Preparation of PEGylated antibacterial peptide and application of PEGylated antibacterial peptide in bacterial infection

Abstract

The invention provides preparation of a PEGylated antibacterial peptide and application of the PEGylated antibacterial peptide in bacterial infection, belongs to the technical field of biomedical polypeptides, and particularly relates to PEGylation and preparation of amino acid sites containing amino groups in an antibacterial peptide sequence and application of the PEGylated antibacterial peptide in a preparation for preventing or treating bacterial infection related diseases. The PEGylated polypeptide designed in the invention is simple and convenient to operate, low in production cost and strong in antibacterial activity, can greatly improve the stability of the antibacterial peptide, can be used for preventing and treating diseases caused by various microbial infections, can be applied to various drug administration modes such as spraying, intraperitoneal injection, intravenous injection, nasal drop administration, spray administration and the like, and has a wide application prospect. The traditional Chinese medicine has a good treatment effect on diseases such as pneumonia, decayed teeth and the like caused by skin wound infection, sepsis and bacterial infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical polypeptides, and specifically relates to the application of antimicrobial peptides obtained by PEGylation in the prevention and treatment of diseases caused by various microbial infections, and in the treatment of skin wound infections, sepsis, pneumonia caused by bacterial infections, and dental caries. 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, reduce toxicity and enhance stability of polypeptides, 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 polypeptide for antimicrobial peptides has great practical significance for improving their drugability.

[0003] Based on this, taking the modified body 1 (named Lyco-13) in the embodiment of our previous patent application (application number: 2025101311400) as a template for PEG modification, it contains 13 amino acid residues, and its amino acid sequence is: WDapAMDapIDipIDapDipIKDap. Lyco-13 has potent broad-spectrum antibacterial activity, and the MIC against Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Streptococcus mutans is 4 μg / mL. Lyco-13 at 100 μM has almost no hemolytic activity on red blood cells. However, Lyco-13 is a linear polypeptide with poor stability, and it is necessary to modify and transform it to enhance its potential in antibacterial drug development. Based on this, this invention patent introduces small-molecular-weight PEG to optimize the performance such as the stability and preparation yield of the polypeptide while maximizing the retention of the antibacterial activity of the polypeptide, so as to keep the cost relatively low during its production and preparation. The conclusion of this miniPEG modification strategy for improving the toxicity and stability of antibacterial peptides has been confirmed in the previous patent (application number: 2025101311701). At the same time, aiming at the difficulties such as the influence of the position effect on the performance of antibacterial peptides existing in the current PEGylation of polypeptides, we carried out PEG250 modification at the main-chain amino group on the basis of Lyco-13, and carried out miniPEG modification after replacing amino acids with similar properties at the side-chain amino group sites in the sequence. It is expected to develop a promising therapeutic drug for bacterial infections using these antibacterial peptides. Summary of the Invention

[0004] The object of the present invention is to provide a method for improving the stability of antibacterial peptides by PEG modification and maximizing the non-influence on their antibacterial activity, and the application of polypeptides with antibacterial effects in the treatment of bacterial infectious diseases.

[0005] The above object of the invention is achieved by the following technical solutions: Based on the polypeptide Lyco-13 with strong antibacterial activity, miniPEG modification is carried out through site-directed mutagenesis of the sequence or at the side-chain amino group sites on the inherent amino acids in the sequence to design antibacterial peptides, and they are prepared by chemical synthesis for research in antibacterial aspects. The obtained PEG-modified polypeptides have a simple structure, are easy to quality control, can be efficiently prepared by chemical synthesis. More importantly, they have strong antibacterial activity, extremely weak toxicity, and high stability, and can be administered through multiple routes for the treatment of various diseases such as skin wound infections, sepsis, pneumonia caused by bacterial infections, and dental caries caused by bacterial infections.

[0006] The polypeptide provided by the present invention is based on the template peptide Lyco-13, and its amino acid sequence is: WDap 1 AMDap 2 IDipADap 3 DipIK 4 Dap 5Based on the amino acid sequence, PEGylation is carried out at the main-chain amino group and the side-chain amino group site of the Dap residue in the sequence.

[0007] In some embodiments, PEG modification is carried out on the N-terminal main-chain amino group of Lyco-13, and the PEG has an arbitrary molecular weight of ≤250 Da.

[0008] In some embodiments, the Dap at position 1 is selected from Dab, Orn or Lys, and the derivative obtained by carrying out miniPEG modification at this site after replacement.

[0009] In some embodiments, the Dap at position 2 is selected from Dab, Orn or Lys, and the derivative obtained by carrying out miniPEG modification at this site after replacement.

