Low-toxicity broad-spectrum antibacterial peptides containing central PXXP hinge structure and application of low-toxicity broad-spectrum antibacterial peptides

Abstract

The invention discloses a group of low-toxicity broad-spectrum antibacterial peptides containing a central PXXP hinge structure and application of the low-toxicity broad-spectrum antibacterial peptides. The low-toxicity broad-spectrum antibacterial peptide is obtained by adopting a de novo design mode, using lysine (Lys, K) with positive charges and hydrophobic tryptophan (Trp, W), alternately, repeatedly and symmetrically arranging two amino acids, and introducing a PXXP hinge structure containing proline into the symmetric center of an antibacterial peptide sequence. The structural general formula is (KW) nPXXP (WK) n, XX = KK, n = 2-4, or XX = WK, n = 2-3. An in-vitro antibacterial experiment shows that the antibacterial peptide disclosed by the invention has broad-spectrum antibacterial activity; the preferred antibacterial peptide has the advantages of low hemolysis toxicity and high serum stability. Induced drug resistance experiments show that the preferred antibacterial peptide has the characteristic of low drug resistance generation. An in-vivo acute toxicity experiment result shows that compared with a contrast drug Polymyxin B, the preferable antibacterial peptide disclosed by the invention has higher in-vivo safety. Therefore, the compound has a good application prospect in preparation of clinical antibacterial drugs, and is expected to become a novel antibiotic candidate drug.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and relates to a group of low-toxic broad-spectrum antimicrobial peptides containing a central PXXP hinge structure. The invention also relates to the application of the antimicrobial peptides in the preparation of clinical antimicrobial drugs. Background Art

[0002] Traditional antibiotics can effectively treat infectious diseases due to their strong antibacterial activity, but the abuse of antibiotics has led to the emergence of various drug-resistant bacteria, and the prevalence of antimicrobial resistance has risen rapidly, posing a major threat to public health. In particular, the emergence of multidrug-resistant bacteria poses a huge challenge to clinical treatment (Clinical Infectious Diseases. 2019, 68(5): 873-884.). Since the antimicrobial resistance of multidrug-resistant strains can be transferred between strains and spread rapidly around the world, new treatment strategies are urgently needed to overcome the limitations of currently available antimicrobial drugs.

[0003] Antimicrobial peptides (AMPs) are a class of small molecule peptides with broad-spectrum antimicrobial properties. They can quickly kill targets and are part of the immune defense system of organisms. They have become promising candidates for the development of new antibiotics and are widely studied for the treatment of infectious diseases associated with multidrug-resistant pathogens (Nature Reviews Drug Discovery. 2020, 19 (5): 311-332.). The most common structural characteristics of AMPs are cationicity and amphipathicity. The positive charge carried by cationic antimicrobial peptides electrostatically interacts with the negatively charged bacterial surface structure and adsorbs together; the hydrophilic region in the amphipathic structure of the peptide can be inserted into the hydrophobic core of the bacterial cell membrane, destroying the integrity of the bacterial cell membrane, thereby causing cell death (Journal of Molecular Biology. 2019, 431 (18): 3547-3567.). Since the structure and charge properties of the bacterial cell surface cannot be changed, AMPs rarely induce drug resistance and have a significant bactericidal effect on multidrug-resistant bacteria.

