CN108822190B

CN108822190B - Polypeptide and pharmaceutical composition and application thereof

Info

Publication number: CN108822190B
Application number: CN201810499379.3A
Authority: CN
Inventors: 王潮; 钟武; 曹瑞源; 刘克良
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2018-05-23
Filing date: 2018-05-23
Publication date: 2021-11-09
Anticipated expiration: 2038-05-23
Also published as: WO2019223644A1; CN108822190A

Abstract

The present invention provides polypeptides and pharmaceutical compositions and uses thereof, specifically, the present invention provides compounds represented by formula (I) or compounds with at least 80% identity thereto, stereoisomers or pharmaceutically acceptable salts thereof, Pharmaceutical composition and use thereof in the preparation of medicines for anti-influenza virus infection or prevention or treatment of diseases related to influenza virus infection, Ac-[X _a EEX _d X _e KK] _m -L ₁ -K(R ₁ )- _NH2 (I).

Description

Polypeptide and pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of biological medicines, in particular to a polypeptide, a pharmaceutical composition thereof and application thereof.

Background

Influenza Virus (Influenza Virus) belongs to the genus Orthomyxoviridae (Orthomyxoviridae) and Influenza Virus (Influenza Virus) is a single negative strand RNA Virus. Influenza viruses can be classified into A, B, C (i.e., A, B, and C) subtypes according to differences between Nucleoproteins (NPs) and Membrane Proteins (MPs). Influenza A Viruses (IAVs) can infect humans, various birds and other various mammals, cause seasonal Influenza due to their antigenicity being prone to variation, and cause a worldwide pandemic many times, thus being extremely harmful to human health. According to recent estimates by the U.S. centers for disease control and prevention, the world health organization, and global health partners, up to 6-12 million seasonal influenza patients per year by 12 months 2017, causing about 300 to 500 million cases of severe disease, with up to 65 million people dying from respiratory illness caused by seasonal influenza. Currently, there are two main types of drugs clinically used for treating influenza virus infection, neuraminidase inhibitors (such as oseltamivir and zanamivir) and M2 ion channel blockers (such as amantadine). The neuraminidase inhibitor selectively inhibits the activity of neuraminidase, prevents viruses from being released from infected cells to adjacent uninfected cells, and has an inhibition effect on A, B type influenza viruses; m2 ion channel blockers prevent the virus from uncoating and releasing the genetic material RNA into the host cytoplasm, rendering the virus incapable of replication and effective only against influenza A viruses. Because the current H1N1 and H3N2 influenza virus strains are resistant to amantadine drugs, oseltamivir also induces the generation of H1N1 virus resistant strains, and the research and development of novel anti-influenza virus drugs aiming at new targets to overcome the drug resistance of viruses have great practical significance.

The influenza virus surface spinous process is composed of Hemagglutinin (HA) and Neuraminidase (NA). Hemagglutinin HA mediates the process of virus adsorption and entry into host cells during the influenza virus life cycle. It is currently believed that when the virus approaches the host cell, the HA1 subunit of HA will first bind to the α -2,3 or α -2,6 sialic acid receptor at the glycoprotein/glycolipid end of the host cell membrane, and the virus will enter the host cell by endocytosis and be encapsulated in the endosome. Under acidic conditions of endosomes (pH 5.5), the HA1 region, which is originally a closed "clip" like structure under neutral pH conditions, undergoes conformational rearrangement and gradually dissociates from the HA2 region. The HA2 undergoes conformational rearrangement, the fusion peptide hidden in the protein is exposed and inserted on the endosome membrane, then the loop-to-helix transition of the loop domain of HA2 is carried out, so that the NHR (N-terminal helices Repeat) region on the loop domain forms a longer coiled triple helix kernel, finally the CHR (C-terminal helices Repeat) region containing a short alpha helix and a long alpha helix turns back through a V shape and acts on tripolymer formed by NHR to form a hydrophobic groove which forms a Six-strand helix (Six helix bundle,6HB) which is stable like a hairpin, and the membrane fusion process is completed.

