WO2019223644A1 - Polypeptide, composition pharmaceutique et utilisation associées - Google Patents

Polypeptide, composition pharmaceutique et utilisation associées Download PDF

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WO2019223644A1
WO2019223644A1 PCT/CN2019/087595 CN2019087595W WO2019223644A1 WO 2019223644 A1 WO2019223644 A1 WO 2019223644A1 CN 2019087595 W CN2019087595 W CN 2019087595W WO 2019223644 A1 WO2019223644 A1 WO 2019223644A1
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ieeiqkk
compound
ieeiskk
ieeiykk
influenza virus
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PCT/CN2019/087595
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English (en)
Chinese (zh)
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王潮
钟武
曹瑞源
刘克良
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中国人民解放军军事科学院军事医学研究院
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Publication of WO2019223644A1 publication Critical patent/WO2019223644A1/fr

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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
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present application relates to the field of biomedicine, and in particular, to polypeptides and pharmaceutical compositions and uses thereof.
  • Influenza Virus belongs to the Orthomyxoviridae family of influenza viruses (Influenza Virus) and is a single-stranded negative-strand RNA virus. According to the differences between Nucleoprotein (NP) and Membrane Protei (MP), influenza viruses can be divided into three subtypes: A, B, and C (ie, A, B, and C). Influenza A Virus (IAVs) can infect humans, various birds, and other mammals. Due to the susceptibility to mutations in its antigenicity, it can cause seasonal influenza, and it has repeatedly caused worldwide pandemics. The health hazard is enormous. According to the latest estimates from the U.S. Centers for Disease Control and Prevention, the World Health Organization, and global health partners, as of December 2017, there are 6 to 1.2 billion seasonal flu patients each year, causing about 3 to 5 million serious illnesses, many of which Up to 650,000 people have died of respiratory diseases caused by seasonal flu.
  • IAVs Influenza A Virus
  • influenza virus infection-neuraminidase inhibitors such as oseltamivir and zanamivir
  • M2 ion channel blockers such as amantadine.
  • Neuraminidase inhibitors selectively inhibit the activity of neuraminidase, prevent the virus from being released from infected cells to nearby uninfected cells, and have an inhibitory effect on influenza A and B viruses
  • M2 ion channel blockers prevent the virus from unshelling Releases genetic material RNA into the host cytoplasm, making the virus unable to replicate and is only effective against influenza A viruses.
  • H1N1 and H3N2 influenza virus strains are resistant to amantadines, oseltamivir has also induced the generation of H1N1 virus resistant strains.
  • the development of new anti-influenza virus drugs targeting new targets to overcome the virus The drug resistance has great practical significance.
  • the surface spikes of influenza virus are composed of Hemagglutinin (HA) and Neuraminidase (NA).
  • Hemagglutinin HA mediates the process of virus adsorption and entry into host cells during the life cycle of the influenza virus.
  • Current research suggests that after the virus approaches the host cell, the HA1 subunit of HA will first bind to the ⁇ -2,3 or ⁇ -2,6 sialic acid receptor on the glycoprotein / glycolipid end of the host cell membrane, and the virus enters through endocytosis The host cell is enclosed in an endosome.
  • the loop domain of HA2 undergoes a "loop-to-helix transition" conformational transition, causing it to be NHR Repeat) region forms a long coiled triple helix core, and finally a CHR (C-terminal Heptads Repeat) region containing a short alpha helix and a long alpha helix is folded back in a "V" shape and acts on a trimer hydrophobic groove formed by NHR A six-helix spiral (Six Helix Bundle, 6HB) is formed, which completes the process of membrane fusion.
  • NHR C-terminal Heptads Repeat
  • fusion inhibitors designed based on the 6HB formation mechanism have been reported in a variety of type I enveloped viruses, among them, T20 peptides also serve as the first clinically marketed fusion inhibitors in the treatment of AIDS.
  • T20 peptides also serve as the first clinically marketed fusion inhibitors in the treatment of AIDS.
  • fusion inhibitors against influenza viruses that can directly inhibit the HA-mediated membrane fusion process from peptides derived from the HA2CHR region, which may be related to the complex fusion mechanism of the influenza virus itself.
  • influenza virus fusion needs to undergo endocytosis first, triggering the fusion process under low pH conditions in the cell inclusion body.
