Preparation of a PEG-Modified Antimicrobial Peptide and Its Application in Bacterial Infections

Abstract

The present invention provides a preparation of PEG-modified antimicrobial peptides and their application in bacterial infections, belonging to the technical field of biomedical polypeptides, and specifically relates to the preparation of PEG-modified antimicrobial peptides and their application in preparations for preventing or treating diseases related to bacterial infections. The PEG-modified polypeptides designed in the present invention are easy to operate, have low production costs, strong antibacterial activity, and can greatly optimize the toxicity and stability of antimicrobial peptides. They can be used for the prevention and treatment of diseases caused by various microbial infections, and can be administered through various routes such as spraying, intraperitoneal injection, intravenous injection, intranasal administration, and aerosol administration, and have good effects on the treatment of diseases such as skin wound infections, sepsis, and pneumonia caused by bacterial infections.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical polypeptides, and specifically relates to an antimicrobial peptide obtained by a PEG modification method for the prevention and treatment of diseases caused by various microbial infections, and the application of the antimicrobial peptide in the treatment of skin wound infections, sepsis, pneumonia caused by bacterial infections, and the like. Background Art

[0002] Bacterial infection seriously threatens global public health security and is one of the main causes of death. Antibiotics, as the most effective method for treating bacterial infections, face the problems of easy drug resistance and high toxicity. The development of safe and efficient antibacterial molecules is imminent. Antimicrobial peptides have attracted widespread attention in recent years due to their potential therapeutic effects. Compared with traditional antibiotics, antimicrobial peptides are not easy to develop drug resistance, low toxicity, biodiversity and direct aggressiveness, and are considered to be the most promising new generation of antibacterial drugs in the post-antibiotic era. However, there are also problems such as high preparation cost, weak antibacterial activity and strong cytotoxicity. PEG is a linear polymer with good solubility in water, good biocompatibility and low toxicity. PEG modification can enhance the solubility of peptides, reduce toxicity and enhance stability, but the introduction of high molecular weight PEG will reduce activity. Therefore, the use of PEG with a suitable molecular weight and PEG modification at a suitable site of the peptide is of great practical significance for improving its drugability.

[0003] Based on this, the modified body 1 (named Lydp-1) in the embodiment of our previous patent application was used as a template for PEG modification, which contains 13 amino acid residues, and its amino acid sequence is: WDapAMDapIDipIDapDipIKDap. Lydp-1 has strong broad-spectrum antibacterial activity, and its MIC for Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Streptococcus mutans is 4 μg / mL, and 100 μM Lydp-1 has almost no hemolytic activity on red blood cells. However, Lydp-1 has certain toxicity to normal cells and is not very stable. It is necessary to modify and transform it to enhance its potential in the development of antibacterial drugs. Based on this, the patent of this invention introduces PEG modification to maintain the antibacterial activity of the polypeptide while greatly optimizing the polypeptide, such as greatly reducing mammalian cell toxicity, increasing stability, etc., and keeping its production and preparation costs low. In view of the difficulties of existing PEG modification of peptides, such as affecting the antibacterial activity of peptides and complex modification strategies, we used non-natural amino acids or natural amino acids to modify the main chain of peptide mutants based on Lydp-1. N -Terminals are modified with PEG of different molecular weights, and these antimicrobial peptides are expected to be used to develop a promising drug for the treatment of bacterial infections. Summary of the invention

[0004] The purpose of the present invention is to provide a method for improving the toxicity and stability of antimicrobial peptides by PEG modification without affecting their antimicrobial activity, and the use of polypeptides with antimicrobial effects in treating bacterial infections.

[0005] The above invention object is achieved through the following technical scheme: Based on the polypeptide Lydp-1 with strong antibacterial activity, a series of site-directed mutagenesis is performed on the main chain of the mutant N -Terminals are modified with PEG of different molecular weights to design antimicrobial peptides, which are prepared by chemical synthesis for antibacterial research. The obtained PEG-modified peptides have simple structures, are easy to control in quality, can be efficiently prepared by chemical synthesis, and more importantly, have strong antibacterial activity, extremely weak toxicity, and high stability. They can be administered through various routes and used to treat skin trauma infections, sepsis, pneumonia caused by bacterial infections, and other diseases caused by various bacterial infections.

[0006] The polypeptide provided by the present invention is based on the amino acid sequence of the template peptide Lydp-1, by replacing the amino acid at a specific position, and the amino acid sequence is: WDapY 1 Y 2 DapY 3 DipY 4 DapDipY 5 Y 6 Dap, where Y 1 -Y 6 Each independently selected from Dap, Dab, Orn or Lys, based on these mutants, in the main chain of the mutant N -Terminals were modified with PEG of different molecular weights.

