Antimicrobial peptide SAMP4 or its derivatives, pharmaceutical compositions and applications

Abstract

The present invention discloses an antimicrobial peptide SAMP4 or its derivatives, a pharmaceutical composition and application, belonging to the field of biotechnology. The amino acid sequence of the antimicrobial peptide SAMP4 is shown in SEQ ID NO: 1, and has a broad-spectrum antibacterial effect, and has a strong antibacterial effect on various Gram-positive bacteria such as Staphylococcus aureus, Enterococcus and Streptococcus, and various Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa, with strong antibacterial activity, a broad antibacterial spectrum and low toxicity. The present invention also provides derivatives and salts of the antimicrobial peptide SAMP4, and the antimicrobial peptide SAMP4, the derivatives and salts of the antimicrobial peptide SAMP4 are used in the research and development of various antimicrobial products or / antimicrobial drugs, and are used in the treatment of bacterial infectious diseases, with significant advantages and broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to an antimicrobial peptide SAMP4 or a derivative thereof, a pharmaceutical composition and an application thereof. Background Art

[0002] As the problem of bacterial resistance to current antibiotics becomes more and more serious, antimicrobial peptides have gradually come into people's attention as a new anti-infection drug, and some antimicrobial peptides have entered the clinical stage. As an alternative to antibiotic therapy, antimicrobial peptides have become people's hope for solving the problem of bacterial resistance. Antimicrobial peptides are a class of polypeptides with natural biological activity that have inhibitory effects on bacteria, viruses, and fungi. Antimicrobial peptides are widely present in animals and plants. They are the natural immune barrier of organisms (such as amphibians, mammals, insects, and plants) against pathogen invasion. They even exist in the microorganisms' own system and are called the "second defense system." Antimicrobial peptides have the characteristics of high antimicrobial activity, broad antimicrobial spectrum, many types, and low resistance. Therefore, they have broad application prospects in combating drug-resistant pathogen infections. Antimicrobial peptides can also be used in combination with existing drugs as a combination therapy to produce synergistic or even superimposed effects. In addition, antimicrobial peptides can also be used as immunomodulators to mobilize the body's immune response. The above characteristics determine that antimicrobial peptides have great potential for clinical application.

[0003] Although antimicrobial peptides have advantages over current drug classes, their clinical application still faces challenges. For example, the mass production cost of antimicrobial peptides is high; peptide drugs are easily hydrolyzed by proteases and have poor in vivo stability; they are highly cytotoxic and cause hemolysis of red blood cells. Therefore, it is necessary to synthesize new antimicrobial peptides through artificial design and develop stable, low-toxic, and highly effective broad-spectrum antimicrobial drugs to effectively solve the problem of drug resistance of pathogenic bacteria. Summary of the invention

[0004] The purpose of the present invention is to provide an antimicrobial peptide SAMP4 or its derivatives, a pharmaceutical composition and applications.

[0005] According to one aspect of the present invention, an antimicrobial peptide SAMP4 is provided, wherein the amino acid sequence of the antimicrobial peptide SAMP4 is:

[0006] a1) SEQ ID NO: 1; or

[0007] a2) an amino acid sequence in which one or more amino acid sequences of SEQ ID NO: 1 are substituted and / or deleted and / or added and has the same function as SEQ ID NO: 1; or

[0008] a3) an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology to SEQ ID NO: 1 and has the same function as SEQ ID NO: 1.

[0009] In some embodiments, the configuration of the antimicrobial peptide SAMP4 includes an L-type polypeptide, a D-type polypeptide, a Retro-inverso polypeptide or a cyclic peptide.

[0010] According to another aspect of the present invention, a biological derivative of the antimicrobial peptide SAMP4 is provided, which is a chemically modified product of the amino acid sequence of the above-mentioned antimicrobial peptide SAMP4, and the chemical modification includes acetylation, amination, methylation, phosphorylation, glycosylation, liposylation, ubiquitination, biotin labeling or fluorescent protein labeling.