[0004] However, in order to further apply antimicrobial peptides in clinical practice, it is necessary to overcome the shortcomings of natural AMPs, such as poor stability, high synthesis cost, and high toxicity. Common modification strategies include improving the amphiphilicity of peptides, improving hydrophobicity, introducing fatty acids or non-natural amino acid substitutions, etc. Wang (Journal of medicinal chemistry, 2018, 61 (9): 3889-3907.) et al. reported an imperfect amphiphilic palindromic peptide I6 (RRIRIIIRIRR-NH2) that exhibited strong antibacterial activity against fungi and bacteria. At the same time, the study showed that most AMPs with incomplete amphiphilic conformations exhibited better broad-spectrum antibacterial activity. Di (Science advances, 2020, 6 (18): eaay6817.) et al. replaced L-Val with D-Val to increase resistance to protease hydrolysis and reduce toxicity to mammalian cells. In addition, the secondary structure is also an important parameter affecting the activity of antimicrobial peptides. On the one hand, the secondary structure makes an important contribution to maintaining the antibacterial activity of AMPs. On the other hand, regular and stable secondary structures usually lead to increased hemolytic activity and cytotoxicity of AMPs (Frontiers inmicrobiology, 2018, 9: 2832). Proline (Pro) can be used as an ideal secondary structure disruptor, and the selectivity of AMPs may be improved by inserting Pro into the peptide sequence. Tripathi et al. (Acta biomaterialia, 2017, 57: 170-86.) used Pro to selectively replace Phe in α-helical peptides in an attempt to improve cell selectivity. The results showed that Pro substitution can effectively reduce the cytotoxicity of AMP to mammalian cells without affecting its antibacterial and anti-endotoxin properties. On this basis, the applicant independently designed an amphiphilic antimicrobial peptide formed by alternating repetitions of positively charged Lys and hydrophobic Trp, and introduced a hinge structure containing Pro at its central position to improve the selectivity of the antimicrobial peptide, in order to obtain an antimicrobial peptide with high activity, low toxicity and high stability. Summary of the invention

[0005] The object of the present invention is to provide a group of low-toxic broad-spectrum antimicrobial peptides containing a central PXXP hinge structure.

[0006] Another object of the present invention is to provide the use of the above antimicrobial peptide in the preparation of clinical antimicrobial drugs.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] (I) Design of low-toxic broad-spectrum antimicrobial peptides containing a central PXXP hinge structure

[0009] The low-toxic broad-spectrum antimicrobial peptide provided by the present invention contains a central PXXP hinge structure, and its general structural formula is (KW) n PXXP(WK) n (where XX=KK, n=2-4 or XX=WK, n=2-3).

[0010] Specifically, the present invention provides a group of broad-spectrum antimicrobial peptides containing a central hinge structure, and the amino acid sequences thereof are as follows:

[0011] Lys-Trp-Lys-Trp-Pro-Lys-Lys-Pro-Trp-Lys-Trp-Lys-NH2, labeled (KW)2PK;

[0012] or Lys-Trp-Lys-Trp-Lys-Trp-Pro-Lys-Lys-Pro-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Lys-NH2, labeled (KW)3PK;

[0013] Or: Lys-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Pro-Lys-Lys-Pro-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Lys-NH2, labeled (KW)4PK;

[0014] or Lys-Trp-Lys-Trp-Pro-Trp-Lys-Pro-Trp-Lys-Trp-Lys-Trp-Lys-NH2, labeled (KW)2PWK;

[0015] or Lys-Trp-Lys-Trp-Lys-Trp-Pro-Trp-Lys-Pro-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Lys-NH2, labeled (KW)3PWK.

[0016] (II) Synthesis of low-toxic broad-spectrum antimicrobial peptides containing a central PXXP hinge structure

[0017] The low-toxic broad-spectrum antimicrobial peptide containing a central PXXP hinge structure is synthesized using Rink-MBHA Resin as a raw material and a classical polypeptide Fmoc solid phase synthesis method. In the polypeptide synthesis process, amino acid coupling is performed using HOBt / HBTU as a condensing agent, secondary amine is detected using a ninhydrin colorimetric method, and polypeptides connected to MBHA resins are sequentially coupled according to the polypeptide sequence, and the polypeptides are cut from the MBHA resin and purified by HPLC to obtain the peptides.

[0018] (III) Application of low-toxic broad-spectrum antimicrobial peptides containing a central PXXP hinge structure

[0019] 1. In vitro antibacterial experiment

[0020] The minimum inhibitory concentration (MIC) of antimicrobial peptides against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii) was determined by the classic micro-serial two-fold dilution method. The antibiotics Rifampin, Vancomycin, and Polymyxin B were used as positive controls, and the experiment was repeated three times in parallel. The results are shown in Table 1.

