Isopeptide bond-containing antibacterial peptide analogue and application thereof

Abstract

The invention belongs to the technical field of polypeptide related drugs, and discloses an isopeptide bond-containing antibacterial peptide analogue and application thereof, based on the structural characteristics of the conventional lysine-containing antibacterial peptide sequence VWRKWRRFWKR-NH2, the sequence contains two Lys residues which are easy to become restriction enzyme cutting sites of some specific proteases, so that the sequence is unstable in vivo and easy to hydrolyze. Based on the defect, an isopeptide bond is introduced into a sequence by changing a connection mode of two amino groups contained in a lysine residue at a specific site in the sequence, but the size, the sequence sequence, the charge and the molecular weight of the whole antibacterial peptide sequence are not changed, so that the method aims at improving the proteolysis resistance of the antibacterial peptide, and improving the proteolysis resistance of the antibacterial peptide. The toxicity and the hemolysis are reduced. The problem that the antibacterial peptide is easily subjected to enzymolysis is solved from the source, and the key bottleneck of oral medication is overcome, so that the problem of high cost in the production process of preparing an anti-enzymolysis antibacterial peptide preparation is relieved, and the clinical application of the antibacterial peptide is promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide-related drugs and relates to an antimicrobial peptide analog containing an isopeptide bond and its application. Background Art

[0002] The abuse and misuse of traditional antibiotics have led to the emergence of many multidrug-resistant strains, which seriously threaten human health. Therefore, finding new structural categories of antibiotics that kill pathogenic microorganisms with new mechanisms and are less likely to develop drug resistance has become one of the current research hotspots.

[0003] Antimicrobial peptides have a unique selective biofilm action mechanism, showing characteristics such as low toxicity and side effects on host cells, strong specificity, and low resistance to drug resistance, which has attracted widespread attention from researchers.

[0004] Traditional antimicrobial peptides are primarily composed of naturally occurring amino acids linked by peptide bonds. However, this structure can, in some cases, limit their stability and bioactivity. Antimicrobial peptides are easily degraded by blood-borne proteases, have rapid renal clearance, and exhibit a short circulation half-life, limiting their clinical application as antimicrobial agents. While oral administration offers advantages such as ease of use and low production costs, the ease of enzymatic degradation of antimicrobial peptides limits their use as oral medications.

[0005] To overcome these limitations, researchers have begun to explore chemical modifications and structural modifications of antimicrobial peptides to improve their antimicrobial properties, stability, and bioavailability. Among them, the introduction of isopeptide bonds (i.e., peptide bonds between non-natural amino acids) has become an effective strategy.

[0006] The introduction of isopeptide bonds, which can be achieved through chemical or biosynthesis, can alter the secondary and tertiary structures of antimicrobial peptides, thereby affecting their interaction with bacterial cell membranes and antimicrobial mechanisms. This structural modification not only helps enhance the antimicrobial peptide's selective bactericidal ability against specific bacterial species but also has the potential to reduce its toxicity to host cells.

[0007] The research and application of antimicrobial peptides still face several challenges. For example, ensuring the stability and bioavailability of antimicrobial peptides in vivo and precisely targeting specific bacterial species are crucial. Furthermore, with the emergence and increasing prevalence of bacterial resistance, the development of antimicrobial peptide analogs with novel antimicrobial mechanisms and broader antimicrobial activity is becoming increasingly important.

[0008] Currently, several antimicrobial peptide analogs containing isopeptide bonds have been reported and exhibit promising antimicrobial properties and bioactivity. However, these analogs still exhibit differences and limitations in terms of structure, synthesis methods, application scope, and antimicrobial mechanism. Therefore, continued research and development of antimicrobial peptide analogs containing isopeptide bonds with enhanced performance is of great significance. Summary of the Invention

