Antimicrobial short peptides and their disulfide cyclization and applications

Abstract

The application discloses a group of antimicrobial short peptides and disulfide bond cyclization products thereof. The amino acid sequence of the short peptide is 13 amino acids in length, each sequence contains two cysteine residues, and can form an intramolecular disulfide bond. The minimum inhibitory concentration experiment verifies that the short peptide has inhibitory effect on Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and antimicrobial peptide technology, specifically relating to a group of short peptides with antimicrobial activity, intramolecular disulfide cyclizations of said short peptides, compositions containing said short peptides, and the use of said short peptides in the preparation of antibacterial and / or antifungal preparations. Background Technology

[0002] Bacterial and fungal infections have long been a major challenge in clinical medicine, public health, food safety, and biomaterials. Although the widespread use of antibiotics and antifungal drugs has significantly reduced the incidence and mortality of infectious diseases over the past few decades, the widespread and even overuse of antimicrobial drugs has led to the emergence and rapid spread of drug-resistant microorganisms, resulting in a significant decline in the effectiveness of existing antimicrobial drugs in an increasing number of clinical scenarios. The World Health Organization has listed drug-resistant bacterial infections as one of the top ten global public health threats, among which Acinetobacter baumannii (Acinetobacter baumannii) is a significant culprit. Acinetobacter baumannii ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Staphylococcus aureus ( Staphylococcus aureus Pathogens such as [list of pathogens] are listed as key pathogens for which new antimicrobial drugs urgently need to be developed. Therefore, developing antimicrobial active substances with novel mechanisms of action, novel chemical structures, and that are less likely to induce drug resistance has significant clinical value and strategic importance.

[0003] Antimicrobial peptides (AMPs) are important components of the organism's innate immune system. They typically consist of 12-50 amino acids, most of which are cationic and amphiphilic. They exert rapid bactericidal / bacteriostatic effects primarily by disrupting the integrity of microbial cell membranes or acting on intracellular targets. Compared to traditional antibiotics, AMPs, due to their mechanisms of action often involving physical membrane perturbation, are less likely to induce high levels of drug resistance and are considered highly promising next-generation anti-infective candidates.

[0004] Short peptides (<20 amino acids in length) offer significant advantages in drug development: they can be prepared efficiently and at low cost using mature solid-phase synthesis techniques, and are easily modified through end-capture, cyclization, or PEGylation. Quality control is also convenient, making them more suitable for industrial production. However, the antimicrobial activity of short peptides is not solely determined by length, but rather by a complex synergistic effect of multiple factors, including amino acid composition, residue arrangement, net charge, hydrophobicity, amphiphilicity, and conformational flexibility. Notably, even with identical amino acid compositions, differences in residue order can lead to significant variations in activity, selectivity, and stability. Therefore, it is impossible to accurately predict the effectiveness of a specific short peptide sequence based on conventional physicochemical parameters.

[0005] Disulfide bonds can form conformational constraints in cysteine-containing peptides. Different cysteine ​​positions and the size of the resulting rings can alter the peptide's conformation, protease sensitivity, and antimicrobial activity. However, even two short peptides with similar amino acid compositions may exhibit different antimicrobial activities due to differences in their residue arrangements or disulfide bond positions. Therefore, it is difficult to accurately predict whether a specific short peptide sequence and its cyclization form possess effective antimicrobial activity based solely on conventional physicochemical properties.

[0006] Therefore, there is still a need in the field to provide short peptides with novel sequences and experimentally verified antimicrobial activity. Summary of the Invention

[0007] This invention experimentally verified the synergistic inhibitory effects of a group of short peptides with novel amino acid sequences and their intramolecular disulfide cyclizations on Gram-positive bacteria (including but not limited to Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Staphylococcus epidermidis) and fungi (including but not limited to Candida albicans and Candida glabrata). This invention also provides antimicrobial compositions comprising the aforementioned short peptides or their disulfide cyclizations and their applications.

