D-amino acid-based antibacterial peptide and application thereof

Abstract

The application discloses a D-type amino acid-based antibacterial peptide and application thereof, and has the amino acid sequence of w-r-r-w-r-r-w-w-r-k-r (dTrp-dArg-dArg-dTrp-dArg-dArg-dTrp-dTrp-dArg-dLys-dArg). The antibacterial peptide can be synthesized by Fmoc solid-phase chemical synthesis, has low synthesis difficulty, and has good in-vivo antibacterial activity. The antibacterial peptide has broad-spectrum killing activity on carbapenem-resistant pseudomonas aeruginosa, pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli, methicillin-resistant staphylococcus aureus and staphylococcus aureus, has low hemolytic toxicity, can reach more than half of the cell survival rate at a medium or low concentration, has almost no toxic effect on cells, has good stability, has strong operability, and is low in cost.

Description

Technical Field

[0001] This invention relates to an antimicrobial peptide based on D-amino acids and its applications, and relates to the field of polypeptides. Background Technology

[0002] Bacterial infections are a common public health problem. With the overuse of antibiotics, antibiotic resistance is constantly rising, and various multidrug-resistant bacteria have emerged, posing a serious threat to global public health. Data from the Antimicrobial Resistance Surveillance Network shows that Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae, among others, have shown varying degrees of resistance to common antibiotics, with some exhibiting resistance rates exceeding 80%. Therefore, there is an urgent need for novel antimicrobial drugs to alleviate the problem of antibiotic resistance.

[0003] Antimicrobial peptides (AMPs) are a class of short peptides found in all organisms. They are cationic and amphiphilic with diverse structures and are crucial components of the innate immune system in combating pathogen invasion, maintaining stability and high efficiency throughout biological evolution. Antimicrobial peptides are widely distributed in animals, plants, and microorganisms. Based on their structure, AMPs possess three main structures: α-helix, β-sheet, and extended linear chains. Most antimicrobial peptides exhibit good water solubility, are thermally stable (retaining high antimicrobial activity even after heating at 100°C for 10 minutes), and demonstrate good stability in solutions with varying pH and salt ions. Furthermore, some antimicrobial peptides resist protease degradation. Their mechanisms of action primarily involve membrane permeation and non-membrane permeation mechanisms, which allow for diverse target sites and reduce the likelihood of bacterial resistance. Therefore, AMPs show promising potential as novel antimicrobial drug candidates. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide an antimicrobial peptide based on D-amino acids. The present invention also provides applications of this antimicrobial peptide, which exhibits strong antimicrobial activity, low cost, and low hemolytic toxicity.

[0005] To achieve the above objectives, the technical solution of the present invention is: an antimicrobial peptide based on D-type amino acids, characterized in that the amino acid sequence is: wrrwrrwwrkr(dTrp-dArg-dArg-dTrp-dArg-dArg-dTrp-dTrp-dArg-dLys-dArg).

[0006] The structural formula is:

[0007] .

[0008] The application of the aforementioned D-amino acid-based antimicrobial peptide in the preparation of antimicrobial drugs.

[0009] Preferred strains include multidrug-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, and standard strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Staphylococcus aureus.

[0010] Preferred bacteria include carbapenem-resistant Pseudomonas aeruginosa, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Staphylococcus aureus.

[0011] The application of the aforementioned D-amino acid-based antimicrobial peptide as an antimicrobial substance in personal care products, food preservatives, antimicrobial agents for medical consumables, and cosmetics.

[0012] Compared to traditional L-type antimicrobial peptides, D-type antimicrobial peptides exhibit better resistance to enzymatic degradation and greater stability. D-type antimicrobial peptides have excellent targeting properties against bacterial biofilms, capable of disrupting existing biofilms and inhibiting the development of antibiotic resistance.

[0013] Antimicrobial peptides contain 4-6 positively charged amino acids, and a certain degree of hydrophobicity is essential for their activity. They have strong cell membrane penetration ability and significant antimicrobial activity.

