An antibacterial peptide derivative, a synthetic method thereof and application thereof in resisting drug-resistant bacteria

Abstract

The application belongs to the technical field of medicines, and particularly relates to a polypeptide derivative based on polypeptide sC18 4b or a pharmaceutically acceptable salt thereof. Based on antibacterial peptide sC18 4b , hydrophobic modification and amino acid substitution are carried out, a series of derivative peptides with high antibacterial activity and low cytotoxicity are designed and synthesized, and the derivative peptides have good application prospects in resisting drug-resistant bacteria.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an antimicrobial peptide derivative, its synthesis method, and its application in combating drug-resistant bacteria. Background Technology

[0002] The overuse of antibiotics has led to the accelerated emergence of multidrug-resistant bacteria, making multidrug-resistant bacterial infections one of the greatest threats to human health. Therefore, strict and effective measures must be taken to curb the spread of drug-resistant strains and prevent their threat to human health. In terms of measures to curb drug-resistant infections, in addition to the rational use of antibiotics, the development of antimicrobial drugs that are less likely to induce bacterial resistance is particularly crucial. Unlike traditional antibiotics that target specific protein sites, antimicrobial peptides primarily exert their antimicrobial effect by directly disrupting the cell membrane structure, causing leakage of bacterial contents. This mechanism is rapid, does not require receptor mediation, and has broad-spectrum and lethal effects on bacteria. Compared to specific protein targets in bacteria, the bacterial cell membrane structure is more conserved and less prone to major mutations, making it difficult for bacteria to develop resistance to antimicrobial peptides. In summary, antimicrobial peptides possess broad-spectrum antimicrobial activity and are less likely to induce resistance, making them a promising next-generation antimicrobial drug to replace traditional antibiotics.

[0003] Most antimicrobial peptides are rich in cationic and hydrophobic amino acids, exhibiting an amphiphilic structure. Studies have shown that, within a certain range, the stronger the hydrophobicity and amphiphilicity of an antimicrobial peptide, the stronger its membrane-breaking and antibacterial abilities. Therefore, increasing the hydrophobicity (H) and hydrophobic torque (M) of antimicrobial peptides is beneficial. H Hydrophobicity and amphiphilicity are important means to enhance the antimicrobial activity of antimicrobial peptides. However, higher hydrophobicity and amphiphilicity are not always better; excessively high levels of these properties can increase the cytotoxicity and hemolytic toxicity of antimicrobial peptides, significantly reducing their safety. How to enhance the activity of antimicrobial peptides while maintaining or improving their safety is one of the key issues in the optimized design of antimicrobial peptides. Summary of the Invention

[0004] This invention provides a method based on the antimicrobial peptide sC18 4b The polypeptide derivative or its pharmaceutically acceptable salt, said polypeptide derivative having an amino acid sequence as shown in general formulas (1), (2), (3) and / or (4):

[0005] Z-O1X1Y1Y2Y3X2Y4Y5X3X4O2Y6X5Y7X6-B(1)

[0006] Z-O1X1Y1Y2X2X3Y3Y4X4X5O2Y5X6Y6Y7–B(2)

[0007] Z-O1X1X2Y1X3X4Y2Y3X5X6O2Y4X7Y5Y6–B(3)

[0008] Z-O1X1X2Y1X3X4Y2Y3X5X6O2X7X8Y4Y5–B(4)

[0009] Among them, the polypeptide derivative having the amino acid sequence of general formula (1) includes: 7 basic amino acids or amino acid derivatives with amino groups in the side chain, 6 hydrophobic amino acids or hydrophobic amino acid derivatives, 2 neutral and hydrophilic amino acids or neutral and hydrophilic amino acid derivatives, and five hydrophobic amino acids or hydrophobic amino acid derivatives are continuously distributed on the hydrophobic interface of the polypeptide derivative.