Although fusion inhibitors designed based on the 6HB formation mechanism have been reported in many type I enveloped viruses, the T20 polypeptide also plays a role as the first clinically marketed fusion inhibitor in the treatment of AIDS. However, no report that polypeptides derived from the HA2CHR region can directly inhibit the HA-mediated membrane fusion process HAs been reported for fusion inhibitors of influenza virus, which may be related to the complex fusion mechanism of influenza virus itself. Unlike HIV-1 virus, which directly fuses on the surface of host cell membranes, influenza virus fusion requires first undergoing endocytosis, triggering the fusion process under low pH conditions in the cell inclusion bodies.

Disclosure of Invention

Based on the virus-host cell membrane fusion mechanism of IAVs and the essential characteristics of six-strand alpha helix bundle (6HB), artificially designed alpha helix peptide capable of targeting IAVs NHR region is conjugated with lipid molecule with membrane anchoring function, and the designed lipopeptide can effectively inhibit influenza A virus H1N1 and H3N 2.

Accordingly, in one aspect the present invention provides a compound of formula (I) or a compound having at least 80% identity thereto, a stereoisomer or a pharmaceutically acceptable salt thereof,

Ac-[X_aEEX_dX_eKK]_m-L₁-K(R₁)-NH₂

(I)

wherein, X_aRepresents a hydrophobic amino acid, each X_aThe same or different;

X_drepresents a hydrophobic amino acid, each X_dThe same or different;

X_ean amino acid selected from the group consisting of: ser, Asn, Gln, Glu, Asp, Lys, Arg, His, Tyr, Trp, Met and Cys, each X_eThe same or different;

e is Glu;

k is Lys;

R₁denotes cholesterol, steroids, sphingosine and fatty acids (e.g. C)_6-24Fatty acids);

L₁selected from the group consisting of Gly, beta-alanine (. beta. -Ala), gamma-aminobutyric acid (GABA), 6-aminocaproic acid (6-Aca) and NH₂-(CH₂CH₂-O)_n-CH₂CH₂-COOH, wherein n is an integer selected from 1 to 25;

m is selected from 2,3, 4, 5, 6, 7, 8, 9 and 10.

In certain preferred embodiments, X_aIs a region of a transmembrane subunit NHR of a fusion protein which can be fused with the type I enveloped virusAmino acid residues in which hydrophobic interactions occur in the domain.

In certain preferred embodiments, X_aSelected from Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp and Met, each X_aThe same or different.

In certain preferred embodiments, X_aSelected from Ala, Val, Leu, Ile, Phe and Tyr, each X_aThe same or different.

In certain preferred embodiments, X_aIs Ile.

In certain preferred embodiments, X_dIs an amino acid residue which can generate hydrophobic interaction with a transmembrane subunit NHR region of the type I enveloped virus fusion protein.

In certain preferred embodiments, X_dSelected from Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp and Met, each X_dThe same or different.

In certain preferred embodiments, X_dSelected from Ala, Val, Leu, Ile, Phe and Tyr, each X_dThe same or different.

In certain preferred embodiments, X_dIs Ile.

In certain preferred embodiments, X_eIs an amino acid residue which can have polar interaction with the NHR region of the transmembrane subunit of the type I enveloped virus fusion protein.

In certain preferred embodiments, X_eSelected from Ser, Gln, Glu, Lys, His, Tyr and Trp, each X_eThe same or different.

In certain preferred embodiments, X_eGln, Ser or Tyr.

In certain preferred embodiments, X_eIs Gln.

In certain preferred embodiments, R₁Is selected from C_6-24A saturated fatty acid.

In certain preferred embodiments, R₁Selected from caprylic acid, capric acid, lauric acid, myristic acid and palmitic acid.

In certain preferred embodiments, L₁Selected from Gly, beta-alanine (beta-Ala)) 6-aminocaproic acid (6-Aca) and NH₂-(CH₂CH₂-O)_n-CH₂CH₂-COOH, wherein n ═ 1, 2,3, 4, 5, 7, 9, 11, or 23.

In certain preferred embodiments, L₁Selected from Gly, beta-alanine (beta-Ala), 6-aminocaproic acid (6-Aca) and NH₂-(CH₂CH₂-O)₇-CH₂CH₂-COOH。

In certain preferred embodiments, L₁Selected from Gly, beta-alanine (. beta. -Ala) and 6-aminocaproic acid (6-Aca).

In certain preferred embodiments, L₁Is beta-alanine (beta-Ala).

In certain preferred embodiments, m is selected from 3, 4, 5, 6, 7 and 8.