  • the artificially designed alpha helix peptides that can target the IAVs and NHR regions were conjugated with lipid molecules with membrane anchoring functions. Lipopeptide can effectively inhibit influenza A H1N1 and H3N2 influenza viruses.
  • the present application provides a compound of formula (I) or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof,
  • X a represents a hydrophobic amino acid, and each X a is the same or different;
  • X d represents a hydrophobic amino acid, and each X d is the same or different;
  • X e is selected from the following amino acids: Ser, Asn, Gln, Glu, Asp, Lys, Arg, His, Tyr, Trp, Met, and Cys, each X e being the same or different;
  • E is Glu
  • R 1 represents cholesterol, steroids, sphingosine, and fatty acids (such as C 6-24 fatty acids);
  • L 1 is selected from Gly, ⁇ -alanine ( ⁇ -Ala), ⁇ -aminobutyric acid (GABA), 6-aminohexanoic acid (6-Aca), and NH 2- (CH 2 CH 2 -O) n- CH 2 CH 2 -COOH, where n is an integer selected from 1-25;
  • n is selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • X a is an amino acid residue that can have a hydrophobic interaction with the transmembrane subunit NHR region of a type I enveloped virus fusion protein.
  • X a is selected from Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, and Met, each X a being the same or different.
  • X a is selected from Ala, Val, Leu, Ile, Phe, and Tyr, each X a being the same or different.
  • X a is Ile.
  • X d is an amino acid residue that can have a hydrophobic interaction with the transmembrane subunit NHR region of a type I enveloped virus fusion protein.
  • X d is selected from Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp and Met, each X d being the same or different.
  • X d is selected from Ala, Val, Leu, Ile, Phe, and Tyr, each X d being the same or different.
  • X d is Ile.
  • X e is an amino acid residue that can interact polarly with the NMR region of a transmembrane subunit of a type I enveloped virus fusion protein.
  • X e is selected from Ser, Gln, Glu, Lys, His, Tyr, and Trp, each X e being the same or different.
  • X e is Gln, Ser or Tyr.
  • X e is Gln.
  • R 1 is selected from C 6-24 saturated fatty acids.
  • R 1 is selected from caprylic acid, capric acid, lauric acid, myristic acid, and palmitic acid.
  • L 1 is selected from Gly, ⁇ -alanine ( ⁇ -Ala), 6-aminocaproic acid (6-Aca), and NH 2- (CH 2 CH 2 -O) 7- CH 2 CH 2 -COOH.
  • L 1 is selected from Gly, ⁇ -alanine ( ⁇ -Ala), and 6-aminocaproic acid (6-Aca).
  • L 1 is ⁇ -alanine ( ⁇ -Ala).
  • m is selected from 3, 4, 5, 6, 7, and 8.
  • m is selected from 4, 5, and 6.
  • n is 5.
  • X a is Ile;
  • Xd is Ile;
  • X e is Gln, Ser or Tyr;
  • R 1 is palmitic acid;
  • L 1 is Gly, ⁇ -alanine ( ⁇ -Ala), 6 -Aminohexanoic acid (6-Aca) or NH 2- (CH 2 CH 2 -O) 7 -CH 2 CH 2 -COOH;
  • m is 5.
  • the compound is selected from:
  • a is ⁇ -Ala
  • z is 6-Aca
  • p is NH 2- (CH 2 CH 2 -O) 7 -CH 2 CH 2 -COOH
  • G is Gly
  • C 8 is caprylic acid
  • C 10 is capric acid
  • C 12 is lauric acid
  • C 14 is myristic acid
  • C 16 is palmitic acid.
  • the compound of the invention has at least 90% identity with the compound of formula (I), preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% Identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I) or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof, and a One or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical composition of the present application can be made into tablets, sustained-release tablets, controlled-release tablets, dragees, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules , Syrups or elixirs, drip pills, pellets, or oral solutions.
  • Pharmaceutical compositions for oral use may also contain, for example, one or more colorants, sweeteners, flavoring agents, and / or preservatives.
  • Suitable excipients for tablets include, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate; disintegrants such as corn starch and alginic acid; binders such as starch; lubricants such as magnesium stearate , Stearic acid or talc; preservatives such as ethyl or propyl paraben; and antioxidants such as ascorbic acid and the like.