[0009] In order to improve the stability of antimicrobial peptides in vivo, the present invention intends to be based on the structural characteristics of the previous lysine-containing antimicrobial peptide sequence VWRKWRRFWKR-NH2. This sequence is a modified peptide of the Chinese forest frog skin antimicrobial peptide. Its sequence contains two Lys residues, which are easy to become the enzymatic cleavage sites of certain specific proteases (such as trypsin, etc.). Therefore, it is unstable in the body and easily hydrolyzed. Based on this defect, the present invention introduces an isopeptide bond into the sequence by changing the connection mode of the two amino groups contained in the lysine residues at specific sites in the sequence, but the size, sequence order, charge, and molecular weight of the overall antimicrobial peptide sequence are not changed. The purpose of this method is to improve the antimicrobial peptide's ability to resist proteolysis and reduce toxicity and hemolysis. Solve the problem of easy enzymatic hydrolysis of antimicrobial peptides from the source, overcome the key bottleneck of oral medication, thereby alleviating the high cost problem in the production process of antimicrobial peptide preparations resistant to enzymatic hydrolysis, and promote the clinical application of antimicrobial peptides.

[0010] The present invention provides an antimicrobial peptide analog containing an isopeptide bond and its application, which is achieved through the following technical solutions:

[0011] An antimicrobial peptide analog containing an isopeptide bond, the structure of which is shown in B, C, or D:

[0012] Analogue B:

[0013]

[0014] Analog C:

[0015]

[0016] Analog D:

[0017]

[0018] The invention relates to an application of an antimicrobial peptide analog containing an isopeptide bond, which acts against Gram-positive bacteria, Gram-negative bacteria or fungi.

[0019] Furthermore, Gram-positive bacteria include drug-resistant Staphylococcus aureus, drug-resistant Staphylococcus epidermidis, and methyl-resistant Staphylococcus aureus.

[0020] Furthermore, Gram-negative bacteria include drug-resistant Escherichia coli, drug-resistant Acinetobacter baumannii, drug-resistant Pseudomonas aeruginosa, and drug-resistant Klebsiella pneumoniae.

[0021] Furthermore, the fungus includes Candida albicans. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1RT-HPLC was used to test the stability of a series of modified peptide analogs containing isopeptide bonds in a pancreatic enzyme environment. a represents analog B, b represents analog C, and c represents analog D.

[0023] Figure 2 It is a line graph of the hemolytic activity of antimicrobial peptide analogs B, C and D containing isopeptide bonds;

[0024] Figure 3 This is a line graph showing the cytotoxicity of antimicrobial peptide analogs B, C, and D containing isopeptide bonds to RAW264.7 cells;

[0025] Figure 4 The morphological effects of analogue C on MRSA (ATCC 43300) were observed by scanning electron microscopy. A is untreated, B is untreated (partial magnification), C is treated with 1xMIC modified peptide C, and D is treated with 1xMIC modified peptide C (partial magnification).

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The antimicrobial peptide containing isopeptide bonds in the present invention is artificially synthesized and has the advantages of small molecular weight, strong antimicrobial activity against multidrug-resistant bacteria, good anti-enzymatic activity, etc. It is easy to further develop and utilize and has broad application prospects. DETAILED DESCRIPTION

[0028] The present invention is described in detail below by specific examples, but the scope of protection of the present invention is not limited. Unless otherwise specified, the experimental methods adopted in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0029] Example 1

[0030] Previous studies have found that the sequence VWRKWRRFWKR-NH2 has good broad-spectrum antimicrobial activity, but its susceptibility to enzymatic hydrolysis limits its clinical application. Based on the structural characteristics of the known antimicrobial peptide VWRKWRRFWKR-NH2, which contains two lysine residues, antimicrobial peptide analogs containing a single lysine isopeptide bond or two lysine isopeptide bonds were designed. Antimicrobial peptide analogs are single compounds and cannot be sequenced. Based on the designed sequence, the full sequence was synthesized using solid-phase synthesis (Fmoc). The specific steps are as follows:

[0031] (1) Using Fmoc-Arg(pbf)-Wang Resin S = 0.33 mmol / g and the Fmoc / tbu process, amino acids were condensed sequentially from the C-terminus to the N-terminus (from right to left) according to the following peptide sequence using the method in Table 1 to form this peptide resin:

[0032] Table 1

[0033]

[0034] The following amino acids were coupled sequentially:

[0035] A-01Fmoc-Lys(Boc)-OH, A-02Fmoc-Trp(Boc)-OH, A-03Fmoc-Phe-OH, A-04Fmoc-Arg(Pbf)-OH, A-05Fmoc-Arg(Pbf)-OH, A-06Fmoc-Trp(Boc)-OH, A-07Fmoc-Lys(Boc)-OH, A-08Fmoc-Arg(Pbf)-OH, A-09Fmoc-Tr p(Boc)-OH, A-10Fmoc-Val-OH. The peptide resin was washed, transferred, and dried to constant weight for cleavage.

[0036] (2) Peptide resin cleavage: Preparation of cleavage reagent: Calculate the amount of cleavage reagent based on the volume of 1 g peptide resin to 10 mL ± 2 mL: TFA: H2O: EDT: TIS = 95:1:2:2. Place the required cleavage reagents H2O, TFA, EDT, and TIS in the cleavage reaction bottle in sequence. Control the temperature of the cleavage reagent at 0-10°C. Add the cleavage reagent to the peptide resin under stirring. After the system temperature stabilizes, control the temperature at 25-30°C and stir the reaction for 2.5 hours.

[0037] The lysate was filtered out and precipitated with 5 times the volume of icy ether. The precipitate was filtered out and washed three times with 3 times the volume of icy ether, and then dried under reduced pressure at room temperature to obtain a crude solid product.

[0038] (3) Purification and lyophilization: Grind the crude product into powder, prepare purified water, and slowly add the ground crude product while stirring. Simultaneously, add acetonitrile aqueous solution dropwise. After the crude product is added and completely dissolved, filter it with a 0.45 μm microporous filter membrane. Purify the crude product using a C-18 column packing preparative column, mobile phase: A 0.1% TFA / H2O, B 0.1% TFA / ACN. Separate and purify the product at room temperature using an appropriate gradient. Collect the target product, analyze, test, and classify. The impurity purity is required to be ≥95%. Collect the unqualified target product and separate and purify it again using an appropriate gradient. The qualified main peak is then freeze-dried under reduced pressure to obtain a powdered purified polypeptide.

[0039] The product was purified by high performance liquid chromatography and its molecular weight and purity were identified by ESI-MS, as shown in Table 2.

[0040] Table 2 Amino acid sequences and physicochemical properties of lysine isopeptide bond modified peptides

[0041]

[0042] Sequence A:

[0043]

[0044] The structure of analog B is as follows:

[0045]

[0046] The structure of analog C is as follows:

[0047]

[0048] The structure of analog D is as follows:

[0049]

[0050] Example 2 Antimicrobial activity of antimicrobial peptide analogs containing isopeptide bonds

[0051] The experimental strains were cultured overnight at 37°C in a shaking incubator and diluted to 2×10 5 CFU / mL bacterial suspension is ready for use;

[0052] The experimental groups were as follows: the first group was the antimicrobial peptide analogue and bacterial solution as the experimental group; the second group was the antimicrobial peptide analogue and LB culture medium; the third group was the bacterial solution and ultrapure water as the negative control group; the fourth group was ultrapure water and LB culture medium as the blank control group.

[0053] Gentamicin was used as an antibiotic control. The drug was dissolved in autoclaved ultrapure water and prepared using a half-dilution method to create a series of concentration gradients (1.56 mM to 100 mM). 50 mL of each solution was added to a 96-well plate. After mixing, the mixture was incubated overnight at 37°C. The OD value of each sample at 600 nm was measured using a microplate reader. The minimum inhibitory concentration (MIC) was defined as the antibiotic concentration that resulted in an inhibition rate ≥95%.

[0054] The antibacterial results are shown in Table 3.