[0010] In some embodiments, the Dap at position 3 is selected from Dab, Orn or Lys, and the derivative obtained by carrying out miniPEG modification at this site after replacement.

[0011] In some embodiments, the Lys at position 4 is selected from Dap, Dab or Orn, and the derivative obtained by carrying out miniPEG modification at this site after replacement.

[0012] In some embodiments, the PEGylated polypeptide design composition includes any combination of all the PEGylated polypeptides.

[0013] On the one hand, the present invention relates to a composition of PEGylated polypeptides, which comprises any of the polypeptides as described above in the present invention.

[0014] On the other hand, the present invention relates to a PEGylated antibacterial peptide and its application in the preparation of a preparation for preventing or treating diseases related to bacterial infections.

[0015] In some embodiments, the bacteria include Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Streptococcus mutans. The bacterial infections include but are not limited to skin wound infections, sepsis, pneumonia caused by bacterial infections and dental caries and other diseases. The beneficial effects of the present invention are: different from most other PEG-modified antibacterial peptides, the polypeptide PEGylation of the present invention is simple in operation, short in sequence, and has the advantage of greatly reducing production costs. It can produce significant optimization effects in terms of antibacterial peptide stability, preparation yield, etc. Description of the Drawings

[0016] Figure 1 : Chemical structure diagrams of amino acids Dap, Dab, Orn and Lys.

[0017] Figure 2: Chemical structural diagrams of miniPEG and PEG250.

[0018] Figure 3 : Analysis of MIC and hemolytic activity after PEG250 modification of the main-chain amino group of Lyco-13.

[0019] Figure 4 : Analysis of MIC and hemolytic activity after miniPEG modification of the side-chain amino groups of Dap and Lys in the Lyco-13 sequence. Detailed implementation mode

[0020] The PEGylated polypeptides disclosed in the present invention can exist in the form of their hydrates or in the form containing their solvents (such as ethanol, DMSO, etc.), and can be used for crystallization. The compounds disclosed in the present invention can inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, the compounds of the present invention include solvated and unsolvated forms.

[0021] For amino acid substitution of the polypeptides of the present invention, natural amino acids in the polypeptides can be replaced with unnatural amino acids, and the unnatural amino acids include but are not limited to 2,3-diaminopropionic acid (Dap), 3,3-diphenylalanine (Dip), 2,4-diaminobutyric acid (Dab), 2,5-diaminopentanoic acid (Orn), and 4-phenylphenylalanine (Bip).

[0022] The term "PEG" refers to polyethylene glycol, which is a high molecular polymer and is the general term for ethylene glycol polymers containing α, ω-bis-terminal hydroxyl groups. The chemical formula is HO(CH2CH2O)nH. The PEG used in this patent for invention includes miniPEG (with a molecular weight of 163 Da, and the molecular structural formula is as Figure 2 shown) and PEG250 (with a molecular weight of 250 Da, and the molecular structural formula is as Figure 2 shown).

[0023] Any one of the 20 common L-amino acids in peptides synthesized in nature, namely 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).

[0024] The term "natural amino acid" refers to any one of the 20 L-amino acids commonly found in proteins and peptides existing in nature, namely 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). Examples

[0025] The present invention provides a total of 6 examples. In Example 1, PEG250 modification was carried out at the N-terminal main chain of the antimicrobial peptide Lyco-13, and the obtained modified body was named Lyco-13A. Examples 2-6 were respectively carrying out miniPEG modification on the side-chain amino groups at positions 1, 2, 3, 4, and 5 of Lyco-13, and the obtained modified bodies were named Lyco-13B - Lyco-13F respectively, where positions 1, 2, 3, and 5 are Dap residues, and position 4 is a Lys residue. The specific information of the said examples is shown in the following table.

[0026]

[0027] The above 6 modified bodies were efficiently prepared by solid-phase chemical synthesis of polypeptides, with a purity > 95%. The measured molecular weight of the modified bodies was consistent with the theoretical molecular weight, indicating that they can be efficiently prepared by chemical synthesis. The minimum inhibitory concentration (MIC) against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Streptococcus mutans, and the hemolytic activity of mouse red blood cells were systematically tested.

