链，更 优选含8-16个碳原子的脂肪酸链。
在本发明的实施方案中，所述多肽的N末端连接乙酰基(CH3CO-)，和/或C末端连接酰胺基(-CONH2)。
本发明第二方面涉及核酸分子，其编码本发明第一方面任一项所述的多肽。
本发明第三方面涉及重组载体，其含有本发明第二方面所述的核酸分子。
根据本发明第三方面任一项的重组载体，其中所述载体为克隆载体或表达载体，所述表达载体例如为原核表达载体或真核表达载体。
本发明第四方面涉及重组细胞，其含有本发明第三方面所述的重组载体。
根据本发明第四方面任一项的重组细胞，其中所述细胞为原核细胞(例如大肠杆菌)或真核细胞(例如酵母细胞、哺乳动物细胞)。
本发明的第五方面涉及本发明第一方面任一项的多肽直接或通过连接物与其它分子共价连接得到的偶联物。
根据本发明第五方面任一项的偶联物，其中所述其它分子例如为提高靶向性的分子(例如为抗体)、提高稳定性或延长半衰期的分子(例如PEG分子)、或其它生物活性分子。
本发明的另一方面涉及一种药物组合物，其包含本发明第一方面任一项的多肽、其衍生物、或其可药用盐、或者本发明第二方面任一项的核酸分子、或者本发明第三方面任一项的重组载体、或者本发明第四方面任一项的重组细胞、或者本发明第五方面任一项的偶联物；任选地，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％的本发明第一方面任一 项的多肽、其衍生物、或其可药用盐、或者本发明第二方面任一项的核酸分子、或者本发明第三方面任一项的重组载体、或者本发明第四方面任一项的重组细胞、或者本发明第五方面任一项的偶联物。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，可将本发明第一方面任一项的多肽、其衍生物、或其可药用盐、或者本发明第二方面任一项的核酸分子、或者本发明第三方面任一项的重组载体、或者本发明第四方面任一项的重组细胞、或者本发明第五方面任一项的偶联物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。
本发明的再一方面涉及本发明第一方面任一项的多肽、其衍生物、或其可药用盐、或者本发明第二方面任一项的核酸分子、或者本发明第三方面任一项的重组载体、或者本发明第四方面任一项的重组细胞、或者本发明第五方面任一项的偶联物在制备治疗和/或预防和/或辅助治疗包膜类病毒感染所致疾病的药物中的用途。
在本发明的实施方案中，所述包膜类病毒为人免疫缺陷病毒(HIV)，例如为HIV-1。
在本发明的实施方案中，所述包膜类病毒感染所致的疾病为艾滋病，例如为I型艾滋病。
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所致疾病的方法，包括给予有需要的受试者有效量的本发明第一方面任一项的多肽、其衍生物、或其可药用盐、或者本发明第二方面任一项的核酸分子、或者本发明第三方面任一项的重组载体、或者本发明第四方面任一项的重组细胞、或者本发明第五方面任一项的偶联物的步骤。
在本发明的实施方案中，所述包膜类病毒为人免疫缺陷病毒(HIV)，例如为HIV-1。
在本发明的实施方案中，所述包膜类病毒感染所致的疾病为艾滋 病，例如为I型艾滋病。
本发明的再一方面涉及本发明第一方面任一项的多肽、其衍生物、或其可药用盐、或者本发明第二方面任一项的核酸分子、或者本发明第三方面任一项的重组载体、或者本发明第四方面任一项的重组细胞、或者本发明第五方面任一项的偶联物在制备作为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本发明第一方面任一项的多肽、其衍生物、或其可药用盐、或者本发明第二方面任一项的核酸分子、或者本发明第三方面任一项的重组载体、或者本发明第四方面任一项的重组细胞、或者本发明第五方面任一项的偶联物，其用于治疗和/或预防和/或辅助治疗包膜类病毒感染所致疾病。
在本发明的实施方案中，所述包膜类病毒为人免疫缺陷病毒(HIV)，例如为HIV-1。
在本发明的实施方案中，所述包膜类病毒感染所致的疾病为艾滋病，例如为I型艾滋病。
本发明的再一方面涉及一种在体内或体外抑制HIV的方法，包括给予有效量的本发明第一方面任一项的多肽、其衍生物、或其可药用盐、或者本发明第二方面任一项的核酸分子、或者本发明第三方面任一项的重组载体、或者本发明第四方面任一项的重组细胞、或者本发明第五方面任一项的偶联物的步骤。
在本发明的实施方案中，当用于在体内抑制HIV时，给予对象为有需要的受试者，即感染HIV的受试者；当用于在体外抑制HIV时，给予对象为有需要的细胞，即感染HIV的细胞。
