CN108484727B

CN108484727B - Oligopeptide, and derivative and application thereof

Info

Publication number: CN108484727B
Application number: CN201810245759.4A
Authority: CN
Inventors: 牛淼淼; 徐寒梅
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2018-03-23
Filing date: 2018-03-23
Publication date: 2020-08-28
Anticipated expiration: 2038-03-23
Also published as: CN108484727A

Abstract

The invention discloses an oligopeptide, a derivative thereof and application thereof, and belongs to the technical field of biological medicines. The oligopeptide sequence comprises a structural domain X-Y-Leu-Arg-Ala, and the structural general formula of the derivative is C₁₇H₃₅CO-X-Y-Leu-Arg-Ala, wherein X is a positively charged amino acid (e.g., Lys, His, Arg, etc.); y is hydrophobic or positively charged amino acid (such as Pro, Lys, Leu and the like), the number of the screened polypeptide amino acid is less than 7, and experimental results show that the polypeptide can obviously inhibit the proliferation of the anti-influenza virus, and the polypeptide has important development and application values in anti-influenza virus treatment, so that the polypeptide can be used for preparing anti-influenza drugs.

Description

Oligopeptide, and derivative and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an oligopeptide, and a derivative and application thereof.

Background

Influenza, as an acute respiratory infectious disease seriously harming human health, affects about 5 hundred million people worldwide each year, wherein 25-50 ten thousand people die, and the disease rate in China is about 10% -30% each year. More alarming is the increasing number of highly pathogenic, highly lethal influenza virus subtypes H5N1 and H1N1 infected in recent years, and the mortality rate is quite high. One of the major reasons for influenza virus abuse is its antigen variability, which limits vaccine immunoprotection, and moreover, vaccines only protect against known subtypes of influenza virus, but are ineffective against new influenza viruses that develop as a result of antigenic drift or shift.

The anti-influenza drugs commonly used at present mainly comprise two main classes of ion channel blockers and neuraminidase inhibitors. The ion channel blocker is represented by amantadine and rimantadine, and mainly achieves the purpose of inhibiting the proliferation of the influenza A virus by inhibiting the matrix protein-2 of the influenza A virus, preventing the virus from penetrating into host cells and releasing nucleic acid. However, amantadine can cause significant gastrointestinal adverse effects, central nervous toxic side effects, and cross-drug resistance. Thus, experts in WHO have suggested discontinuing the use of ion channel blockers as anti-influenza drugs. Neuraminidase (NA) inhibitors, mainly including Zanamivir (Zanamivir) and Oseltamivir (Oseltamivir). The action mechanism of the medicine is that the medicine specifically binds to NA of the virus to block the activity of the virus, so that the virus cannot be released from the surface of a host cell and the self-aggregation of the virus is promoted, thereby playing the role of resisting influenza virus. However, research shows that the NA inhibitor is easy to generate drug resistance and cross-drug resistance in the process of therapeutic application, so that the development of a novel anti-influenza virus drug is particularly urgent and important.

Hemagglutinin (HA) and Neuraminidase (NA) present on the influenza virus envelope play important roles in the influenza virus infection process and virus budding from host cells, respectively. The former binds to sialic acid receptors on host cell membranes, and after the virus enters the endosome, the virus fuses with the cell through a change in its conformation. At the end of infection, NA acts to cleave sialic acid residues on itself or the host cell membrane as virions bud or dissociate from the host cell, thereby ensuring release of the new virus. Therefore, the HA protein can be a potential target of anti-influenza virus medicines. Through the specific binding of the HA protein, the binding of the HA protein and a host cell membrane receptor is inhibited, and further the effect of preventing the infection of the influenza virus is achieved. Currently, many studies report that polymers containing sialic acid are effective in inhibiting the entry of influenza virus into host cells. The polypeptide can effectively inhibit the interaction of HA protein and receptor protein. Therefore, it is important to select polypeptides that specifically bind to the HA protein and effectively inhibit its action.

Disclosure of Invention

1. Problems to be solved

Aiming at the problems of large molecular weight and difficult synthesis of the existing anti-influenza virus peptides, the invention provides an oligopeptide, a derivative thereof and application thereof, wherein the number of amino acids of the polypeptide is less than 7, the molecular weight is small, the polypeptide is easy to synthesize, has the activity of inhibiting influenza virus, can be specifically combined with a hemagglutinin site, and has important development and application values in anti-influenza virus treatment.

2. Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

an oligopeptide, the sequence of which comprises an X-Y-Leu-Arg-Ala domain, wherein X is a positively charged amino acid; y is a hydrophobic or positively charged amino acid.