[0028] The chemical structure diagrams of the amino acids Dap, Dab, Orn, and Lys are as Figure 1 shown. The side chains of the said amino acids are all amino groups and are all positively charged amino acids. Theoretically, these 4 amino acids all contain two amino groups and one carboxyl group. Their α-amino groups and side-chain amino groups tend to be protonated and carry a positive charge at physiological pH, while the carboxyl group tends to dissociate and carry a negative charge. Therefore, the overall net charge is +1. It is generally recognized in the art that the overall charge of an antimicrobial peptide has an important impact on its activity. If the theoretical overall charge does not change, its activity usually does not change. As Figure 2As shown, the molecular weight of PEG used for PEGylation in the present invention is 250 Da, and the modification site is located at the N-terminal site of the antimicrobial peptide backbone. In the previous patent application (application number: 2025101311701), we confirmed that the miniPEG modification of Lyco-13 significantly prolonged the plasma stability of the antimicrobial peptide Lyco-13, and it is a generally recognized conclusion in the industry that PEG modification prolongs the stability of antimicrobial peptides. Therefore, in this invention patent, PEG modification has the effect of increasing the stability of polypeptides without significantly changing the activity of antimicrobial peptides. More importantly, under laboratory conditions, compared with the 20% yield of the preparation and synthesis sample of the main-chain miniPEG modification of Lyco-13 in the previous patent, the yield of the preparation after side-chain miniPEG modification has been significantly improved, between 25% and 35%, showing significant advantages.

[0029] The antibacterial experiment of the present invention fully shows that, as Figure 3 shown, the antimicrobial peptide Lyco-13A can kill a variety of bacteria, including Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Streptococcus mutans, and the MIC of the action is between 4 μg / mL, indicating that it has good antibacterial effects on these bacteria. Moreover, the hemolytic activity of 100 μM Lyco-13A on mouse red blood cells is relatively weak, and this property is the same as that of miniPEG-modified Lyco-13 in the previous patent. These results show that when PEG with a molecular weight less than or equal to 250 Da is used to modify the antimicrobial peptide Lyco-13, its antibacterial and hemolytic effects are not significant, proving that any molecular weight PEG with a molecular weight less than or equal to 250 Da can be used for modification at this site of the polypeptide. Then, as Figure 4 shown, we carried out miniPEG modification on the Dap and Lys sites in the Lyco-13 sequence, and measured the MIC and hemolytic activity of these examples. The antimicrobial peptides Lyco-13B~F can kill a variety of bacteria, including Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Streptococcus mutans, and the MIC of the action is between 4-16 μg / mL, indicating that they have good antibacterial effects on these bacteria. Moreover, the hemolytic activity of 100 μM of the polypeptide on mouse red blood cells is relatively weak (<10%). It is worth noting that when miniPEG modification is carried out at the Dap site at position 5, its activity against Escherichia coli is greatly weakened (MIC>128 μg / mL), which indicates that the effect of the above-mentioned examples is not produced by carrying out miniPEG modification at this site. In summary, the Dap, Dab, Orn, and Lys residues with amino side chains and positive charges can be substituted for the Dap at positions 1-3 and Lys at position 4 in the sequence, and miniPEG modification can be carried out. Research methods

[0030] 1. Solid-phase peptide chemical synthesis of antimicrobial peptides.

[0031] The present invention uses the Fmoc solid-phase peptide synthesis method to synthesize linear peptides. That is, when synthesizing, Rink resin is used as a carrier, and the synthesis is carried out in sequence from the C-terminus to the N-terminus. Fmoc (9-fluorenylmethoxycarbonyl) is used as the amino protecting group of amino acids to synthesize peptides. For PEGylation modification, the PEG modification of the polypeptide backbone is the same as the above amino acid steps. For the PEG modification of the amino side chain sites, amino acids protected by protecting groups such as Boc need to be selected. After the main chain synthesis is completed, the amino protecting group at this specific site is removed with hydrazine hydrate, and the side chain amino group is exposed to carry out miniPEG modification. After the coupling is completed, the newly synthesized peptide is cleaved from the Rink resin with a cleavage solution, and then precipitated with ice-cold diethyl ether and identified by mass spectrometry. According to the polypeptide synthesis scale of 0.1 mmol, the specific steps are as follows: (1)Swelling of the resin. Weigh 0.1 mmol of Rink resin and put it into a synthesis tube, add 3 mL of DMF, swell for 1 h and filter off the DMF in the tube by suction.

[0032] (2)Deprotection of the resin. Add 3 mL of 20% piperidine to the swollen resin and deprotect on a rotary shaker for 7 min, then suck out the piperidine. Add another 3 mL of 20% piperidine and deprotect on a rotary shaker for the second time for 8 min. After deprotection, suck dry the piperidine, and then wash with DMF 8 times.

[0033] (3)Activation of amino acids. While carrying out the first deprotection, activate the amino acids. Take one portion each of HATU and HOBT, add 0.75 mL of N-methylmorpholine to each, mix them and then add to the pre-weighed amino acids, and mix on a rotary mixer. The activation time is 15 - 20 min.