本发明的再一方面涉及一种在体内或体外抑制HIV-1Env介导的细胞融合的方法，包括给予有效量的本发明第一方面任一项的多肽、其 衍生物、或其可药用盐、或者本发明第二方面任一项的核酸分子、或者本发明第三方面任一项的重组载体、或者本发明第四方面任一项的重组细胞、或者本发明第五方面任一项的偶联物的步骤。
在本发明的实施方案中，当用于在体内抑制HIV时，给予对象为有需要的受试者，即感染HIV的受试者；当用于在体外抑制HIV时，给予对象为有需要的细胞，即感染HIV的细胞。
本发明中使用的术语“可药用盐”意指在制药上可接受的并且具有母体化合物的所需药理学活性的本发明化合物的盐。这类盐包括：与无机酸或与有机酸形成的酸加成的盐，所述的无机酸诸如盐酸，氢溴酸，硫酸，硝酸，磷酸等；所述的有机酸诸如乙酸，丙酸，己酸，环戊丙酸，乙醇酸，丙酮酸，乳酸，丙二酸，琥珀酸，苹果酸，马来酸，富马酸，酒石酸，柠檬酸，苯甲酸，肉桂酸，扁桃酸，甲磺酸，乙磺酸，苯磺酸，萘磺酸，樟脑磺酸，葡庚糖酸，葡糖酸，谷氨酸，羟基萘甲酸，水杨酸，硬脂酸，粘康酸等；或在母体化合物上存在的酸性质子被金属离子，例如碱金属离子或碱土金属离子取代时形成的盐；或与有机碱形成的配位化合物，所述的有机碱诸如乙醇胺，二乙醇胺，三乙醇胺，N-甲基葡糖胺等。
本发明的药物组合物包括有效剂量的本发明的多肽、其衍生物、可药用盐、核酸分子、重组载体、重组细胞、偶联物，和一种或多种适宜的药学上可接受的载体。这里的药用载体包括但不限于：离子交换剂，氧化铝，硬脂酸铝，卵磷脂，血清蛋白如人血白蛋白，缓冲物质如磷酸盐，甘油，山梨酸，山梨酸钾，饱和植物脂肪酸的部分甘油酯混合物，水，盐或电解质，如硫酸鱼精蛋白，磷酸氢二钠，磷酸氢钾，氯化钠，锌盐，胶态氧化硅，三硅酸镁，聚乙烯吡咯烷酮，纤维素物质，聚乙二醇，羧甲基纤维素钠，聚丙烯酸酯，蜂蜡，羊毛脂。
本发明化合物的药物组合物可以以下面的任意方式施用：口服，喷雾吸入，直肠用药，鼻腔用药，颊部用药，局部用药，非肠道用药，如皮下，静脉，肌内，腹膜内，鞘内，心室内，胸骨内和颅内注射或 输入，或借助一种外植储器用药。其中优选静脉内给药方式。
本发明的药物组合物可以以无菌注射制剂形式用药，包括无菌注射水或油悬浮液或无菌注射溶液。其中，可使用的载体和溶剂包括水、林格氏溶液和等渗氯化钠溶液。另外，灭菌的非挥发油也可用作溶剂或悬浮介质，如单甘油酯或二甘油酯。
对受试者给予的本发明多肽的量取决于所述疾病或病况的类型和严重程度以及受试者的特征，如一般健康状况、年龄、性别、体重和对药物的耐受度，还取决于制剂的类型和药物的给药方式，以及给药周期或时间间隔等因素。本领域技术人员能够根据这些因素和其它因素来确定适当的剂量。
在本发明中，所述受试者为脊椎动物，例如为哺乳动物，例如为人、猴、猪、牛、马、狗、羊、鼠。
发明的有益效果
本发明涉及的多肽长度为36-38个天然氨基酸残基，其活性较原始序列大大提高，其中最佳活性序列较原始序列在活性上提高了近100倍，达到了39nM。这些多肽作为直接衍生于NHR的单体融合抑制剂，具有很好的应用价值，且其合成成本大大降低，具有非常好的开发前景。同时，这些多肽水溶性好，在溶液状态下可形成稳定的螺旋结构，对于改善药物水溶性和研究此类药物作用的机制非常有利。
附图说明
图1多肽1单独溶液及其与CHR混合溶液的CD谱
图2多肽2单独溶液及其与CHR混合溶液的CD谱
图3多肽3单独溶液及其与CHR混合溶液的CD谱
图4多肽4单独溶液及其与CHR混合溶液的CD谱
图5多肽5单独溶液及其与CHR混合溶液的CD谱
图6多肽6单独溶液及其与CHR混合溶液的CD谱
图7多肽7单独溶液及其与CHR混合溶液的CD谱
图1-图7中，横坐标为波长(nm)，纵坐标为θ(deg·cm2·dmol-1)；其中的1-7分别为多肽的编号。
图8多肽1的CD变温扫描曲线
图9多肽2的CD变温扫描曲线
图10多肽3的CD变温扫描曲线
图11多肽4的CD变温扫描曲线
图12多肽5的CD变温扫描曲线
图13多肽6的CD变温扫描曲线
图14多肽7的CD变温扫描曲线
图8-图14中，横坐标为温度(℃)，纵坐标为θ(deg·cm2·dmol-1)；其中的1-7分别为多肽的编号。
图15多肽1-8与CHR衍生多肽相互作用的N-PAGE图
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：
AID S(Acquired Immure Deficiency Syndrome)获得性免疫缺陷综合症
Ala(Alanine，A)丙氨酸
Arg(Arginine，R)精氨酸
Asn(Asparagine，N)天冬酰胺
Asp(Asparticacid，D)天冬氨酸
CD(Cycurlar Dicroism)圆二色性
DCM(Dichloromethane)二氯甲烷
DMF(N，N-Dimethyl formamide)二甲基甲酰胺
DMSO二甲亚砜
Env(Envelope glycoprotein)包膜糖蛋白