[0021] Table 1 Minimum inhibitory concentration of the antimicrobial peptides of the present invention against common standard strains

[0022]

[0023] The results in Table 1 show that the antimicrobial peptide of the present invention has a strong inhibitory effect on both Gram-positive bacteria and Gram-negative bacteria, showing a broad-spectrum antimicrobial activity. The effect on Gram-negative bacteria is better than Vancomycin; the effect on Gram-positive bacteria is similar to that of Polymyxin B.

[0024] 2. Hemolytic activity test

[0025] In order to investigate the toxicity of the antimicrobial peptide synthesized in the present invention to normal mammalian cells, the hemolysis of the antimicrobial peptide synthesized in the present invention and mouse red blood cells after incubation for 1 hour was tested. Figure 6 .

[0026] Figure 6 The results showed that the peptides containing the PKKP hinge structure showed no hemolytic activity, and the hemolytic activity was close to 0 even at a concentration of 256 μM, indicating good safety. The peptide containing the PWKP hinge structure showed obvious hemolytic activity when the (KW) unit was repeated 4 times. However, when the (KW) unit was repeated 2 and 3 times, the hemolytic activity was less than 10% at a concentration of 256 μM, indicating good safety. The results of hemolytic activity prove that the antimicrobial peptides (KW)2PK, (KW)3PK, (KW)4PK, (KW)2PWK and (KW)3PWK of the present invention have good safety.

[0027] 3. Serum stability test

[0028] In order to investigate the stability of the preferred antimicrobial peptides (KW)3PK and (KW)3PWK in serum, RP-HPLC was used to monitor the degradation of hinge-containing antimicrobial peptides (KW)3PK and (KW)3PWK at different time points when they were co-incubated with mouse serum at 37°C, and the serum half-life was determined. The results are shown in Table 1. Figure 7 .

[0029] Figure 7 The results showed that the serum half-life of (KW)3PK was 18.32h, and the serum half-life of (KW)3PWK was 15.74h. This result shows that the antimicrobial peptide containing the central PXXP hinge structure synthesized by the present invention has good stability in mouse serum and is not easily degraded by enzymes in the serum and loses its antimicrobial activity.

[0030] 4. Induced resistance experiment

[0031] A major problem with the clinical use of antibiotics is that they can easily cause bacteria to develop drug resistance. Due to the membrane dissolving mechanism of antimicrobial peptides, it is difficult for bacteria to develop drug resistance. Therefore, in order to study whether the antimicrobial peptides of the present invention have the characteristic of being less likely to develop drug resistance, the induced drug resistance of the antimicrobial peptides (KW) 3PK and (KW) 3PWK after continuous action with Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC27853 for 20 days was measured, and the antibiotics Gentamicin and Polymyxin B were used as controls. The results are shown in Figure 8 and Fig. 9 .

[0032] Generally speaking, it is normal for MIC to fluctuate within the range of 1-4 times. If the MIC is greater than 4 times, it indicates that antibiotic resistance has developed. Figure 8 The results showed that the antimicrobial peptides (KW)3PK and (KW)3PWK did not develop resistance after 20 days of continuous action with Staphylococcus aureus ATCC 25923, while the antibiotic Gentamicin quickly developed resistance, with the MIC increasing by 32768 times after 20 days, and the MIC of Polymyxin B increased by 8 times after 20 days. Fig. 9 The results showed that the antimicrobial peptides (KW) 3PK and (KW) 3PWK did not develop drug resistance after continuous action with Pseudomonas aeruginosa ATCC 27853 for 20 days, while the antibiotic Gentamicin quickly developed drug resistance, with the MIC increasing by 32768 times after 20 days, and the MIC of Polymyxin B increased by 64 times after 20 days. It is worth noting that the mixture of antimicrobial peptides (KW) 3PK and (KW) 3PWK and the antibiotic Gentamicin in a 1:1 ratio did not develop drug resistance. This result shows that compared with traditional antibiotics, the antimicrobial peptides synthesized by the present invention are not easy to induce bacteria to develop drug resistance, and when used in combination with traditional antibiotics, they can also reduce the development of antibiotic resistance.

