CN108409834A - A kind of oligopeptides and its derivative and application - Google Patents

Abstract

The invention discloses a kind of oligopeptides and its derivative and applications, belong to biomedicine technical field.Oligopeptide sequence of the present invention includes X Arg Leu Y Arg structural domains, and the general structure of derivative is C₁₇H₃₅CO X Arg Leu Y Arg, wherein X is hydrophobic amino acid (such as Leu, Ala etc.)；Y is hydrophobic amino acid (such as Pro, Leu etc.), experimental result is shown, such polypeptide can significantly inhibit the proliferation of resisiting influenza virus, prompt it in resisiting influenza virus treatment to there is important development and application to be worth, therefore can be used for preparing Tamiflu.

Description

A kind of oligopeptide and its derivative and application

technical field

The invention belongs to the technical field of biomedicine, and more specifically relates to an oligopeptide and its derivatives and applications.

Background technique

Influenza, as an acute respiratory infectious disease that seriously endangers human health, causes about 500 million people to become sick each year, of which 250,000 to 500,000 people die, and the annual incidence rate in my country is about 10% to 30%. What is even more worrying is that in recent years, the number of human infections with highly pathogenic and fatal influenza virus H5N1 and H1N1 subtypes has been increasing, and the mortality rate is quite high. One of the main reasons for the raging influenza virus is its strong ability to mutate antigens, which makes the immune protection of the vaccine limited. Moreover, the vaccine can only prevent known influenza virus subtypes, and it is not effective against those caused by antigenic drift or conversion. The resulting novel influenza virus is ineffective.

Currently commonly used anti-influenza drugs mainly include ion channel blockers and neuraminidase inhibitors. Ion channel blockers are represented by amantadine and rimantadine, which mainly inhibit the proliferation of influenza A virus by inhibiting influenza A virus matrix protein-2, preventing the virus from penetrating into host cells and releasing nucleic acids . However, amantadine can cause significant gastrointestinal adverse reactions, central nervous system toxicity and cross-resistance. Therefore, WHO experts have recommended to stop using ion channel blockers as anti-influenza drugs. Neuraminidase (NA) inhibitors mainly include Zanamivir and Oseltamivir. The mechanism of action of this type of drug is to block its activity by specifically binding to the NA of the virus, resulting in the inability of the virus to be released from the surface of the host cell and promoting the self-agglutination of the virus, thereby exerting an anti-influenza virus effect. However, studies have shown that NA inhibitors are prone to drug resistance and cross-resistance during therapeutic application, so it is particularly urgent and important to develop a new anti-influenza virus drug.

Hemagglutinin (HA) and neuraminidase (NA) present on the envelope of influenza virus play an important role in the process of influenza virus infection and virus budding from host cells, respectively. The former binds to the sialic acid receptor on the host cell membrane, and when the virus enters the endosome, the virus and the cell fuse through the change of its conformation. At the end of infection, NA acts to cut sialic acid residues on itself or on the host cell membrane when the virus particles bud or dissociate from the host cell, thereby ensuring the release of new viruses. Therefore, HA protein can be a potential target for anti-influenza virus drugs. By specifically binding to HA protein to inhibit its binding to host cell membrane receptors, the effect of preventing influenza virus infection is achieved. At present, many studies have reported that polymers containing sialic acid can effectively inhibit the entry of influenza virus into host cells. The polypeptide can effectively inhibit the interaction between HA protein and receptor protein. Therefore, it is particularly important to screen a polypeptide that specifically binds to the HA protein and effectively inhibits its action.

Contents of the invention

1. The problem to be solved

Aiming at the problem that existing anti-influenza virus peptides have large molecular weight and are difficult to synthesize, the present invention provides an oligopeptide and its derivatives and applications. The number of amino acids of this type of oligopeptide is less than 7, the molecular weight is small, easy to synthesize and It has the activity of inhibiting influenza virus and can specifically bind to the hemagglutinin site, suggesting that it has important development and application value in anti-influenza virus treatment.

2. Technical solution

In order to solve the above problems, the technical scheme adopted in the present invention is as follows:

An oligopeptide whose sequence contains an X-Arg-Leu-Y-Arg domain, wherein X is a hydrophobic amino acid; Y is a hydrophobic amino acid.