MALDI-TOF-MS(Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry)基质辅助激光解析飞行时间质谱
Fmoc(Fluorenylmethoxycarbonyl)芴甲氧羰基
Gly(Glycine，G)甘氨酸
Gln(Glutamine，Q)谷酰胺
Glu(Glutamic acid，E)谷氨酸
6-HB(six-helix bundle)六螺旋体
HBTU 2-(1H-1-羟基苯并三唑)-1，1，3，3-四甲基六氟磷酸
His(Histidine，H)组氨酸
HOBt(1-Hydroxyl benzotiazole anhydrous)1-羟基苯并三氮唑
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-11型人免疫缺陷病毒
HPLC(high performance liquid chromatography)高效液相色谱
Ile(Isoleucine，I)异亮氨酸
Leu(Leucine，L)亮氨酸
Met(Methionine，M)甲硫氨酸
N-PAGE(Native Poiy-Acrylamide Gel Electrophoresis)非变性聚丙烯酰胺凝胶电泳
Lys(Lysine，K)赖氨酸
Phe(Phenylalanine，F)苯丙氨酸
Ser(Serine，S)丝氨酸
TFA(Trifluoroacetic acid)三氟乙酸
Thr(Threonie，T)苏氨酸
Tyr(Tyrosine，Y)酪氨酸
Val(Valine，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、HOBt、DIEA及Fmoc保护的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-甲基吡咯烷酮(NMP)为ACROS公司产品；TFA、DMF、DCM为北京博迈科技有限公司产品；色谱纯乙腈为Fisher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。所有多肽序列均按照多肽合成的常规将C-端酰胺化，N-端乙酰化。选用Rink Amide树脂，肽类由C-端向N-端延长。缩合剂为HBTU/HOBt/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％。基质辅助激光解析飞行时间质谱(MALDI-TOF-MS)确定多肽分子量。
微波多肽合成：氨基酸：0.2M的Fmoc保护氨基酸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(0.25mm01)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遍后用无水甲醇收缩，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500ml茄形瓶中，冰浴，电磁搅拌。按1g肽树脂加入10ml的量配制裂解液(体积比：三氟乙酸∶乙二硫醇∶间甲苯酚∶水＝82.8∶10∶5∶2.5)。TFA需预先冰浴降温30min或者预先放于冰箱中使用。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C8柱，洗脱液为乙腈，水及少量乙酸。具体操作步骤：称取粗肽1g，加水20ml，乙腈5ml溶解，用0.22μm微孔滤膜过滤，取滤液上样。色谱柱预先用15％乙腈/水/0.1％TFA溶液200ml平衡，上样后继续用200ml同样洗脱液平衡，继而根据程序设定洗脱梯度逐步升高乙腈含量进行组分分离，根据紫外吸收峰收集不同的洗脱液，高效液相检测洗脱液成分合并同组分洗脱液，旋转蒸发除去大部分溶剂，冻干得纯肽，即多肽1，HPLC检测含量＞90％。
多肽1经MALDI-TOF-MS质谱确定其分子量(见表2)。
实施例2～实施例10：多肽2-10的制备
参照实施例1中的方法进行制备得到多肽2-10。多肽1-10的具体氨基酸序列分别如表1中所示，其分别对应于SEQ ID NO：1-SEQ ID NO：10。其计算分子量和实测分子量如下面的表2所示。
下面的实施例11-实施例12所用多肽样品为实施例1-实施例10所合成的多肽1-10。
表1 多肽序列
Figure PCTCN2015070570-appb-000001
Figure PCTCN2015070570-appb-000002
表2 合成多肽的分子量
Figure PCTCN2015070570-appb-000003
实施例11：多肽的抗HIV-1融合活性检测