[0033] 5. Acute toxicity test in mice

[0034] The experimental mice were BALB / C mice, male, 18-22 g, and fed according to the Lanzhou University Laboratory Animal Ethics Management Methods.

[0035] Preliminary acute toxicity experiment in mice: 3 mice in each group were intraperitoneally injected with antimicrobial peptides (KW)3PK and (KW)3PWK dissolved in physiological saline, and the mortality was observed for 7 days. Polymyxin B was used as a control to explore the minimum dose that caused 100% mortality in mice and the maximum dose that caused 0% mortality, and to determine the upper and lower limits of the dose.

[0036] Acute toxicity test in mice: The LD of (KW)3PK was obtained based on the results of the preliminary experiment 50 The dose range is 41.5-80 mg / kg, and the LD of (KW)3PWK 50 The dose range is 58.5-100 mg / kg, and the LD of the control drug Polymyxin B is 50 The dosage range is 15-24 mg / kg. According to the dosage range of (KW)3PK, the common ratio of its grouping is calculated to be 1.3884, and it is divided into 3 groups. The dosages from low to high are 41.5 mg / kg, 57.7 mg / kg, and 80 mg / kg. The common ratio of (KW)3PWK is 1.3074, and it is divided into 3 groups. The dosages from low to high are 58.5 mg / kg, 76.6 mg / kg, and 100 mg / kg. The common ratio of the control drug Polymyxin B is 1.2649, and it is divided into 3 groups. The dosages from low to high are 15 mg / kg, 18.9 mg / kg, and 24 mg / kg. Eight mice were administered each dose of each drug, and each group of mice was intraperitoneally injected with the corresponding dose of (KW)3PK, (KW)3PWK, and Polymyxin B once according to the grouping situation. The death was observed within 7 days, and the LD 50 The results are shown in Table 2. Fig.10 , Fig.11 and Fig.12 .

[0037] Table 2 LD of antimicrobial peptides and Polymyxin B in BALB / C mice 50

[0038]

[0039] Fig.10 The results showed that after intraperitoneal injection of 80 mg / kg of (KW)3PK to mice, all 8 mice died within 2 days, with a mortality rate of 100%. After intraperitoneal injection of 57.7 mg / kg of (KW)3PK to mice, 6 of 8 mice died within 7 days, with a mortality rate of 75%. After intraperitoneal injection of 41.5 mg / kg of (KW)3PK to mice, all mice survived within 7 days. Fig.11The results showed that after intraperitoneal injection of 100 mg / kg of (KW)3PWK to mice, all 8 mice died within 2 days, with a mortality rate of 100%. After intraperitoneal injection of 76.6 mg / kg of (KW)3PWK to mice, 3 of the 8 mice died within 7 days, with a mortality rate of 37.5%. After intraperitoneal injection of 58.5 mg / kg of (KW)3PWK to mice, all mice survived within 7 days. Fig.12 The results showed that after the mice were intraperitoneally injected with 24 mg / kg of the control drug Polymyxin B, all 8 mice died within 1 hour, with a mortality rate of 100%. After the mice were intraperitoneally injected with 18.9 mg / kg of the control drug Polymyxin B, 2 of the 8 mice died within 7 days, with a mortality rate of 25%. After the mice were intraperitoneally injected with 15 mg / kg of the control drug Polymyxin B, all the mice survived within 7 days. The results in Table 2 show the calculated LD values ​​of the acute toxicity experiments of the antimicrobial peptides (KW)3PK, (KW)3PWK and the control drug Polymyxin B in BALB / C mice. 50 The LD value of the antimicrobial peptide (KW) 3PK of the present invention 50 The value is about 2.65 times that of the control drug Polymyxin B. The LD 50 The value is about 3.95 times that of the control drug Polymyxin B. The results show that the antimicrobial peptide of the present invention has higher safety in mice than the control drug Polymyxin B.