[0014] This invention relates to a D-type antimicrobial peptide designed based on the natural antimicrobial peptide configuration. It exhibits good antimicrobial activity against a variety of common Gram-negative and Gram-positive bacteria. Safety evaluation experiments have demonstrated its high safety profile, solving the problems of easy drug resistance and insufficient safety of natural AMP, and thus showing great promise for clinical application.

[0015] Beneficial effects:

[0016] The antimicrobial peptide provided by this invention exhibits good antimicrobial activity against a variety of pathogenic bacteria. This antimicrobial peptide can be synthesized via the Fmoc solid-phase chemical method, which is easy to synthesize and provides good in vivo antimicrobial activity. In particular, this antimicrobial peptide has broad-spectrum killing activity against carbapenem-resistant Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Staphylococcus aureus. It exhibits low hemolytic toxicity, and at low to medium concentrations (2 μg / mL to 32 μg / mL), the cell survival rate can reach more than 50%. It has no cellular toxicity, good stability, high operability, and low cost. Attached Figure Description

[0017] Figure 1 This is the high-performance liquid chromatography of the antimicrobial peptide of the present invention.

[0018] Figure 2 This is the mass spectrometry of the antimicrobial peptide of the present invention.

[0019] Figure 3 This is a graph showing the results of an antimicrobial peptide safety experiment.

[0020] Figure 4 This is a graph showing the hemolytic toxicity results of the antimicrobial peptides.

[0021] Figure 5 This is a graph showing the results of the temperature tolerance evaluation of antimicrobial peptides.

[0022] Figure 6 The graph shows the results of the antimicrobial peptides' resistance to enzymatic hydrolysis. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments and accompanying drawings:

[0024] Example 1

[0025] Synthesis of antimicrobial peptides

[0026] The target product, Pep01, has the following sequence:

[0027] Pep01:wrrwrrwwrkr(dTrp-dArg-dArg-dTrp-dArg-dArg-dTrp-dTrp-dArg-dLys-dArg).

[0028] The structural formula is:

[0029]

[0030] Simultaneously, products Pep02-Pep08 were designed, with the following sequence:

[0031] Pep02: wrrwrrfkr(dTrp-dArg-dArg-dTrp-dArg-dArg-dPhe-dLys-dArg).

[0032] Pep03:frrwrrfkr(dPhe-dArg-dArg-dTrp-dArg-dArg-dPhe-dLys-dArg).

[0033] Pep04:wkrwrrwwrr(dTrp-dLys-dArg-dTrp-dArg-dArg-dTrp-dTrp-dArg-dArg).

[0034] Pep05: wrrfrrfkr(dTrp-dArg-dArg-dPhe-dArg-dArg-dPhe-dLys-dArg).

[0035] Pep06: wrrwkrfkr(dTrp-dArg-dArg-dTrp-dLys-dArg-dPhe-dLys-dArg).

[0036] Pep07: wrrwkrwwrr(dTrp-dArg-dArg-dTrp-dLys-dArg-dTrp-dTrp-dArg-dArg).

[0037] Pep08: wrrwrrawkr(dTrp-dArg-dArg-dTrp-dArg-dArg-dAla-dTrp-dLys-dArg).

[0038] The structural formulas are as follows:

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] .

[0046] Pep01-Pep08 correspond to sequences 1-8 in the sequence list, respectively.

[0047] Antimicrobial peptides were artificially synthesized using the solid-phase synthesis method for polypeptides.

[0048] The synthesized product was purified using a high-purity silica gel column (SHIMADZU Inertsil ODS-SP). Mobile phases A and B were 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution, respectively.

[0049] Synthesis method:

[0050] 1. Resin activation and pretreatment

[0051] Weigh 1g of dichloromethane resin (0.3mmol / g) and place it in a polypeptide solid-phase synthesizer. Shake and swell the resin in dichloromethane (DCM) for 30 minutes. Test the resin color using the ninhydrin colorimetric method. A colorless and transparent resin indicates that it is normal.