In certain preferred embodiments, m is selected from 4, 5 and 6.

In certain preferred embodiments, m is 5.

In certain preferred embodiments, X_aIs Ile; xd is Ile; x_eGln, Ser or Tyr; r₁Is palmitic acid; l is₁Is Gly, beta-alanine (beta-Ala), 6-aminocaproic acid (6-Aca) or NH₂-(CH₂CH₂-O)₇-CH₂CH₂-COOH; m is 5.

In certain preferred embodiments, the compound is selected from:

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C₈)，

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C₁₀)，

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C₁₂)，

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C₁₄)，

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C₁₆)，

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-G-K(C₁₆)，

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-z-K(C₁₆)，

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-p-K(C₁₆)，

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-a-K(C₁₆)，

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-G-K(C₁₆)，

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-z-K(C₁₆)，

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-p-K(C₁₆)，

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-a-K(C₁₆)，

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-G-K(C₁₆)，

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-z-K(C₁₆)，

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-p-K(C₁₆)，

IEEIQKK IEEISKK IEEISKKIEEISKK IEEIQKK-a-K(C₁₆) And are and

IEEIQKK IEEIYKK IEEIYKK IEEIYKK IEEIQKK-a-K(C₁₆)；

wherein a is beta-Ala, z is 6-Aca, p is NH₂-(CH₂CH₂-O)₇-CH₂CH₂-COOH, G is Gly, C₈Is octanoic acid, C₁₀Is capric acid, C₁₂Is lauric acid, C₁₄Is myristic acid, C₁₆Is palmitic acid.

In certain preferred embodiments of the invention, a compound of the invention has at least 90% identity, 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 with a compound of formula (I).

In another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention, or a compound having at least 80% identity thereto, a stereoisomer, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

When orally administered, the pharmaceutical composition of the present invention may be formulated into tablets, sustained-release tablets, controlled-release tablets, troches, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs, dripping pills, pellets or oral solutions. Thus, compositions for oral use may contain, for example, one or more coloring, sweetening, flavoring and/or preservative agents.

Suitable pharmaceutically acceptable excipients for tablets include inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate; disintegrating agents 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. Tablets may be uncoated or they may be coated to modify their disintegration and subsequent absorption of the active ingredient in the gastrointestinal tract or to improve their stability and/or appearance, in any event using conventional coating agents and procedures well known in the art.

Suitable pharmaceutically acceptable excipients for use in hard capsules include inert solid diluents such as calcium carbonate, calcium phosphate or kaolin, and the like. Suitable pharmaceutically acceptable 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 finely divided form together with one or more dispersing, wetting or suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth, gum acacia, and the like; dispersing or wetting agents, for example, condensation products of lecithin or an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaoxyethylene cetyl alcohol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives (e.g., ethyl or propyl paraben), antioxidants (e.g., ascorbic acid), coloring, flavoring, and/or sweetening agents (e.g., sucrose, saccharin and aspartame), and the like.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may also contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to enhance the taste of the oral dosage form. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

The pharmaceutical compositions of the present invention may also take the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures thereof. Suitable emulsifying agents may be, for example, natural gums such as gum acacia or gum tragacanth, natural phosphatides such as soya bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate), and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening agent, flavoring agent, and antiseptic.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may contain demulcents, preservatives, flavouring and/or colouring agents and the like.

When administered parenterally (e.g., intravenously, subcutaneously, or intramuscularly), the pharmaceutical composition may be formulated as a sterile aqueous or oily solution, sterile powder, liposome, emulsion, microemulsion, nanoemulsion, or microcapsule.

The pharmaceutical compositions 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 and/or suspending agents, such as those 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 parenterally acceptable, for example as a solution in 1, 3-butanediol.

For further information on the formulation, reference may be made to Comprehensive Medicinal Chemistry, Vol.5, chapter 25.2 (Corwin Hanschl; Chairman of Editorial Board), Pergamon Press 1990.

The amount of active ingredient that is combined with one or more excipients to produce a single dosage form may vary depending upon the host treated and the particular route of administration. For example, formulations intended for oral administration to humans typically contain, for example, 0.5mg to 2g of the active ingredient, together with suitable and conventional amounts of excipients (about 5 to 98% by weight of the total composition). Unit preparations typically contain about 1mg to about 500mg of the active ingredient. For further information on the route of administration and the dosage regimen, reference is made to Comprehensive Medicinal Chemistry, Vol.5, chapter 25.3 (Corwin Hanschl; Chairman of Editorial Board), Pergamon Press 1990.