[0007] In some embodiments, the Y 1 and Y 6 Each independently selected from Dap, Dab, Orn or Lys is replaced in the polypeptide main chain N -Derivatives obtained by carrying out PEG modification of different molecular weights at the end.

[0008] In some embodiments, the Y 2 After substitution with Dap, Dab, Orn or Lys, the main chain of the polypeptide N -Derivatives obtained by carrying out PEG modification of different molecular weights at the end.

[0009] In some embodiments, the Y 3 After substitution with Dap, Dab, Orn or Lys, the main chain of the polypeptide N -Derivatives obtained by carrying out PEG modification of different molecular weights at the end.

[0010] In some embodiments, the Y 4After substitution with Dap, Dab, Orn or Lys, the main chain of the polypeptide N -Derivatives obtained by carrying out PEG modification of different molecular weights at the end.

[0011] In some embodiments, the Y 5 The derivatives are selected from the group consisting of Dap, Dab, Orn or Lys, and then PEG-modified with different molecular weights at the site.

[0012] In some embodiments, the one PEG-modified polypeptide design composition includes any combination of all the PEG-modified polypeptides.

[0013] In one aspect, the present invention relates to a PEG-modified polypeptide composition, which comprises any of the polypeptides described above. In another aspect, the present invention relates to a PEG-modified antimicrobial peptide and its use in preparing a preparation for preventing or treating bacterial infection-related diseases.

[0014] In some embodiments, the bacteria include but are not limited to Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Streptococcus mutans. The bacterial infection includes but is not limited to skin wound infection, sepsis, pneumonia caused by bacterial infection and other diseases.

[0015] The beneficial effects of the present invention are as follows: different from most other PEG-modified antimicrobial peptides, the polypeptide PEG modification of the present invention is simple to operate, has a short sequence, and has the advantage of greatly reducing production costs. It can kill bacteria with a relatively broad spectrum, has a large diameter of the inhibition zone, and can effectively solve the defects of poor stability and high toxicity of existing antimicrobial peptides. More importantly, it has no effect on the antimicrobial activity of antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Reverse phase high performance liquid chromatography (RP-HPLC) and mass spectra of template peptide Lydp-1 and modified form 1.

[0017] Figure 2 :MIC of template peptide Lydp-1 and modified peptide 1 against bacteria.

[0018] Figure 3 :Analysis of the hemolytic activity of template peptide Lydp-1 and modified form 1 on mouse erythrocytes.

[0019] Figure 4 :Analysis of cytotoxic activity of template peptide Lydp-1 and modified form 1 on normal cells.

[0020] Figure 5 : Plasma stability analysis of template peptide Lydp-1 and modified form 1. DETAILED DESCRIPTION

[0021] The PEG modified polypeptides disclosed in the present invention can exist in the form of their hydrates or in the form of solvents (e.g., ethanol, DMSO, etc.) and can be used for crystallization. The compounds disclosed in the present invention can form solvates with pharmaceutically acceptable solvents (including water) inherently or by design; therefore, the compounds of the present invention include solvated and unsolvated forms.

[0022] For amino acid replacement of the polypeptide of the present invention, non-natural amino acids can be used to replace the natural amino acids in the polypeptide, and non-natural amino acids include but are not limited to 2,3-diaminopropionic acid (Dap), 3,3-diphenylalanine (Dip), 2,4-diaminobutyric acid (Dab), and 2,5-diaminopentanoic acid (Orn).

[0023] The term "PEG" refers to polyethylene glycol, which is a high molecular weight polymer, and is the general term for ethylene glycol polymers containing α, ω-dihydroxyl groups, and its chemical formula is HO(CH2CH2O)nH. The PEG used in the present invention includes but is not limited to PEG molecules of any molecular weight such as miniPEG (molecular weight of 163 Da, molecular structure as shown in Example 1).

[0024] The term "natural amino acid" refers to any of the 20 L-amino acids commonly found in proteins and peptides found in nature, i.e., the L-isomers of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0025] Example 1.

[0026] Preparation of Example 1 (modified body 1).

[0027] Transformation 1 is a miniPEG modified with an amino group on the N-terminal main chain of the template peptide Lydp-1, and its chemical structure is as follows:

[0028]

[0029] The above-mentioned modified body was efficiently prepared by peptide solid phase chemical synthesis, and the antibacterial properties of modified body 1 were characterized by testing the minimum inhibitory concentration (MIC) against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Streptococcus mutans, the hemolytic activity against mouse erythrocytes, the toxicity to normal cells such as L929 and the plasma stability.