Oligopeptide derivative prepared by modifying the above polypeptides with stearic acidPeptide sequence of formula C₁₇H₃₅CO-X-Y-Leu-Arg-Ala, wherein X is a positively charged amino acid; y is a hydrophobic or positively charged amino acid.

Furthermore, X is one of Lys, His and Arg; y is one of Pro, Lys and Leu.

Further, the oligopeptide derivatives are respectively:

oligopeptides I, C₁₇H₃₅CO-Lys-Pro-Leu-Arg-Ala; or

Oligopeptides II, C₁₇H₃₅CO-His-Pro-Leu-Arg-Ala; or

Oligopeptides III, C₁₇H₃₅CO-Arg-Lys-Leu-Arg-Ala; or

Oligopeptides IV, C₁₇H₃₅CO-Arg-Leu-Leu-Arg-Ala。

The use of the above-mentioned oligopeptides or oligopeptide derivatives for the manufacture of a medicament.

The oligopeptide or oligopeptide derivative is applied to the preparation of the drugs for preventing or treating influenza diseases.

Furthermore, the influenza disease comprises influenza diseases caused by one or more of H1N1, H3N2, H5N1 and H7N9 influenza viruses.

A medicine for preventing or treating influenza diseases comprises one or more of the oligopeptide derivatives and pharmaceutically acceptable auxiliary materials.

Furthermore, the dosage form of the medicine comprises injection, freeze-dried powder injection, microspheres, powder spray, capsules, tablets, pills, nasal spray, aerosol, enteric coating, nanospheres, microemulsion or multiple emulsion.

A medicine for preventing or treating influenza diseases comprises the oligopeptide and pharmaceutically acceptable auxiliary materials.

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) the oligopeptide derivative has anti-influenza virus activity and can be specifically combined with a hemagglutinin site;

(2) the number of amino acids of the polypeptide with anti-influenza virus activity obtained by screening is less than 7, and experimental results show that the polypeptide can obviously inhibit the proliferation of anti-influenza virus, so that the polypeptide has important development and application values in anti-influenza virus treatment, and can be used for preparing anti-influenza drugs;

(3) compared with the anti-influenza virus peptides reported in the existing documents, the existing anti-influenza virus peptides have larger molecular weight and are difficult to synthesize; the oligopeptide synthesized by the invention is composed of natural amino acids, has small molecular weight, is easy to synthesize, has the activity of inhibiting influenza viruses, and is an efficient and ideal antiviral medicament.

Drawings

FIG. 1 is a graph showing the results of the in vivo anti-influenza pharmacodynamic test of an oligopeptide I in the present invention;

FIG. 2 is a schematic diagram showing the binding pattern of oligopeptide I and hemagglutinin;

FIG. 3 is a schematic diagram showing the binding pattern of oligopeptide II and hemagglutinin in the present invention;

FIG. 4 is a schematic diagram showing the binding pattern of oligopeptide III and hemagglutinin;

FIG. 5 is a schematic diagram of the binding mode of oligopeptide IV and hemagglutinin in the present invention.

Detailed Description

The invention is further described with reference to specific examples.

Example 1

Viral virulence assay (TCID)₅₀)

Influenza virus antigens H1N1(FM1), H3N2(N3), H5N1 and H7N9 used in the invention are virus inactivated antigens with hemagglutination activity, which are all pathogenic, H1N1 and H3N2 are human influenza, H5N1 and H7N9 are avian influenza, and H7N9 is taken as an example, virus virulence determination is carried out to prove the activity of the influenza virus antigens, and the specific experimental steps and results are as follows:

after MDCK cells (influenza virus-susceptible canine kidney passage cell strains) in logarithmic growth phase are spotted and layered into a monolayer, the influenza virus H1N1 is continuously diluted by 10 times, and 10^-2～10^-10The diluted virus of (4) was inoculated into MDCK cells, adsorbed at 37 ℃ for 1 hour, washed 2 times with PBS, and cultured with a maintenance solution, and 4 replicate wells were set for normal cell controls. Observing cytopathic effect under inverted microscope every day, recording pathological change degree and number of wells, taking culture wells with cytopathic rate of 50% or more as pathological change wells, and calculating virus TCID according to Reed-Muench method₅₀(median tissue cultureinfective dosage)。

TABLE 1 Virus virulence assay (TCID)₅₀) Results

Distance ratio (percentage above 50% rate-50%)/(percentage above 50% rate-percentage below 50% rate) ═ 60-50)/(60-20) ═ 10/40 ═ 0.25;

lgTCID₅₀distance ratio × difference between log of dilutions + log of dilutions above 50% morbidity rate 0.25 × (-1) + (-3) — 3.25

TCID₅₀＝10^-3.25/0.1mL

The meaning is as follows: diluting the virus 10^3.25Inoculation with 50uL resulted in 50% of the cells becoming diseased.