An oligopeptide derivative obtained by modifying the above polypeptide sequence with stearic acid, its structural formula is C ₁₇ H ₃₅ CO-X-Arg-Leu-Y-Arg, wherein X is a hydrophobic amino acid; Y is a hydrophobic amino acid. .

Furthermore, said X is one of Leu and Ala; Y is one of Pro and Leu.

Further, the oligopeptide derivatives are respectively:

Oligopeptide I, C ₁₇ H ₃₅ CO-Leu-Arg-Leu-Pro-Arg; or

Oligopeptide II, C ₁₇ H ₃₅ CO-Ala-Arg-Leu-Leu-Arg.

Application of the above-mentioned oligopeptide or oligopeptide derivative in the preparation of medicine.

Application of the above-mentioned oligopeptide or oligopeptide derivative in the preparation of medicine for preventing or treating influenza disease.

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

A medicine for preventing or treating influenza disease, comprising one or more mixtures of the above-mentioned oligopeptide derivatives, and pharmaceutically acceptable auxiliary materials.

Furthermore, the dosage form of the drug includes injection, lyophilized powder injection, microsphere, powder, powder mist, capsule, tablet, pill, nasal spray, aerosol, enteric coating, nanosphere, microemulsion or Double milk.

A medicine for preventing or treating influenza disease, comprising the above-mentioned oligopeptide, and pharmaceutically acceptable auxiliary materials.

3. Beneficial effect

Compared with the prior art, the beneficial effects of the present invention are:

(1) The oligopeptide derivative of the present invention has anti-influenza virus activity and can specifically bind to the hemagglutinin site;

(2) A class of polypeptides with anti-influenza virus activity screened by the present invention, the number of amino acids of this class of polypeptides is less than 7, the experimental results show that this class of polypeptides can significantly inhibit the proliferation of anti-influenza viruses, suggesting that it is effective in anti-influenza virus It has important development and application value in virus treatment, so it can be used to prepare anti-influenza drugs;

(3) Compared with the anti-influenza virus peptides reported in the existing literature, the present invention has relatively large molecular weights and is difficult to synthesize; and the synthetic oligopeptides of the present invention are composed of natural amino acids with small molecular weights and are easy to synthesize. It is synthesized and has the activity of inhibiting influenza virus. It is an efficient and ideal antiviral drug.

Description of drawings

Fig. 1 is the result figure of anti-influenza pharmacodynamics test in vivo of oligopeptide I in the present invention;

Fig. 2 is a schematic diagram of the mutual binding mode of oligopeptide I and hemagglutinin in the present invention;

Fig. 3 is a schematic diagram of the mutual binding mode of oligopeptide II and hemagglutinin in the present invention.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments.

Example 1

Virus Virulence Assay (TCID ₅₀ )

Influenza H1N1 (FM1), H3N2 (N3), H5N1 and H7N9 virus antigens used in the present invention are virus inactivated antigens with hemagglutination activity, all can cause disease, H1N1, H3N2 are human influenza, H5N1 and H7N9 It is bird flu. Taking H7N9 as an example, the virus virulence was tested to prove its activity. The specific experimental steps and results are as follows:

After the MDCK cells in the logarithmic growth phase (canine kidney passage cell strain susceptible to influenza virus) were plated as a single layer, the influenza virus H1N1 was serially diluted 10 times, and the dilution of 10 ^-2 to 10 ^-10 The high-degree virus was inoculated in MDCK cells, after being adsorbed at 37°C for 1 hour, washed twice with PBS, and then replaced with maintenance medium to continue culturing. Normal cells were used as controls, all with 4 replicate wells. The cell lesion was observed under an inverted microscope every day, and the degree of lesion and the number of wells were recorded. The culture wells with a cell lesion rate of 50% or more were counted as lesion wells, and the TCID ₅₀ (median tissue culture infectious dosage) of the virus was calculated according to the Reed-Muench method.

Table 1 virus virulence assay (TCID ₅₀ ) results

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

lgTCID ₅₀ = difference between distance ratio x log of dilution + log of dilution above 50% lesion rate = 0.25 x (-1) + (-3) = -3.25

TCID ₅₀ ＝10 ^-3 . ²⁵ /0.1mL

Meaning: Dilute the virus by 10 ^3.25 and inoculate 50uL can make 50% of the cells pathological.