  • the tablets may be uncoated or they may be coated to alter their disintegrating effect and subsequent absorption of the active ingredient in the gastrointestinal tract or to improve their stability and / or appearance, which can be used in any case Conventional coating agents and methods well known in the art.
  • Suitable excipients for hard capsules include inert solid diluents such as calcium carbonate, calcium phosphate or kaolin, and the like.
  • Suitable excipients for soft capsules include water or oils such as peanut oil, liquid paraffin or olive oil, and the like.
  • Aqueous suspensions generally contain the active ingredient in the form of micronized powder and one or more dispersants, wetting agents or suspending agents, such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl ester Cellulose, sodium alginate, polyvinyl-pyrrolidone, tragacanth and gum arabic; etc .; dispersants or wetting agents, such as condensates of lecithin or alkenyl oxide with fatty acids (such as polyoxyethylene stearate) ), Or the condensation product of ethylene oxide with a long-chain fatty alcohol, such as ethylene cetyl heptyl alcohol, or the condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol, such as polyoxygenation Ethylene sorbitol monooleate, or the condensation product of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitan monoo
  • Aqueous suspensions may also contain one or more preservatives (e.g., ethyl or propyl parabens), antioxidants (e.g., ascorbic acid), colorants, flavoring agents, and / or sweeteners (e.g., Sucrose, saccharin and aspartame).
  • preservatives e.g., ethyl or propyl parabens
  • antioxidants e.g., ascorbic acid
  • colorants e.g., ascorbic acid
  • flavoring agents e.g., ascorbic acid
  • sweeteners e.g., Sucrose, saccharin and aspartame
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or a mineral oil (such as liquid paraffin). Oily suspensions may also contain thickening agents such as beeswax, solid paraffin or cetyl alcohol. Sweeteners and flavoring agents as described above may be added to enhance the mouthfeel of the oral formulation.
  • the pharmaceutical composition can be preserved by the addition of an antioxidant such as ascorbic acid.
  • the pharmaceutical composition of the present application may also take the form of an oil-in-water emulsion.
  • the oily phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin or a mixture thereof.
  • Suitable emulsifiers may be, for example, natural gums such as acacia or astragalus gum, natural phospholipids such as soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides (such as sorbitan monooleate), And a condensation product of the partial ester and ethylene oxide, such as polyethylene oxide sorbitan monooleate.
  • Emulsions may also contain sweeteners, flavoring agents, preservatives, and the like.
  • Syrups and elixirs may be formulated with sweeteners (such as glycerol, propylene glycol, sorbitol, aspartame, or sucrose), and may also contain a demulcent, a preservative, a flavoring agent, and / or a colorant.
  • sweeteners such as glycerol, propylene glycol, sorbitol, aspartame, or sucrose
  • the pharmaceutical composition When administered parenterally (e.g., intravenously, subcutaneously or intramuscularly), the pharmaceutical composition can be prepared as a sterile aqueous or oily solution, sterile powder, liposome, emulsion, microemulsion, Nanoemulsions or microcapsules.
  • the pharmaceutical composition may also be in the form of a sterile aqueous or oily suspension for injection, which may be formulated according to known methods using one or more suitable dispersing, wetting agents and / or suspending agents, these The reagents are as described above.
  • the sterile injectable preparation may also be a sterile aqueous or oleaginous suspension for injection in a diluent or solvent, which is non-toxic and gastrointestinal acceptable, such as a solution in 1,3-butanediol .
  • the amount of active ingredient which is combined with one or more excipients to produce a single dosage form can be determined depending on the host treated and the particular route of administration.
  • formulations for oral administration to humans generally contain, for example, 0.5 mg to 2 g of active ingredient and appropriate and conventional amounts of excipients (approximately 5-98% of the total weight of the composition).
  • a unit preparation generally contains about 1 mg to 500 mg of the active ingredient.
  • the dosage of the pharmaceutical composition for therapeutic or preventive purposes should be adjusted according to the nature and severity of the disorder, the age and sex of the animal or patient, and the route of administration.
  • the pharmaceutical composition When used for treatment or prevention, it is generally administered in a daily dose in the range of, for example, 1 mg to 100 mg / kg of body weight, and divided doses may be administered if necessary.
