Antibacterial peptide SAMP4 or derivative thereof, pharmaceutical composition and application

Abstract

The invention discloses an antibacterial peptide SAMP4 or a derivative thereof, a pharmaceutical composition and application, and belongs to the technical field of biology, the amino acid sequence of the antibacterial peptide SAMP4 is as shown in SEQ ID NO: 1, and the antibacterial peptide SAMP4 has a broad-spectrum antibacterial effect and can be used for preparing antibacterial peptide SAMP4. The compound has a strong bacteriostatic 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, and is strong in antibacterial activity, wide in antibacterial spectrum and low in toxicity. The invention also provides a derivative and a salt of the antibacterial peptide SAMP4 and application of the antibacterial peptide SAMP4 and the derivative and the salt of the antibacterial peptide SAMP4 to research, development and preparation of various antibacterial products or / and antibacterial drugs and treatment of bacterial infectious diseases, and the antibacterial peptide SAMP4 and the derivative and the salt of the antibacterial peptide SAMP4 are remarkable in advantages and wide in application prospect.

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 bacterial resistance to current antibiotics becomes increasingly serious, antimicrobial peptides (AMPs) have gradually gained attention as a new class of anti-infective agents, with some already entering clinical trials. As an alternative to antibiotic therapy, AMPs offer a promising approach to addressing bacterial resistance. AMPs are a class of naturally bioactive polypeptides with inhibitory effects against bacteria, viruses, and fungi. AMPs are widely found in animals and plants, serving as a natural immune barrier against pathogens in organisms (such as amphibians, mammals, insects, and plants). They even exist within microorganisms themselves, earning them the nickname "secondary defense system." AMPs possess high antimicrobial activity, a broad antimicrobial spectrum, and a low resistance to developing drug resistance. Therefore, they hold great promise for combating infections caused by drug-resistant pathogens. AMPs can also be used in combination with existing drugs as combination therapies, generating synergistic or even additive effects. Furthermore, AMPs can act as immunomodulators, mobilizing the body's immune response. These characteristics guarantee their significant potential for clinical application.

[0003] Despite their significant advantages over existing drug classes, antimicrobial peptides face challenges in clinical application. For example, their large-scale production is expensive; peptide drugs are susceptible to proteolysis and have poor in vivo stability; and they are highly cytotoxic, leading to hemolysis of erythrocytes. Therefore, it is necessary to artificially design and synthesize new antimicrobial peptides to develop stable, low-toxic, and highly effective broad-spectrum antimicrobial drugs to effectively address the problem of drug resistance in pathogens. 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. The amino acid sequence of the antimicrobial peptide SAMP4 is: a1) SEQ ID NO: 1; or a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acid sequences in SEQ ID NO: 1 and having the same function as SEQ ID NO: 1; or a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% identical to SEQ ID NO: 1 and has the same function as SEQ ID NO: 1.

[0006] 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.

[0007] 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 antimicrobial peptide SAMP4, wherein the chemical modification includes acetylation, amination, methylation, phosphorylation, glycosylation, lipidation, ubiquitination, biotin labeling or fluorescent protein labeling.

[0008] 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.

[0009] According to a 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 an antibacterial product or / and 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.

[0010] 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, a derivative of the antimicrobial peptide SAMP4, and a salt of the antimicrobial peptide SAMP4.

[0011] In some embodiments, the pharmaceutical composition is in the form of an aerosol, spray, powder, pill, tablet, film, ointment, suppository, paste, solution, injection, mixture, lotion, or liniment.

[0012] According to a sixth aspect of the present invention, there is provided 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 medicament for preventing or treating Staphylococcus aureus infection.

[0013] 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 for the treatment of Staphylococcus aureus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the mass spectrum of the antimicrobial peptide SAMP4 in Example 1 of the present invention.

[0015] Figure 2 This is a liquid chromatogram of the antimicrobial peptide SAMP4 in Example 1 of the present invention.

[0016] Figure 3 This is a graph showing the in vitro bactericidal kinetics of the antimicrobial peptide SAMP4 in Example 3 of the present invention.