[0010] The polypeptide derivative having the amino acid sequence of general formula (2) includes: 7 basic amino acids or amino acid derivatives with amino groups in the side chain, 6 hydrophobic amino acids or hydrophobic amino acid derivatives, 2 neutral and hydrophilic amino acids or neutral and hydrophilic amino acid derivatives, wherein the six hydrophobic amino acids or hydrophobic amino acid derivatives are continuously distributed on the hydrophobic interface of the polypeptide derivative.

[0011] The polypeptide derivative having the amino acid sequence of general formula (3) includes: 6 basic amino acids or amino acid derivatives with amino groups in the side chain, 7 hydrophobic amino acids or hydrophobic amino acid derivatives, 2 neutral and hydrophilic amino acids or neutral and hydrophilic amino acid derivatives, wherein the seven hydrophobic amino acids or hydrophobic amino acid derivatives are continuously distributed on the hydrophobic interface of the polypeptide derivative.

[0012] The polypeptide derivative having the amino acid sequence of general formula (4) includes: 5 basic amino acids or amino acid derivatives with amino groups in the side chain, 8 hydrophobic amino acids or hydrophobic amino acid derivatives, 2 neutral and hydrophilic amino acids or neutral and hydrophilic amino acid derivatives, wherein eight hydrophobic amino acids or hydrophobic amino acid derivatives are continuously distributed on the hydrophobic interface of the polypeptide derivative.

[0013] Wherein, X1 to X8 may be the same or different, and each is independently selected from hydrophobic amino acids or hydrophobic amino acid derivatives; the hydrophobic amino acids or hydrophobic amino acid derivatives may be selected from one of Leu (L), Ile (I), Trp (W), Pro (P), Val (V), Ala (A), Met (M), naphthylalanine, 6-aminohexanoic acid or γ-aminobutyric acid;

[0014] Y1 to Y7 may be the same or different, and each is independently selected from basic amino acids or amino acid derivatives with an amino group in their side chain; the basic amino acids or amino acid derivatives with an amino group in their side chain may be selected from Lys(K), Arg(R), His(H), homoarginine or ornithine.

[0015] O1 and O2 may be the same or different, and each is independently selected from neutral, hydrophilic amino acids or neutral, hydrophilic amino acid derivatives; the neutral, hydrophilic amino acids or neutral, hydrophilic amino acid derivatives may be selected from Cys(C), Ser(S), Gly(G), Thr(T), Tyr(Y), Gln(Q), Asn(N), Lys(K), Arg(R), His(H), homoarginine or ornithine;

[0016] Z represents an N-terminal group, for example, Z is selected from NH2 or C. 1-20 Alkyl amide groups (e.g., AcNH), etc.;

[0017] B represents a C-terminal group, for example, B is selected from COOH or carboxyl derivatives, such as B being CONH2, etc.

[0018] According to an embodiment of the present invention, the polypeptide derivative or its pharmaceutically acceptable salt has the following amino acid sequence:

[0019] P-α: Ac-GLRKFLRKFFNKIKR-NH2;

[0020] P-β: Ac-GLFKFLRKFFNKIKR-NH2;

[0021] P-α-01: Ac-GLRKVLRKFVNKIKR-NH2;

[0022] P-α-02: Ac-GLRKWLRKFWNKIKR-NH2;

[0023] P-α-03: Ac-GLRKILRKFINKIKR-NH2;

[0024] P-α-04: Ac-GLRKLLRKFLNKIKR-NH2;

[0025] P-α-05: Ac-GLRKALRKFANKIKR-NH2;

[0026] P-α-06: Ac-GLRKMLRKFMNKIKR-NH2.

