Low-toxic broad-spectrum antimicrobial peptide containing hydroxyl amino acid and application thereof

Abstract

This invention discloses a low-toxicity, broad-spectrum antimicrobial peptide containing hydroxyl amino acids and its applications. The amino acid sequence of the antimicrobial peptide is KWLKKWWYKL-NH2, labeled as peptide 21, and its amino acid sequence is shown in SEQ ID No. 1. Replacing all L-type amino acids in the peptide 21 sequence with D-type amino acids yields the antimicrobial peptide. D K D W D L D K D K D W D W D Y D K D L-NH2, labeled as peptide D-21. In vitro antibacterial experiments showed that peptide 21 and D-21 have broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, while exhibiting extremely low hemolytic toxicity and low likelihood of inducing drug resistance. In vivo acute toxicity experiments showed that peptide 21 and D-21 have higher in vivo safety compared to the control drug Polymyxin B. Therefore, they can be used in the preparation of clinical antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to novel low-toxicity broad-spectrum antimicrobial peptides containing hydroxy amino acids. This invention also relates to the application of said antimicrobial peptides in the preparation of antimicrobial drugs. Background Technology

[0002] Traditional antibiotics have played a vital role in treating infectious diseases, but their widespread overuse has led to the emergence of drug-resistant bacteria. Antimicrobial resistance is rapidly increasing globally, posing a serious threat to public health. In particular, the emergence of multidrug-resistant strains has brought enormous challenges to clinical treatment (Bangladesh Medical Research Council Bulletin, 2021, 47: 101–102). Therefore, there is an urgent need to develop novel antimicrobial strategies to overcome the limitations of existing antimicrobial drugs.

[0003] Antimicrobial peptides (AMPs), as key components of the organism's innate immune defense system, operate on a core mechanism that utilizes their cationic properties and amphiphilic structure to preferentially recognize and bind to negatively charged bacterial cell membranes via electrostatic attraction. This leads to physical disruption of the membrane's integrity, a process independent of interaction with specific protein targets, making it difficult for bacteria to develop resistance through simple mutations (The European Physical Journal: SpecialTopics, 2024, 233: 2981–2996). Furthermore, antimicrobial peptides possess broad-spectrum and rapid bactericidal properties, exhibiting significant activity against a variety of pathogens, including multidrug-resistant bacteria. These advantages make them highly promising candidates in the field of novel antibiotics (Molecules 2025, 30: 1529).

[0004] However, naturally occurring and traditionally designed antimicrobial peptides often suffer from drawbacks such as high hemolytic activity, significant cytotoxicity, and poor in vivo stability, limiting their clinical application (Military Medical Research, 2021, 8: 48). Therefore, a systematic sequence engineering design strategy is urgently needed to achieve a balance between high activity, low toxicity, and high stability. Studies have shown that the bioactivity of antimicrobial peptides is closely related to their physicochemical parameters such as net charge, hydrophobicity, and amphiphilicity. Precisely controlling these parameters through sequence engineering is key to achieving a balance between high activity and low toxicity (International Journal of Molecular Sciences, 2024, 25: 10821). The introduction of hydroxy amino acids (such as serine, threonine, and tyrosine) can effectively regulate the hydrophilicity and biocompatibility of peptides, while the introduction of D-type amino acids can improve the in vivo stability of peptides (ACS Omega, 2024, 9:26030−26049; Acta Biochimica et Biophysica Sinica, 2017, 49(10): 916–925). Based on this, the applicant designed a novel low-toxicity, broad-spectrum antimicrobial peptide containing hydroxy amino acids and further prepared its D-type enantiomer in order to obtain an antimicrobial peptide with high activity, low toxicity, and high stability. Summary of the Invention

[0005] One of the objectives of this invention is to provide a low-toxicity, broad-spectrum antimicrobial peptide containing hydroxy amino acids.

[0006] A second objective of this invention is to provide the D-enantiomer of the aforementioned antimicrobial peptide.

[0007] A third objective of this invention is to provide the application of the above-mentioned antimicrobial peptides in the preparation of antimicrobial drugs.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: (I) Design of a low-toxicity, broad-spectrum antimicrobial peptide containing hydroxy amino acids This invention first provides a class of antimicrobial peptides designed de novo based on a full-leucine backbone. The total amino acid sequence length of this series of antimicrobial peptides is 10 amino acids, and the basic backbone structure is a linear full-leucine sequence. Structurally, positions 1, 4, 5, 8, and 9 of the sequence are designated as hydrophilic, while the remaining positions (positions 2, 3, 6, 7, and 10) are hydrophobic.