[0008] In a first aspect, the present invention provides an antimicrobial short peptide comprising the amino acid sequence shown in SEQ ID NO:1, namely RWRCWRMRGRRCW.

[0009] In one embodiment, the amino acid sequence of the short peptide is shown in SEQ ID NO:1, and it is 13 amino acids in length, with two cysteine ​​residues located at positions 4 and 12, respectively.

[0010] In one embodiment, the short peptide is in a linear form, wherein both of its cysteine ​​residues exist as free thiol groups and do not form intramolecular disulfide bonds.

[0011] In one embodiment, the short peptide is in a disulfide cyclized form, with an intramolecular disulfide bond formed between its two cysteine ​​residues.

[0012] Furthermore, for the short peptide shown in SEQ ID NO:1, its disulfide bond linkage is Cys 4 —S—S—Cys 12 .

[0013] In a second aspect, the present invention provides an antimicrobial composition comprising an effective amount of the short peptide and / or its disulfide cyclization as described in the first aspect, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0014] In one embodiment, the composition further comprises one or more auxiliary ingredients selected from the group consisting of: penetration enhancers, stabilizers, antioxidants, solubilizers, preservatives, buffers, and thickeners.

[0015] In one embodiment, the dosage form of the composition is selected from solutions, sprays, lyophilized powder injections, creams, gels, lotions, coating liquids, or solid dispersions.

[0016] Thirdly, the present invention provides the use of the short peptides and / or their disulfide cyclizations described in the first aspect in the preparation of antimicrobial agents.

[0017] In one embodiment, the microorganism is selected from one or more of the following: Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, or Candida albicans.

[0018] In one embodiment, the strain is selected from one or more of the following: Acinetobacter baumannii (… A. baumannii ATCC 19606, Candida albicans ( C. albicans ATCC 10231, Staphylococcus aureus ( S. aureus ATCC 6538, Pseudomonas aeruginosa ( P. aeruginosa ATCC 27853 or E. coli ( E. coli ATCC8739. In one embodiment, the formulation is a pharmaceutical preparation, a disinfectant, a medical device coating agent, a food preservative, or a cosmetic additive.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) Three novel short peptide sequences of 13 amino acids each, SEQ ID NO:1, are provided, whose amino acid composition, arrangement and cysteine ​​position are different from the published antimicrobial peptide sequences.

[0020] (2) The short peptides and their disulfide cyclizations can be obtained by solid-phase polypeptide synthesis. The purity of the peptides is above 95% as determined by reversed-phase high-performance liquid chromatography. The measured molecular weight is consistent with the theoretical value as determined by mass spectrometry, and the structure is clear.

[0021] (3) The minimum inhibitory concentration experiment showed that the linear form and / or disulfide cyclized form of the short peptide had a measurable inhibitory effect on one or more microorganisms selected from Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans.

[0022] (4) For most tested strains, the MIC value of the disulfide bond cyclized form was lower than that of the corresponding linear form, indicating that intramolecular disulfide bond cyclization can enhance the antimicrobial activity of the short peptide.

[0023] (5) The short peptide is only 13 amino acids long and can be prepared on a large scale by chemical synthesis. The synthesis cost is low, the quality control is convenient, and it has good prospects for industrial application. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the process for obtaining and screening the antimicrobial short peptides of the present invention.

[0025] Figure 2 This is a schematic diagram of the structure of the short peptide and its disulfide cyclization product of the present invention.

[0026] Figure 3 This is a reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram for the purity identification of the short peptide P1 of this invention.

[0027] Figure 4 This is a reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram for the purity identification of the disulfide bond cyclized short peptide P1-Cyc of this invention.

[0028] Figure 5 This is a mass spectrometry molecular weight identification spectrum of the short peptide P1 of this invention.

[0029] Figure 6 This is a mass spectrometry molecular weight identification spectrum of the disulfide bond cyclized short peptide P1-Cyc of the present invention.