[0027] The present invention is based on antimicrobial peptide sC18 4b The synthesis of polypeptide derivatives includes at least one of the following three aspects:

[0028] 1) Replace the antimicrobial peptide sC18 4b The positions of arginine at position 15 and phenylalanine at position 5 in (Ac-GLRKRLRKFFNKIKF-NH2) were adjusted, thus altering the sC18 position. 4bThe spatial arrangement of hydrophobic and hydrophilic interfaces results in a larger hydrophobic moment (M). H The polypeptide Peptide Alpha (P-α);

[0029] 2) Replacing arginine at the third position in p-α with phenylalanine yields a compound with greater hydrophobicity (H) and hydrophobic torque (M). H The polypeptide Peptide Beta (P-β);

[0030] 3) By replacing the phenylalanine at positions 5 and 10 in P-α with different hydrophobic amino acids or hydrophobic amino acid derivatives, the hydrophobicity (H) and hydrophobic torque (M) are obtained. H Different peptides, such as Peptide Alpha n (P-α-n);

[0031] The general formulas for the sequences of polypeptides P-α, P-β, and P-α-n are as follows:

[0032] P-α:Ac-GLRKFLRKFFNKIKR-NH2

[0033] P-β:Ac-GLFKFLRKFFNKIKR-NH2

[0034] P-α-n: Ac-GLRKXLRKFXNKIKR-NH2

[0035] In P-α-n, when X is V (valine), n = 01; when X is W (tryptophan), n = 02; when X is I (isoleucine), n = 03; when X is L (leucine), n = 04; when X is A (alanine), n = 05; and when X is M (methionine), n = 06.

[0036] According to an embodiment of the present invention, the antimicrobial peptide sC18-based 4b The polypeptide derivatives were synthesized using a solid-phase synthesis method.

[0037] The present invention also provides the use of the said polypeptide derivative or its pharmaceutically acceptable salt in any of the following S1)-S4):

[0038] S1) Prepare products for the prevention and / or treatment of diseases caused by bacterial infections;

[0039] S2) Prevention and / or treatment of diseases caused by bacterial infections;

[0040] S3) Prepare products that are antibacterial, bactericidal, or antimicrobial;

[0041] S4) Antibacterial, bactericidal, or antimicrobial.

[0042] The present invention also provides a method for treating bacterial infection, comprising administering a patient a therapeutically effective amount of the polypeptide derivative or a pharmaceutically acceptable salt thereof, thereby treating the bacterial infection.

[0043] According to an embodiment of the present invention, the bacteria may be selected from at least one of Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Citrobacter freundii.

[0044] According to an embodiment of the present invention, the bacteria may be a multidrug-resistant strain selected from at least one of Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Citrobacter freundii.

[0045] The present invention also provides a method for antibacterial, sterilization or bacteriostatic treatment, comprising administering to a patient a therapeutically effective amount of the polypeptide derivative or a pharmaceutically acceptable salt thereof.

[0046] According to an embodiment of the present invention, the bacteria may be selected from at least one of Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Citrobacter freundii.

[0047] According to an embodiment of the present invention, the bacteria may be selected from at least one multidrug-resistant strain of Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Citrobacter freundii.

[0048] The present invention also provides an antibacterial pharmaceutical composition comprising the polypeptide derivative or a pharmaceutically acceptable salt thereof.

[0049] According to an embodiment of the present invention, the antimicrobial composition further includes at least one pharmaceutically acceptable carrier.

[0050] Beneficial effects

[0051] P-α and P-β compared to sC18 4b These studies achieved enhanced antibacterial activity against drug-resistant bacteria. P-α, in particular, exhibited lower cytotoxicity and hemolytic toxicity while simultaneously increasing activity. P-α-02 and P-α-04 further enhanced antibacterial activity compared to P-α, with P-α-04 maintaining low levels of cytotoxicity while increasing activity, achieving a balance between antibacterial activity and safety, and showing promising application prospects in the treatment of drug-resistant bacteria. Attached Figure Description

[0052] Figure 1 : peptide sC18 4b Spiral wheel diagrams of P-α, P-β, and P-α-n.

[0053] Figure 2 sC18 4b Results of the proliferation toxicity of P-α and P-β on 293T cells.

[0054] Figure 3 Results of the cytotoxic effects of P-α, P-α-02, and P-α-04 on the proliferation of 293T cells. Detailed Implementation

[0055] The technical solutions of this disclosure will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of this disclosure and should not be construed as limiting the scope of protection of this disclosure. All technologies implemented based on the above content of this disclosure are covered within the scope of protection intended by this disclosure.