[0009] The specific design rules are as follows: 1. The hydrophilic surface is introduced with 1 to 3 hydroxy amino acids (threonine, serine, or tyrosine), and the hydrophilic surface contains 2 to 4 lysine amino acids, satisfying the following combination: When there is one hydroxy amino acid, there can be two, three, or four lysines; when there are two hydroxy amino acids, there can be two or three lysines; when there are three hydroxy amino acids, there are two lysines; the remaining positions in the hydrophilic surface, except for the hydroxy amino acid and lysine, are filled with histidine. 2. The amino acid composition of the hydrophobic side is dynamically adjusted according to the amount of lysine on the hydrophilic side: When there are 2 lysine residues, the hydrophobic surface is entirely composed of leucine; when there are 3 lysine residues, 1 tryptophan residue is introduced into the hydrophobic surface, and the rest are leucine; when there are 4 lysine residues, 3 tryptophan residues are introduced into the hydrophobic surface, and the rest are leucine. The overall hydrophobicity (H) was calculated using the HeliQuest website, and the design threshold H was set to be ≥ 0.6 in this invention.

[0010] Based on the above rules, a total of 27 candidate peptides were designed and obtained.

[0011] (ii) Preferred antimicrobial peptides and their D-enantiomers After screening for in vitro antibacterial activity and toxicity (screening criteria: minimum inhibitory concentration (MIC) ≤ 16 μM against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, and drug concentration ≥ 100 μM that causes 10% hemolysis in mouse erythrocytes), a preferred antimicrobial peptide was obtained, named peptide 21, with the amino acid sequence: Lys-Trp-Leu-Lys-Lys-Trp-Trp-Tyr-Lys-Leu-NH2 (SEQ ID No. 1).

[0012] To further improve the in vivo stability of the antimicrobial peptide, this invention provides the full D-enantiomer of peptide 21, named D-21, with the following amino acid sequence: D-Lys-D-Trp-D-Leu-D-Lys-D-Lys-D-Trp-D-Trp-D-Tyr-D-Lys-D-Leu-NH2.

[0013] (III) Synthesis of antimicrobial peptides The antimicrobial peptide of this invention is synthesized using the classic Fmoc solid-phase synthesis method. Rink Amide MBHA resin is used as the raw material, and corresponding Fmoc-protected amino acids are sequentially coupled from the C-terminus to the N-terminus of the peptide chain (Fmoc-D-type amino acids are used for the synthesis of D-21). The coupling reagent is an HBTU / HOBt / DIEA system, and secondary amines are determined using the ninhydrin colorimetric method. After peptide synthesis, the peptide is cleaved from the resin using a cleavage reagent (TFA:TIS:H2O=95:2.5:2.5), precipitated with diethyl ether, and freeze-dried to obtain the crude peptide. Finally, the crude peptide is purified by reversed-phase high-performance liquid chromatography to obtain the target antimicrobial peptide with a purity ≥ 95%.

[0014] (iv) Application of low-toxicity broad-spectrum antimicrobial peptides containing hydroxy amino acids in the preparation of antimicrobial drugs 1. In vitro antibacterial test The minimum inhibitory concentration (MIC) of antimicrobial peptides against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) was determined using the micro-broth dilution method. Polymyxin B and vancomycin were used as positive controls. The experiment was repeated in triplicate, and the results are shown in Table 1.

[0015] Table 1. Minimum inhibitory concentrations of the antimicrobial peptides of this invention against common standard strains. Table 1 shows that the antimicrobial peptide 21 and its D-isomer D-21 of the present invention exhibit strong inhibitory effects on all tested Gram-positive and Gram-negative bacteria, with a minimum inhibitory concentration in the range of 2-16 μM, and have broad-spectrum antimicrobial activity.

[0016] 2. Hemolytic activity test To evaluate the toxicity of the synthesized antimicrobial peptides to mammalian erythrocytes, fresh anticoagulated blood was collected from mice, and an 8% erythrocyte suspension was prepared. This suspension was incubated with different concentrations (1-256 μM) of the antimicrobial peptides at 37 °C for 1 hour, and the absorbance at 490 nm was measured to calculate the hemolysis rate. 1% Triton X-100 was used as a positive control (100% hemolysis), and PBS was used as a negative control (0% hemolysis). Results are shown below. Figure 3 .

[0017] Figure 3 The results showed that even at a concentration of 256 μM, the hemolysis rate of peptide 21 and D-21 on mouse erythrocytes was less than 5%, indicating extremely low hemolytic toxicity and good biocompatibility.