[0030] Figure 7 Relative cell viability of HaCaT cells after treatment with different concentrations of P1 and P1-Cyc for 24 hours. The x-axis represents the concentration of the short peptide (μg / mL), and the y-axis represents the percentage of cell viability relative to the untreated control group (%). Data are expressed as mean ± standard deviation.

[0031] Figure 8 The apparent hemolysis percentage after treatment of human erythrocytes with different concentrations of P1 and P1-Cyc is shown. The horizontal axis represents the short peptide concentration (μg / mL), and the vertical axis represents the apparent hemolysis percentage (%). Data are expressed as mean ± standard deviation. Detailed Implementation

[0032] The technical solution of the present invention will be described in more detail below with reference to specific embodiments and accompanying drawings. In implementing the present invention, except for the experimental steps, conditions, and methods described in detail below, all other techniques and common knowledge in the art are employed, and no additional special limitations are imposed.

[0033] It should be noted that the embodiments described herein are only some typical examples of the present invention, and not all implementation methods. The following description of exemplary embodiments is for illustrative purposes only and does not constitute any limitation on the present invention or its application scenarios.

[0034] In the demonstration and discussion of the various embodiments, the specific numerical values ​​and parameters involved are for illustrative purposes only and are not intended to be limiting. Therefore, other embodiments based on the concept of this invention can have their relevant numerical values ​​adjusted according to actual needs.

[0035] Materials, instruments and measurement conditions used in the examples Unless otherwise specified, the amino acid monomers, solid-phase resins, coupling reagents, organic solvents, and buffer salts used in the following examples were all commercially available analytical grade or HPLC grade reagents, purchased from a primary peptide synthesis reagent supplier. Test strain: Acinetobacter baumannii (… Acinetobacter baumannii ATCC 19606, Candida albicans ( Candida albicans ATCC 10231, Staphylococcus aureus ( Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ( Pseudomonas aeruginosa ATCC 27853, Escherichia coli ( Escherichia coli ATCC 8739 was purchased from the American Type Culture Collection (ATCC). Positive controls vancomycin, colistin, and ketoconazole were purchased from the standards supplier.

[0036] Example 1: Obtaining candidate short peptide sequences This embodiment is used to illustrate the design source of the short peptide sequence of the present invention, but the short peptide products of the present invention are not limited to obtaining them in this way.

[0037] The short peptide sequences described in this invention are obtained using an AI model generation method. For details on the acquisition and screening process, please refer to [link to relevant documentation]. Figure 1The process involves five steps: Step 1 is merging and cleaning the publicly available antimicrobial peptide sequence database; Step 2 is generating the peptide sequence generation model under certain conditions; Step 3 is cleaning, deduplicating, and screening candidate sequences based on their physicochemical properties; Step 4 is scoring the MIC prediction model; and Step 5 is selecting the sequences with the highest predicted activity. Specifically, referring to the model architecture and generation method disclosed in Chinese patent application CN120932734A (Peptide Sequence Generation Model and Generation Method Using Intermediate Sequence MSA and Diffusion Masking Mechanism), candidate peptide sequences are generated using the conditional sequence generation mode of the model, with active sequences from the organized publicly available antimicrobial peptide databases (APD6, DRAMP V4.0, dbAMP 2.0, DBAASP v3, YADAMP, DADP) as seeds.

[0038] The generated candidate sequences undergo quality control to remove sequences containing illegal amino acid characters, sequences that do not meet the 13-amino acid length requirement, or sequences that are repetitive or highly repetitive. Subsequently, candidate sequences are screened based on one or more of the following physicochemical properties: sequence length, net charge, hydrophobicity, isoelectric point, proportion of hydrophobic amino acids, number and position of cysteine ​​residues, likelihood of forming intramolecular disulfide bonds, and solubility or aggregation risk.