[0056] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0057] Example:

[0058] Peptide synthesis:

[0059] This design uses solid-phase peptide synthesis (SPPS) to synthesize peptides, and the specific operation is as follows.

[0060] 1. Reagent preparation:

[0061] Silanization (SBA) solution: The silanizing agent is obtained by thoroughly mixing hexamethyldisilane, trimethylchlorosilane and pyridine in a volume ratio of 3:1:9.

[0062] Deprotection solution: 20% piperidine / N,N-dimethylformamide (DMF) solution.

[0063] Phenol solution: A phenol solution is obtained by mixing redistilled anhydrous phenol with anhydrous ethanol in a 4:1 ratio.

[0064] Ninhydrin solution: Weigh 0.5g of ninhydrin, transfer it to a 10ml volumetric flask, add anhydrous ethanol to the mark, and mix well to obtain ninhydrin solution.

[0065] Pyridine solution: Pyridine is redistilled to remove water.

[0066] Peptide lysis buffer: This reagent is prepared fresh before use. Add 82.5 ml of trifluoroacetic acid, 5 ml of anisole, 5 ml of m-methylphenol, 5 ml of water, and 2.5 ml of ethylenedithiol to an anhydrous reagent bottle and mix thoroughly to obtain the lysis buffer.

[0067] Amino acids and coupling solution: Weigh three times the amount of the resin (0.75 mmol) of the corresponding amino acid and 0.1 g of 1-hydroxybenzotriazole (HOBT) and 0.27 g of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) into a 10 mL centrifuge tube, add 5 mL of DMF and 150 μl of N,N-diisopropylethylamine (DIEA) and mix well.

[0068] 2. Synthesis process:

[0069] Silanization: A magnetic stirrer connected to a vacuum pump is placed into a clean reactor, which is then fixed to an iron stand with the magnetic stirrer positioned below. The silanizing reagent is added to the reactor and allowed to stand for 2 hours. The solution is then discarded, and the reactor is repeatedly rinsed with DMF.

[0070] Swelling of resin: Weigh 0.472 g (0.25 mmol) of Rink amide resin (loading capacity of 0.53 mmol / g) and add it to the reactor. Add 10 ml of dichloromethane (DCM) and stir to swell for 10 min. After the resin has expanded, turn on the vacuum pump to filter and dry it.

[0071] Deprotection: Add deprotecting agent, stir for 5 min, and then dry under vacuum. Add deprotecting agent again, stir for 20 min, and remove the Fmoc protecting group from the resin to expose the amino group. Wash the container and resin three times each with DMF, methanol, and DCM solutions alternately (the same applies below).

[0072] Amino acid detection: Dip a small amount of resin into a centrifuge tube using a capillary tube, add one drop each of phenol solution, ninhydrin solution, and pyridine solution, and place in a 110°C metal bath heating device for 3 minutes. Remove and observe. If the resin turns blue, the deprotection is successful and proceed to the next step. If there is no color change, the deprotection has failed. Add the deprotection reagent again and repeat the above steps.

[0073] Condensation: After the amino acid and coupling solution are thoroughly mixed and activated, the mixture is added to the reactor. The reactor is then stirred and sealed, and the reaction is carried out at room temperature for 4 hours. After the reaction is complete, the reaction solution is dried, and the resin is cleaned with DMF, methanol, and DCM. The detection process is repeated. If the resin shows no color reaction, the amino acid condensation is successful. If the resin turns blue, the condensation has failed or the reaction is incomplete. The reaction is repeated until the amino acid condensation is successful. Then, the deprotection process is repeated to couple the next cis amino acid until the last amino acid is condensed.

[0074] N-terminal acetylation: After removing the Fmoc protecting group, wash the resin three times and then acetylate the exposed N-terminal amino group. Add 2 ml of acetic anhydride and DIEA to the reactor, ensuring the resin is submerged. Stir and react at room temperature for 30 min. Then, dry the mixture and wash the resin. Repeat the testing process; if the resin becomes colorless, the acetylation is complete.