[0018] 3. Serum stability test To investigate the stability of the antimicrobial peptides of this invention in mouse serum, RP-HPLC was used to monitor the degradation of peptides 21 and D-21 with mouse serum (10% serum and 100% serum) at different time points after co-incubation at 37 °C, and the serum half-life was calculated. The results are shown in [Figure number missing]. Figure 4 and Figure 5 .

[0019] Figure 4 and Figure 5The results showed that the half-life of peptide 21 in 10% serum was 14.49 h, and the half-life in 100% serum was 2.44 h. Compared with L-peptide 21, the all-D-type antimicrobial peptide D-21 had a half-life greater than 24 h in both 10% and 100% serum, which was significantly better than that of L-peptide, demonstrating excellent serum stability.

[0020] 4. Induction of drug resistance experiment To investigate whether the antimicrobial peptides synthesized in this invention can induce bacterial resistance, the resistance-inducing effect of antimicrobial peptides 21 and D-21 on Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 was determined after 20 days of continuous treatment. Antibiotics Rifampin, Gentamicin, Vancomycin, and Polymyxin B were used as controls. The results are shown in [Figure number missing]. Figure 6 and Figure 7 .

[0021] Figure 6 The results showed that antimicrobial peptides 21 and D-21 did not induce resistance to Staphylococcus aureus ATCC 25923 after 20 days of continuous treatment. However, Rifampin and Gentamicin rapidly developed resistance. Rifampin's MIC increased by 4096 times after 20 days, and Gentamicin's MIC increased by 1024 times after 20 days. Figure 7 The results showed that antimicrobial peptides 21 and D-21 did not induce resistance in *Escherichia coli* ATCC 25922 after 20 days of continuous treatment, while Rifampin and Gentamicin rapidly developed resistance, with the MIC increasing 128-fold after 20 days. The resistance induction experiment results indicated that, compared with commonly used clinical antibiotics rifampin and gentamicin, peptides 21 and D-21 showed no significant change in MIC values ​​(change fold < 4-fold) after 20 days of continuous treatment with *Staphylococcus aureus* or *Escherichia coli*, indicating that the antimicrobial peptides of this invention are unlikely to induce bacterial resistance.

[0022] 5. Acute toxicity test in mice The experimental mice were male BALB / c mice, weighing 18-22g, and were fed in accordance with the Lanzhou University Laboratory Animal Ethics Management Regulations.

[0023] Acute toxicity LD50 50 Preliminary experiment: Three animals per group were given a single intraperitoneal injection of antimicrobial peptides 21 and D-21 dissolved in physiological saline. Mortality was observed within 7 days. Polymyxin B was used as a control to explore the minimum dose that caused 100% mortality and the maximum dose that caused 0% mortality, and to determine the upper and lower limits of the dose.

[0024] Acute toxicity LD50 50Formal Experiment: Based on preliminary experiments, we measured the LD of peptide 21. 50 The dosage range is 60-120 mg / kg, and the LD50 of peptide D-21 is... 50 The dosage range for peptide 21 was 30-70 mg / kg, and the dosage range for the control drug Polymyxin B was 15-20 mg / kg. Therefore, based on the dosage range of peptide 21, we calculated the common ratio of its groups to be 1.4142, dividing it into three groups of 60, 84.8, and 120 mg / kg. The common ratio of peptide D-21 groups was 1.5275, dividing it into three groups of 30, 45.8, and 70 mg / kg. The common ratio of the control drug Polymyxin B groups was 1.1547, dividing it into three groups of 15, 17.3, and 20 mg / kg, which were then used in the subsequent formal experiments. According to the grouping, we administered a single intraperitoneal injection of the corresponding dose of peptide 21, D-21, or Polymyxin B to each group of mice. Mortality was observed within 7 days, and the LD50 was calculated. 50 The results are shown in Table 2. Figure 8 , Figure 9 and Figure 10 .