The amount of the pharmaceutical composition to be administered for therapeutic or prophylactic purposes should be adjusted depending on the nature and severity of the condition, the age and sex of the animal or patient, the route of administration, and the like.

In the case of therapeutic or prophylactic use of the pharmaceutical compositions, they are generally administered in daily doses in the range of, for example, 1mg to 100mg per kg of body weight, and if desired in divided doses. Generally, lower doses are employed when administered parenterally, for example intravenously, typically in the range of, for example, 1mg to 10mg per kg of body weight.

In another aspect, the present invention provides the use of a compound of the present invention, or a compound having at least 80% identity thereto, a stereoisomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition, in the manufacture of a medicament for inhibiting fusion of an influenza virus with a target cell membrane.

In certain preferred embodiments, the influenza virus is an influenza a virus.

In certain preferred embodiments, the influenza virus is selected from H1N1 and H3N 2.

In certain preferred embodiments, the target cell is a cell line or a cell from a subject.

In another aspect, the invention provides the use of a compound of the invention, or a compound having at least 80% identity thereto, a stereoisomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition in the manufacture of a medicament for the prevention or treatment of a disease associated with influenza virus infection or a medicament against influenza virus.

In certain preferred embodiments, the influenza virus is an influenza a virus.

In certain preferred embodiments, the disease associated with influenza virus infection is selected from influenza a H1N1 and influenza a H3N 2.

In another aspect, the present invention provides a compound of the present invention, or a compound having at least 80% identity thereto, a stereoisomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in inhibiting fusion of an influenza virus with a target cell membrane.

In certain preferred embodiments, the influenza virus is an influenza a virus.

In another aspect, the present invention provides a compound of the present invention or a compound having at least 80% identity thereto, a stereoisomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use in the prevention or treatment of a disease associated with influenza virus infection or against influenza virus.

In certain preferred embodiments, the influenza virus is an influenza a virus.

In another aspect, the present invention provides a method of inhibiting fusion of an influenza virus with a target cell membrane, comprising administering to a cell an effective amount of a compound of the present invention or a compound having at least 80% identity thereto, a stereoisomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition.

In certain preferred embodiments, the influenza virus is an influenza a virus.

In another aspect, the present invention provides an antiviral method comprising administering to a subject in need thereof an effective amount of a compound of the present invention or a compound having at least 80% identity thereto, a stereoisomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition.

In certain preferred embodiments, the influenza virus is an influenza a virus.

In another aspect, the present invention 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 the present invention or a compound, stereoisomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition having at least 80% identity thereto.

In certain preferred embodiments, the influenza virus is an influenza a virus.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "fatty acid" refers to an aliphatic carbon chain containing one carboxyl group at one end. Can be classified into saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids according to the degree of saturation of the carbon chain, wherein the saturated fatty acids have C_xH_2x+1The general structural formula of COOH. And can be divided into short chain fatty acid (the number of carbon atoms on the carbon chain is less than 6), medium chain fatty acid (the number of carbon atoms on the carbon chain is 6-12) and long chain fatty acid (the number of carbon atoms on the carbon chain is more than 12) according to the length of the carbon chain.

As used herein, the term "hydrophobic amino acid" includes primarily tyrosine, tryptophan, phenylalanine, valine, leucine, isoleucine, proline, methionine, and alanine.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then 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 positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, Needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of E.Meyers and W.Miller (Compout.appl biosci., 4:11-17(1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48: 444-.