[0017] Figure 4 This is a diagram showing the drug resistance development of the antimicrobial peptide SAMP4 in Example 4 of the present invention.

[0018] 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.

[0019] Figure 6 This is a comparison chart of the bacterial load in the wound tissue of mice with Staphylococcus aureus skin infection after administration in Example 6 of the present invention. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail through specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention by those skilled in the art are all within the scope of the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents available on the conventional market.

[0021] Example 1 The antimicrobial peptide SAMP4, whose amino acid sequence is AKKWWKLLWKLWKKAK (SEQ ID NO. 1), 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 FIG. 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 in Figure 2 As shown, liquid chromatography detection conditions are as follows: chromatographic column: 4.6*150mm, kromasil C18-5, mobile phase A: 0.1% trifluoroacetic acid in acetonitrile, mobile phase B: 0.1% trifluoroacetic acid in water, 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.

[0022] 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 isolated and purified.

[0023] Example 2 MIC and MBC determination of antimicrobial peptide SAMP4 The antimicrobial peptide SAMP4 obtained in Example 1 was subjected to minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination. 600 The test bacterial solution at a concentration of 0.1 was diluted 1:100 with MH medium and then used. The concentration of the antimicrobial peptide SAMP4 was adjusted to 256 μg / mL. 100 μL of the bacterial solution was added to columns 1 to 11 of a 96-well plate. 100 μL of the antimicrobial peptide SAMP4 solution was added to the first column. 100 μL of the bacterial solution from the first well was serially diluted to well 10. 100 μL of sterile MHB was added to well 12. Three replicates were placed for each test compound and the plates were incubated at 37°C for 24 h. The minimum inhibitory concentration (MIC) was defined as the antimicrobial peptide concentration at which no bacteria grew. After determining the minimum inhibitory concentration (MIC), 0.1 mL of the mixture from all wells showing no bacterial growth was added to drug-free nutrient agar medium and incubated overnight at 37°C. If the number of colonies growing on the plate was ≤0.1% of the inoculum size, the antimicrobial peptide concentration was considered the minimum bactericidal concentration (MBC). When MBC / MIC ≤ 2, the antimicrobial peptide was considered to have a bactericidal effect; when MBC / MIC ≥ 4, the antimicrobial peptide was considered to have an antibacterial effect. Specific test results are shown in Table 1.

[0024] Table 1 Antibacterial activity test of antimicrobial peptide SAMP4

[0025] 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.

[0026] Example 3 In vitro bactericidal kinetics of antimicrobial peptide SAMP4 The frozen Staphylococcus aureus ATCC29213 was streaked on a TSA plate and then cultured in a 37°C incubator. A single colony was picked and inoculated into MH broth for overnight enrichment. The OD value was adjusted with MH broth the next day. 600 Set the value to 0.1 for future use. Based on the MIC values ​​(4 µg / ml) of the test bacteria in Example 2, prepare antimicrobial peptide SAMP4 working solutions at concentrations of 10×MIC, 20×MIC, 40×MIC, 80×MIC, 160×MIC, and 320×MIC. Add 0.6 ml of bacterial solution and 0.6 ml of antimicrobial peptide SAMP4 working solution to 6 ml of MHB system to achieve final concentrations of the antimicrobial peptide SAMP4 to be tested: 1×MIC, 2×MIC, 4×MIC, 8×MIC, 16×MIC, and 32×MIC. Incubate the cultures on a shaker at 37°C. Bacterial solutions without antimicrobial peptide SAMP4 served as controls, and colony counts were performed. At 30 min, 1 h, 2 h, 3 h, and 5 h, 0.1 mL of sample was taken from all tubes. After 10-fold and 100-fold dilution, the sample was added to the surface of drug-free nutrient agar medium and evenly spread using a disposable spreader stick. All plates were placed in a 36°C incubator for overnight incubation for 18 h. The colonies were counted the next day to draw a time-kill curve. Figure 3 .

[0027] like Figure 3 As shown in the figure, at a concentration of 0.5×MIC, the concentration of the antimicrobial peptide SAMP4 increased dramatically, while at a concentration of 1×MIC, the antimicrobial peptide SAMP4 inhibited 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 contact with Staphylococcus aureus for 30 minutes and 2 hours, respectively, demonstrating a rapid bactericidal effect.