[0039] Candidate sequences selected based on physicochemical properties were input into a trained machine learning activity evaluation model (referring to the model training and scoring method described in CN120932734A) to obtain the predicted minimum inhibitory concentration (MIC) scores for each sequence against representative Gram-positive bacteria, Gram-negative bacteria, and fungi. Sequences were then ranked comprehensively based on predicted activity, physicochemical properties, sequence novelty, and feasibility of chemical synthesis, ultimately selecting sequence P1 (SEQ ID NO:1) for chemical synthesis and experimental verification. The sequences of this short peptide and its disulfide cyclization are shown in Table 1. Figure 2 This is a schematic diagram of the structure of the disulfide cyclization P1-Cyc of this short peptide.

[0040] Table 1. Sequence information of short peptides and their disulfide cyclizations

[0041] Example 2: Solid-phase synthesis of linear short peptides The linear short peptide RWRCWRMRGRRCW was prepared using the Fmoc solid-phase peptide synthesis method.

[0042] Weigh an appropriate amount of Fmoc-Cys(Trt)-Wang resin (degree of substitution 0.3–0.5 mmol / g) and place it in a peptide synthesis reactor. Add DMF to swell the resin for 30 minutes. Couple the Fmoc-protected amino acid monomers sequentially from the C-terminus to the N-terminus. Add 3 equivalents of Fmoc-amino acid, 3 equivalents of HBTU / HOBt, and 6 equivalents of DIEA to each coupling reaction system, and react at room temperature for 45–60 minutes. After each round of coupling, remove the Fmoc protecting group with a 20% piperidine / DMF solution (react at room temperature for 20 minutes), then wash the resin 3–5 times alternately with DMF and DCM. Monitor the coupling efficiency using the ninhydrin colorimetric method; for incomplete coupling, perform a second coupling.

[0043] After full sequence assembly, the peptide was lysed from the resin using TFA lysis buffer containing 2.5% TIS and 2.5% H2O, simultaneously removing side-chain protecting groups. The reaction was carried out at room temperature for 2.5 hours. The resin was removed by filtration, and the filtrate was concentrated under reduced pressure. Pre-cooled diethyl ether was added to precipitate the crude peptide, and the precipitate was collected by centrifugation and dried at room temperature.

[0044] The crude product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) under the following conditions: C18 preparative column (10 μm, 250 mm × 21.2 mm); mobile phase A: 0.1% TFA / water; mobile phase B: 0.1% TFA / acetonitrile; gradient elution (B%: 5%–65%, 30 min); flow rate: 10 mL / min; detection wavelengths: 220 nm and 280 nm. The target peak fractions were collected, combined, and lyophilized to obtain the linear short peptide P1 with a free carboxylic acid terminus at the C-terminus.

[0045] Example 3: Preparation of disulfide bond cyclized short peptides The linear short peptide P1 prepared in Example 2 was dissolved in 20% DMSO / water solution, and the final peptide concentration was adjusted to 0.5–1.0 mg / mL. The pH was adjusted to 7.5–8.5 with 0.1 M Tris-HCl buffer. The reaction was allowed to proceed at room temperature for 24–48 hours, and intramolecular disulfide bond formation was promoted by air oxidation. The cyclization reaction process was monitored using analytical reversed-phase high-performance liquid chromatography.

[0046] After the reaction was completed, the reaction solution was diluted 5 times with 0.1% TFA / water to terminate the reaction and reduce the organic solvent content. The cyclized product was separated by preparative reversed-phase high-performance liquid chromatography under the same chromatographic conditions as in Example 2. The target peak fraction was collected, lyophilized, and the disulfide-bonded cyclized short peptide P1-Cyc was obtained.

[0047] Compared with the corresponding linear short peptide, the theoretical molecular weight decreases by about 2 Da after the formation of an intramolecular disulfide bond, and the measured molecular weight decreases by 2.31 Da, which is consistent with the theoretical expectation (Table 2).

[0048] Example 4: Identification of the purity and molecular weight of short peptides The linear short peptides and their disulfide cyclizations obtained in Examples 2 and 3 were identified in terms of purity and molecular weight, respectively.