[0025] Table 2. LD50 of the antimicrobial peptides and Polymyxin B of this invention in BALB / c mice. 50 Figure 8 The results showed that after intraperitoneal injection of 120 mg / kg peptide 21 into mice, all 10 mice died within 2 days, with a mortality rate of 100%. After intraperitoneal injection of 84.8 mg / kg peptide 21 into mice, 4 out of 10 mice died within 7 days, with a mortality rate of 40%. After intraperitoneal injection of 60 mg / kg peptide 21 into mice, all mice survived within 7 days. Figure 9 The results showed that after intraperitoneal injection of 70 mg / kg peptide D-21 into mice, all 10 mice died within 2 days, with a mortality rate of 100%. After intraperitoneal injection of 45.8 mg / kg peptide D-21 into mice, 5 out of 10 mice died within 7 days, with a mortality rate of 50%. After intraperitoneal injection of 30 mg / kg peptide D-21 into mice, 1 out of 10 mice died within 7 days, with a mortality rate of 10%. Figure 10The results showed that after intraperitoneal injection of 20 mg / kg of the control drug Polymyxin B into mice, all 10 mice died within 20 minutes, with a mortality rate of 100%. After intraperitoneal injection of 17.3 mg / kg of the control drug Polymyxin B into mice, 5 out of 10 mice died within 7 days, with a mortality rate of 50%. After intraperitoneal injection of 15 mg / kg of the control drug Polymyxin B into mice, 2 out of 10 mice died within 7 days, with a mortality rate of 20%. Table 2 shows the calculated LD50 of the antimicrobial peptides 21, D-21, and the control drug Polymyxin B in BALB / c mice during the acute toxicity test. 50 Value, LD value of the antimicrobial peptide 21 of the present invention 50 The LD50 value is approximately 5.25 times that of the control drug Polymyxin B, indicating that the antimicrobial peptide D-21 of this invention has an LD50 value of approximately 5.25 times that of the control drug Polymyxin B. 50 The value was approximately 2.60 times that of the control drug Polymyxin B, indicating that the antimicrobial peptide of the present invention has higher safety in mice compared with the control drug Polymyxin B.

[0026] As can be seen from the above experiments and data, the low-toxicity broad-spectrum antimicrobial peptide containing hydroxy amino acids provided by the present invention can be used in the preparation of drugs for the prevention or treatment of bacterial infections, where the bacteria are Gram-positive or Gram-negative bacteria.

[0027] Furthermore, the low-toxicity broad-spectrum antimicrobial peptide containing hydroxy amino acids provided by this invention can be used to prepare drugs for treating bacterial drug-resistant infections.

[0028] Furthermore, based on the above experimental data, the present invention also provides a pharmaceutical composition or pharmaceutical preparation for the prevention or treatment of bacterial infections, wherein the pharmaceutical composition or pharmaceutical preparation includes the peptide 21 or D-21 described in the present invention, and pharmaceutically applicable excipients; the bacteria are Gram-positive or Gram-negative bacteria; and the pharmaceutical preparation is preferably an injection solution.

[0029] In summary, this invention provides a low-toxicity, broad-spectrum antimicrobial peptide, peptide 21, containing a hydroxy amino acid, and its full D-enantiomer, D-21. In vitro antibacterial experiments show that the antimicrobial peptide of this invention exhibits broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, while possessing extremely low hemolytic toxicity and low likelihood of inducing drug resistance. In vivo acute toxicity experiments indicate that the antimicrobial peptides 21 and D-21 of this invention have higher in vivo safety compared to the control drug Polymyxin B. Therefore, the antimicrobial peptide of this invention can be used in the preparation of clinical antimicrobial drugs. Attached Figure Description

[0030] Figure 1 This is the mass spectrum of antimicrobial peptide 21; Figure 2This is the mass spectrum of the antimicrobial peptide D-21; Figure 3 Results of the hemolytic activity of antimicrobial peptide 21 and D-21 on mouse erythrocytes; Figure 4 Stability curves of antimicrobial peptides 21 and D-21 in 10% mouse serum; Figure 5 Stability curves of antimicrobial peptides 21 and D-21 in 100% mouse serum; Figure 6 The results show the induced drug resistance after continuous treatment of antimicrobial peptides 21 and D-21 with Staphylococcus aureus ATCC 25923 for 20 days. Figure 7 The results of inducing drug resistance after continuous treatment of antimicrobial peptides 21 and D-21 of the present invention with Escherichia coli ATCC 25922 for 20 days; Figure 8 The results of an in vivo acute toxicity test of antimicrobial peptide 21 in BALB / c mice; Figure 9 The results of an in vivo acute toxicity test of antimicrobial peptide D-21 in BALB / c mice; Figure 10 This is the result of an in vivo acute toxicity test of the control drug Polymyxin B in BALB / c mice. Detailed Implementation

[0031] The present invention will be further described below through specific embodiments.

[0032] Example 1: Synthesis of antimicrobial peptide 21 (1) Activation of resin Weigh 0.43 g of Rink Amide MBHA resin (substitution value 0.46 mmol / g), add it to a polypeptide solid-phase synthesizer, swell with dichloromethane (DCM) for 30 min, wash three times with N,N-dimethylformamide (DMF), and confirm the resin is qualified (colorless) by ninhydrin colorimetric method.