本发明人用HIV-1Env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-bl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CD4T-细胞受体和趋化因子辅助受体CCR5和CXCR4，可被HIV-1Env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-1Env，由Env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℃下在含有5％CO2的培养箱中单独培养。两种细胞均为贴壁细胞，细胞用胰酶/EDTA消化后收获或传代。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50μl加入96孔细胞培养板中(3.75万/孔)，5％CO2，37℃下培养24小时。
将多肽或阳性对照样品(T20)溶于磷酸盐缓冲溶液(PBS)中，或加入适量DMSO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。
其中，T20(Enfuvirtide，商品名Fuzeon)序列：(gp41aa 638-673)
Ac-NNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-NH2(SEQ ID NO：11)
配制150万/ml的HL2/3效应细胞。
将20μl/孔的等比稀释的多肽抑制剂加入前一天培养的TZM-bl细胞中，然后加入50μl/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％CO2，37℃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细胞裂解液根据用量用双蒸水稀释至1x裂解液，室温下放置；用底物缓冲液溶解底物，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200μl/孔PBS洗两次，尽量去除清洗液；然后以50μl/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40μl/孔加入96孔化学发光检测用酶标板(Corning)中，加样时尽量避免引入气泡；避光下将底物以40μl/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融合的有效性，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(IC50)，阳性对照样品的IC50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1-10的细胞融合抑制活性列于表3，阳性对照T20的IC50为5.42nM，与文献报道相符(Wild，C.T.，et a1.，Proceedings of the National Academy of Sciences of the United States of America，1994.91(21)：p.9770-9774.)。结果显示，本发明的多肽具有良好的HIV-1Env介导的 细胞融合抑制活性，能够作为HIV融合抑制剂。
表3 多肽的细胞融合抑制活性
Figure PCTCN2015070570-appb-000004
实施例12：圆二色谱研究多肽1-10与CHR的相互作用
本发明人用圆二色谱(CD)研究多肽1-10与gp41CHR的相互作用，本发明人选用C36MN、C36、C36MC分别对应的与N38MC系列(SEQ ID NO：1-5)、N38Mut系列(SEQ ID NO：6-8)、N38MN系列(SEQ ID NO：9-10)结合，其序列如下所示，上述多肽的N末端乙酰化，C末端成酰胺，其制备方法可以参考实施例1。
C36MN：
Ac-QIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKN-NH2(SEQ ID NO：12)
C36：
Ac-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL-NH2(SEQ ID NO：13)
C36MC：
Ac-INNYTSLIHSLIEESQNQQEKNEQELLELDKWASLW-NH2(SEQ ID NO：14)
圆二色谱仪为Bio-logic MOS450谱仪。将测定CHR多肽(即 C36MN、C36、C36MC)溶于PBS中，NHR多肽(即多肽1-10)溶于双蒸水中，根据溶液在280nm下的紫外吸收确定母液浓度；然后稀释配制成20μM的多肽PBS溶液。