[0040] In summary, the present invention adopts a de novo design approach, uses positively charged lysine (Lys, K) and hydrophobic tryptophan (Trp, W), and arranges the two amino acids alternately and repeatedly symmetrically, and introduces a proline-containing PXXP hinge structure at the symmetry center of the antimicrobial peptide sequence to obtain a low-toxic broad-spectrum antimicrobial peptide. In vitro antibacterial experiments show that the antimicrobial peptide of the present invention has a broad-spectrum antimicrobial activity. The preferred peptides (KW) 3PK and (KW) 3PWK have the advantages of low hemolytic toxicity, high serum stability, and low drug resistance. The results of in vivo acute toxicity experiments show that the antimicrobial peptides (KW) 3PK and (KW) 3PWK of the present invention have higher in vivo safety than the control drug Polymyxin B. Therefore, it has a good application prospect in the preparation of clinical antibacterial drugs and is expected to become a new antibiotic candidate. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the mass spectrum of the antimicrobial peptide (KW) 2PK of the present invention;

[0042] Figure 2 is the mass spectrum of the antimicrobial peptide (KW) 3PK of the present invention;

[0043] Figure 3 is the mass spectrum of the antimicrobial peptide (KW) 4PK of the present invention;

[0044] Figure 4 is the mass spectrum of the antimicrobial peptide (KW) 2PWK of the present invention;

[0045] Figure 5 is the mass spectrum of the antimicrobial peptide (KW) 3PWK of the present invention;

[0046] Figure 6 The results of the hemolytic activity of the antimicrobial peptide of the present invention on mouse erythrocytes after incubation with erythrocytes for 1 hour;

[0047] Figure 7 The serum stability results after the antimicrobial peptide of the present invention was co-incubated with mouse serum;

[0048] Figure 8 The results of drug resistance induction after the antimicrobial peptide of the present invention and the antimicrobial peptide of the present invention mixed with the antibiotic Gentamicin acted on Staphylococcus aureus ATCC 25923 for 20 consecutive days;

[0049] Fig. 9 The results of drug resistance induction after the antimicrobial peptide of the present invention and the antimicrobial peptide of the present invention mixed with the antibiotic Gentamicin and continuously acted on Pseudomonas aeruginosa ATCC 27853 for 20 days;

[0050] Fig.10 This is a graph showing the results of an in vivo acute toxicity experiment of the antimicrobial peptide (KW) 3PK of the present invention on BALB / C mice;

[0051] Fig.11 This is a graph showing the results of an in vivo acute toxicity experiment of the antimicrobial peptide (KW) 3PWK of the present invention on BALB / C mice;

[0052] Fig.12 This is a graph showing the results of an in vivo acute toxicity experiment of the control drug Polymyxin B on BALB / C mice. DETAILED DESCRIPTION

[0053] The synthesis of the low-toxic broad-spectrum antimicrobial peptide containing a central PXXP hinge structure of the present invention is further described below through specific examples.

[0054] Example 1: Synthesis of antimicrobial peptide (KW) 2PK

[0055] (1) Activation of resin

[0056] Weigh 0.42 g of MBHA resin (0.48 mmol / g), put it into the bottom of the solid phase synthesizer, add DCM to swell for 30 min, drain, wash with DMF 3 times, drain, and identify the resin with ninhydrin colorimetry. If the resin is colorless, it can be used normally.

[0057] (2) Synthesis of Fmoc-(KW)2PK-MBHA

[0058] The swollen resin was washed 3 times with a DMF solution containing 20% ​​piperidine, and then drained. After washing 3 times with DMF, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Leu, HOBt, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that Lys condensation was successful, and Fmoc-Lys-MBHA was obtained.

[0059] According to the above method, Lys, Trp, Lys, Trp, Pro, Lys, Lys, Pro, Trp, Lys, Trp, and Lys are condensed in sequence to obtain Fmoc-Lys-Trp-Lys-Trp-Pro-Lys-Lys-Pro-Trp-Lys-Trp-Lys-Trp-Lys-MBHA.