Example 2

The present invention uses the protein database (Protein Data Bank) hemagglutinin protein (4BSE) crystal structure as research basis, screens out a series of oligopeptides with antiviral activity, its sequence contains X-Arg-Leu-Y-Arg structure The domain is modified with stearic acid on the basis of the short peptide X-Arg-Leu-Y-Arg to obtain a class of oligopeptide derivatives. The general structural formula of such oligopeptide derivatives is C ₁₇ H ₃₅ CO-X-Arg-Leu-Y-Arg, wherein X is a hydrophobic amino acid (such as Leu, Ala, etc.); Y is a hydrophobic amino acid (such as Pro, Leu, etc.). These oligopeptide derivatives all have antiviral activity and are expected to be developed into ideal antiviral drugs. The specific structure of oligopeptide derivatives can be:

Oligopeptide I, C ₁₇ H ₃₅ CO-Leu-Arg-Leu-Pro-Arg; or

Oligopeptide II, C ₁₇ H ₃₅ CO-Ala-Arg-Leu-Leu-Arg.

Determination of Inhibitory Influenza Virus Hemagglutination Activity in Vitro:

After the resuscitated H1N1, H3N2, H5N1 and H7N9 influenza viruses were inactivated, their virus titers were measured by conventional hemagglutination tests (refer to the WHO influenza virus hemagglutination titer standard method), and then according to reference 1 (Jeremyt CJ, ErikW .S., Curtis RB and Stacey SCIndentification of the minimal active sequence of an anti-influenza virus peptide. Antimicrobial agents and chemotherapy, 2011,55(4):1810-1813) and literature 2 (Teruhiko M., Ai Onishi., Tomomi S ., Aki S., etal.Sialic acid-mimic peptides as hemagglutinin inhibitors for anti-influenzatherapy.J.Med.Chem.2010,53,4441-4449) method will contain 4 hemagglutination titers of influenza virus with different Mix the peptides at a certain concentration and act for 60 minutes at 37°C, then measure the virus titer of each sample after treatment, evaluate the antiviral activity of each sample according to the decline of virus titer, and take the lowest concentration that inhibits the hemagglutination activity of influenza virus as the IC of the sample ₅₀ .

Table 2 The minimum effective concentration (μM) of oligopeptide derivatives inhibiting influenza virus agglutination of erythrocytes

In this experiment, the hemagglutination inhibition test method was used to test the hemagglutination activity of peptides inhibiting the hemagglutination of 4 subtypes of influenza virus. Red blood cells have inhibitory activity.

Example 3

Anti-influenza virus efficacy test method:

Take cells in good logarithmic growth phase, add 0.25% trypsin digestion solution, blow evenly after digestion and fall off, count, 2×10 ⁵ cells/well, inoculate in 96-well cell culture plate, and place in constant temperature CO ₂ Cultivate in the incubator for 16-24h. Set up normal cell control group, virus infection group, positive drug control group and test drug group respectively. Discard the culture medium, rinse with PBS three times, add 100 TCID ₅₀ infectious influenza virus to each well except the normal control group, put it in a constant temperature _CO2 incubator for 1 hour, discard unadsorbed virus, and rinse twice with PBS , and then add drug-containing maintenance solutions with different concentrations. There were 6 doses in the test drug group, each with 3 replicate wells. Then continue to cultivate the cell culture plate with the above-mentioned scheme at 35° C. for 72 h, observe the cytopathy, and when the cytopathy of the virus control hole reaches more than 75%, adopt the MTT method or crystal violet staining method to measure the absorbance value (A value) of each group. . Calculate the antiviral efficiency (effective rate, ER) of the tested drug, and calculate the half effective concentration (EC ₅₀ ) of the drug. ER=(A value of the drug test group-A value of the virus control group)/(A value of the normal cell control group-A value of the virus control group)×100%. Pretreatment test method: mix the influenza virus containing 100TCD ₅₀ with the hemagglutination polypeptide according to the concentration gradient, and then treat it in a warm bath at 35°C for 60min, then infect MDCK cells according to the above method, add maintenance solution and continue to culture for 72h, and then observe with crystal violet staining Cytopathic results, taking the H7N9 virus as an example, the experiment was carried out, and the experimental results are shown in Table 3.