[0011] According to another aspect of the present invention, a salt of the antimicrobial peptide SAMP4 is provided, including a metal salt of the antimicrobial peptide SAMP4, a salt formed by the antimicrobial peptide SAMP4 and an inorganic acid or an organic acid, wherein the metal includes lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum, the inorganic acid includes hydrochloric acid, sulfuric acid, boric acid or carbonic acid, and the organic acid includes acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid or oxalic acid.

[0012] According to the fourth aspect of the present invention, there is provided the use of the above-mentioned antimicrobial peptide SAMP4, derivatives of the antimicrobial peptide SAMP4, and salts of the antimicrobial peptide SAMP4 in the preparation of antibacterial products or / antibacterial drugs, wherein the bacteria are one or more of Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Streptococcus suis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, Salmonella, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

[0013] According to a fifth aspect of the present invention, there is provided a pharmaceutical composition comprising at least one of the above-mentioned antimicrobial peptide SAMP4, derivatives of antimicrobial peptide SAMP4, and salts of antimicrobial peptide SAMP4.

[0014] In some embodiments, the dosage form of the pharmaceutical composition is any one of an aerosol, a spray, a powder, a pill, a tablet, a film, an ointment, a suppository, a paste, a solution, an injection, a mixture, a lotion, or a liniment.

[0015] According to a sixth aspect of the present invention, provided is the use of the antimicrobial peptide SAMP4, a derivative of the antimicrobial peptide SAMP4, a salt of the antimicrobial peptide SAMP4 or a pharmaceutical composition in the preparation of a drug for preventing or treating Staphylococcus aureus infection.

[0016] Beneficial effects of the present invention: The antimicrobial peptide SAMP4 of the present invention has a broad-spectrum antibacterial effect, and has a strong antibacterial effect on various Gram-positive bacteria such as Staphylococcus aureus, Enterococcus and Streptococcus, and various Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa. It has strong antibacterial activity, a broad antibacterial spectrum, and low toxicity. It can be used in the research and development of various anti-Staphylococcus aureus drugs, and can be used for the treatment of Staphylococcus aureus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the mass spectrum of the antimicrobial peptide SAMP4 in Example 1 of the present invention.

[0018] Figure 2 It is the liquid chromatogram of the antimicrobial peptide SAMP4 in Example 1 of the present invention.

[0019] Figure 3 This is a graph showing the in vitro bactericidal kinetics of the antimicrobial peptide SAMP4 in Example 3 of the present invention.

[0020] Figure 4 This is a diagram showing the drug resistance development of the antimicrobial peptide SAMP4 in Example 4 of the present invention.

[0021] Figure 5 This is a graph showing the survival rates of Hep-2, Caco-2 and HGE cells when the antimicrobial peptide SAMP4 is added in Example 5 of the present invention.

[0022] Figure 6 This is a comparison chart of the bacterial load in the wound tissue of the skin infection of Staphylococcus aureus in mice after administration in Example 6 of the present invention. DETAILED DESCRIPTION

[0023] The present invention is further described in detail through specific implementation cases, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art are all limited by the claims attached to this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0024] Example 1

[0025] The antimicrobial peptide SAMP4 has an amino acid sequence of AKKWWKLLWKLWKKAK (SEQ ID NO. 1). The antimicrobial peptide was synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd. (product name: TG-LG-16242 SAMP4, batch number: TP-20067840, molecular weight: 2141.74) with a purity greater than 95%. Its mass spectrum is shown in Figure 1As shown, the mass spectrometry detection conditions are: flow rate: 0.2 mL / min, running time: 1 min, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid acetonitrile solution; the HPLC chromatogram is shown Figure 2 As shown, liquid chromatography detection conditions: chromatographic column: 4.6*150mm, kromasil C18-5, mobile phase A: 0.1% trifluoroacetic acid in acetonitrile solution, mobile phase B: 0.1% trifluoroacetic acid aqueous solution, elution conditions: 0-0.01min mobile phase A 5%, mobile phase B 95%; 0.01-25min mobile phase A increased from 5% to 50%, mobile phase B decreased from 95% to 50%; 25-30min mobile phase A increased from 50% to 90%, mobile phase B decreased from 50% to 10%, flow rate: 1mL / min, wavelength: 214nm, injection volume: 20μL.