  • lower dosages are used for parenteral administration, for example, intravenous administration generally uses dosages in the range of, for example, 1 mg to 10 mg / kg of body weight.
  • the present application provides a compound of formula (I) or a compound having at least 80% identity, a stereoisomer, or a pharmaceutically acceptable salt or pharmaceutical composition thereof for the preparation of an influenza virus and Use of a target cell membrane fusion drug.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the target cells are cell lines or cells from a subject.
  • the present application provides a compound of formula (I), or a compound having at least 80% identity, a stereoisomer or a pharmaceutically acceptable salt thereof, that inhibits the fusion of an influenza virus with a target cell membrane in vitro. use.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the target cells are cell lines or cells from a subject.
  • the present application provides a compound of formula (I) or a compound having at least 80% identity, a stereoisomer or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the preparation of a prophylactic or therapeutic agent and Use of medicine or anti-flu virus medicine for diseases related to influenza virus infection.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the disease associated with influenza virus infection is selected from the group consisting of H1N1 influenza and H3N2 influenza.
  • the application provides a compound of formula (I) or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt or pharmaceutical composition thereof for inhibiting influenza virus Fusion with target cell membrane.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the target cells are cell lines or cells from a subject.
  • the compound, or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt or pharmaceutical composition thereof is used in an in vivo method.
  • the present application provides a compound of formula (I) as described above or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt or pharmaceutical composition thereof for use in prevention or treatment Diseases related to influenza virus infection or anti-influenza virus.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the disease associated with influenza virus infection is selected from the group consisting of H1N1 influenza and H3N2 influenza.
  • the present application provides a method for inhibiting fusion of an influenza virus with a target cell membrane, which comprises administering to a cell an effective amount of a compound of formula (I) as described above or a compound having at least 80% identity with the same, a steric difference Or a pharmaceutically acceptable salt or pharmaceutical composition.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the target cells are cell lines or cells from a subject.
  • the method is performed in vivo.
  • the method is performed in vitro.
  • the application provides an antiviral method comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a compound having at least 80% identity therewith, Isomers or pharmaceutically acceptable salts or pharmaceutical compositions.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the application provides a method of preventing or treating a disease associated with an influenza virus infection, comprising administering to a subject in need thereof an effective amount of a compound of formula (I) % Identity compounds, stereoisomers or pharmaceutically acceptable salts or pharmaceutical compositions thereof.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the disease associated with influenza virus infection is selected from the group consisting of H1N1 influenza and H3N2 influenza.
  • fatty acid refers to an aliphatic carbon chain containing one carboxyl group at one end. According to the degree of carbon chain saturation, it can be divided into saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids. Among them, the saturated fatty acids have the structural formula of C x H 2x + 1 COOH. According to the length of the carbon chain, it can be divided into short-chain fatty acids (the number of carbon atoms on the carbon chain is less than 6), medium-chain fatty acids (the number of carbon atoms on the carbon chain is 6-12), and long-chain fatty acids (the carbon on the carbon chain) The number of atoms is greater than 12).
  • hydrophobic amino acid mainly includes tyrosine, tryptophan, phenylalanine, valine, leucine, isoleucine, proline, methionine and Alanine.
  • the term "identity" is used to refer to a sequence match between two polypeptides or between two nucleic acids.
  • a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or two Each position of the polypeptide is occupied by lysine)
  • the molecules are identical at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of compared positions x 100. For example, if 6 of the 10 positions of two sequences match, the two sequences are 60% identical.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 positions out of a total of 6 positions match).
  • comparisons are made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc).
  • the algorithm of E.Meyers and W.Miller Comput.Appl. Biosci., 4: 11-17 (1988)
  • the PAM120 weight residue table is used.
  • the term "subject" refers to an animal, particularly a mammal, preferably a human.
  • the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect.
  • a prophylactically effective amount refers to an amount sufficient to prevent, prevent, or delay the onset of a disease
  • a therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent a disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such an effective amount.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the manner in which the drug is administered, and other treatments administered concurrently and many more.
  • Figure 1 shows how NBD-IIQ16 enters MDCK cells and its distribution in inclusion bodies as observed by a laser confocal microscope.