[0052] 2. Synthesis of peptide chains

[0053] The Fmoc protecting group on the resin was removed using a DMF solution containing 20% ​​piperidine. The resin color was tested using the ninhydrin colorimetric method; a blue-purple color indicated that the protecting group had been removed. Fmoc-Trp-OH (300 mg), HOBT (137 mg of 1-hydroxybenzotriazole), HBTU (271 mg of O-benzotriazole-tetramethylurea hexafluorophosphate), and DIEA (193 mg of diisopropylethylamine) were dissolved and mixed in 20 mL of DMF (N,N-dimethylformamide). This mixture was then added to the synthesizer and mixed with the deprotected dichloro resin. The condensation reaction was carried out for 1 hour. The resin was tested using the ninhydrin colorimetric method; a colorless and transparent color indicated successful condensation, yielding Fmoc-Trp-resin. The subsequent amino acid, Fmoc-Arg(Pbf)-OH, was condensed sequentially using the same method. Add 20 mL of 20% DMF and react for 30 min. The amounts of HOBT and HBTU are the same as above. The peptide chain obtained above was deprotected with a 1% TFA (trifluoroacetic acid) solution in DCM (dichloromethane). The ninhydrin colorimetric method was used to test the resin. The resin turned blue-purple, indicating that the protecting group had been removed.

[0054] 3. Peptide cleavage

[0055] The peptide chain obtained in the above steps was cleaved using a mixed solution containing 95% TFA, 2.5% Tis (p-toluenesulfonyl isocyanate) and 2.5% H2O as the cleavage reagent, extracted with ice-cold ether and water, and freeze-dried to obtain crude peptide lyophilized powder.

[0056] 4. Purification

[0057] The obtained crude peptide lyophilized powder was purified by separation using a high-purity silica gel column (SHIMADZU Inertsil ODS-SP), the eluent was collected, and then freeze-dried. The high-performance liquid chromatogram is shown below. Figure 1 During purification, mobile phases A and B were 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution, respectively. The synthesis of Pep02-Pep08 was carried out according to this method.

[0058] 5. Structural identification

[0059] The purified crude peptide lyophilized powder was dissolved in 50% acetonitrile solution, and its structure was identified by triple-quadrupole liquid chromatography-mass spectrometry (LC-MS / MS). The molecular weight was determined to be 1828.5. The mass spectrum is shown below. Figure 2 .

[0060] Experimental Example 1

[0061] Antimicrobial activity testing of minimum inhibitory concentration (MIC) of antimicrobial peptides.

[0062] Standard bacterial strains: Acinetobacter baumannii (ATCC19606), Pseudomonas aeruginosa (ATCC27853), Klebsiella pneumoniae (ATCC700603), Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC29213).

[0063] Drug-resistant strains: Multidrug-resistant Acinetobacter baumannii (MDRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and methicillin-resistant Staphylococcus aureus (MRSA). The activity testing method is briefly described below:

[0064] After removing the bacterial culture from the -80℃ freezer, the bacterial suspension was revived and streaked onto LB solid medium. The culture was incubated at 37℃ for 18 hours. Single colonies were then picked and incubated overnight at 37℃ in LB liquid medium. The culture was then diluted with physiological saline to a concentration of 1×10⁻⁶. 8 CFU / mL, then diluted to 1×10⁻⁶ with MHB medium. 5 CFU / mL. The MIC value of the antimicrobial peptide was determined using the microdilution method. 100 μL of an initial concentration of 256 μg / mL antimicrobial peptide solution was added to a 96-well plate, and then serially diluted (256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL). 100 μL of diluted bacterial culture in MHB medium was added to each well. To avoid edge effects, the outermost wells were used as blank wells. After incubation at 37℃ for 18–24 h, the absorbance of each well was measured at 600 nm using a microplate reader. The experiment was repeated three times.