[0026] Depend on Figure 1 and Figure 2 It can be seen that the antimicrobial peptide SAMP4 of the present invention, whose sequence is shown in SEQ ID NO: 1, was successfully synthesized and separated and purified.

[0027] Example 2 MIC and MBC determination of antimicrobial peptide SAMP4

[0028] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the antimicrobial peptide SAMP4 obtained in Example 1 were determined. 600 The test bacterial solution of 0.1 was diluted with MH medium at 1:100 for later use. The concentration of antimicrobial peptide SAMP4 was adjusted to 256 μg / mL, and 100 μL of bacterial solution was added to the 1st to 11th columns of the 96-well plate. 100 μL of antimicrobial peptide SAMP4 solution was added to the first column, and 100 μL of bacterial solution was drawn from the first well and diluted to the 10th well in turn. 100 μL of sterile MHB was added to the 12th well. Each test compound was repeated 3 wells, and the cells were cultured in a 37°C incubator for 24 h. The antimicrobial peptide concentration at which no bacteria grew was taken as the minimum inhibitory concentration. After the minimum inhibitory concentration (MIC) results were read, 0.1 mL of the mixed solution in all wells where no bacteria grew was added to the drug-free nutrient agar medium, and the cells were cultured in a 37°C incubator overnight. If the number of colonies grown by the test bacteria on the plate was ≤0.1% of the inoculated bacteria, the antimicrobial peptide concentration was considered to be the minimum bactericidal concentration (MBC). When MBC / MIC≤2, the antimicrobial peptide is considered to have bactericidal effect; when MBC / MIC≥4, the antimicrobial peptide is considered to have antibacterial effect. The specific test results are shown in Table 1.

[0029] Table 1 Antibacterial activity test table of antimicrobial peptide SAMP4

[0030]

[0031] As shown in Table 1, the antimicrobial peptide SAMP4 has good antibacterial and bactericidal activities against both Gram-positive and Gram-negative bacteria, showing a broad-spectrum antibacterial effect.

[0032] Example 3 In vitro bactericidal kinetics of antimicrobial peptide SAMP4

[0033] Draw a line on the frozen Staphylococcus aureus ATCC29213 plate and place it in a 37℃ incubator for culture. Pick a single colony and inoculate it in MH broth for overnight enrichment. Adjust the OD value with MH broth the next day. 600 The value is 0.1 for standby use. According to the MIC value (4µg / ml) of the test bacteria in Example 2, the antimicrobial peptide SAMP4 working solution with a concentration of 10×MIC, 20×MIC, 40×MIC, 80×MIC, 160×MIC, and 320×MIC was prepared. 0.6ml of bacterial solution and 0.6ml of antimicrobial peptide SAMP4 working solution were added to the 6ml MHB system to make the final concentration of the antimicrobial peptide SAMP4 to be tested 1×MIC, 2×MIC, 4×MIC, 8×MIC, 16×MIC, and 32×MIC, and placed in a 37°C shaking incubator for shaking culture. The bacterial solution without antimicrobial peptide SAMP4 was used as the control group, and the colony count was performed. 0.1 mL of the sample was taken from all tubes at 30 min, 1 h, 2 h, 3 h, and 5 h, and diluted 10-fold and 100-fold respectively, and then evenly spread on the surface of drug-free nutrient agar medium using a disposable spreader stick. All plates were placed in a 36°C incubator for overnight culture for 18 h. The colonies were counted the next day to draw a time-killing curve, see Figure 3 .

[0034] like Figure 3 As shown in the figure, at a concentration of 0.5×MIC, the concentration of the antimicrobial peptide SAMP4 increased sharply, while at a concentration of 1×MIC, the antimicrobial peptide SAMP4 showed an inhibitory effect on the growth of Staphylococcus aureus. In contrast, at concentrations of 2×MIC and 4×MIC, the antimicrobial peptide SAMP4 was able to completely kill Staphylococcus aureus after contacting with Staphylococcus aureus for 30 min and 2 h, respectively, showing a rapid bactericidal effect.