[0030] 1. Solid phase peptide chemical synthesis.

[0031] The present invention adopts Fmoc solid phase peptide synthesis method to synthesize linear peptides, that is, Rink resin is used as a carrier during synthesis, and the peptides are synthesized in order from C-terminus to N-terminus, and Fmoc (9-fluorenylmethoxycarbonyl) is used as the amino protecting group of amino acids to synthesize peptides. To carry out PEGylation modification, the PEG modification of the polypeptide main chain is the same as the above-mentioned amino acid steps. After the coupling is completed, the newly synthesized polypeptide is cleaved from the Rink resin with a cleavage solution, and then precipitated with ice ether and identified by mass spectrometry. According to the scale of 0.1 mmol polypeptide synthesis, the specific steps are as follows:

[0032] (1) Expansion of resin.

[0033] Weigh 0.1 mmol of Rink resin into a synthesis tube, add 3 mL of DMF, swell for 1 h and filter out the DMF in the tube.

[0034] (2) Deprotection of the resin. After the resin was expanded, 3 mL of 20% piperidine was added and deprotected on a rotator for 7 min. The piperidine was then removed and 3 mL of 20% piperidine was added to the rotator for a second deprotection for 8 min. After deprotection, the piperidine was removed and washed with DMF 8 times.

[0035] (3) Activation of amino acids.

[0036] While the first deprotection is being performed, the amino acid is activated. Take one portion of HATU and one portion of HOBT, add 0.75 mL of N-methylmorpholine to each, mix the two, and then add them to the pre-weighed amino acid. Mix on a rotary mixer. The activation time is 15-20 min.

[0037] (4) Coupling of amino acids.

[0038] Add the activated amino acid to the resin in step (2), shake to mix the resin and amino acid solution thoroughly, and carry out coupling reaction on a rotator for 1 hour.

[0039] (5) Extension of the peptide chain.

[0040] Repeat steps (2)-(4) until the last amino acid coupling reaction is completed.

[0041] (6) Cleavage of peptide chains.

[0042] After the last amino acid coupling reaction is completed, use 20% piperidine to deprotect, then wash with DMF 8 times, and wash with anhydrous methanol 4-5 times. After washing, drain and add 6 mL of lysate. React in a rotary mixer for 2 h. Collect the lysate in a 50 mL centrifuge tube, add ice ether to the lysate for precipitation twice, and the lower precipitate obtained is the crude polypeptide. The crude polypeptide after precipitation is placed in a fume hood for 2-3 minutes to volatilize the ether contained in the tube, and then seal and store at 4 °C.

[0043] 2. Isolation and purification of modified body 1 ( Figure 1 ).

[0044] The cleaved peptide crude product was dissolved in an appropriate amount of ultrapure water, centrifuged at 10,000 rpm for 10 min, and filtered with a 0.22 μm filter. RP-HPLC purification was performed on a semi-preparative reversed-phase high-performance liquid chromatography (Hanbang) using a C18 reversed-phase column (10 mm × 250 mm, 5 μm, Yuexu), with a detection wavelength of 215 nm or 280 nm, a column temperature of 25 ± 5 °C, and a phase A (H 2 O +0.1% TFA), phase B (ACN + 0.1% TFA). The concentration of phase B varied from 10% to 54%, the elution gradient was 2% / min, the elution rate was 3 mL / min, and the collected target peaks were freeze-dried.

[0045] 3. Determination of molecular weight of modified body 1 ( Figure 1 ).

[0046] The molecular weight of peptides was determined using an AB SCIEX-TOF / TOFTM 5800 mass spectrometer produced by ABI, USA, using MALDI-TOF for detection. 1 μL of peptide sample was spotted on the sample plate, dried at room temperature, and the operation was repeated 3-4 times. Then 1 μL of CCA saturated solution was spotted to cover the sample and the molecular weight was determined.

[0047] 4. Activity determination of transformant 1.

[0048] (1) Minimum inhibitory concentration (MIC) determination Figure 2 ).

[0049] The MIC of the peptide was determined by broth microdilution method. The bacteria tested included Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Streptococcus mutans. When the bacteria grew to the logarithmic phase in liquid culture medium, the bacterial solution was diluted to 1×10 7CFU / mL, add 1 μL of diluted bacterial solution to a 96-well plate, and dilute the peptide in the culture medium until the final concentration is 1-128 μg / mL. Add 99 μL of each concentration of peptide dilution solution to a 96-well plate, and place the 96-well plate in a 37 °C anaerobic incubator for 18-24 h. Use a microplate reader (BioTek, USA) to detect the absorbance of the above 96-well plate at 600 nm. Calculate the inhibition rate of the peptide on bacteria based on the absorbance OD 600.