  • IAVs Type A (type A) influenza virus
  • MALDI-TOF-MS Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • NHR N-terminal heptad repeat
  • NMP N-Methyl pyrrrolidone
  • TFA trifluoroacetic acid
  • Trp Tryptophan
  • the Rink amide resin used as a solid-phase synthesis carrier in the examples is a product of Tianjin Nankai Synthesis Co., Ltd.
  • HBTU, HOBt, DIEA and Fmoc protected natural amino acids or D-type unnatural amino acids are the products of Shanghai Gill Biochemical Co., Ltd. and Beijing Okinas Technology Co., Ltd.
  • NMP N-methylpyrrolidone
  • TFA trifluoroacetic acid
  • DMF and DCM are products of Sinopharm Chemical Reagent Co., Ltd.
  • Chromatographically pure acetonitrile is a Fisher product.
  • Other reagents are domestically produced analytical products unless otherwise specified.
  • Peptide synthesis was performed using standard Fmoc solid-phase methods. With Rink Amide resin, the peptide chain is extended from the C-terminus to the N-terminus.
  • the condensing agent is HBTU / HOBt / DIEA.
  • the deprotecting agent was a piperidine / DMF solution.
  • the lysate was trifluoroacetic acid (TFA).
  • the crude peptide was dissolved in water and lyophilized. It was separated and purified by medium pressure liquid chromatography or high pressure liquid chromatography (HPLC), and the pure peptide content was greater than 90%.
  • Matrix-assisted laser analytical time-of-flight mass spectrometry MALDI-TOF-MS determines the molecular weight of peptide sequences.
  • the peptide sequence was synthesized using a CEM microwave peptide synthesizer.
  • Blocking reagent 20% v / v acetic anhydride in DMF.
  • Rink Amide resin 0.23 g (0.1 mmol) of Rink Amide resin was weighed and placed in the reactor of CEM microwave peptide synthesizer, and then protected amino acids, condensation reagents, activated bases, deprotection reagents, and blocking reagents were configured according to the above concentrations. Automatic peptide synthesizer for synthesis. After completion, the peptide resin was transferred to a peptide solid-phase synthesis reactor, washed twice with DMF, anhydrous methanol, and DCM, respectively, and dried under vacuum at room temperature to obtain 1.25 g of the peptide resin.
  • Peptide resin [containing Lys (Dde) special amino acid)] swell resin with a little DCM for 20 min, and drained.
  • Dde protection group removal reagent 2ml of hydrazine hydrate is dissolved in 40ml of DMF in a volume ratio of 20: 1. The deprotection reagent was added to the resin, stirred at room temperature for 3 minutes, dried, and repeated 4 times to remove the reaction.
  • Peptide resin lysis Weigh 1.31g of peptide resin synthesized by a microwave synthesizer, put it into a 250ml eggplant-shaped bottle, and ice bath. A lysate was prepared by adding 10 ml of 1 g of peptide resin. The TFA needs to be cooled in the ice bath for 30 minutes or stored in the refrigerator before use; add the prepared lysate to the peptide resin under the ice bath condition, and slowly stir the electromagnetically, the resin turns orange-red, and react for 30 minutes in the ice bath condition, and then Remove the ice bath and continue to stir the reaction for 150 minutes. The reaction is complete. Add 200 ml of ice ether cooled at 4 ° C under vigorous stirring to precipitate a white precipitate.
  • Crude peptides are purified by medium pressure or high pressure chromatography.
  • the chromatographic column was a C8 column, and the eluents were acetonitrile, water, and a small amount of TFA.
  • the column was equilibrated with 200 ml of 20% acetonitrile / water / 0.1% TFA solution in advance.
  • a ⁇ -Ala; z: 6-Aca; p: NH 2- (CH 2 CH 2 -O) 7 -CH 2 CH 2 -COOH; G: Gly; C 8 : caprylic acid, C 10 : capric acid , C 12 : lauric acid, C 14 : myristic acid, C 16 : palmitic acid.
  • the peptides were respectively incubated with the target peptide N66 derived from the H3N2HA region (residues 40-105 of the N3 region of the H3N2 fusion protein). Incubation method: The peptide was mixed with N66 equimolarly and incubated at 30 ° C for 30 min. Sodium acetate buffer (pH 5.0) The mixture was diluted to a final concentration of 10 ⁇ M for testing.