[0035] Example 4 Evaluation of the drug resistance of antimicrobial peptide SAMP4 to Staphylococcus aureus

[0036] According to the minimum inhibitory concentration (MIC) results of Staphylococcus aureus in Example 2, 0.5×MIC of Staphylococcus aureus in a 96-well plate was inoculated into MH broth with a final concentration of 0.5×MIC, incubated at 37°C for 24 hours and used for the next MIC determination. Ciprofloxacin was used as a control. After incubation at 37°C for 24 hours, a new MIC value was read. The experiment lasted for 28 generations. The results are shown in Figure 4 .

[0037] Depend on Figure 4 It can be seen that after 28 generations of culture at subinhibitory concentrations, the MIC value of the antimicrobial peptide SAMP4 increased by no more than 2 times, while the MIC value of ciprofloxacin increased by 64 times, indicating that the antimicrobial peptide SAMP4 can avoid inducing drug resistance in Staphylococcus aureus.

[0038] Example 5 Cytotoxicity assay of antimicrobial peptide SAMP4

[0039] HEp-2 cells, Caco-2 cells and HGE cells frozen in liquid nitrogen were revived and inoculated in DMEM medium containing 10% fetal bovine serum and subcultured at 37°C and 5% CO2. A 10 cm plate that had grown to 90% or a dense monolayer was selected, the medium was discarded and washed 1-2 times with PBS, and trypsin digestion solution was added to digest the adherent cells to round them. DMEM medium was added to terminate the digestion, and the adherent cells were washed with a pipette and transferred to a 10 ml centrifuge tube. The supernatant was discarded at 1500 rpm for 4 min, and new DMEM medium was added to make a cell suspension. After counting with a cell counting plate, 100 μL of the cell suspension was inoculated in a 96-well plate, with 5000 cells in each well, and the plate was placed in a 37°C incubator overnight. Different final concentrations of the antimicrobial peptide SAMP4 solution were added to intervene in the cells for 48 h. The culture medium in the wells was discarded, 100 µL of DMEM culture medium without antibiotics was added, 10 µL of CCK8 solution was added, and the 96-well plate was placed in a 37°C incubator for 40 min-60 min. The absorbance at 450 nm was measured using an ELISA reader, and the wells without antimicrobial peptide SAMP4 were used as the control group, and the wells without antimicrobial peptide SAMP4 and cells were used as the blank group. The cell survival rate was calculated based on the OD value.

[0040] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%,

[0041] Among them, As is the OD value of the experimental group, Ac is the OD value of the control group, and Ab is the OD value of the blank well. The experiment was repeated three times, and the results are shown in Tables 2 and Figure 5 .

[0042] Table 2 Results of in vitro toxicity test of SAMP4 on mammalian cells

[0043]

[0044] From Table 2 and Figure 5It can be seen that at a concentration of 32 µg / mL, the survival rates of HEp-2 cells, Caco-2 cells and HGE cells were 65.17%, 40.57% and 84.34%, respectively, indicating that there is no obvious cytotoxicity within the range of its inhibitory concentration, and the toxicity to gingival epithelial cells is relatively low.

[0045] Example 6 Antimicrobial peptide SAMP4 treats Staphylococcus aureus skin infection in mice

[0046] The frozen methicillin-resistant Staphylococcus aureus YZSA-24 was inoculated into TSB broth and cultured overnight at 37°C. The mixture was transferred to 10 mL preheated TSB at a ratio of 3%, and cultured in a constant temperature shaker until the logarithmic phase. The OD value was adjusted to about 0.1 with TSB broth for later use (the corresponding concentration is about 10 7 CFU / mL). The antimicrobial peptide SAMP4 was prepared into a stock solution, which was diluted with PBS to a final concentration of 1% (W / V) antimicrobial peptide SAMP4 solution. A final concentration of 2.5% (W / V) ampicillin solution was used as a control drug, and PBS was used as a solvent control. 18 ICR mice were randomly divided into 3 groups and intraperitoneally injected with 0.2 ml of cyclophosphamide solution on the 4th day (150 mg / kg) and the 1st day (100 mg / kg) before infection. The mice were anesthetized with isoflurane, and the hair was shaved to expose the skin, which was then disinfected with alcohol. The surface skin was polished with sterile sandpaper to make the skin visibly damaged but without bleeding. This produced a 1.5 cm 2 of infection and treatment area.