[0028] Example 4 Evaluation of the drug resistance of antimicrobial peptide SAMP4 against Staphylococcus aureus 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 then used for the next MIC determination. Ciprofloxacin was used as a control. After incubation at 37°C for 24 hours, the new MIC value was read. The experiment lasted for 28 generations. The results are shown in FIG. Figure 4 .

[0029] Depend on Figure 4It can be seen that after 28 generations of passage under subinhibitory concentration, 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.

[0030] Example 5 Cytotoxicity Assay of Antimicrobial Peptide SAMP4 HEp-2, Caco-2, and HGE cells frozen in liquid nitrogen were revived and seeded in DMEM medium supplemented with 10% fetal bovine serum. The cells were subcultured at 37°C in 5% CO2. 10 cm plates were selected when the cells had grown to a 90% or dense monolayer. The medium was discarded and the cells were washed one to two times with PBS. Trypsin was added to round the adherent cells. DMEM medium was added to terminate the digestion. Adherent cells were rinsed with a pipette and transferred to a 10 ml centrifuge tube. The supernatant was discarded and fresh DMEM medium was added to prepare a cell suspension. After counting with a cell counter, 100 µL of the cell suspension was plated into a 96-well plate at 5,000 cells per well and incubated overnight at 37°C. The cells were treated with various final concentrations of the antimicrobial peptide SAMP4 for 48 hours. Discard the culture medium in the wells and add 100 µL of antibiotic-free DMEM medium and 10 µL of CCK8 solution. Incubate the 96-well plate in a 37°C incubator for 40–60 minutes. Measure the absorbance at 450 nm using a microplate reader. Wells without the antimicrobial peptide SAMP4 serve as controls, while wells without the antimicrobial peptide SAMP4 and cells serve as blank controls. Calculate cell viability based on OD values.

[0031] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%, 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 Table 2 and Figure 5 .

[0032] Table 2 Results of in vitro toxicity test of SAMP4 on mammalian cells

[0033] From Table 2 and Figure 5 It 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 its inhibitory concentration range and its toxicity to gingival epithelial cells is relatively low.

[0034] Example 6: Antimicrobial Peptide SAMP4 Treats Staphylococcus aureus Skin Infection in Mice Frozen methicillin-resistant Staphylococcus aureus YZSA-24 was inoculated into TSB broth and cultured overnight at 37°C. 3% of the inoculation ratio was transferred to 10 mL of preheated TSB and cultured in a constant temperature shaker until the logarithmic phase. The OD value was then adjusted to approximately 0.1 with TSB broth (corresponding to a concentration of approximately 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, the hair was shaved to expose the skin, and then disinfected with alcohol. The surface skin was polished with sterile sandpaper to make the skin obviously damaged but without bleeding. This produced a 1.5 cm 2 of the infected and treated area.

[0035] Infection was initiated by aspirating 10 µL of bacterial solution onto the skin surface. One hour after infection, 40 µL of each of the antimicrobial peptide SAMP4 solution, ampicillin solution, and solvent control solutions were applied to the infected areas of three groups of mice. Treatments were then administered 4, 8, 12, 20, and 24 hours after infection. Mice were sacrificed by cervical dislocation 25 hours after infection. The wounds were gently cleaned with alcohol cotton balls, excised, minced, and placed in a 10 mL tube. 4 mL of PBS was added and vortexed to mix thoroughly. After a 10-fold dilution, 100 µL of the solution was plated on a TSA plate for viable bacterial count.

[0036] The results are as follows Figure 6 It was shown that by topical administration to skin wounds infected with Staphylococcus aureus, the antimicrobial peptide SAMP4 was able to reduce the bacterial load in the wound by 1.5 logarithmic levels compared with the solvent control, indicating that SAMP4 has a positive therapeutic effect on skin infections caused by Staphylococcus aureus.

[0037] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all fall within the scope of protection 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; or 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 a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homologous to SEQ ID NO: 1 and has the same function as SEQ ID NO:

1.

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

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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