配制要检测的多肽样品：将C36MN和多肽1-5(或C36和多肽6-7)以1∶1体积比混合得二者混合样品；如果是单独多肽样品，将20μM的样品与缓冲溶液以1∶1混合。样品在37℃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。
将配制好的样品在圆二色谱仪上测定，仪器扫描波长范围为190-260nm，波长间隔为1nm，扫描速度为100nm/min，扫描3次取平均值。先用缓冲溶液扫描得到空白，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。
通过样品的CD谱特征峰可以确定其溶液二级结构，所有NHR衍生多肽单独溶液以及与CHR衍生多肽的混合溶液在208nm和222nm处均显示负峰，提示其多肽的溶液状态呈α螺旋结构。不同的溶液显示的负峰高度不一，提示其螺旋结构的含量有所不同。(如图1-图7所示)
此外，本发明人还通过CD温度扫描测定多肽1-5与C36MN(以及6-7与C36)形成的螺旋结构的热稳定性。具体方法如下：将样品稀释到1μM，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(见图8-图14)。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4)，即样品的热转变温度。
表4：多肽的Tm值
Figure PCTCN2015070570-appb-000005
实施例13：N-PAGE研究多肽1-8与NHR靶点的相互作用
本发明人用N-PAGE研究了多肽1-8与CHR衍生多肽的相互作用。选用C36MN、C36分别对应的与N38MC系列(多肽1-5)、N38Mut系列(多肽6-8)结合，其序列如实施例12所示，其制备方法可以参考实施例1。同时以N38MC、N38原始序列做对照，其氨基酸序列如下，制备方法参考实施例1。
N38MC：Ac-Q QNNLLRA IEAQQHL LQLTVWG IKQLQAR ILAVERY LK-NH2(SEQ ID NO：15)
N38：Ac-L LSGIVQQ QNNLLRA IEAQQHL LQLTVWG IKQLQAR IL-NH2(SEQ ID NO：16)
N-PAGE实验使用BG-Power3500多用电泳仪(北京百晶生物技术有限公司)完成。
将多肽1-8溶于双蒸水中，C36MN、C36溶于PBS中，根据280nm下的紫外吸收强度确定母液浓度；然后稀释配制成200μM的多肽溶液。将C36MN和多肽1-5(C36和多肽6-7)以1∶1体积比混合得二者混合样品；如果是单独多肽样品，将20μM的样品与缓冲溶液以1∶1混合。样品在37℃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。结束后，每个样品中加入等体积的N-PAGE 2X上样缓冲液(Invitrogen公司)，混匀后待用。
配制分离胶(16％)：30％丙烯酰胺溶液8ml，4X分离胶缓冲液3.75ml，水3.1ml，10％AP溶液100μl，TEMED 10μl，震荡混合。小心将上述溶液，两层玻璃板之间，上层留有1-2cm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1ml，4X浓缩胶缓冲液2ml，水4.8ml，加入10％AP溶液100μl，TEMED 10μl，震荡混合。用上述溶液注满玻璃板，插入上样梳子。
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电泳缓冲液稀释到1X，注满电容 槽。电压约150V，电流约为25mA，预电泳20min，关掉电源。使用缓冲液清洗上样槽，使用加样枪或微量注射器将所配样品溶液缓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)。
取下凝胶，用双蒸水洗三遍，各5分钟。加入BioRad G250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。
结果显示(图15)：多肽序列1-8均可以和靶点CHR衍生肽结合形成新的条带，说明所发明多肽1-8与gp41CHR间存在相互作用，提示其抗病毒活性机制可能与CHR靶点相互作用有关。
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 (20)

  1. 一种多肽、其衍生物、或其可药用盐，其中，所述多肽包含选自SEQ ID NO：1～SEQ ID NO：10所示的氨基酸序列：
    QWKNLLEAIKAQQELLKLTVEGIKQLQERIKAVERYLK(SEQ ID NO：1)；
    QINNLLRAIEALQHLLQLLVWGIKQLQARILAVERYLK(SEQ ID NO：2)；