Example 2

The invention takes the crystal structure of Protein Data Bank hemagglutinin Protein (4BSE) as the research basis, screens out a series of oligopeptides with antiviral activity, the sequence of which comprises X-Y-Leu-Arg-Ala structural domain, and modifies with stearic acid on the basis of short peptide X-Y-Leu-Arg-Ala to obtain the oligopeptide derivatives. The general structural formula of the oligopeptide derivative is C₁₇H₃₅CO-X-Y-Leu-Arg-Ala, wherein X is a positively charged amino acid (e.g., Lys, His, Arg, etc.); y is a hydrophobic or positively charged amino acid (e.g., Pro, Lys, Leu, etc.). The oligopeptide derivatives have antiviral activityAnd the compound is hopeful to be developed into an ideal antiviral drug. The specific structure of the oligopeptide derivative can be as follows:

oligopeptides I, C₁₇H₃₅CO-Lys-Pro-Leu-Arg-Ala; or

Oligopeptides II, C₁₇H₃₅CO-His-Pro-Leu-Arg-Ala; or

Oligopeptides III, C₁₇H₃₅CO-Arg-Lys-Leu-Arg-Ala; or

Oligopeptides IV, C₁₇H₃₅CO-Arg-Leu-Leu-Arg-Ala。

Determination of in vitro inhibition of influenza virus hemagglutination activity:

the virus titer of the recovered H1N1, H3N2, H5N1 and H7N9 influenza viruses is determined by a conventional hemagglutination test (referring to a WHO influenza virus hemagglutination titer determination standard method), influenza viruses containing 4 titers were then mixed well with different concentrations of polypeptides by the methods of reference 1(Jeremyt C.J., ErikW.S., Curtis R.B.and Standard S.C.Industention of the minor active sequence of an anti-influenza virus peptide, anti-microbial agents and chemotherapy,2011,55(4):1810 @) and reference 2(Teruhiko M., Ai Onishi, Tomomi S. Aki S., ethyl. simple acid-peptides as heparin inhibitors for anti-influenza virus J.Med.Chem.2010. um., 53,4441-, then, the virus titer of each sample after treatment is determined, and the antiviral activity of each sample is evaluated according to the reduction of the virus titer, and the lowest concentration for inhibiting the hemagglutination activity of the influenza virus is taken as the IC of the sample.₅₀。

TABLE 2 minimum effective concentration (. mu.M) of oligopeptide derivative for inhibiting agglutination of erythrocytes by influenza virus

The experiment adopts a hemagglutination inhibition test method to test the hemagglutination activity of the polypeptide for inhibiting the subtype 4 influenza virus, and the result shows that the oligopeptide I and oligopeptide II polypeptides have the inhibition activity on the hemagglutination erythrocyte action of the subtype H1N1 and H7N9 influenza viruses, but have no obvious inhibition on the hemagglutination erythrocyte action of the subtype H3N2 and H5N1 influenza viruses; wherein oligopeptide III has an inhibitory effect on hemagglutination of H7N9 subtype influenza virus of 0.71 μ M, and oligopeptide IV has an inhibitory effect on hemagglutination of H1N1 subtype influenza virus of 3.12 μ M.

Example 3

The method for testing the anti-influenza virus drug effect comprises the following steps:

taking cells in logarithmic growth phase, adding 0.25% trypsin digestion solution, digesting, blowing uniformly, counting according to 2 × 10⁵Inoculating into 96-well cell culture plate, and placing in constant temperature CO₂Culturing in an incubator for 16-24 h. A normal cell control group, a virus infection group, a positive drug control group and a tested drug group are respectively arranged. The culture medium was discarded, washed three times with PBS, and 100TCID was added to each well except the normal control group₅₀Influenza virus of infectious amount, placing in constant temperature CO₂Standing the incubator for 1h, discarding unadsorbed virus, washing with PBS twice, and adding drug-containing maintenance solution with different concentrations. The tested drug groups are provided with 6 doses and 3 compound holes. And then continuously culturing the cell culture plate which is added with the medicine according to the scheme for 72h at 35 ℃, observing cytopathic effect, and determining the light absorption value (A value) of each group by adopting an MTT method or a crystal violet staining method when the cytopathic effect of the virus control hole reaches more than 75%. Calculating the antiviral Efficiency (ER) of the tested drug, calculating the half Effective Concentration (EC) of the drug₅₀) ER × 100% (drug test group a value-viral control group a value)/(normal cell control group a value-viral control group a value) pretreatment test method 100 TCD-containing solution₅₀Mixing the influenza virus and the hemagglutination polypeptide according to the concentration gradient, carrying out warm bath treatment at 35 ℃ for 60min, then infecting MDCK cells according to the method, adding a maintenance solution, continuing to culture for 72H, then adopting a crystal violet method to dye and observe the cytopathic result, taking H7N9 virus as an example, carrying out experiments, and the experimental results are shown in Table 3.