[0049] Purity determination was performed using analytical reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic conditions were as follows: C18 analytical column (5 μm, 250 mm × 4.6 mm); mobile phase A: 0.1% TFA / water; mobile phase B: 0.1% TFA / acetonitrile; gradient elution (B%: 10%–70%, 25 min); flow rate: 1 mL / min; detection wavelength: 220 nm; injection volume: 20 μL. Purity was calculated using the peak area normalization method. The HPLC purity identification chromatograms of the linear short peptide P1 and its disulfide cyclized derivatives are shown below. Figure 3 and Figure 4 .

[0050] Molecular weight determination was performed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). The matrix was α-cyano-4-hydroxycinnamic acid (CHCA), and the instrument was operated in reflectance positive ion mode. The mass detection range was m / z 500–4000. The mass spectrometry molecular weight identification spectra of the linear short peptide P1 and its disulfide cyclized derivatives are shown below. Figure 5 and Figure 6 The relative deviations between the measured and theoretical mass spectrometry values ​​were all less than 0.05%, and the main signal peaks of each sample were all attributed to the expected molecular ion peaks ([M+H]). + Or [M+2H] 2+ The identification results are shown in Table 2.

[0051] Table 2. Summary Table of Short Peptide Sequence Information

[0052] The results showed that the measured molecular weight of the synthesized short peptide deviated from the theoretical value by less than 1 Da, and the HPLC purity was greater than 95%, which met the quality requirements for subsequent antimicrobial activity experiments.

[0053] Example 5: Determination of Minimum Inhibitory Concentration (MIC) The minimum inhibitory concentration (MIC) of each short peptide was determined using the broth microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI).

[0054] 5.1 Experimental Materials 5.1.1 Test strains Acinetobacter baumannii ATCC 19606; Candida albicans ATCC 10231; Staphylococcus aureus ATCC 6538; Pseudomonas aeruginosa ATCC 27853; Escherichia coli ATCC 8739.

[0055] 5.1.2 Positive control drug Vancomycin is used to treat Staphylococcus aureus. Colistin is used to treat Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. Ketoconazole is used to treat Candida albicans.

[0056] 5.1.3 Culture medium MH broth (Mueller-Hinton Broth): used for bacterial culture; RPMI 1640 medium: for Candida albicans culture.

[0057] 5.2 Experimental Methods 5.2.1 Preparation of bacterial suspension After activating each tested bacterial strain by streaking it onto the corresponding solid culture medium, a single colony was picked and inoculated into liquid culture medium. The culture was then incubated until the logarithmic growth phase, and the bacterial suspension concentration was adjusted to 1×10⁻⁶. 6 CFU / mL, for later use.

[0058] 5.2.2 Sample Dilution Take an appropriate amount of each short peptide sample, dissolve it in sterile water to prepare a stock solution, filter it to remove bacteria, and then dilute it with culture medium to the set concentration.

[0059] 5.2.3 Sample addition and incubation Take a 96-well plate and add 50 μL of bacterial suspension and an equal volume of sample solution of different concentrations to each well, so that the final volume of each well is 100 μL. The final sample concentration ranges from 0.78 to 200 μg / mL. Also include control wells without sample and blank wells without culture medium. Add vancomycin, colistin, or ketoconazole to the positive control wells, respectively.

[0060] Incubate the 96-well plate at 37°C (bacteria) or 35°C (Candida albicans) for 18–24 hours (bacteria) or 48 hours (fungi).

[0061] 5.2.4 Result Judgment The lowest sample concentration at which no visible microbial growth can be observed is the minimum inhibitory concentration (MIC).

[0062] 5.3 Experimental Results The MIC determination results of each short peptide against the five tested microorganisms are shown in Table 3.

[0063] Table 3. Minimum inhibitory concentration (MIC) of each short peptide against the tested microorganisms (unit: μg / mL)

[0064] Note: "—" indicates that it was not measured.

[0065] As shown in Table 3: (1) The linear form and / or disulfide bond cyclized form of the short peptide of the present invention have inhibitory activity against at least one selected from Acinetobacter baumannii, Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.