[0065] Table 1. Antimicrobial activity of 8 antimicrobial peptides against common Gram-negative bacteria

[0066]

[0067] The results are shown in Table 1. Pep01 exhibited good antibacterial activity, especially against CRPA, ATCC 27853, and ATCC700603. It also showed strong antibacterial activity against other common Gram-negative bacteria and drug-resistant strains.

[0068] The antimicrobial activity of the four antimicrobial peptides against Gram-positive bacteria was tested using the same method as above.

[0069] Table 2 Antimicrobial activity of antimicrobial peptides against common Gram-positive bacteria

[0070]

[0071] The results are shown in Table 2. The antimicrobial peptide Pep01 has strong antimicrobial activity against both standard strains and drug-resistant strains of Staphylococcus aureus.

[0072] Experiment Example 2

[0073] Antimicrobial peptide safety testing

[0074] The cytotoxicity of the antimicrobial peptide Pep01 against H9C2 (rat cardiomyocytes), THLE-3 (human immortalized liver cells), Vero (African green monkey kidney cells), and NCM460 (normal human colonic epithelial cells) was evaluated using the CCK-8 assay. The experimental procedures are briefly described below:

[0075] 1. Cell resuscitation

[0076] H9C2, THLE-3, Vero, and NCM460 cell lines were removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath. H9C2, Vero, and THLE-3 cells were added to DMEM medium, centrifuged, and then seeded into cell culture flasks. NCM-460 cells were added to RPMI-1640 medium, centrifuged, and then seeded into cell culture flasks. Cells were cultured in a 37°C CO2 incubator until adherence. After adherence, when changing the medium, dead suspended cells were washed away with PBS before continuing culture.

[0077] 2. Cell passage

[0078] When the cells in the culture flask reach 90% confluence, passage the cells. Discard the culture medium, wash 2-3 times with PBS, add 2 mL of 0.25% trypsin-EDTA solution and digest for 2-3 minutes, observing the digestion process under a microscope. After digestion is complete, add 6 mL of culture medium to stop digestion, centrifuge and discard the supernatant. Add 2 mL of fresh culture medium to the precipitate, gently aspirate and mix, then divide into flasks and incubate at 37℃ in a CO2 incubator.

[0079] 3. Cell counting

[0080] Mix 10 μL of cell suspension with 10 μL of trypan blue solution, add the mixture to a hemocytometer, and count the cells under a microscope.

[0081] 4. CCK8 method for detection

[0082] Cells cultured to passage 3 were used for experiments. The old culture medium was discarded, the cells were washed with PBS, digested with trypsin, and then counted. The cell suspension density was adjusted to 4 × 10⁻⁶. 4 Cells / mL. The adjusted cell suspension was evenly spread into 90 μL per well of a 96-well plate. To avoid edge effects, the outermost well of each 96-well plate was blocked with 100 μL of PBS solution and incubated overnight at 37°C in a CO2 incubator. After incubation, 10 μL of different concentrations of antimicrobial peptide solution (256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL) was added to each well. Three replicates were set up for each group, with each replicate containing one blank control and one negative control. The blank control contained only culture medium and no cells, while the negative control contained only cells and culture medium. The 96-well plates were incubated for another 10 h. After incubation, 10 µL of CCK-8 solution was added to each well, and the plates were incubated for 2 h. The OD values ​​were then measured using a microplate reader. 450 Value. The formula for calculating cell viability is:

[0083]