As used herein, the term "subject" refers to an animal, particularly a mammal, preferably a human.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a prophylactically effective amount is an amount sufficient to prevent, or delay the onset of disease; a therapeutically effective amount is an amount sufficient to cure or at least partially arrest the 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 effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

Drawings

FIG. 1 shows the entrance of NBD-IIQ16 into MDCK cells and distribution in inclusion bodies as observed by confocal laser microscopy.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The abbreviations used in the present invention have the following meanings:

ala (Alanine, A): alanine

Arg (argine, R): arginine

Asn (Asparagine, N): asparagine

Asp (Aspartic acid, D): aspartic acid

CHR (C-terminal header repeat): c terminal repeat sequence

Cys (Cysteine, C): cysteine

Dcm (dichromethane): dichloroethane

DMF (N, N-Dimethyl malonate): dimethyl formamide

Fmoc (fluoroxylmethod) of: fmoc group

Gln (Glutamine, Q): glutamine

Glu (Glutamic acid, E): glutamic acid

Gly (Glycine, G): glycine

HBTU: 2- (1H-1-hydroxybenzotriazole) -1,1,3, 3-tetramethyluronium hexafluorophosphate

His (Histidine, H): histidine

HOBt (1-Hydroxybenzotriazol anhydrous): 1-hydroxybenzotriazole

Hplc (high performance liquid chromatography): high performance liquid chromatography

IAVs: influenza A virus (type A)

Ile (Isoleucine, I): isoleucine

Leu (Leucine, L): leucine

Lys (Lysine, K): lysine

MALDI-TOF-MS: matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Met (Methionine, M): methionine

NHR (N-tertiary header repeat): n-terminal repeat sequence

NMP (N-Methyl pyrollidone): n-methyl pyrrolidone

Phe (phenyl alanine, F): phenylalanine

Pro (Proline, P): proline

Ser (Serine, S): serine

Tfa (trifluoroacetic acid): trifluoroacetic acid

Thr (Threonine, T): threonine

Trp (Tryptophan, W): tryptophan

Tyr (Tyrosine, Y): tyrosine

Val (Valine, V): valine

The solid phase synthesis carrier Rink amide resin used in the examples is a product of Tianjin Nankai Synthesis responsibility Co. HBTU, HOBt, DIEA and Fmoc protected natural amino acids or D-type unnatural amino acids are products of Shanghai Jier Biochemical company and Beijing Oakunas science and technology Limited. N-methylpyrrolidone (NMP) and trifluoroacetic acid (TFA) are products of Bailingwei technologies, Beijing. DMF and DCM are products of chemical reagents of national drug group, Inc. The chromatographic pure acetonitrile is a product of Fisher company. Other reagents are domestic analytical pure products unless otherwise specified.

Example 1: preparation of the Compounds

Polypeptide synthesis was performed using standard Fmoc solid phase methods. Rink Amide resin is selected, and a peptide chain is extended from a C end to an N end. The condensing agent is HBTU/HOBt/DIEA. The deprotection agent is piperidine/DMF solution. The lysate is trifluoroacetic acid (TFA), and the crude peptide is dissolved in water and then freeze-dried for storage. Separating and purifying by medium pressure liquid chromatography or High Pressure Liquid Chromatography (HPLC), wherein the pure peptide content is more than 90%. And determining the molecular weight of the peptide sequence by matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS).

Peptide sequences were synthesized using a CEM microwave polypeptide synthesizer.

The synthesis conditions were as follows:

protection of amino acids: 0.2M DMF solution of the protected amino acid,

condensation reagent: 0.45M HBTU/HOBt in DMF,

activating alkali: a 2M solution of DIEA in NMP,

deprotection reagents: 20% v/v piperidine in DMF,

blocking reagent: 20% v/v acetic anhydride in DMF.

Weighing 0.23g (0.1mmol) of Rink Amide resin, placing the Rink Amide resin in a reactor of a CEM microwave polypeptide synthesizer, preparing protected amino acid, a condensation reagent, an activated base, a deprotection reagent and a blocking reagent according to the concentration, and synthesizing by using the CEM microwave full-automatic polypeptide synthesizer. After completion, the peptide resin was transferred to a polypeptide solid phase synthesis reactor, washed twice with DMF, absolute methanol, DCM, respectively, and vacuum dried at room temperature to obtain 1.25g of peptide resin.