    QWNNLLEAIKALQELLKLLVEGIKQLQERIKAVERYLK(SEQ ID NO：3)；
    QWKNLLEAIKALQELLKLLVEGIKQLQERIKAVERYLK(SEQ ID NO：4)；
    WKNLLEAIKALQELLKLLVEGIKQLQERIKAVERYL(SEQ ID NO：5)；
    LLSGIVQQINNLLRAIEALQHLLQLIVWGIKQLQARIL(SEQ ID NO：6)；
    LLKGIVEQIKKLLEAIKALQELLKKIVEGIKWLQERIL(SEQ ID NO：7)；
    LLKGIVEQIKNLLEAIKALQELLKLIVEGIKWLQERIK(SEQ ID NO：8)；
    LWEQIRKLLSGIVQQINNLLRAIEALQHLLQKIVEGIK(SEQ ID NO：9)；
    LWKQIRELLKGIVEQIKNLLEAIKALQELLKLIVEGIK(SEQ ID NO：10)。
  2. 权利要求1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、亲脂性基团、或胆固醇等；
    优选地，所述寡肽序列为1-10个氨基酸残基组成的寡肽序列(例如 EEE、KKK、GQAV、GEEE等)；
    优选地，所述亲脂性基团为含有3到20个碳原子的脂肪酸链，更优选含8-16个碳原子的脂肪酸链。
  3. 核酸分子，其编码权利要求1的多肽。
  4. 重组载体，其含有权利要求3的核酸分子。
  5. 重组细胞，其含有权利要求4的重组载体。
  6. 权利要求1或2的多肽直接或通过连接物与其它分子共价连接得到的偶联物。
  7. 权利要求6的偶联物，其中所述其它分子例如为提高靶向性的分子(例如为抗体)、提高稳定性或延长半衰期的分子(例如PEG分子)、或其它生物活性分子。
  8. 药物组合物，其包含权利要求1或2的多肽、其衍生物、或其可药用盐、权利要求3的核酸分子、权利要求4的重组载体、权利要求5的重组细胞或权利要求6或7的偶联物；任选地，还包含药学上可接受的载体或辅料。
  9. 权利要求1或2的多肽、其衍生物、或其可药用盐、权利要求3的核酸分子、权利要求4的重组载体、权利要求5的重组细胞或权利要求6或7的偶联物在制备治疗和/或预防和/或辅助治疗包膜类病毒感染所致疾病的药物中的用途。
  10. 权利要求9的用途，其中所述包膜类病毒为人免疫缺陷病毒(HIV)，例如为HIV-1。
  11. 权利要求9的用途，其中所述包膜类病毒感染所致的疾病为艾滋病，例如为I型艾滋病。
  12. 权利要求1或2的多肽、其衍生物、或其可药用盐、权利要求3的核酸分子、权利要求4的重组载体、权利要求5的重组细胞或权利要求6或7的偶联物在制备作为HIV融合抑制剂或者抗HIV药物中的用途。
  13. 一种治疗和/或预防和/或辅助治疗包膜类病毒感染所致疾病的方法，包括给予有需要的受试者有效量的权利要求1或2的多肽、其衍生物、或其可药用盐、或者权利要求3的核酸分子、或者权利要求4的重组载体、或者权利要求5的重组细胞、或者权利要求6或7的偶联物的步骤。
  14. 权利要求13所述的方法，其中所述包膜类病毒为人免疫缺陷病毒(HIV)，例如为HIV-1。
  15. 权利要求13所述的方法，其中所述包膜类病毒感染所致的疾病为艾滋病，例如为I型艾滋病。
  16. 权利要求1或2的多肽、其衍生物、或其可药用盐、或者权利要求3的核酸分子、或者权利要求4的重组载体、或者权利要求5的重组细胞或者权利要求6或7的偶联物，其用于治疗和/或预防和/或辅助治疗包膜类病毒感染所致疾病。
  17. 权利要求16所述的多肽、其衍生物、或其可药用盐、或者核酸分子、或者重组载体、或者重组细胞或者偶联物，其中所述包膜类病毒为人免疫缺陷病毒(HIV)，例如为HIV-1。
  18. 权利要求16所述的多肽、其衍生物、或其可药用盐、或者核酸 分子、或者重组载体、或者重组细胞，其中所述包膜类病毒感染所致的疾病为艾滋病，例如为I型艾滋病。
  19. 一种在体内或体外抑制HIV的方法，包括给予有效量的权利要求1或2的多肽、其衍生物、或其可药用盐、或者权利要求3的核酸分子、或者权利要求4的重组载体、或者权利要求5的重组细胞或权利要求6或7的偶联物的步骤。
  20. 一种在体内或体外抑制HIV-1Env介导的细胞融合的方法，包括给予有效量的权利要求1或2的多肽、其衍生物、或其可药用盐、或者权利要求3的核酸分子、或者权利要求4的重组载体、或者权利要求5的重组细胞或权利要求6或7的偶联物的步骤。
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