[0055] Table 3 Antimicrobial activity of antimicrobial peptide analogs containing isopeptide bonds

[0056]

[0057] The results, shown in Table 3, show that antimicrobial peptide analogs containing isopeptide bonds exhibit strong antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as fungi, including a large number of clinically isolated pathogens. For example, analog C exhibited a minimum MIC of 3.13 mM against methicillin-resistant Staphylococcus aureus and a minimum MIC of 3.13 mM against clinically isolated multidrug-resistant Staphylococcus aureus, demonstrating that antimicrobial peptide analogs containing isopeptide bonds can inhibit the growth of Staphylococci at extremely low concentrations. In particular, analog C exhibited strong antimicrobial activity against Gram-negative, multidrug-resistant Acinetobacter baumannii and Escherichia coli, with MICs of 6.25 and 3.13 mM, respectively.

[0058] Example 3 Enzymatic stability

[0059] Analogs B, C, and D with strong antimicrobial activity were selected to investigate their stability in a pancreatic enzyme environment. A 0.2 mg / mL pancreatic enzyme solution and a 10 mM isopeptide-bonded antimicrobial peptide analog were prepared. 10 mL of the 10 mM antimicrobial peptide analog was added to 150 mL of the pancreatic enzyme solution and incubated in a 37°C incubator for 0 min, 30 min, and 180 min, respectively. After incubation, 320 μL each of 15% acetonitrile and TFA were added to the sample, mixed evenly, and centrifuged at 4°C, 4000 rpm / min, for 10 min. The supernatant was filtered through a membrane for later use. The supernatant was collected at a flow rate of 1 mL / min, with phase A containing 0.1% TFA and phase B containing acetonitrile containing 0.1% TFA. The data were collected using a C18 column (5 mm, 0.46 × 25 cm) for analysis.

[0060] like Figure 1 As shown, the antimicrobial peptide analogs containing isopeptide bonds degraded to varying degrees over time during incubation, and the degree of degradation was proportional to the time. The HPLC data show that analogs C and D exhibited a high resistance to enzymatic degradation by pancreatic enzymes, with virtually no degradation occurring. Analog B, on the other hand, experienced a small amount of degradation, with its characteristic peak area decreasing significantly over time. We can clearly see that the elution peak area at around 9 minutes decreased significantly over time. This indicates that lysine isopeptide bond modification is helpful in improving the stability of antimicrobial peptides in the presence of pancreatic enzymes. Analog D, which had lysine isopeptide bond modifications at two sites, exhibited high stability in the presence of pancreatic enzymes.

[0061] Example 4 Determination of Hemolytic Activity of Antimicrobial Peptide Analogs B, C, and D Containing Isopeptide Bonds

[0062] Fresh human blood was allowed to stand to retain blood cells and then diluted with 0.9% saline to a 2% red blood cell suspension (v / v). Antimicrobial peptide analogs containing isopeptide bonds were dissolved in 0.9% saline and diluted halfway to a final concentration of 200 mM to 12.5 mM. Equal volumes of 750 mL of the analog dilution were mixed with 750 mL of human red blood cells to form the experimental group. A negative control (750 mL of 0.9% saline) and a positive control (750 mL of 0.1% Triton X-100) were used. Each group was incubated in a 37°C water bath for 1 hour, removed, and centrifuged at 1500 rpm / min for 10 minutes. 200 mL of the supernatant from each sample was added to a 96-well plate, and the absorbance of each well at 414 nm was measured using a microplate reader according to the following formula:

[0063] Hemolytic activity = {(F n -F0) / (F t -F0)}×100%(F n is the absorbance of the experimental group, F t The absorbance of F0 was the absorbance of the positive control group, and the absorbance of F1 was the absorbance of the negative control group. The hemolytic activity was calculated.

[0064] The results are as follows Figure 2 As shown by Figure 2 It can be seen that the designed series of antimicrobial peptides will not cause hemolysis in human blood even at a high concentration of 200 μM.