[0066] (2) The MIC values ​​of the disulfide bond cyclized form are generally lower than those of the corresponding linear form, indicating that cyclization can enhance the antimicrobial activity of some peptide-strain combinations.

[0067] (3) P1-Cyc showed certain inhibitory activity against all five tested microorganisms, with MIC values ​​of 6.25 to 25 μg / mL. The MIC against Staphylococcus aureus ATCC 6538 was 6.25 μg / mL, the MIC against Acinetobacter baumannii ATCC 19606 and Pseudomonas aeruginosa ATCC 27853 was 25 μg / mL, the MIC against Candida albicans ATCC 10231 was 25 μg / mL, and the MIC against Escherichia coli ATCC 8739 was 50 μg / mL.

[0068] Example 6: Effects of short peptides on HaCaT cell viability This embodiment is used to evaluate the cytotoxicity of the short peptide P1 and its disulfide cyclization P1-Cyc of the present invention on human immortalized keratinocytes HaCaT.

[0069] 6.1 Experimental Materials HaCaT cell line was purchased from ATCC; high glucose DMEM medium, fetal bovine serum, and penicillin-streptomycin mixture were purchased from Gibco; CCK-8 assay kit was purchased from Dojindo.

[0070] 6.2 Cell Culture HaCaT cells were cultured in high-glucose DMEM complete medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin mixture at 37°C, 5% CO2 and saturated humidity.

[0071] When the cells reached 80%–90% confluence, they were digested with 0.25% trypsin-EDTA to prepare a cell suspension. Five × 10⁶ cells were then placed in each well. 3 Cells were seeded at a density of 100 μL in each well of a 96-well cell culture plate, and incubated overnight at 37°C and 5% CO2 to allow the cells to adhere fully.

[0072] 6.3 Short peptide treatment P1 and P1-Cyc were dissolved in sterile water to prepare stock solutions, which were then diluted to the required concentrations using complete culture medium.

[0073] After cell adhesion, the original culture medium was aspirated, and 100 μL of complete culture medium containing the corresponding short peptide was added to each well, resulting in final peptide concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg / mL. Wells without added short peptides served as negative controls (cell viability set at 100%), and wells containing only culture medium without cells served as blank controls. Three replicates were set up for each concentration.

[0074] The culture plate was incubated at 37°C and 5% CO2 for 24 hours.

[0075] 6.4 Cell viability assay After short peptide treatment, 10 μL of CCK-8 assay reagent was added to each well, and the mixture was incubated at 37°C in the dark for 2 hours. The absorbance at 450 nm (A450) was measured using a microplate reader.

[0076] Relative cell viability is calculated using the following formula: In the formula: Absorbance of cell pores treated with short peptides; The absorbance of the cell pores without the addition of short peptides; The absorbance of the blank well containing only culture medium and detection reagents, and no cells.

[0077] 6.5 Experimental Results The results are as follows Figure 7 As shown in the figure, within the test concentration range of 6.25–200 μg / mL, the relative viability of HaCaT cells treated with P1 remained above 86%, and no significant concentration-dependent decrease in cell viability was observed. The relative viability of cells treated with P1-Cyc within the concentration range of 6.25–100 μg / mL was 90%–103%; when the concentration was increased to 200 μg / mL, the relative viability was approximately 78%.

[0078] The above results indicate that, under the test conditions of this embodiment, the short peptides and their disulfide cyclizations of the present invention did not show significant cytotoxicity against HaCaT cells within the main antimicrobial activity concentration range (MIC ≤ 50 μg / mL). P1-Cyc only showed a certain degree of cell viability reduction at the highest test concentration of 200 μg / mL (far higher than its MIC values ​​of 6.25–100 μg / mL for each tested strain).

[0079] Example 7: Evaluation of the hemolytic activity of short peptides on erythrocytes This embodiment is used to evaluate the in vitro blood compatibility of the short peptide P1 and its disulfide cyclization P1-Cyc of the present invention.