[0050] (2) Hemolytic activity assay ( Figure 3 ).

[0051] Fresh mouse erythrocytes were extracted and diluted 25 times with PBS buffer (pH=7.4) to prepare about 4% mouse erythrocyte suspension. Different concentrations of peptide solutions were prepared with PBS solution and placed in a 1.5 mL sterile centrifuge tube with a volume of 200 μL. In this tube, 200 μL of the above mouse erythrocyte suspension was added. After incubation at 37 °C for 1 h and centrifugation at 3500 rpm for 5 minutes, 100 μL of supernatant was collected on a 96-well plate and the absorbance at 490 nm was measured with an enzyme reader. Untreated erythrocyte suspension and 0.1% Triton X-100-treated erythrocyte suspension in equal volumes of PBS solution were used as negative and positive controls, respectively.

[0052] (3) Cytotoxicity assay ( Figure 4 ).

[0053] L929 cells were cultured in 96-well plates using DMEM containing 10% FBS and 1% antibiotics, with each well containing approximately 1 × 10 5 After culturing the cells in a carbon dioxide incubator for 24 hours, different concentrations of peptides were added to the corresponding wells and incubated for another 24 hours under the same conditions. CCK-8 solution was then added and incubated for 1-4 hours to determine cell viability. The absorbance of the 96-well plate at A450 nm was measured using an ELISA reader, and the IC of the peptides on the cells was calculated using GraphPad Prism 10 software. 50 value.

[0054] 5. Plasma stability test ( Figure 5 ).

[0055] Mouse blood was obtained and centrifuged at 10,000 rpm for 5 min to prepare plasma, which was then filtered with a 0.22 μm filter for later use. Transformant 1 and Lydp-1 were incubated in mouse plasma at 37 °C for 0 to 12 h. Samples were taken at 0, 4, 8, and 12 h to determine the MIC of the peptide against Acinetobacter baumannii after incubation in plasma, and the change in MIC value was used to measure the stability of the peptide. The method for determining the peptide MIC was as described above.

[0056] Study Results

[0057] After solid phase chemical synthesis of modified compound 1, it was separated by RP-HPLC and identified by mass spectrometry. Figure 1 As shown, its purity is >95%, and the measured molecular weight of the modified form 1 is consistent with the theoretical molecular weight, indicating that it can be efficiently prepared by chemical synthesis.

[0058] like Figure 2 As shown, the antibacterial experiment of the present invention fully shows that the antimicrobial peptide modified body 1 of the present invention can kill a variety of bacteria, including Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Streptococcus mutans, and the MIC of the action is 4 μg / mL. Compared with the template peptide Lydp-1 (the MIC of the action on the above bacteria is 4 μg / mL), the activity of the template peptide is not reduced after miniPEG modification in modified body 1.

[0059] 100 μM modified peptide 1 had no significant effect on mouse erythrocytes, and its hemolytic activity was similar to that of the template peptide Lydp-1, indicating that its hemolytic activity was low ( Figure 3 ). Compared with the template peptide Lydp-1, it has stronger toxicity to mouse epithelial fibroblast L929 cells (IC 50 The IC value of the modified substance 1 on L929 cells was 19.2 μM. 50 The results of further plasma stability tests showed that PEG modification can reduce the mammalian cell toxicity of antimicrobial peptides (Figure 4). Figure 5 As shown in the figure, after incubation with mouse plasma for 0-8 h, the MIC value of the modified body 1 against Acinetobacter baumannii remained unchanged, and its MIC value gradually increased after 8 h, indicating that the modified body 1 can remain stable in mouse plasma within 8 h; for the template peptide Lydp-1, it can remain stable in mouse plasma within 4 h, indicating that the stability of the peptide after miniPEG modification has been significantly improved. In summary, the PEG-modified antimicrobial peptide of the present invention can maintain strong antibacterial activity while greatly improving the stability of the peptide and reducing the toxicity of the peptide, and has good drugability.

[0060] One embodiment of the present invention has been specifically described above, but the present invention is not limited to the described embodiment. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A PEG-modified polypeptide, characterized in that: The polypeptide is modified with miniPEG on the N-terminal amino group of the main chain based on the amino acid sequence of the antimicrobial peptide Lydp-1. The molecular weight of the miniPEG is 163Da. The sequence of the obtained antimicrobial peptide modification is: miniPEG-WDapAMDapIDipADapDipIKDap.

2. The PEG-modified polypeptide according to claim 1, characterized in that The antibacterial drug can be prepared for preventing or treating bacterial infectious diseases, and the bacteria include Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Streptococcus mutans.

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