[0084] Experimental results showed that the antimicrobial peptide Pep01 maintained a cell survival rate of over 50% at low to medium concentrations (2 μg / mL to 32 μg / mL) and exhibited almost no cellular toxicity. Safety assays demonstrated that the antimicrobial peptide had good safety profiles and no significant toxicity. Pep01 maintained a cell viability of over 60% for H9C2 cell lines within the concentration range of 2 μg / mL to 128 μg / mL, but this dropped sharply to below 20% at 256 μg / mL. For NCM460 cell lines, the cell viability remained above 80% within the 256 μg / mL range. For THLE-3 cell lines, the viability remained around 90% within the concentration range of 2 μg / mL to 32 μg / mL, but dropped sharply to below 50% at 64 μg / mL, and almost no cell survival was observed at 128 μg / mL and 256 μg / mL. For Vero cell lines, the cell viability remained above 50% within the concentration range of 2 μg / mL to 64 μg / mL, but dropped sharply to less than 10% at 128 μg / mL and 256 μg / mL. Therefore, Pep01 is non-toxic to cells at concentrations of 2 μg / mL to 32 μg / mL, but exhibits significant cytotoxicity at concentrations of 64 μg / mL to 256 μg / mL. Pep04 maintained cell viability above 80% for all four cell lines within a concentration range of 2 μg / mL to 32 μg / mL, around 50% at 64 μg / mL, and dropped sharply to below 20% at 128 μg / mL and 256 μg / mL. Pep06 maintained cell viability above 90% for H9C2, THLE-3, and Vero cell lines within a concentration range of 2 μg / mL to 256 μg / mL. Cell viability was slightly lower for the NCM460 cell line, but still maintained around 80%, only dropping to 70% at 256 μg / mL. Pep07 maintained cell viability of over 80% for H9C2 cell lines within a concentration range of 2 μg / mL to 256 μg / mL; for NCM460 cell lines, cell viability remained between 50% and 80% within a concentration range of 2 μg / mL to 128 μg / mL, decreasing to 40% at 256 μg / mL; for THLE-3 cell lines, cell viability remained above 70% within a concentration range of 2 μg / mL to 128 μg / mL, dropping sharply to less than 10% at 256 μg / mL; and for Vero cell lines, cell viability remained above 70% within a concentration range of 2 μg / mL to 128 μg / mL, decreasing to below 40% at 256 μg / mL. The experimental results show that the cell survival rate of all four antimicrobial peptides remained above 50% at low to medium concentrations (2 μg / mL to 32 μg / mL), exhibiting low to almost no toxicity. However, toxicity increased at medium to high concentrations (128 μg / mL to 256 μg / mL), particularly with Pep01 and Pep04, which showed stronger cytotoxicity. See attached figure for experimental results. Figure 3 .

[0085] Experimental Example 3

[0086] Take an appropriate amount of fresh sterile sheep blood and centrifuge it at 4℃, 3000 rpm / min for 5 min in a low-temperature centrifuge. Discard the supernatant, resuspend in PBS, and centrifuge again under the same conditions. Wash three times until the supernatant is colorless and discard the supernatant. Take 0.1 mL of hematocrited red blood cells and add 4.9 mL of PBS to prepare a 2% red blood cell suspension for later use. Dilute the initial concentration of 256 μg / mL to a final concentration of 1 μg / mL using a serial dilution method. Add 800 µL of the diluted antimicrobial peptide solution to each tube, followed by 200 µL of the 2% red blood cell suspension, and incubate in a 37℃ biochemical incubator for 1 h. After incubation, centrifuge at 4℃, 3000 rpm / min for 5 min, and aspirate 100 µL of the supernatant into a 96-well plate to measure the OD540 value. Calculate the hemolysis rate of the antimicrobial peptide using the following formula. In the experiment, the negative control group was an equal volume of PBS solution, and the positive control was an equal volume of 0.1% Triton-X100 solution. This experiment was repeated three times.