Determination of the hemolytic Activity in vitro:

carrying out 2-fold gradient dilution on the oligopeptide derivative from 50 mu M, then mixing 50 mu L of sample with 50 mu L of 0.5% chicken erythrocyte, carrying out action for 60min at 37 ℃, centrifuging, taking supernatant, and measuring the OD value of 385nm wavelength by using a TECAN multifunctional microplate reader, wherein the specific result is shown in Table 3.

TABLE 3 oligopeptide derivatives in vitro anti-influenza pharmacodynamic test results

As can be seen from Table 3, oligopeptides I and III have some hemolytic activity at a concentration of 12.5. mu.M, whereas oligopeptides II and IV show hemolytic activity at a concentration of 25. mu.M.

As can be seen from FIG. 3, the results of MTT assay showed that the samples had oligopeptides I, II and III close to CC₀Has certain protective effect on the pathological changes caused by the infection of MDCK cells by influenza strains under the concentration condition. In order to further examine the efficacy of the polypeptides against influenza in vitro, a pretreatment method is adopted, and from table 3, it can be seen that oligopeptide I and oligopeptide II have the effect of directly inhibiting influenza virus from infecting MDCK cells under the condition of 10 mu M concentration or oligopeptide IV under the condition of 50 mu M concentration.

In order to verify the effects of the oligopeptides I, II, III and IV on H1N1, H3N2 and H5N1 viruses, the same method is adopted for carrying out experiments, and the results show that the oligopeptides I to IV have the effect of directly inhibiting influenza viruses from infecting MDCK cells.

Example 4

In vivo pharmacodynamic evaluation against influenza virus:

an in vivo pharmacodynamic evaluation experiment of anti-influenza virus was performed using oligopeptide i sequence as a representative. Mice were randomly divided into a virus Control group (Control), a positive drug Ribavirin (Ribavirin) group and an oligopeptide I administration group, 10 mice in each group were anesthetized with ether, 40. mu.L of stock solution of H1N1 avian influenza virus was nasally dropped with a pipette, and the administration was carried out 2H after infection. The virus control groups are all subjected to intraperitoneal injection of normal saline (10mL/kg), the positive drug group is subjected to intraperitoneal injection of ribavirin (50mg/kg), the oligopeptide I administration group (50mg/kg) is subjected to intramuscular injection for administration 2 times a day for 16 days, the death condition of each group of mice is observed and recorded every 24 hours, and the experimental result is shown in figure 1.

As can be seen from FIG. 1, the survival rate of mice in the group to which oligopeptide I was administered after 16 days of virus inoculation was maintained at 80%, and the life extension rate was significantly higher than that of mice in the virus control group and the positive drug group, so that it was found that oligopeptide I had significant preventive and therapeutic effects on influenza virus-infected mice.

The oligopeptide II, oligopeptide III and oligopeptide IV sequences are used for carrying out an in vivo pharmacodynamic evaluation experiment of the influenza virus according to the method, and the experiment result shows that the life prolonging rate of mice in an administration group (an experiment group which is respectively injected with the oligopeptide II, oligopeptide III and oligopeptide IV polypeptide) is obviously higher than that of mice in a virus control group and a positive drug group, so that the oligopeptides II, oligopeptide III and oligopeptide IV have obvious effects of preventing and treating influenza virus infected mice.

Example 5

Analysis of interaction Pattern of oligopeptide I with hemagglutinin

To further analyze the interaction of oligopeptide I with the hemagglutinin crystal complex (PDB ID:4BSE), the oligopeptide I was docked to the active site of hemagglutinin using Triangle mather docking module in MOE, as shown in FIG. 2. The oligopeptide I can form hydrogen bond interaction with key amino acids such as Gly135, Asn137 and Ser136 in the active site of hemagglutinin. The action mode of the oligopeptide I and the hemagglutinin predicts that the oligopeptide I has the function of inhibiting the hemagglutinin to a certain extent, and provides an important reference for further researching the hemagglutinin inhibitor.