[0075] Pyrolysis: After repeated cleaning of the reactor and resin following the reaction, anhydrous diethyl ether was added, and the mixture was washed three times. The resin was then thoroughly dried under vacuum. The resin was transferred to a round-bottom flask, and peptide lysis buffer (10 mL / g) was added according to the mass of the resin after the reaction. The flask was capped and stirred. To prevent the reaction from becoming too vigorous, an ice bath was used for the first 30 minutes of the reaction, after which the ice bath was removed. The reaction was stirred at room temperature for 2.5-3 hours. After lysis, anhydrous diethyl ether (100 mL / g) was added according to the peptide mass, and the mixture was stirred rapidly for 0.5-1 hour. The mixture was then allowed to stand until the peptides were completely precipitated and poured into a G4 funnel. The resin and residual lysis buffer in the peptides were repeatedly washed with diethyl ether under vacuum. A new, clean vacuum flask was used, and the peptides in the funnel were dissolved in small amounts several times with double-distilled water until the peptides in the funnel were mostly dissolved and the remaining resin was in a sandy state.

[0076] Freeze-drying: Collect the crude peptide solution in the filtration flask, transfer it to a centrifuge tube and freeze it in liquid nitrogen. Then freeze-dry it in a freeze dryer for 24-48 hours until it is completely freeze-dried into powder to obtain crude peptide.

[0077] 3. Analysis and purification

[0078] Molecular weight determination: 0.1 mg of lyophilized crude peptide powder was dissolved in 100 μl of ultrapure water and analyzed by analytical high performance liquid chromatography (HPLC). The peak tip of the main peak was collected, and the molecular weight of the peptide was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOFMS). After confirming the correct molecular weight, the peptide was purified.

[0079] Peptide purification: Preparative high-performance liquid chromatography (HPLC) was used to purify the peptides, and analytical HPLC was used to analyze the purified peptides.

[0080] Chromatographic column selection: analytical high performance liquid chromatography column (C18), preparative high performance liquid chromatography column (C18).

[0081] Mobile phase preparation: Mobile phase A is a 1‰ trifluoroacetic acid (TFA) / water solution. After mixing, filter it through a 0.45μm aqueous phase filter membrane to remove impurities, and then place it in an ultrasonic instrument for 10 minutes to remove air bubbles.

[0082] Mobile phase B is a 1‰ TFA / (acetonitrile:water = 7:3) solution. After mixing, it is filtered through a 0.45μm organic phase filter membrane to remove impurities and then placed in an ultrasonic instrument for 10 minutes to remove air bubbles.

[0083] Based on the physicochemical properties of each target peptide, the experimental conditions for analysis and purification were selected and adjusted. Preparative high-performance liquid chromatography (HPLC) was used for gradient elution at room temperature to collect the target peptides, which were then further purified using analytical HPLC.

[0084] Purity analysis was performed by collecting peptide solutions with a purity greater than 95%, removing acetonitrile using a rotary evaporator at 37°C to obtain a pure peptide aqueous solution, which was then collected in a lyophilization bottle, lyophilized, and used to obtain pure peptide powder. The powder was collected, weighed, sealed in centrifuge tubes, and stored at -40°C.

[0085] The relevant information of the polypeptides prepared in this invention is shown in Table 1:

[0086] Table 1

[0087]

[0088] In vitro antibacterial activity evaluation: The in vitro antibacterial activity of the peptide against multidrug-resistant bacteria (MDR) was evaluated using the dilution method to determine the activity of sC18. 4b The minimum inhibitory concentration (MIC) of the derived peptides in Bacillus subtilis, Escherichia coli, Staphylococcus aureus (MDR), Acinetobacter baumannii (MDR), Klebsiella pneumoniae (MDR), Pseudomonas aeruginosa (MDR), and Citrobacter freundii (MDR).