  • a MOS-450 circular dichroic analyzer was used for spectral scanning of the peptide solution, and the parameters were set: the scanning wavelength was 180-280nm, the scanning optical path was 1mm, the scanning unit was 1nm, and the scanning repetition number of each sample was 3 times.
  • Polypeptides with a typical ⁇ -helical structure appear on the CD spectrum as negative peaks at 208 nm and 222 nm and positive peaks at 195 nm, while ⁇ -helicality is based on a molar ellipticity of -33,000 (deg ⁇ cm 2 at 222 nm).
  • Dmol -1 is 100% alpha helicity to calculate the percentage of helix content of the polypeptide and complex to be tested.
  • the stability of the polypeptide described herein to interact with the target N66 and form a conjugate is determined by a CD temperature scan.
  • the specific method is as follows: transfer the above-mentioned polypeptide for measuring the CD signal into the sample cell (can also be reconfigured), set the CD instrument program to temperature scanning, the detection wavelength is 220nm, and the scanning range is 20-98 ° C. Obtain the change curve of CD signal with temperature, and calculate the Tm value according to the curve. The results of circular dichroism are shown in Table 2.
  • Peptide samples were diluted at a ratio of 0.2% BSA in DMEM and incubated with 100TCID 50 / mL of influenza virus A / Puerto Rico / 8/34 (H1N1) or A / Aichi / 2/68 (H3N2) for 30 minutes. After 30 min, the virus and peptide mixture was added to the cells to adsorb the virus. After 1 hour, the virus was adsorbed, the virus mixture was removed, and 3 ml of a 1% agarose-containing maintenance solution (1 ⁇ g / ml TPCK-trypsin) was added to cover the cells.
  • a normal MDCK cell negative control group, a virus infection positive control group and an antiviral drug oseltamivir control group were set up. After incubation at 37 ° C for 5 hours with 5% CO 2 , the morphological changes of the cells were observed daily under an inverted microscope.
  • the antiviral activity was measured by the MTT method: the cell plate after 48 h of culture was removed, the medium was removed, and 100 ⁇ l of a medium containing 0.5 mg / ml MTT was added, and cultured at 37 ° C and 5% CO 2 in the dark for 4 h; then the medium was discarded Add 150 ⁇ l of DMSO to dissolve and shake for 10 min, determine the OD value at 570 nm, calculate the IC 50 of the compound using Prism 5.01, and repeat the test three times. The results of the activity test are shown in Table 3.
  • the cells selected in this experiment were the target cells MDCK cells in the influenza virus infection model.
  • the peptides were labeled with the green fluorescent label NBD, and the situation of NBD-IIQ16 entering MDCK cells and the distribution of inclusion bodies were directly observed by a laser confocal microscope. After incubating the peptide with MDCK cells, they were thoroughly washed with PBS, and the cells were fixed after LysoTracher-red labeling. Peptides that emit green fluorescence can enter cells and are widely distributed in the red area of inclusion bodies labeled with LysoTracher (green fluorescence and red fluorescence are superimposed to form yellow fluorescence). The ability to penetrate the membrane and enter inclusion bodies may be an important reason why lipopeptides inhibit the fusion of influenza virus membranes.
  • the N-terminus of IIQ16 was covalently conjugated to the fluorescent molecule NBD and named NBD-IIQ16.
  • Experiment MDCK cells prior to administration 24h 10 4 cells were seeded in small NEST 15mm dish and incubated at 37 °C 5% CO 2 incubator. After 24 hours, the culture solution was aspirated, and 1 mL of NBD-IIQ16 DMEM culture solution was added, and incubated at 37 ° C for 2 hours. After that, the culture solution was aspirated, washed three times with PBS, and then 1 ml of 50 nM LysoTracker Red / DMEM solution was added, followed by incubation at 37 ° C for 30 minutes.
  • LysoTracker Red has an excitation wavelength of 577 nm. The results are shown in Figure 1.