Determination of hemolytic activity in vitro:

The oligopeptide derivatives were diluted 2-fold starting from 50 μM, and then 50 μL samples were mixed with 50 μL 0.5% chicken red blood cells, and reacted at 37 ° C for 60 min, centrifuged, and the supernatant was taken, and the wavelength of 385 nm was measured by TECAN multi-functional microplate reader OD value, the specific results are shown in Table 3.

Table 3 oligopeptide derivatives anti-influenza pharmacodynamics test results in vitro

From Table 3, we can see that oligopeptide I has no obvious hemolytic activity under the condition of 50 μM concentration, while oligopeptide II shows hemolytic activity only under the condition of 12.5 μM concentration.

From Table 3, we can see that the results of MTT assay show that oligopeptide I and oligopeptide II have a certain protective effect on MDCK cells infected with influenza strains at a concentration close to CC ₀ .

In order to verify the effects of oligopeptide I and oligopeptide II on H1N1, H3N2 and H5N1 viruses, the same method as above was used for experiments, and the results showed that both oligopeptide I and oligopeptide II could directly inhibit influenza virus from infecting MDCK cells.

Example 4

In vivo pharmacodynamic evaluation of anti-influenza virus:

Taking the oligopeptide I sequence as a representative, the in vivo pharmacodynamic evaluation experiment of anti-influenza virus was carried out. The mice were randomly divided into virus control group (Control), positive drug ribavirin (Ribavirin) group and oligopeptide I administration group, with 10 mice in each group. Inject 40 μL of H1N1 avian influenza virus stock solution, and administer it 2 hours after infection. The virus control group was given intraperitoneal injection of normal saline (10mL/kg), the positive drug group was given intraperitoneal injection of ribavirin (50mg/kg), and the oligopeptide I administration group (50mg/kg) was given intramuscular injection, 2 days a day. times, a total of 16 days, observed once every 24h and recorded the death of mice in each group, the experimental results are shown in Figure 1.

It can be seen from Figure 1 that the survival rate of the mice in the oligopeptide I administration group remained at 75% 16 days after inoculation with the virus, and the life extension rate was higher than that of the mice in the virus control group and the positive drug group, so it can be seen that the oligopeptide I It has obvious preventive and therapeutic effects on mice infected with influenza virus.

The anti-influenza virus in vivo pharmacodynamic evaluation experiment was carried out with the oligopeptide II sequence according to the above method. The experimental results showed that the life extension rate of the mice in the administration group (the experimental group injected with the oligopeptide II polypeptide) was significantly higher than that of the virus control group and the virus control group. The mice in the positive drug group showed that oligopeptide II had obvious preventive and therapeutic effects on mice infected with influenza virus.

Example 5

Analysis of the interaction mode between oligopeptide Ⅰ and hemagglutinin

In order to further analyze the interaction between oligopeptide I and hemagglutinin crystal complex (PDB ID: 4BSE), the Triangle Matcher placement module in MOE was used to dock oligopeptide I to the active site of hemagglutinin. The results are shown in Figure 2 Show. Oligopeptide I can form hydrogen bond interactions with key amino acids such as Thr155, Gly135 and Ser136 in the hemagglutinin active site. Referring to the same method, the oligopeptide II sequence was docked to the active site of hemagglutinin, and the oligopeptide II could form hydrogen bond interactions with key amino acids such as Thr187 and Ser136 in the active site of hemagglutinin (as shown in Figure 3 ). The above mode of action between oligopeptide I and hemagglutinin indicates to a certain extent that oligopeptide I has the function of inhibiting hemagglutinin, which provides an important reference for further research on hemagglutinin inhibitors.