[0060] (3) Peptide cleavage

[0061] The obtained Fmoc-Lys-Trp-Lys-Trp-Pro-Lys-Lys-Pro-Trp-Lys-Trp-Lys-MBHA was washed 3 times with a DMF solution containing 20% ​​piperidine, and then dried. After washing with DMF 3 times, the resin was dried and identified by the ninhydrin colorimetric method. If the resin was dark blue-purple, it indicated that the Fmoc protecting group had been removed. After the resin was washed alternately with DCM and methanol 3 times, it was pumped with a vacuum pump for 1 hour. After the resin was dried into dry particles, 10mL of a cleavage reagent (TFA:Tris:water=9.5:0.25:0.25 (v:v:v)) was added to react for 3 hours, and stirred slowly every 20 minutes. After the reaction time was reached, the cleavage reagent was collected, extracted with ether, and freeze-dried.

[0062] (4) Peptide purification

[0063] The freeze-dried peptide was purified by RP-HPLC. The RP-HPLC purification conditions were as follows: mobile phase A: 0.1% TFA / acetonitrile, mobile phase B: 0.1% TFA / water, linear gradient elution was used, the target peak effluent was collected, and freeze-dried to obtain the antimicrobial peptide (KW) 2PK, the mass spectrum of which is shown in Figure 1 shown.

[0064] The theoretical calculation result of the molecular weight of (KW)2PK is 1725, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

[0065] Example 2: Synthesis of antimicrobial peptide (KW) 3PK

[0066] (1) Activation of resin

[0067] Same as Example 1.

[0068] (2) Synthesis of Fmoc-(KW)3PK-MBHA

[0069] The same method as in Example 1 was used to sequentially condense Lys, Trp, Lys, Trp, Lys, Trp, Pro, Lys, Lys, Pro, Trp, Lys, Trp, Lys, Trp, Lys, to obtain

[0070] Fmoc-Lys-Trp-Lys-Trp-Lys-Trp-Pro-Lys-Lys-Pro-Trp-Lys-Trp-Lys-Trp-Lys-MBHA.

[0071] (3) Peptide cleavage

[0072] Same as Example 1.

[0073] (4) Peptide purification

[0074] The same method as in Example 1 was used to obtain (KW)3PK, the mass spectrum of which is shown in Figure 2 shown.

[0075] The theoretical calculation result of the molecular weight of (KW)3PK is 2353, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

[0076] Example 3: Synthesis of antimicrobial peptide (KW) 4PK

[0077] (1) Activation of resin

[0078] Same as Example 1.

[0079] (2) Synthesis of Fmoc-(KW)4PK-MBHA

[0080] The same method as in Example 1 was used to sequentially condense Lys, Trp, Lys, Trp, Lys, Trp, Lys, Trp, Pro, Lys, Lys, Pro, Trp, Lys, Trp, Lys, Trp, Lys, Trp, Lys, Trp, Lys to obtain

[0081] Fmoc-Lys-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Pro-Lys-Lys-Pro-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Lys-MBH A.

[0082] (3) Peptide cleavage

[0083] Same as Example 1.

[0084] (4) Peptide purification

[0085] Same as Example 1, (KW)4PK was obtained, and its mass spectrum is as follows Figure 3 shown.

[0086] The theoretical calculation result of the molecular weight of (KW)4PK is 2982, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

[0087] Example 4: Synthesis of antimicrobial peptide (KW) 2PWK

[0088] (1) Activation of resin

[0089] Same as Example 1.

[0090] (2) Synthesis of Fmoc-(KW)2PWK-MBHA

[0091] Lys, Trp, Lys, Trp, Pro, Lys, Trp, Pro, Trp, Lys, Trp, Lys, Trp, Lys were condensed in sequence according to the method of Example 1 to obtain Fmoc-Lys-Trp-Lys-Trp-Pro-Trp-Lys-Pro-Trp-Lys-Trp-Lys-Trp-Lys-MBHA.

[0092] (3) Peptide cleavage

[0093] Same as Example 1.

[0094] (4) Peptide purification

[0095] Same as in Example 1, (KW)2PWK was obtained, and its mass spectrum is as follows Figure 4 shown.

[0096] The theoretical calculation result of the molecular weight of (KW)2PWK is 1783, which is consistent with the mass spectrometry identification result, proving that the structure of the antimicrobial peptide is correct.