Abstract

L'invention concerne un polypeptide, une composition pharmaceutique et une utilisation associées. En particulier, l'invention concerne un composé représenté par la formule (I) ou un composé ayant au moins 80 % d'identité à celui-ci, un stéréoisomère ou un sel pharmaceutiquement acceptable du composé, une composition pharmaceutique du composé, et l'utilisation du composé dans la préparation de médicaments contre des infections par le virus de la grippe ou des médicaments pour prévenir ou traiter des maladies associées aux infections par le virus de la grippe, Ac-[XaEEXdXeKK]m-L1-K(R1)-NH2 (I).
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007326781A (ja) * 2004-09-02 2007-12-20 Kyoto Univ 抗ｓａｒｓウイルス剤
WO2009108261A2 (fr) * 2008-01-23 2009-09-03 Dana Farber Cancer Institute Compositions et méthodes de traitement des infections virales
WO2009110952A2 (fr) * 2007-12-31 2009-09-11 New York University Contrôle de la fusion de membrane hôte virale avec des hélices artificielles à base de succédané de liaison hydrogène
WO2009155789A1 (fr) * 2008-06-25 2009-12-30 中国人民解放军军事医学科学院毒物药物研究所 Polypeptides et leurs dérivés inhibant l’infection par le vih
WO2010089129A1 (fr) * 2009-02-06 2010-08-12 Cormus Srl Inhibiteurs de fusion virale et leurs utilisations
CN104628869A (zh) * 2015-01-28 2015-05-20 南方医科大学 一类兼具抗菌和抗流感病毒活性的融合肽衍生物
CN106749558A (zh) * 2017-02-04 2017-05-31 中国医学科学院病原生物学研究所 广谱抑制hiv的脂肽、其衍生物、其药物组合物及其用途
CN108659105A (zh) * 2018-05-23 2018-10-16 中国人民解放军军事科学院军事医学研究院 抗病毒多肽及其药物组合物和用途
CN108822190A (zh) * 2018-05-23 2018-11-16 中国人民解放军军事科学院军事医学研究院 多肽及其药物组合物和用途

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3058930A1 (fr) * 2017-04-18 2018-10-25 Institute Of Pathogen Biology, Chinese Academy Of Medical Sciences Lipopeptide servant a inhiber le vih de maniere potente, derive associe,composition pharmaceutique associee et son utilisation

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007326781A (ja) * 2004-09-02 2007-12-20 Kyoto Univ 抗ｓａｒｓウイルス剤
WO2009110952A2 (fr) * 2007-12-31 2009-09-11 New York University Contrôle de la fusion de membrane hôte virale avec des hélices artificielles à base de succédané de liaison hydrogène
WO2009108261A2 (fr) * 2008-01-23 2009-09-03 Dana Farber Cancer Institute Compositions et méthodes de traitement des infections virales
WO2009155789A1 (fr) * 2008-06-25 2009-12-30 中国人民解放军军事医学科学院毒物药物研究所 Polypeptides et leurs dérivés inhibant l’infection par le vih
WO2010089129A1 (fr) * 2009-02-06 2010-08-12 Cormus Srl Inhibiteurs de fusion virale et leurs utilisations
CN104628869A (zh) * 2015-01-28 2015-05-20 南方医科大学 一类兼具抗菌和抗流感病毒活性的融合肽衍生物
CN106749558A (zh) * 2017-02-04 2017-05-31 中国医学科学院病原生物学研究所 广谱抑制hiv的脂肽、其衍生物、其药物组合物及其用途
CN108659105A (zh) * 2018-05-23 2018-10-16 中国人民解放军军事科学院军事医学研究院 抗病毒多肽及其药物组合物和用途
CN108822190A (zh) * 2018-05-23 2018-11-16 中国人民解放军军事科学院军事医学研究院 多肽及其药物组合物和用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEE, K.K.: "Capturing a Fusion Intermediate of Influenza Hemagglutinin with a Cholesterol-conjugated Peptide, a New Antiviral Strategy for Influenza Virus", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 286, no. 49, 9 December 2011 (2011-12-09), pages 42141 - 42149, XP055076229 *
MENG, GUANGPENG ET AL.: "Membrane fusion mechanism based anti-class I enveloped virus peptides: Research Advances", JOURNAL OF INTERNATIONAL PHARMACEUTICAL RESEARCH, vol. 44, no. 11, 30 November 2017 (2017-11-30), pages 995 - 1005 *

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