[0080] 7.1 Experimental Materials Fresh, healthy adult whole blood was purchased from the city's blood center; PBS buffer (pH 7.4) was prepared according to standard procedures; Triton X-100 was purchased from Sigma-Aldrich.

[0081] 7.2 Preparation of Red Blood Cell Suspension Collect fresh anticoagulated whole blood and centrifuge at 1000 rpm for 5 minutes, discarding the supernatant plasma. Add PBS buffer (pH 7.4) to the red blood cell pellet, mix gently, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Repeat the washing process 3 times until the supernatant is clear. Prepare a 2% (v / v) red blood cell suspension with PBS buffer for later use.

[0082] 7.3 Short peptide treatment P1 and P1-Cyc were dissolved separately in PBS buffer and serially diluted twofold with PBS. Each concentration of short peptide solution was mixed with an equal volume of 2% erythrocyte suspension to make the final concentrations of short peptides 3.125, 6.25, 12.5, 25, 50, 100 and 200 μg / mL.

[0083] PBS was used as a negative control instead of the short peptide solution (0 μg / mL), and Triton X-100 at a final concentration of 1% (v / v) was used as a positive control (complete hemolysis). Each group had 3 replicates.

[0084] 7.4 Hemolysis rate determination Each group of samples was incubated at 37°C for 30 minutes. After incubation, the samples were centrifuged at 1000 rpm for 5 minutes to allow unlysaturated red blood cells to settle. The supernatant was carefully transferred to a 96-well plate, and the absorbance at 540 nm (A540) was measured using a microplate reader.

[0085] The percentage of apparent hemolysis (%) is calculated using the following formula: In the formula: The absorbance of the supernatant from the short peptide treatment group or the 0 μg / mL negative control group at 540 nm; The absorbance of the supernatant from the 1% Triton X-100 fully hemolyzed group at 540 nm.

[0086] The "apparent hemolysis percentage" referred to in this invention includes hemoglobin release caused by short peptides, spontaneous hemolysis of red blood cells, and background of the detection system, and is not equivalent to the corrected hemolysis rate after deducting the background of the negative control.

[0087] 7.5 Experimental Results Experimental results are as follows Figure 8 As shown in the figure, within the test concentration range of 3.125–200 μg / mL, the hemolysis rate of both the P1 and P1-Cyc treatment groups was less than 5%, and the overall level of each concentration treatment group was close to that of the negative control.

Claims

1. An antimicrobial short peptide, characterized in that, The amino acid sequence of the short peptide is shown in SEQ ID NO:

1.

2. The short peptide according to claim 1, characterized in that, The short peptide is in a linear form, and both of its cysteine ​​residues exist as free thiol groups without forming intramolecular disulfide bonds.

3. The short peptide according to claim 2, characterized in that: In the short peptide shown in SEQ ID NO:1, an intramolecular disulfide bond is formed between the 4th and 12th cysteine ​​residues, and the disulfide bond connection mode is Cys 4 —S—S—Cys 12 .

4. An antimicrobial composition, characterized in that, The composition comprises an effective amount of the short peptide and / or its disulfide cyclization as described in any one of claims 1 to 3, and a pharmaceutically acceptable carrier, excipient, or diluent.

5. The composition according to claim 4, characterized in that, The composition further comprises one or more auxiliary ingredients selected from the following: penetration enhancers, stabilizers, antioxidants, solubilizers, preservatives, buffers, and thickeners.

6. The composition according to claim 4, characterized in that, The dosage form of the composition is selected from solutions, sprays, lyophilized powder injections, creams, gels, lotions, coating liquids, or solid dispersions.

7. The use of the short peptide and / or its disulfide cyclized derivative as described in any one of claims 1 to 3 in the preparation of antimicrobial agents.

8. The application according to claim 7, characterized in that, The microorganisms are selected from one or more of the following: Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, or Candida albicans.

9. The application according to claim 7, characterized in that, The preparation is a pharmaceutical preparation, a disinfectant preparation, a medical device coating agent, a food preservative, or a cosmetic additive.

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