[0097] Example 5: Synthesis of antimicrobial peptide (KW) 3PWK

[0098] (1) Activation of resin

[0099] Same as Example 1.

[0100] (2) Synthesis of Fmoc-(KW)3PWK-MBHA

[0101] The same method as in Example 1 was used to sequentially condense Lys, Trp, Lys, Trp, Lys, Trp, Pro, Lys, Trp, Pro, Trp, Lys, Trp, Lys, Trp, Lys, to obtain

[0102] Fmoc-Lys-Trp-Lys-Trp-Lys-Trp-Pro-Trp-Lys-Pro-Trp-Lys-Trp-Lys-Trp-Lys-MBHA.

[0103] (3) Peptide cleavage

[0104] Same as Example 1.

[0105] (4) Peptide purification

[0106] Same as Example 1, (KW)3PWK was obtained, and its mass spectrum is as follows Figure 5 shown.

[0107] The theoretical calculation result of the molecular weight of (KW)3PWK is 2411, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

Claims

1. A group of low-toxic broad-spectrum antimicrobial peptides containing a central PXXP hinge structure, characterized in that: The low-toxic broad-spectrum antimicrobial peptide is obtained by alternating and repeating positively charged Lys and hydrophobic Trp to form an amphiphilic antimicrobial peptide, and then introducing a hinge structure containing Pro at the center thereof. The general structural formula is: (KW) n PXXP(WK) n , where XX=KK, n=2-4 or XX=WK, n=2-3; When XX=KK, it is recorded as (KW) n PK, n=2-4, whose amino acid sequence is shown in SEQ ID No.1-3; When XX=WK, it is recorded as (KW) n PWK, n=2-3, its amino acid sequence is shown in SEQ ID No.4-5.

2. A group of low-toxic broad-spectrum antimicrobial peptides containing a central PXXP hinge structure as claimed in claim 1, characterized in that: The low-toxic broad-spectrum antimicrobial peptide is (KW)3PK or (KW)3PWK.

3. A group of low-toxic broad-spectrum antimicrobial peptides containing a central PXXP hinge structure as claimed in claim 2, characterized in that: The low-toxic broad-spectrum antimicrobial peptide is (KW)3PK.

4. Use of the low-toxic broad-spectrum antimicrobial peptide containing a central PXXP hinge structure as described in any one of claims 1 to 3 in the preparation of clinical antimicrobial drugs.

5. Use of the low-toxic broad-spectrum antimicrobial peptide containing a central PXXP hinge structure as claimed in claim 4 in the preparation of clinical antimicrobial drugs, characterized in that: The bacteria inhibited by the low-toxic broad-spectrum antimicrobial peptide are Gram-positive bacteria or Gram-negative bacteria.

6. Use of the low-toxic broad-spectrum antimicrobial peptide containing a central PXXP hinge structure as claimed in claim 5 in the preparation of clinical antimicrobial drugs, characterized in that: The Gram-positive bacteria are Staphylococcus aureus and Bacillus subtilis, and the Gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii.

7. Use of the low-toxic broad-spectrum antimicrobial peptide containing a central PXXP hinge structure as claimed in claim 5 in the preparation of clinical antimicrobial drugs, characterized in that: The bacteria inhibited by the low-toxic broad-spectrum antimicrobial peptide are Gram-negative bacteria.

8. Use of a low-toxic broad-spectrum antimicrobial peptide containing a central PXXP hinge structure as claimed in any one of claims 4 to 6 in the preparation of clinical antimicrobial drugs, characterized in that: The antimicrobial peptide (KW)3PK or (KW)3PWK is used in combination with the antibiotic Gentamicin to reduce the development of Gentamicin resistance.

9. Use of the low-toxic broad-spectrum antimicrobial peptide containing a central PXXP hinge structure as claimed in claim 8 in the preparation of clinical antimicrobial drugs, characterized in that: When the antimicrobial peptide (KW)3PK or (KW)3PWK is used in combination with the antibiotic Gentamicin, the mixing ratio is 1:1.

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