[0047] 10 µL of bacterial solution was dripped on the skin surface to start infection. 1 h after infection, 40 µL of antimicrobial peptide SAMP4 solution, ampicillin solution, and solvent control were applied to the infected areas of the three groups of mice, and then the drugs were administered at 4 h, 8 h, 12 h, 20 h, and 24 h. 25 h after infection, the mice were killed by dislocation of the neck, the wound was gently wiped with alcohol cotton balls, excised, cut into pieces and placed in a 10 ml tube, 4 mL PBS was added and vortexed to mix, and 100 µL was applied to the TSA plate after 10-fold dilution for viable bacterial count.

[0048] The results are as follows Figure 6 The results showed that the antimicrobial peptide SAMP4 could reduce the bacterial load in wounds by 1.5 logarithmic levels compared with the solvent control by topical administration to skin wounds infected with Staphylococcus aureus, indicating that SAMP4 has a positive therapeutic effect on skin infections caused by Staphylococcus aureus.

[0049] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. Antimicrobial peptide SAMP4, characterized in that The amino acid sequence of the antimicrobial peptide SAMP4 is: a1) SEQ ID NO:

1.

2. The antimicrobial peptide SAMP4 according to claim 1, characterized in that The configuration of the antimicrobial peptide SAMP4 includes an L-type polypeptide or a D-type polypeptide.

3. A derivative of the antimicrobial peptide SAMP4, characterized in that: The derivative is a chemically modified product of the amino acid sequence of the antimicrobial peptide SAMP4 according to claim 1, and the chemical modification includes acetylation, amination, methylation, phosphorylation, glycosylation, liposylation, ubiquitination, biotin labeling or fluorescent protein labeling.

4. A salt of the antimicrobial peptide SAMP4, characterized in that The invention comprises a metal salt of the antimicrobial peptide SAMP4 according to claim 1, or a salt formed by the antimicrobial peptide SAMP4 according to claim 1 and an inorganic acid or an organic acid, wherein the metal comprises lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum, the inorganic acid comprises hydrochloric acid, sulfuric acid, boric acid or carbonic acid, and the organic acid comprises acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid or oxalic acid.

5. Use of the antimicrobial peptide SAMP4 according to any one of claims 1 to 2, the derivative of the antimicrobial peptide SAMP4 according to claim 3, or the salt of the antimicrobial peptide SAMP4 according to claim 4 in the preparation of an antibacterial product or / and an antibacterial drug, wherein the bacteria is one or more of Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Streptococcus suis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, Salmonella, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

6. A pharmaceutical composition, characterized in that The method comprises at least one of the antimicrobial peptide SAMP4 according to any one of claims 1 to 2, the derivative of the antimicrobial peptide SAMP4 according to claim 3, and the salt of the antimicrobial peptide SAMP4 according to claim 4.

7. A pharmaceutical composition according to claim 6, characterized in that: The dosage form of the pharmaceutical composition is any one of aerosol, spray, powder, pill, tablet, film, ointment, suppository, paste, solution, injection, mixture, lotion or liniment.

8. Use of the antimicrobial peptide SAMP4 according to any one of claims 1 to 2, the derivative of the antimicrobial peptide SAMP4 according to claim 3, the salt of the antimicrobial peptide SAMP4 according to claim 4, or the pharmaceutical composition according to any one of claims 6 to 7 in the preparation of a medicament for preventing or treating Staphylococcus aureus infection.

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