[0089] Pipettes 50 μl of the drug solution into rows 2-8 of a 96-well plate. Take a pre-prepared 200 μM antimicrobial peptide solution, pipette 100 μl into each well of the first row, then use a twofold dilution method, pipetting 50 μl of the peptide solution from the first row into the second row, mixing well, and then pipetting the solution from the second row into the third row. Repeat this operation until the eighth row. Finally, take a new sterile sample container and add the diluted bacterial solution (concentration 5 × 10⁻⁶). 5 (CFU / mL) Use a pipette to add 50 μl of the solution sequentially to rows 1-8 of a 96-well plate. Mix the solution thoroughly with the bacterial culture, incubate at 37°C with shaking for 16 h at 180 rpm, and observe. The MIC value is defined as the lowest concentration of peptide in which no visible bacterial growth is observed in the well when the plate is placed under fluorescent light.

[0090] The experimental results are shown in Table 2.

[0091] Table 2

[0092]

[0093] Cell proliferation toxicity assay:

[0094] Human kidney epithelial cells (293T) were used in the cell proliferation toxicity assay.

[0095] Cells were cultured to the logarithmic growth phase to obtain a concentration of 10. 5Cell suspension per ml. Add 100 μl of cell suspension to 96-well cell culture plates and incubate at 37°C in a 5% CO2 sterile cell culture incubator for 24 h. After 24 h, aspirate the original culture medium from the wells, wash 1-2 times with PBS, add the prepared peptide solutions in basal medium, and incubate at 37°C for 12 h.

[0096] After 12 hours, remove the culture plate, pipette the peptide culture medium mixture, and wash three times with PBS. Protect from light, add CCK-8 working solution: 10% (v / v) CCK-8 / basal culture medium solution, mix thoroughly, and add 100 μl to each well of a 96-cell empty plate. Cover with aluminum foil and protect from light, then incubate for 1 hour. After the procedure, measure the absorbance (OD) value at 450 nm using a microplate reader in absorbance (Abs) mode. The cell viability percentage, or cell viability, is calculated using the following formula.

[0097] Cell survival percentage = [OD(pep) - OD(bla) / OD(neg) - OD(bla)] * 100%

[0098] OD(pep): Represents the absorbance value corresponding to the cell pore used for drug delivery.

[0099] OD(neg): Indicates the absorbance value of the negative control well.

[0100] OD(bla): Represents the absorbance value of the blank control well.

[0101] The experimental results are shown in Figure 2 , Figure 3 .

[0102] The above description provides an exemplary account of the implementation methods of the technical solution disclosed herein. It should be understood that the scope of protection of this disclosure is not limited to the above-described embodiments. Any modifications, equivalent substitutions, or improvements made by those skilled in the art within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this application.

Claims

1. The polypeptide or its pharmaceutically acceptable salt as described below, characterized in that, The polypeptide is selected from the amino acid sequence shown below: P-α: Ac-GLRKFLRKFFNKIKR-NH2; P-β: Ac-GLFKFLRKFFNKIKR-NH2; P-α-01: Ac-GLRKVLRKFVNKIKR-NH2; P-α-02: Ac-GLRKWLRKFWNKIKR-NH2; P-α-03: Ac-GLRKILRKFINKIKR-NH2; P-α-04: Ac-GLRKLLRKFLNKIKR-NH2; P-α-05: Ac-GLRKALRKFANKIKR-NH2; P-α-06: Ac-GLRKMLRKFMNKIKR-NH2.

2. The method for synthesizing the polypeptide or its pharmaceutically acceptable salt according to claim 1, wherein the polypeptide is synthesized by solid-phase synthesis.

3. The use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof in any of the following: S1) Prepare products for the prevention and / or treatment of diseases caused by bacterial infections; S3) Prepare products that are antibacterial, bactericidal, or antimicrobial; The bacteria are selected from at least one of Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Citrobacter freundii.

4. The application according to claim 3, characterized in that, The bacteria are selected from at least one multidrug-resistant strain of Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Citrobacter freundii.

5. An antibacterial pharmaceutical composition comprising the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof.

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