Referring to the same method, oligopeptide II, oligopeptide III and oligopeptide IV are respectively aligned to the active site of hemagglutinin, and the oligopeptide II can form hydrogen bond interaction with key amino acids such as Ser219, Ser227 and Gln189 in the active site of hemagglutinin (shown in figure 3). Oligopeptide III can form hydrogen bond interaction with key amino acids such as Asn133, Gly135, Ser136, Glu190, Ser219 and Ser186 in the active site of hemagglutinin (shown in figure 4). The oligopeptide IV can form hydrogen bond interaction with key amino acids such as Asn137, Ser145, Ser136, Ser219 and Ser227 in the active site of the hemagglutinin (as shown in figure 5), and the result indicates that the oligopeptide IV has the function of inhibiting the hemagglutinin to a certain extent.

Example 6

The oligopeptides I, II, III and IV have anti-influenza virus activity, can obviously inhibit the proliferation of anti-influenza virus, and can be prepared into anti-influenza medicaments by combining with pharmaceutically acceptable auxiliary materials or carriers. A "pharmaceutically acceptable" excipient is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., a material that has a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Sufficient details regarding pharmaceutically acceptable carriers can be found in Remington's pharmaceutical Sciences (Mack pub. co., n.j.1991). Pharmaceutically acceptable carriers in the compositions may comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.

The term "pharmaceutically acceptable adjuvant" refers to any suitable pharmaceutically acceptable adjuvant, carrier, diluent, preservative, etc., used in pharmaceutical formulations. For exemplary purposes only, known adjuvants include, but are not limited to, e.g., complete freund's adjuvant, incomplete freund's adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lyso-soft phospholipids, pluronic polyols, polyanions, peptides, oil emulsions, hydrocarbon emulsions, keyhole limpet hemocyanin, and the like. Known carriers include, but are not limited to, sterile liquids, such as water, oils, or mixtures of water and oils, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Known diluents include, but are not limited to, water, saline, dextrose, ethanol, glycerol, and the like. Known preservatives include, but are not limited to, thimerosal, EDTA and the like. The selection of pharmaceutically acceptable excipients can be achieved by techniques known in the art, and the skilled person can select suitable pharmaceutically acceptable excipients based on the prior art according to the polypeptide pharmaceutical dosage form to be prepared. For example, for preparing oral liquid preparations (e.g., suspensions, microemulsions or multiple emulsions), the adjuvants selected may include, for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like. For another example, for preparing an oral solid preparation (e.g., powder, aerosol, capsule or tablet), the selected excipients may include, for example, starch, saccharides, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like. In addition, if necessary, the polypeptide drug of the present invention may be prepared as a sugar coating or an enteric coating, or a controlled release formulation.

According to another aspect of the present invention, there is provided a method for the control and/or prevention of influenza virus, which comprises administering to an animal in need thereof an effective amount of an oligopeptide derivative of the present invention, the term "effective amount" referring to an amount which elicits an antiviral response in the animal to which it is administered.

Sequence listing

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Claims

1. An oligopeptide derivative, which is characterized in that: the oligopeptide derivative is obtained by modifying a polypeptide sequence with stearic acid, and the oligopeptide derivative comprises:

oligopeptides I, C₁₇H₃₅CO-Lys-Pro-Leu-Arg-Ala; or

Oligopeptides II, C₁₇H₃₅CO-His-Pro-Leu-Arg-Ala; or

Oligopeptides III, C₁₇H₃₅CO-Arg-Lys-Leu-Arg-Ala。

2. The use of oligopeptide I or oligopeptide II derivatives according to claim 1 in the preparation of a medicament for the prevention or treatment of influenza diseases caused by one or more influenza viruses H1N1 and H7N 9.

3. The use of the oligopeptide derivative oligopeptide III according to claim 1 for the preparation of a medicament for the prevention or treatment of influenza disease caused by influenza virus type H7N 9.

4. A medicament for preventing or treating H1N1 influenza, which is characterized in that: comprises one or more of oligopeptide I or oligopeptide II in the oligopeptide derivative as defined in claim 1 and pharmaceutically acceptable auxiliary materials.

5. A medicament for preventing or treating H7N9 influenza, which is characterized in that: comprises one or more of oligopeptide I, oligopeptide II or oligopeptide III in the oligopeptide derivative as defined in claim 1, and pharmaceutically acceptable auxiliary materials.

6. The medicament according to claim 4 or 5, wherein: the dosage form of the medicine comprises injection, freeze-dried powder injection, microspheres, powder spray, capsules, tablets, pills, aerosol, enteric coating or microemulsion.

7. The medicament according to claim 4 or 5, wherein: the dosage form of the medicine comprises powder, nasal spray, nanospheres or compound emulsion.