[0034] (4)Coupling of amino acids. Add the activated amino acids to the resin in step (2), shake to fully mix the resin and the amino acid solution, and carry out the coupling reaction on a rotary shaker for 1 h.

[0035] (5)Extension of the peptide chain. Repeat steps (2)-(4) until the coupling reaction of the last amino acid is completed.

[0036] (6)Cleavage of the peptide chain. After the coupling reaction of the last amino acid is completed, deprotect with 20% piperidine, then wash with DMF 8 times and wash with anhydrous methanol 4 - 5 times. After washing, suck dry and add 6 mL of cleavage solution. React in a rotary mixer for 2 h. Then collect the cleavage solution with a 50 mL centrifuge tube, add ice-cold diethyl ether to precipitate 2 times to obtain the crude polypeptide as the lower layer precipitate. Place the precipitated crude polypeptide in a fume hood for 2 - 3 min to volatilize the diethyl ether contained in the tube. Then, seal and store at 4 °C.

[0037] 2. Determination of minimum inhibitory concentration (MIC) ( Figure 3 and Figure 4 ).

[0038] The MIC of the polypeptide 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 the liquid medium, the bacterial suspension was diluted to 1×10 7 CFU / mL. 1 μL of the diluted bacterial suspension was added to a 96-well plate, and the polypeptide was diluted in the medium until the final concentration was 1 - 128 μg / mL. 99 μL of the polypeptide dilution solution at each concentration was added to the 96-well plate, and the 96-well plate was incubated in an anaerobic incubator at 37 °C for 18 - 24 h. The absorbance of the above 96-well plate at 600 nm was detected with an enzyme-linked immunosorbent assay (ELISA) reader. The inhibition rate of the polypeptide against the bacteria was calculated based on the absorbance OD 600.

[0039] 3. Hemolytic activity determination ( Figure 3 and Figure 4 ).

[0040] Fresh mouse red blood cells were extracted and diluted 25-fold with PBS buffer (pH = 7.4) to prepare a mouse red blood cell suspension of approximately 4%. Different concentrations of polypeptide solutions were prepared with PBS solution and placed in 1.5 mL sterile centrifuge tubes with a volume of 200 μL. 200 μL of the above mouse red blood cell suspension was added to this tube. After incubation at 37 °C for 1 h and centrifugation at 3500 rpm for 5 minutes, 100 μL of the supernatant was collected on a 96-well plate, and the absorbance at 490 nm was measured with an ELISA reader. The untreated red blood cell suspension in an equal volume of PBS solution and the red blood cell suspension treated with 0.1% Triton X-100 were used as negative control and positive control respectively.

Claims

1. A PEGylated antimicrobial peptide or a pharmaceutically acceptable salt thereof, characterized in that: The PEGylated antimicrobial peptide is based on the template peptide Lyco-13, whose sequence is: WDap1AMDap2IDipADap3DipIK4Dap5, and any molecular weight PEG modification with a molecular weight of ≤250 Da is carried out at the amino group of the main chain of the N-terminal end.

2. A PEGylated antimicrobial peptide or a pharmaceutically acceptable salt thereof, characterized in that: The PEGylated antimicrobial peptide is based on the template peptide Lyco-13, whose sequence is: WDap1AMDap2IDipADap3DipIK4Dap5, and the remaining Dap sites and Lys sites except the Dap residue at position 5 are modified with miniPEG (molecular weight of 163 Da).

3. The antimicrobial peptide or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: Except for the Dap residue at position 5, the remaining Dap sites and Lys sites of the antimicrobial peptide are independently selected from positively charged amino acids Dap, Dab, Orn or Lys whose side chains are all amino groups, and amino acid replacement is performed, and miniPEG (molecular weight 163 Da) modification is carried out on the amino acid side chain of the replacement site.

4. A PEGylated polypeptide composition, characterized in that: Comprising the polypeptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

5. The PEGylated polypeptide composition according to claim 4, characterized in that: The composition also includes a pharmaceutically acceptable carrier.

6. The PEGylated polypeptide composition according to claim 4, characterized in that: The composition also includes a pharmaceutically acceptable diluent.

7. The PEGylated polypeptide composition according to claim 4, characterized in that: The composition also includes a pharmaceutically acceptable adjuvant.

8. The PEGylated polypeptide composition according to claim 4, characterized in that The composition also includes a pharmaceutically acceptable vehicle.

9. Use of the PEGylated antimicrobial peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that: The preparation can be prepared for preventing or treating bacterial infectious diseases, and the bacteria include Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Streptococcus mutans.

10. The use of the polypeptide composition according to any one of claims 4 to 8, characterized in that: The preparation 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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