Example 6

Both oligopeptide I and oligopeptide II herein have anti-influenza virus activity, can significantly inhibit the proliferation of anti-influenza virus, and can be prepared into anti-influenza drugs in combination with pharmaceutically acceptable auxiliary materials or carriers. "Pharmaceutically acceptable" excipients are substances that are suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation and allergic reactions), ie substances with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The term refers to pharmaceutical carriers which, by themselves, are not essential active ingredients and which are not unduly toxic upon administration. Suitable vectors are well known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Pharmaceutically acceptable carriers in compositions can contain liquids such as water, saline, glycerol and ethanol. In addition, there may also be auxiliary substances in these carriers, such as lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, and the like.

The term "pharmaceutically acceptable excipient" refers to any suitable pharmaceutically acceptable adjuvant, carrier, diluent, preservative and the like used in pharmaceutical preparations. 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 lysolecithin, complex multivariate Alcohols, polyanions, peptides, oil emulsions, hydrocarbon emulsions, keyhole limpet hemocyanin, etc. Known carriers include, but are not limited to, sterile liquids such as water, oil, or a mixture of water and oil, 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 and EDTA, among others. The selection of pharmaceutically acceptable excipients can be achieved by known techniques in the art, and those skilled in the art can select appropriate pharmaceutically acceptable excipients based on the prior art according to the polypeptide pharmaceutical dosage form to be prepared. For example, for the preparation of oral liquid preparations (eg, suspension, microemulsion or double emulsion), the selected excipients may include, for example, water, oil, alcohols, flavoring agents, preservatives, coloring agents and the like. For another example, for the preparation of oral solid preparations (for example, powders, powder mist, capsules or tablets), the selected auxiliary materials can include, for example, starch, sugars, diluents, granulating agents, lubricants, binders, disintegrants etc. In addition, if necessary, the polypeptide drug of the present invention can also be prepared into sugar-coated or enteric-coated, or controlled-release preparations.

According to another aspect of the present invention, the present invention provides a method for controlling and/or preventing influenza virus, the method comprising administering an effective amount of the oligopeptide derivative of the present invention to an animal in need thereof, and the term "effective amount" means The amount that elicits an antiviral response in the administered animal.

Claims (10)

1. An oligopeptide, characterized in that its sequence contains an X-Arg-Leu-Y-Arg domain, wherein X is a hydrophobic amino acid; Y is a hydrophobic amino acid. 2. An oligopeptide derivative, characterized in that it is obtained by modifying the polypeptide sequence described in claim 1 with stearic acid, and its structural formula is C ₁₇ H ₃₅ CO-X-Arg-Leu-Y-Arg, wherein X Is a hydrophobic amino acid; Y is a hydrophobic amino acid. 3. The oligopeptide derivative according to claim 2, characterized in that: said X is one of Leu and Ala; Y is one of Pro and Leu. 4. according to claim 2 or 3 described oligopeptide derivatives, it is characterized in that: described oligopeptide derivatives are respectively: Oligopeptide I, C ₁₇ H ₃₅ CO-Leu-Arg-Leu-Pro-Arg; or Oligopeptide II, C ₁₇ H ₃₅ CO-Ala-Arg-Leu-Leu-Arg. 5. The application of the oligopeptide according to claim 1 or the oligopeptide derivative described in claim 2 or 3 or 4 in the preparation of medicine. 6. Use of the oligopeptide according to claim 1 or the oligopeptide derivative according to claim 2 or 3 or 4 in the preparation of a medicament for preventing or treating influenza disease. 7. The application of the oligopeptide derivative according to claim 6 in the preparation of a medicament for preventing or treating influenza disease, characterized in that: the influenza disease comprises one of H1N1, H3N2, H5N1, H7N9 influenza virus Influenza illness caused by one or more of them at the same time. 8. A medicine for preventing or treating influenza disease, characterized in that: it comprises one or more mixtures of the oligopeptide derivatives described in claim 2 or 3 or 4, and pharmaceutically acceptable auxiliary materials . 9. The medicine for preventing or treating influenza disease according to claim 8, characterized in that: the dosage forms of the medicine include injections, freeze-dried powder injections, microspheres, powders, powder sprays, capsules, tablets, pills , nasal spray, aerosol, enteric coating, nanosphere, microemulsion or double emulsion. 10. A medicine for preventing or treating influenza disease, characterized in that it comprises the oligopeptide as claimed in claim 1 and pharmaceutically acceptable auxiliary materials.