Peptide resin [ containing Lys (Dde) special amino acid) ] is added with a little DCM to swell the resin for 20min, and then is dried by pumping. Preparing a reagent for removing the Dde protecting group: 2ml of hydrazine hydrate are dissolved in 40ml of DMF in a volume ratio of 20: 1. Adding the deprotection reagent into the resin, stirring for 3min at room temperature, pumping, repeating for 4 times, and finishing the deprotection reaction. 3equiv of saturated fatty acid (caprylic acid, capric acid, lauric acid, myristic acid or palmitic acid), 0.11g of HBTU (3equiv) and 40mg of HOBt (3equiv) were weighed out and dissolved in 6ml of DMF, and added to the reactor, and 320. mu.l of DIEA (6equiv) was added thereto, followed by stirring at room temperature for 1 hour. After the reaction, the reaction solution was drained, the resin was washed twice with DMF, absolute methanol and DCM alternately, the residual saturated fatty acid was washed off with diethyl ether, and the peptide resin 1.31g was obtained by vacuum drying.

Lysis buffer (volume percent): trifluoroacetic acid: m-cresol: thioanisole: water-8.5: 0.5:0.5: 0.5.

Cleavage of peptide resin: weighing 1.31g of peptide resin synthesized by a microwave synthesizer, putting the peptide resin into a 250ml eggplant-shaped bottle, and carrying out ice bath. The lysate was prepared by adding 10ml of 1g of peptide resin. The TFA needs to be cooled for 30min in an ice bath in advance or stored in a refrigerator for use in advance; adding the prepared lysate into peptide resin under ice bath condition, performing slow electromagnetic stirring to change the resin into orange red, reacting for 30min under ice bath condition, removing the ice bath, continuing to stir at the room temperature for 150min, completing the reaction, adding 200ml of diethyl ether which is cooled at 4 ℃ in advance under vigorous stirring, separating out white precipitate, continuing to stir for 30min, and standing for 30 min; the precipitate was filtered off using a G4 sand-core funnel, and the filter cake was washed repeatedly with glacial ethyl ether 3 times and dried. Adding 30ml of double distilled water and 10ml of acetonitrile to fully dissolve the solid, carrying out suction filtration, and freeze-drying the filtrate to obtain 1.04g of crude peptide.

Purification of the crude peptide: the crude peptide is purified by medium or high pressure chromatography. The column was a C8 column, and the eluent was acetonitrile, water and a small amount of TFA. The method comprises the following specific operation steps: 1.00g of crude peptide was weighed, 20ml of water and 5ml of acetonitrile were added to dissolve the solid, the mixture was centrifuged for 10min (3000 rpm) to obtain a supernatant, and the supernatant was filtered through a 0.23 μm sterile filter and then applied. The column was equilibrated with 200ml of 20% acetonitrile/water/0.1% TFA solution. After loading, the sample is washed by 200ml of 20% acetonitrile/water/0.1% TFA solution, and the eluent components are detected by high performance liquid phase. And gradually increasing the acetonitrile content according to the liquid phase detection result until the main peak of the purified polypeptide is eluted. Combining the eluates, rotary evaporating to remove all acetonitrile solvent, freeze-drying pure peptide, detecting content by HPLC (high performance liquid chromatography) to be more than 90%, and confirming molecular weight by MALDI-TOF. The polypeptide sequences are shown in table 1:

TABLE 1 polypeptide sequence information

Wherein, a is beta-Ala; z is 6-Aca; p is NH₂-(CH₂CH₂-O)₇-CH₂CH₂-COOH；G：Gly；C₈: octanoic acid, C₁₀: capric acid, C₁₂: lauric acid, C₁₄: myristic acid, C₁₆: palmitic acid.

Example 2: circular dichroism analysis of polypeptides

The polypeptides were incubated with the target peptide N66 derived from the H3N2HA region (residues 40-105 of the NHR region of the H3N2 fusion protein), respectively, by the following incubation method: the polypeptide was mixed equimolar with N66 and incubated at 30 ℃ for 30min, and the mixture was diluted with sodium acetate buffer (pH 5.0) to a final concentration of 10. mu.M for testing.

The polypeptide solution was subjected to a spectral scan using a MOS-450 circular dichrograph, setting the parameters: the scanning wavelength is 180-280nm, the scanning optical path is 1mm, the scanning unit is 1nm, and the scanning of each sample is repeated for 3 times. The polypeptide with typical alpha helical structure shows negative peaks at 208nm and 222nm and positive peaks at 195nm on a CD spectrogram, and the alpha helicity isThe molar ellipticity at 222nm was determined to be-33000 (deg.cm)²·dmol^-1) The percent helix content of the test polypeptides and complexes is calculated as 100% alpha helicity. Next, the stability of the polypeptides described in the present invention to interact with the target N66 and form conjugates was determined by CD temperature scanning. The specific method comprises the following steps: transferring the polypeptide for determining the CD signal into a sample cell (or reconfiguring), setting the program of the CD instrument into temperature scanning, detecting the wavelength of 220nm, scanning the range of 20-98 ℃, performing program temperature scanning to obtain a curve of the CD signal changing along with the temperature, and calculating the Tm value according to the curve. The results of the circular dichroism chromatography are shown in Table 2.