[0065] Example 5: Detection of the cytotoxicity of antimicrobial peptide analogs B, C, and D containing isopeptide bonds on RAW264.7 cells using the CCK8 assay

[0066] RAW264.7 cells (density 1x10 5 / mL) were inoculated into 96-well plates, 100 μL per well, and cultured for 24 hours; a series of concentration gradients of antimicrobial peptide analogs containing isopeptide bonds (final concentration of 1.56 μM to 100 μM) prepared by half-dilution method were added to the 96-well plates, 5 μL per well, and cultured at 37°C and 5% CO2 for 24 hours; in a light-proof environment, 10 μL / well of CCK8 solution was added and cultured in the dark for 2 hours; the experiment was divided into three groups: blank group: culture medium without cells and CCK8 solution were added; experimental group: cells, culture medium, CCK8 solution and antimicrobial peptide analogs were added; negative control group: cells, culture medium, CCK8 solution were added; three parallel wells for each sample were used, and the OD value of each well at 450 nm was measured using an enzyme-labeled instrument. Calculate cell viability. The calculation formula is as follows: Cell viability = {(F n -F0) / (F t -F0)}×100%(F n is the absorbance of the experimental group, F0 is the absorbance of the blank group, F t is the absorbance of the negative control group). Figure 3 As shown in Figure 2, with the increase of the concentration of the designed series of antimicrobial peptides, the survival rate of RAW264.7 cells gradually decreased. Figure 3 It can be seen that when the concentration of the analogue is less than 50 μM, the cell survival rate can reach more than 80%.

[0067] Example 6 Observation of the effect of analogue C on methicillin-resistant Staphylococcus aureus using scanning electron microscopy

[0068] The bacterial strain MRSAATCC43300 in the logarithmic phase was incubated in a shaker at 37°C and diluted to 2×10 5 CFU / mL, set aside; prepare antimicrobial peptide analog C containing an isopeptide bond with a final concentration of 1×MIC; the experiment was divided into two groups, control group: 1 mL of bacterial solution was added to the EP tube; experimental group: 1 mL of bacterial solution and analog C were added to the EP tube and cultured for 1 hour; after the time was reached, the antimicrobial peptide analog was washed three times with ultrapure water to remove the antimicrobial peptide analog (4000 rpm, 10 minutes); pre-cooled 2.5% glutaraldehyde (pH 7.2-7.4) was added to the precipitate and resuspended, fixed in a 4°C refrigerator for 12 hours, and washed three times with ultrapure water to remove 2.5% glutaraldehyde; the bacteria were dehydrated step by step with ethanol: the ethanol concentrations were 30%, 50%, 70%, 80%, 90% and 100% respectively; the bacterial precipitate was resuspended with anhydrous ethanol, smeared on a glass slide, and dried at room temperature; vacuum gold was sprayed, and the precipitate was observed and photographed with a scanning electron microscope.

[0069] like Figure 4 As shown: In the control group, untreated bacterial cells were morphologically intact, growing well, and exhibiting a smooth, bright membrane surface. However, after treatment with the antimicrobial peptide, bacterial morphology changed significantly. Treatment with the analog at a concentration of 1× the MIC resulted in a roughened bacterial membrane surface, with some bacterial membranes collapsing. Bacterial cells ruptured and collapsed, with their contents leaking out. Scanning electron microscopy results indicate that the analog can kill bacteria by altering their morphology and rendering them inactive.

[0070] The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be all feasible embodiments of the present invention. For those skilled in the art, any obvious modifications made thereto without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. An antimicrobial peptide analog containing an isopeptide bond, characterized in that: The structure is shown in B or C or D: Sequence B: Sequence C: Sequence D:

2. An application of an antimicrobial peptide analog containing an isopeptide bond, characterized in that: Acts against Gram-positive bacteria, Gram-negative bacteria or fungi.

3. The use of the antimicrobial peptide analog containing an isopeptide bond according to claim 2, characterized in that: Gram-positive bacteria include drug-resistant Staphylococcus aureus, drug-resistant Staphylococcus epidermidis, and resistant Staphylococcus aureus.

4. The use of the antimicrobial peptide analog containing an isopeptide bond according to claim 2, characterized in that: Gram-negative bacteria include drug-resistant Escherichia coli, drug-resistant Acinetobacter baumannii, drug-resistant Pseudomonas aeruginosa, and drug-resistant Klebsiella pneumoniae.

5. The use of the antimicrobial peptide analog containing an isopeptide bond according to claim 2, characterized in that: Fungi include Candida albicans.

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