[0087]

[0088] Using a 100% hemolysis rate as the positive control (Triton-X100) and a 0% hemolysis rate as the negative control (PBS), the hemolysis rates of the four antimicrobial peptides were calculated. The results showed that Pep07 exhibited hemolytic toxicity close to 2% in the concentration range of 1 μg / mL to 64 μg / mL, but decreased to below 0.5% at 128 μg / mL. Pep01, Pep04, and Pep06 all showed hemolytic toxicity below 1% in the concentration range of 1 μg / mL to 128 μg / mL. Based on the experimental data, none of the four antimicrobial peptides showed significant hemolytic toxicity. The experimental results are attached. Figure 4 .

[0089] Experiment Example 4

[0090] Temperature tolerance evaluation

[0091] Antimicrobial peptide solutions with a concentration of 256 µg / mL were placed in water baths at different temperatures (0°C ice-water mixture, 37°C, and 100°C) for 30 min. The MIC of the treated antimicrobial peptides against MRSA was determined according to the method described in Experimental Example 1. The control group consisted of untreated antimicrobial peptides. This experiment was repeated three times.

[0092] Temperature tolerance is one of the important indicators for evaluating the stability of antimicrobial peptides. Experimental results showed that the antimicrobial efficacy of Pep01 increased in a gradient from 4 μg / mL to 8 μg / mL at low temperature (0℃) and high temperature (100℃); the antimicrobial activity of Pep06 increased from 2 μg / mL to 4 μg / mL at 0℃. Overall, however, all four antimicrobial peptides showed good stability at different temperatures. The experimental results are attached. Figure 5 .

[0093] Experimental Example 5

[0094] Evaluation of resistance to enzymatic hydrolysis

[0095] 50 μL of an antimicrobial peptide solution with an initial concentration of 512 µg / mL was mixed with 50 μL of different protease solutions (trypsin, pepsin, and proteinase K) at a concentration of 1 mg / mL and reacted in a 37°C water bath for 1 h. After the reaction, the proteases were inactivated in a 60°C water bath, and the MIC was determined according to the method described in Experiment Example 1. The control group was the untreated antimicrobial peptide, and the negative control group was bacterial culture containing different protease solutions. This experiment was repeated three times.

[0096] During the metabolism and absorption of drugs in the body, they are degraded by different enzymes depending on the metabolic site, such as pepsin and trypsin. Therefore, resistance to enzymatic degradation is another important reference indicator for evaluating the in vivo stability of antimicrobial peptides and their potential as drug formulations. Figure 6 The experimental results showed that Pep01, Pep06, and Pep07 exhibited good resistance to enzymatic digestion, with no change in MIC values ​​compared to the control group. Pep04 showed a 2-fold increase in MIC values ​​for trypsin and proteinase K compared to the control group, but no change in MIC values ​​for pepsin. Overall, the four antimicrobial peptides demonstrated good resistance to enzymatic digestion. See the attached table for the experimental results. Figure 6 .

[0097] This invention is not limited to the above embodiments. Those skilled in the art will understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this invention. The scope of this invention is defined by the claims and their equivalents.

Claims

1. An antimicrobial peptide based on D-amino acids, characterized in that, The amino acid sequence is: wrrwrrwwrkr; the structural formula is: 。 2. The application of the antimicrobial peptide based on D-amino acids as described in claim 1 in the preparation of antimicrobial drugs, wherein the bacteria is *Pseudomonas aeruginosa* (…). Pseudomonas aeruginosa Klebsiella pneumoniae ( Klebsiella pneumoniae ), Escherichia coli ( Escherichia coli Staphylococcus aureus ( Staphylococcus aureus ).

3. The application of the antimicrobial peptide based on D-amino acids as described in claim 1 in the preparation of antimicrobial drugs, wherein the bacteria are carbapenem-resistant Pseudomonas aeruginosa (… Carbapenem-resistant Pseudomonas aeruginosa Methicillin-resistant Staphylococcus aureus (MRSA) Methicillin-resistant Staphylococcus aureus ).

4. The application of the antimicrobial peptide based on D-amino acids as described in claim 1 in the preparation of personal care products, food preservatives, antimicrobial agents for medical consumables, and cosmetics.

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