TABLE 2 circular dichroism determination of alpha helix content of polypeptides

SEQ ID NO：	Alpha helicity (%)	Tm(℃)
			1	24	48
2	58	67
			3	32	55
4	38	45
			5	55	77
6	51	67
			7	49	45
8	22	44
			9	44	47
10	38	50
			11	59	69
12	60	65
			13	58	49
14	36	41
			15	35	46
16	60	66
			17	62	63
18	33	22

Circular dichroism analysis showed that in PBS (pH 5.0), the polypeptide can interact with the target N66 to form a typical alpha helix structure. The polypeptide with better antiviral activity and the target N66 form a complex with higher helix content and higher stability. These results indicate that the antiviral activity of the polypeptide is related to helicity and thermostability of its target to form heterologous 6 HB.

Example 3: inhibition of IAVs cytopathic assay (IC) by Compounds₅₀)

At 10⁴Cell/number per well MDCK cells (purchased from American type culture Collection ATCC) were seeded on 96-well culture plates using DMEM medium containing 10% Fetal Bovine Serum (FBS) at 37 ℃ with 5% CO₂And culturing for 24 h. The following day, the culture medium was discarded when the cells grew to 80% -90%, and the residual serum-containing medium was removed by washing 2 times with PBS (pH 7.4). Polypeptide samples were diluted in duplicate using DMEM medium containing 0.2% BSA, with 100TCID beforehand₅₀The influenza virus A/Puerto Rico/8/34(H1N1) or A/Aichi/2/68(H3N2) is incubated for 30min, and after 30min, the virus and polypeptide mixed solution is added into cells to adsorb the virus. After 1h, the virus is adsorbed, the virus mixed solution is removed,cells were overlaid with 3ml of a maintenance solution containing 1% agarose (1. mu.g/ml TPCK-pancreatin). And a normal MDCK cell negative control group, a virus infection positive control group and an antiviral positive drug oseltamivir control group are simultaneously established. 5% CO at 37 ℃₂After 48h of inverted culture, the cells were observed daily under an inverted microscope for morphological changes. Antiviral activity assay with MTT method: the 48h cultured cell plates were removed, the medium removed, and 100. mu.l of medium containing 0.5mg/ml MTT, 5% CO at 37 ℃₂Culturing in dark for 4 h; after that, the medium was discarded, 150. mu.l of DMSO was added thereto, dissolved and shaken for 10min, the OD at 570nm was measured, and IC of the compound was calculated using prism5.01₅₀The test was repeated three times. The results of the activity test are shown in Table 3.

TABLE 3 polypeptide anti-influenza Activity test

As can be seen from the activity results in table 3, all polypeptides were effective in inhibiting replication of influenza a strains, wherein compounds 3, 4, 5, 8, 9, 10, 13, 14, 15 inhibited the activity of H1N1 influenza a strains to a low μ M level comparable to the positive control Oseltamivir acid (compound 19); the activity of compounds 2, 5, 6, 7, 9, 11, 12, 13, 16, 17 in inhibiting influenza a H3N2 was comparable to the positive control Oseltamivir acid.

Example 4: laser confocal microscope imaging experiment research on polypeptide entering cell inclusion body

The cells selected in the experiment are target cells MDCK cells in an influenza virus infection model, the polypeptides are marked by a green fluorescent label NBD, and the condition that NBD-IIQ16 enters the MDCK cells and the distribution of inclusion bodies are directly observed through a laser confocal microscope. The polypeptide was incubated with MDCK cells, washed thoroughly with PBS, labeled with LysoTracher-red, and the cells were fixed. The green fluorescence-emitting polypeptide can enter cells and be widely distributed in the red region of the LysoTracher-labeled inclusion body (green fluorescence and red fluorescence are superposed to form yellow fluorescence). The ability to penetrate the membrane and enter the inclusion body may be an important reason for lipopeptides to inhibit influenza virus membrane fusion.