[0033] (2) Synthesis of peptide chains The Fmoc protecting groups on the swollen resin were removed using a DMF solution containing 20% ​​piperidine. The resin was washed four times with DMF, and the ninhydrin test showed a blue color. Three times the excess of Fmoc-Leu, three times the excess of HOBt and HBTU, and six times the excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred for 1 hour. The ninhydrin test showed that the resin was colorless and transparent, indicating successful condensation, yielding Fmoc-Leu-MBHA.

[0034] Following the above method, Lys, Tyr, Trp, Trp, Lys, Lys, Leu, Trp, Lys are condensed sequentially to obtain Fmoc-Lys-Trp-Leu-Lys-Lys-Trp-Trp-Tyr-Lys-Leu-MBHA.

[0035] (3) Peptide cleavage The obtained Fmoc-Lys-Trp-Leu-Lys-Lys-Trp-Trp-Tyr-Lys-Leu-MBHA was washed with DMF solution containing 20% ​​piperidine to remove the Fmoc protecting group, followed by washing with DCM and methanol sequentially, and the resin was thoroughly dried. 10 mL of cleavage reagent (TFA:TIS:water = 95:2.5:2.5 (v:v:v)) was added and reacted for 3 h. After extraction with diethyl ether, the mixture was freeze-dried to obtain crude peptide 21.

[0036] (4) Polypeptide purification and identification The crude peptide was purified by RP-HPLC under the following conditions: mobile phase A: 0.1% TFA / acetonitrile, mobile phase B: 0.1% TFA / water. A gradient elution method was used at a flow rate of 8 mL / min. The eluent from the target peak was collected, lyophilized, and peptide 21 was obtained. Its mass spectrum is shown below. Figure 1 .

[0037] The theoretical molecular weight of peptide 21 is 1477.85, which is consistent with the mass spectrometry identification result, proving that the structure of the antimicrobial peptide is correct.

[0038] Example 2: Synthesis of antimicrobial peptide D-21 (1) Activation of resin Same as Example 1.

[0039] (2) Synthesis of peptide chains Following the method described in Example 1, D-Leu, D-Lys, D-Tyr, D-Trp, D-Trp, D-Lys, D-Lys, D-Leu, D-Trp, and D-Lys are sequentially condensed to obtain Fmoc-D-Lys-D-Trp-D-Leu-D-Lys-D-Lys-D-Trp-D-Trp-D-Tyr-D-Lys-D -Leu-MBHA.

[0040] (3) Peptide cleavage Same as Example 1.

[0041] (4) Polypeptide purification and identification Same as in Example 1, peptide D-21 was obtained, and its mass spectrum is shown below. Figure 2 As shown.

[0042] The theoretical molecular weight calculation result of peptide D-21 is 1477.85, which is consistent with the mass spectrometry identification result, proving that the structure of the antimicrobial peptide is correct.

Claims

1. A low-toxicity, broad-spectrum antimicrobial peptide containing hydroxy amino acids, characterized in that, The amino acid sequence of the antimicrobial peptide is KWLKKWWYKL-NH2, labeled as peptide 21, and its amino acid sequence is shown in SEQ ID No.

1.

2. The low-toxicity, broad-spectrum antimicrobial peptide containing hydroxyl amino acids as described in claim 1, characterized in that, The antimicrobial peptide is obtained by replacing all L-amino acids in peptide 21 with D-amino acids, and its amino acid sequence is as follows: D K D W D L D K D K D W D W D Y D K D L-NH2, labeled as peptide D-21.

3. The use of the low-toxicity, broad-spectrum antimicrobial peptide containing hydroxyl amino acids as described in any one of claims 1-2 in the preparation of drugs for the prevention or treatment of bacterial infections, characterized in that, The bacteria are either Gram-positive or Gram-negative.

4. The use of the low-toxicity broad-spectrum antimicrobial peptide containing hydroxy amino acids as described in claim 3 in the preparation of a medicament for treating bacterial drug-resistant infections.

5. A pharmaceutical composition for the prevention or treatment of bacterial infections, characterized in that, The pharmaceutical composition comprises the antimicrobial peptide of claim 1 or 2, and pharmaceutically applicable excipients; the bacteria are Gram-positive or Gram-negative bacteria.

6. A pharmaceutical preparation for the prevention or treatment of bacterial infections, characterized in that, The pharmaceutical preparation is an injection containing the antimicrobial peptide of claim 1 or 2, and the bacteria are Gram-positive or Gram-negative bacteria.

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