The N-terminus of IIQ16 (Compound 5) was covalently conjugated to the fluorescent molecule NBD and designated NBD-IIQ 16. MDCK cells were used for the experiments and were seeded in NEST 15mm dishes 24h before administration 10⁴Individual cells, 5% CO at 37 ℃₂Culturing in an incubator. After 24h, the culture medium was aspirated, 1mL of NBD-IIQ16 in DMEM was added, and the mixture was incubated at 37 ℃ for 2 h. Then, the culture medium was aspirated, washed 3 times with PBS, and 1ml of 50nM LysoTracker Red/DMEM solution was added thereto, followed by incubation at 37 ℃ for 30 min. The culture medium was aspirated, washed 3 times with 2mg/ml heparin sodium/PBS solution, 3 times with PBS, and the samples were fixed with 3% paraformaldehyde and observed under a Carl Zeiss LSM510meta laser confocal microscope. The LysoTracker Red excitation wavelength was 577 nm. The results are shown in FIG. 1.

While specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

Ac-[X _a EEX _d X _e KK] _m -L ₁ -K(R ₁ )-NH ₂

(I)

Wherein, X _a is Ile;

X _d is Ile;

X _{e is} selected from the following amino acids: Ser, GIn and Tyr, each X _{e is} the same or different;

E is Glu;

K is Lys;

R ₁ is selected from caprylic acid, capric acid, lauric acid, myristic acid and palmitic acid;

L ₁ is selected from Gly, β-alanine (β-Ala), γ-aminobutyric acid (GABA), 6-aminocaproic acid (6-Aca) and NH ₂ -(CH ₂ CH ₂ -O) _n - CH2CH2 _- COOH, wherein n=1, ₂ , 3, 4, 5, 7, 9, 11 or 23;

m is 5.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X _e is GIn.

3. The compound of claim ₁ or 2, or a pharmaceutically acceptable salt thereof, wherein R1 is palmitic acid.

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein L ₁ is selected from Gly, β-alanine (β-Ala), 6-aminocaproic acid (6-Aca) and NH ₂ -(CH _2CH2 _- O) ₇ _- CH2CH2 _- COOH.

5. The compound of claim ₁ , or a pharmaceutically acceptable salt thereof, wherein L1 is selected from the group consisting of Gly, β-alanine (β-Ala), and 6-aminocaproic acid (6-Aca).

6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK(C ₈ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK(C ₁₀ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK(C ₁₂ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK(C ₁₄ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK(C ₁₆ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-GK(C ₁₆ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-zK(C ₁₆ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-pK(C ₁₆ ),

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-aK(C ₁₆ ),

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-GK (C ₁₆ ),

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-zK(C ₁₆ ),

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-pK(C ₁₆ ),

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-aK (C ₁₆ ),

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-GK (C ₁₆ ),

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-zK(C ₁₆ ),

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-pK(C ₁₆ ),

IEEIQKK IEEISKK IEEISKK IEEISKK IEEIQKK-aK (C ₁₆ ), and

IEEIQKK IEEIYKK IEEIYKK IEEIYKK IEEIQKK-aK (C ₁₆ );

Wherein, a is β-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, and C ₁₀ is capric acid , C ₁₂ is lauric acid, C ₁₄ is myristic acid, C ₁₆ is palmitic acid.

7. A pharmaceutical composition comprising a compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

8. Use of the compound of any one of claims 1-6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 7 in the preparation of a medicament for inhibiting the fusion of influenza virus with a target cell membrane.

9. The use according to claim 8, wherein the influenza virus is influenza A virus.

10. The use of claim 8, wherein the influenza virus is selected from the group consisting of H1N1 and H3N2.

11. Use of the compound of any one of claims 1-6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 7 in the preparation of a medicament for preventing or treating a disease associated with influenza virus infection or an anti-influenza virus medicament .

12. The use of claim 11, wherein the influenza virus is influenza A virus.

13. The use of claim 11, wherein the influenza virus is selected from the group consisting of H1N1 and H3N2.

14. The use of claim 11, wherein the disease associated with influenza virus infection is selected from the group consisting of influenza A (H1N1) and influenza A (H3N2).